The codon usage bias of chloroplast genome affects the genes expression efficiency, which provides important information for chloroplast genetic engineering and species genetic improvement. To determine the codon usage characteristic of the chloroplast genome and its main influencing factors in pineapple (Ananas comosus), the codon usage pattern and its preference of pineapple chloroplast genome were analyzed using bioinformatics softwares. The average GC content of codons of pineapple chloroplast genes was 38.31%, the average GC content of codons from the first position to the third position was 46.78%, 39.61% and 28.53%, respectively, and the average GC contents at the first and second positions of codons were significantly higher than that at the third position. The value range of effective number of codons (ENC) was 38.48—61.00, and its average value was 47.21, indicating weak codon bias. GC1 was significantly correlated with GC2, GCall was significantly positively correlated with GC1, GC2 and GC3, and GC3 was not significantly correlated with GC1 and GC2. ENC was not significantly correlated with GC1, but significantly and extremely significantly correlated with GC2 and GC3, respectively. Codon number (N) was only significantly correlated with GC3, which indicating that the base composition of the third position in the three positions of the codon mainly affects codon number. RSCU analysis showed that among the 29 codons with RSCU>1, 12 codons ended with A, 16 codons ended with U, and 1 codon ended with G. Neutrality plot analysis showed that there was not significant correlation between GC12 and GC3, and the correlation coefficient and regression coefficient was 0.065 and 0.085, respectively. ENC-plot analysis showed most genes were distributed near the standard curve, and most ENC ratios were distributed in the interval of -0.05—0.05. PR2-plot analysis showed that all genes were unevenly distributed in the four regions of the PR2 plan, and the use frequency of pyrimidine T/C at the third position of codon was higher than that of purine A/G. Bias analysis of Neutrality plot, ENC-plot and PR2-plot suggested that natural selection and mutation as the main factors affect the codon usage bias of pineapple chloroplast genome. Analysis of optimal codons and RSCU showed that most of the 29 codons with RSCU>1 and the 18 optimal codons ended with A or U. The results provide scientific basis for codon optimization of exogenous genes and improvement of their expression efficiency.
Pepper (Capsicum spp.) is widely used as food, spice, decoration, and medicine. Multiple cultivated Capsicum species belong to important vegetable and spice crops. The analysis of the chloroplast genome of Capsicum has important theoretical and practical value for clarifying the genetic diversity of Capsicum germplasm resources and cultivating and improving cultivated varieties. The availability of complete chloroplast genome sequences has offered enormous opportunity to researchers to study the details of gene number, characterization, phylogenetic relationships and diversification of Capsicum species. The complete chloroplast genome sequences of 13 Capsicum species were downloaded from the NCBI database, and analyzed by online softwares, such as REPuter, MISA, and mVISTA. The chloroplast genome of Capsicum had a conserved quadripartite structure, a pair of inverted repeats (IRs) separated a large single copy (LSC) region and a small single copy (SSC) region. The size of chloroplast genome sequences ranged from 156 583 bp (C. lycianthoides)-158 077 bp (C. frutescens). A total of 113 unique genes were predicted, including 79 protein-coding genes, 30 tRNA genes, and four rRNA genes. About 60-127 pairs of repeats were identified in Capsicum chloroplast genomes, and the proportion of forward repeats and palindromic repeats and 30-39 bp repeats were all high in the chloroplast genomes. About 150-164 potential SSRs were reported in Capsicum chloroplast genomes, and SSRs mainly consisted of mononucleotide and dinucleotide repeats, and most of the repeats were composed of A or T base. Comparative genomics of the 13 Capsicum species revealed a high sequence similarity in genome-wide organization and a lower sequence divergence in the IRs than LSC and SSC. The fragments of rps18-rpl20, rps4-trnT, ndhF-rpl32-trnL and ycf1 were identified as variable regions in Capsicum species, and the nucleotide variability of LSC and SSC was higher than that of IR. The phylogenetic analysis showed that the main cultivated species C. annuum was closely related to C. tovarii, and C. chinense was closely related to C. frutescens and C. eximium. The SSR and variable hotspots identified could be used as molecular markers for species identification and genetic diversity analysis of Capsicum species. Overall, our study would highlight the taxonomic utility of the chloroplast genomes in Capsicum species, and lay a foundation for the evolution, genetic diversity, conservation and biological breeding of Capsicum species.
Actinomycetes belonging to Streptomyces are the most important antibiotic producing bacteria and also the actinomycete genus with the largest number of species. Many streptomycete type strains can be assigned to distinct evolutionary clades on the 16S rRNA gene phylogenetic tree, and the S. violaceusniger 16S rRNA gene clade is one of them. Members of the clade form a greyish yellow substrate mycelium and a grayish aerial hyphae on oatmeal agar that develop into spiral chains of rugose ornamented spores that become black on prolonged incubation. Members of the S. violaceusniger 16S rRNA gene clade have abundant biological activities and produce antibacterial metabolites, antiparasitic metabolites, antitumour compounds and immunosuppressants as important sources for the development and utilization of new antibiotics. S. solisilvae HNM0141Tis a type strain assigned to the S. violaceusniger 16S rRNA gene clade. Here, we present the complete genome sequence of it, which is generated using the third sequencing technology and consists of a linear chromosome of 11 639 536 bp with a GC content of 71.3%, 9363 protein coding genes(CDS), 18 rRNA genes, and 67 tRNA genes. The identified CDS were classified into 6376, 5886, and 3246 categories based on clusters of orthologous genes of proteins (COG), gene ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) designation, respectively. In addition to the general function prediction only, most of the genes involved in transcription, amino acid transport and metabolism, carbohydrate transport and metabolism and secondary metabolic biosynthesis and metabolism were accounted for 12.91%,10.25%, 8.89% and 7.59% respectively. Forty-eight putative secondary metabolite biosynthetic gene clusters were found in the genome using the online antiSMASH software, including eight PKS, seven NRPS, fourteen hybrid gene clusters, six terpenes, three siderophores, two butyrolactones, one NAPAA, one ectoine, one lanthipeptide-class-i, one ladderane, one indole, one RiPP-like, one hserlactone and one redox-cofactor. Polyketone (PKS) and non-ribosomal peptide (NRPS) gene clusters (including heterozygous types) were abundant and accounted for more than 50% of the total gene clusters. Seven putative gene clusters showed 100% similarities to known gene clusters: pristinol, ectoine, geosmin, desferrioxamin B, echoside A/B/C/D/E, nigericin, and coelichelin gene clusters. Thirty-four putative gene clusters showed 2%-96% similarities to known gene clusters. The remaining seven gene clusters could not be matched to known gene clusters. Genome-wide analysis showed that S. solisilvae HNM0141T had strong ability of the biosynthesis of secondary metabolites, indicated that it should be good research potential for mining of the novel antibiotics.
Cassava (Manihot esculenta Crantz) is an important food and energy crop in the tropics and subtropics, and improving cassava production is essential for the development of the cassava industry. The development of cassava storage root directly affects its yield, and different adversity stresses can affect the development of cassava storage root. Therefore, the analysis of cassava storage root development mechanism can help to achieve high cassava yield through molecular breeding, as well as to obtain excellent germplasm with certain resistance quality and enhance the adaptability of cassava, so as to expand the extension of cassava cultivation. Hexose transport proteins (STPs) are a subfamily of MSTs that regulate plant growth and development as well as abiotic stresses by transporting sugars. The MeSTP7 and MeSTP15 genes have been identified to play an important role in sugar accumulation during tuber development and in response to abiotic stresses. To obtain MeSTP7 MeSTP15 cassava double mutants, CRISPR/Cas9 double gene editing vectors for MeSTP7 and MeSTP15 were successfully constructed by designing the sgRNAs of both target genes, MeSTP7 and MeSTP15, using the online software CRISPR-P v2.0. After transformation of the editing vector into Agrobacterium tumefaciens LBA4404, ‘SC8’ cassava brittle embryonic healing tissues were infiltrated, and MeSTP7 and MeSTP15 were successfully edited by Sanger sequencing analysis, while the potential off-target sites were not edited, indicating that the dual editing vector could edit both MeSTP7 and MeSTP15 genes without causing off-target phenomenon. The rooted seedlings were then screened with 15 mmol/L Hygromycin B to produce rooted seedlings, which were then characterized detection to obtain positive transgenic cassava plants with the expression frame of the double-edited vector. The genomes of positive plants were used as the templates for PCR amplification of 100 bp nucleotide sequences before and after each target site of the two genes, and after Hi-TOM sequencing, the results showed that there were 26 strains with mutations, including 23 MeSTP7/15 double mutants, 2 MeSTP7 single mutants and 1 MeSTP15 single mutant, and the editing types were mostly single. The types of editing were mostly single base deletions or insertions, with a small percentage of deletions of large segments of bases. Our preliminary observations of the mutant variants in histoponic flasks revealed that root growth was inhibited and plants were dwarfed in both the single and double mutants, and that the MeSTP7 and MeSTP15 double mutants were more severely damaged. These results not only lay the foundation for further analysis of the mechanism of cassava tuber development, but also provide material for obtaining new germplasm for cassava disease and stress resistance.
Fusarium oxysporum f. sp. cubense (Foc) causes Fusarium wilt (Panama disease), one of the most devastating diseases of banana (Musa spp.). Foc race 4 (Foc4) is currently known as a major concern in global banana production. Previous studies reported that bikaverin PKS-encoding gene Bik1 encoded a polyketide synthase responsible for the biosynthesis of a red pigment bikaverin in F. fujikuroi, and the ΔBik1 mutant totally lost the ability to produce pigmented mycelia. In this study, the Bik1 orthologous gene (FOIG_14908) in Foc4 was cloned and identified. The objective of this study was to identify the Foc4Bik1 gene (FOIG_14908) in Foc4 which had potential as an endogenous reporter gene of Foc4, which could serve as a promising target gene for assessing the feasibility of newly developed CRISPR/Cas9 gene editing technique in Foc4. Foc4Bik1 is a typical type I fungal multifunctional polyketide synthase (PKS) composed of 2036 amino acids that contains conserved protein domain. SMART revealed that Foc4Bik1 contained conserved protein structure such as acyl-carrier protein (ACP) transacylase (SAT), β-ketoacyl synthase (KS), acyltransferase (AT), dehydratase (DH) and thioesterase (TE). The ΔFoc4Bik1 gene-knockout mutants were obtained by the split-marker homologous recombination technique. After two rounds of PCR amplification, the upstream and downstream recombination fragments required for protoplast transformation were successfully amplified by the split-marker approach. The ΔFoc4Bik1 knockout mutant was obtained via PEG-mediated protoplast transformation, the positive transformants were verified with the PCR method. The ΔFoc4Bik1 gene-knockout mutants showed no significant effects on fungal growth, conidial production, and pathogenicity to banana plantlet (Cavendish, AAA), albeit the ΔFoc4Bik1 gene-knockout mutants totally lost the ability to produce reddish pigmented mycelia, and thus the mycelia turned white when cultured in liquid PDB media. Therefore, Foc4Bik1 could be selected as an endogenous reporter gene to verify whether the feasibility of CRISPR/Cas9 gene editing technology available for the filamentous fungus Foc4 via the plasmid CRISPR/Cas9 system. The F. fujikuroi CRISPR/Cas9 vector pUC-fFuCas9-HTBNLS-hph (Addgene, #121092) was used in this study. Foc4Bik1 gRNA sequence was designed via the online software, and the sgRNA162 sequence was introduced into gene editing plasmid resulted the Foc4 CRISPR/Cas9 gene editing vector, pUC-fFuCas9-HTBNLS-hph-Foc4Bik1, associated with donor plasmid pUC19-Foc4Bik1-HDR served as homology directed repair (HDR) template co-transformation into Foc4 protoplasts. Specifically, ΔFoc4Bik1 mutants totally lost the ability to produce pigmented mycelia appeared white mycelia. Using the white mycelia as the judging phenotype, the candidate ΔFoc4Bik1 (HDR) gene replacement mutants with white mycelia when grown in PDB liquid medium were selected for subsequent PCR detection. However, the white phenotypes were coupled with the positive PCR amplification bands. It is important that the positive ΔFoc4Bik1 (HDR) gene replacement mutants could be picked up according to the white phenotype by eyes without PCR procedures. The results proved that CRISPR/Cas9 system is stable and can efficiently disrupt the genes of interest, and Foc4Bik1 gene can serve as the endogenous reporter gene for further Foc4 molecular biology research.
In higher plants, invertase is widely involved in regulating plant growth, development, biological and abiotic stress response, and plays an important role in controlling the distribution of photosynthates in different bank tissues. It is a key enzyme that determines crop economic yield. In this study, the neutral/alkaline invertase ShINV6 was cloned from the stem of ‘Badila’. The sequence characteristics, expression characteristics and enzymatic characteristics of its encoding protein were analyzed. Bioinformatics analysis found that the full length of ShINV6 cDNA was 2859 bp, including 1254 bp of ORF that encoding 417 amino acids, and the content of arginine (Arg) was 12.7%. The molecular weight of ShINV6 was 46.29 kD and the PI value was 10.89. Evolutionary relationship analysis showed that ShINV6 protein was highly homologous with the neutral / alkaline invertase of monocotyledon crops such as sugarcane, maize and sorghum. It has typical Glyco_hydro_100 domain, multiple sequence alignments revealed that the C-terminal of ShINV6 was about 130 amino acids less than other invertases. Cell localization and hydrophilic analysis predict that ShINV6 may be hydrophilic protein in cytoplasm. During the growth and development of sugarcane, the change of sucrose content in sugarcane stems was observed. It was found that the sucrose content increased gradually with the extension of planting time, and the most sugar accumulation month was from October to November. QRT-PCR results showed that ShINV6 expression reached its maximum in September, and combined with regulation of biological yield growth in sugarcane growing period, it was speculated that ShINV6 might be involved in the unloading process of sucrose which the product of photosynthesis in the peak growth stage of sugarcane stems (library), and sucrose was transformed into hexaccharide for the rapid growth of sugarcane stems.
Natural rubber (NR) is an important strategic material related to national economy, livelihood and national security. It is of great significance to increase the production of natural rubber by focusing on the biosynthesis process of natural rubber. Rubber elongation factors (REFs) are mainly membrane proteins on rubber particles, which play an important role in extending rubber hydrocarbon and stable rubber particles during natural rubber biosynthesis process. It is an important approach for high-yield molecular marker-assisted selection breeding of rubber trees by identifying the key REFs gene that affects natural rubber yield. We found HbREF3 was highly and specifically expressed in latex, and its expression level was just less than that of HbREF1, which is the highest expression in latex in HbREFs gene family. The open reading frame of HbREF3 was 528 bp, encoding 175 amino acids. The molecular weight and theoretical isoelectric point of HbREF3 was 19.62 kDa and 5.28, respectively. Phylogenetic tree analysis showed that HbREF3 and HbREF1 belonged to different branches. All HbREFs proteins contain REF domain, and HbREF3 had one more motif than HbREF1. In silico studies showed that HbREF3 protein was a hydrophilic protein, without transmembrane domain, had 14 phosphorylation sites. The results indicated that HbREF3 may not be attached to the surface of rubber particles in a mosaic form, but directly or indirectly interacted with other proteins which embedded in the lipid monolayer membrane to form a complex attached on the surface of the rubber particles. The predicted localization result is different with HbREF1 which is embedded in the rubber particle monolayer membrane. The surface of rubber particles is composed of a monolayer of phospholipid membrane, the formation and development of rubber particles may be related to lipid synthesis. Subcellular localization analysis revealed that HbREF3 was localized in endoplasmic reticulum, we speculate that HbREF3 might be directly involved in rubber particle formation and latex regeneration by participating in lipid synthesis. Moreover, overexpression of REF/SRPPs family genes in nonrubber-producing plants (Arabidopsis and Capsicum) can significantly improve the drought resistance and accumulate a large number of lipid droplets. Therefore, we speculate that HbREF3 have a similar function to REF/SRPPs family proteins of nonrubber-producing plants in responding to drought in nature rubber harvesting process. We clarified the HbREF3 expression pattern and protein characteristics which would build a foundation for elucidating the molecular mechanism of HbREF3 in latex regeneration in rubber trees.
26 phenotypic traits of 204 plants of Renanthera philippinensis population derived from tissue culture were studied to understand the genetic diversity levels of the tissue culture population. Molecular makers such as ISSR markers were used in the study. The results showed that the genetic diversity index of 7 morphological traits was 1.85, and 19 quantitative traits of flower part diversity index was 1.87. The principal component analysis showed that there were 7 principal components of a cumulative contribution rate 67.887%. The main traits leading to the differences among the individuals were whether all flower branches were on the same surface or on the different faces, the thickness of petal, the length of lateral branch, the length of column bearing stamens and pistils, the width of middle calyx, thenumber of lateral branch, and the number of leaf. Cluster analysis on the data of phenotypic traits revealed that the 204 plants were divided into different four groups. Group I consisted of 78 plants, which had much more leaves. Group II contained 11 plants, which had more branches and flowers. Group III had 31 plants, which had shorter first inflorescence branch and shorter leaf length. Group IV contained 70 plants, which had much shorter and narrower style. Group V contained 14 plantswhich had larger pedicel diameter and wider calyx. ISSR markers were used to confirm the result from cluster analysis, and the results showed that 17 polymorphic bands were amplified by 2 pairs of ISSR primers on 24 plants, on average there were 8.5 polymorphic bands amplified by each pair of primers. The research results would provide a new way to seek genetic variation and an important genetic resource in R. philippinensis variety improvements.
In order to explore the mitigating effects of plant growth regulators on the low temperature injury of litchi plants in spring, different concentrations of plant growth regulators were sprayed on leaves under low temperature in spring, and its effects on physiological characteristics and yield of litchi leaves were studied. The 10-year-old ‘Feizixiao’ litchi trees were used as the experimental material. The plant growth regulator is developed by the Chemical Control Center of Guangdong Ocean University. Its main ingredient is Mepiquat chloride. The treatments A (2.5 mL of plant growth regulator was mixed evenly with 10 kg of water, and then sprayed on a litchi tree), B (5 mL of plant growth regulator was mixed evenly with 10 kg of water, and then sprayed on a litchi tree) and C (7.5 mL of plant growth regulator was mixed evenly with 10 kg of water, and then sprayed on a litchi tree) were set up. Meanwhile, the litchi trees were sprayed with the same amount of water were used as control (CK). The leaf fresh weight, leaf dry weight, chlorophyll relative content (SPAD), net photosynthetic rate (Pn), intercellular CO2 concentration (Ci), stomatal conductance (Gs), transpiration rate (Tr), fruit weight and yield were measured. The results showed that plant growth regulators could alleviate the low temperature injury of litchi leaves, and B treatment was the best. Compared with CK, the leaf dry weight and relative water content of B treatment had no significant changes, but the leaf fresh weight increased by 21.18%. Compared with CK, the SPAD, net photosynthetic rate (Pn) and transpiration rate (Tr) increased significantly by 7.44%, 8.40% and 3.48%, while the intercellular CO2 concentration (Ci), stomatal conductance (Gs) and stomatal limit did not change significantly. Compared with CK, the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX) increased significantly by 2.27%, 18.79%, 71.32% and 80.00%, respectively, while the activity of polyphenoloxidase (PPO) decreased by 10.81%. The content of soluble protein and ascorbic acid (AsA) increased significantly by 9.53% and 15.31%, respectively, in litchi leaves. But the impact on the single fruit weight and yield per plant were not significant. To sum up, under low temperature stress, spraying appropriate amount of plant growth regulators could improve leaves photosynthesis, promote leaves mass accumulation, increase the activities of antioxidant enzymes, increase the content of soluble protein and AsA, reduce the damage of membrane lipid caused by low temperature, and improve the ability of low temperature resistance of litchi leaves.
In order to explore a suitable application of potassium chloride on jackfruit seedlings, this experiment used ‘Malaysia No.1’ grafted seedlings as the test materials to compare the effects of different potassium application on the growth, photosynthesis and nutrition accumulation of jackfruit seedlings. The results showed that with the increase of potassium level, the net photosynthetic rate of leaves, root parameters, biomass and nitrogen, phosphorus and potassium accumulation of organs generally increased first and then decreased, and the ratio of root/shoot increased. Compared with the K0 level, K1 level had the better growth effect which increased plant height, stem diameter, dry weight of leaf, stem, root and total plant, ratio of root/shoot by 25.41%, 16.77%, 13.73%, 56.64%, 230.24%, 60.15% and 157.89%, respectively. The growth of jackfruit was inhibited to varying degrees in excessive potassium application. K1 and K2 treatments also had higher values of net photosynthetic rate compared with the K0 level, which increased by 25.29% and 35.00%, respectively. With the increased application of potassium, photosynthesis was obviously inhibited and stomatal restriction was the main factor limiting photosynthetic efficiency of leaves at K4 level. Compared with the K0 level, K1 level had a significant effect on root growth which increased root length, root surface area, root diameter, root volume and root tip number by 10.65%, 84.34%, 71.92%, 215.25% and 46.84%, respectively. The excessive potassium application inhibited the root system in different degrees. The accumulation of nitrogen, phosphorus, and potassium in roots and leaves increased first and then decreased with the increase of potassium application. The change trend of accumulation of potassium in stems was similar. K1 treatment had a higher accumulation of potassium in organs compared with the K0 level, which increased by 203.84%, 100.77% and 64.32%, respectively. The root indexes were significantly positively correlated with dry weight and potassium accumulation of root except for the total root length. The net photosynthetic rate was significantly positively correlated with root diameter, root volume, root tip number, root dry weight and potassium accumulation of root. Stem and leaf potassium accumulation were significantly positively correlated with dry weight of stem and leaf, and root diameter. In summary, Root architecture is closely related to the uptake and accumulation of potassium in roots, and plays an important role in accumulation of root biomass and leaf photosynthesis. Suitable concentration of potassium fertilizer can improve crop nutrient uptake and biomass accumulation by improving root parameters and leaf photosynthesis. The proper potassium nutrition was 1 g/kg in this experiment which could be used as a reference for jackfruit seedlings fertilization.
In order to explore the effects of different depth of Fenlong tillage on root morphological development, nutrient absorption and yield of newly planted sugarcane and perennial sugarcane, and to explore the suitable depth of Fenlong tillage, so as to provide a theoretical basis for popularizing Fenlong tillage technology. In this study, the field experiment was conducted with ‘Guitang 42’ as the test material. Four treatments were established: conventional shallow tillage, Fenlong shallow tillage, conventional deep tillage and Fenlong deep tillage. The results show that: Under the treatment of Fenlong shallow ridge, the yield of newly planted sugarcane was increased by 24.9% and 12.4% respectively compared with the treatment of conventional shallow tillage and the conventional deep tillage; the yield of ratoon cane was increased by 9.7% compared with the conventional deep tillage treatment. Compared with Fenlong deep tillage, whether it is newly planted sugarcane or ratoon cane, Fenlong shallow ridge shows a certain trend of increasing in yield. Except that the sugar content of newly planted sugarcane in the treatment of Fenlong deep tillage was slightly higher than that of other treatments, there was no significant difference in stem length, stem diameter and sugar content between newly planted sugarcane and ratoon sugarcane. Compared with conventional shallow tillage, the root length, surface area and volume of newly planted sugarcane at the tillering stage, the proportion of lateral roots and branches, and the ratio of middle roots and thick roots under Fenlong tillage are all larger than conventional tillage. The contents of N, P and K in the stems and leaves of newly planted sugarcane under the treatment of Fenlong shallow tillage were higher than those under Fenlong deep tillage, while the contents of N and K in roots were lower than those under Fenlong deep tillage. The five types of calcium, sulfur, magnesium, zinc and manganese in the stems and leaves of ratoon cane under the treatment of Fenlong shallow tillage were lower than those under Fenlong deep tillage, but the content of the trace elements in the roots of the five types was not significantly different from that of the Fenlong deep tillage. The results show that Fenlong can promote the growth and development of sugarcane roots, improve root morphology, promote the absorption of N, P, K and other nutrient elements, and finally improve sugarcane yield. The yield increasing effect of Fenlong shallow tillage is better than Fenlong deep tillage.
The axillary buds, cotyledons and epicotyls which were obtained from young stem segments and seeds of Macadamia integrifolia ‘H2’ were used as the explants, the callus induction and adventitious bud proliferation, and then cytological observations of the different callus by paraffin slice were conducted to analyze and compare the callus properties and adventitious bud differentiation effects induced by different explants. The suitable medium for axillary buds was MS+2.0 mg/L 6-BA+0.5 mg/L KT, which had the large numbers of callus and the adventitious bud regeneration coefficient was 3.49. The suitable medium for cotyledon callus induction was MS+2.0 mg/L TDZ+ 200 mg/L CH, the callus in this medium were green and tight, the induction rate was 82.2%, the browning rate was 8.9%, the cooperation of CH and TDZ not only helpt to improve the induction rate of cotyledon callus, but also reduced the browning rate. The optimal medium for adventitious buds of cotyledon callus differentiation was MS+1.0 mg/L KT+0.2 mg/L NAA+1.0 mg/L GA3, the adventitious buds grew well and the regeneration coefficient was 5.40. The suitable medium for epicotyls adventitious bud differentiation was MS+1.0 mg/L KT+0.5 mg/L NAA, and the regeneration coefficient was 7.37. The adventitious bud differentiation ability of the three explants from high to low was epicotyl>cotyledon>axillary bud, and KT combined with other suitable concentrations of growth regulators could produce better adventitious bud differentiation effect, which is the key to promote adventitious bud differentiation. The cotyledons and axillary buds dedifferentiated into callus, and then redifferentiated into adventitious buds, but the epicotyls could directly differentiate adventitious buds without producing callus. The callus induced by axillary bud and cotyledon showed obvious differences in appearance and cytology. A relevant cytological observation showed that the axillary bud callus had large cell volume, high degree of vacuolation, little cytoplasm, and small nucleus, with scattered and irregular intercellular distribution, showing the characteristics of non-embryonic callus, while the cotyledon callus had compact cell arrangement, large nucleus, and deeply stained thick cytoplasm, showing the characteristics of meristematic cell. In comparison, cotyledon is an good material for callus induction, and epicotyl is the best explants for adventitious bud proliferation, the cotyledon callus have some characteristics of embryogenic callus and high differentiation capability, it can be used as a test material for genetic transformation and polyploid induction of macadamia.
Three local avocado strains in Hainan were used as the test materials. White paper bags and yellow outside and black inside kraft paper bags were selected to bag avocado fruits, and non-bagging was used as the control. 100 days after bagging, samples were collected when avocado was mature to determine the appearance and internal quality of fruit. The effects of bagging on fruit quality and fatty acid composition of avocado were observed and determined. Comprehensive evaluation was carried out by the principal component analysis. The results showed that the skin of fruits treated with yellow outside and black inside kraft paper bags turned yellow, and the skin color of fruits treated with white paper bags had little difference from that without bags. Bagging had no significant effect on the appearance indexes of single fruit quality, fruit transverse diameter, fruit longitudinal diameter, fruit shape index, pulp weight, seed quality, seed longitudinal diameter and seed transverse diameter of avocado fruit, but bagging could reduce the harm of diseases and pests, reduce the damage of branch and leaf friction to avocado fruit surface and improve the appearance quality of avocado fruits. Bagging had no significant effects on the internal indexes of edible rate, water content and soluble sugar content of avocado fruit, but bagging could significantly improve the contents of vitamin C, soluble protein, total phenol and total flavonoids. Through the determination of 8 fatty acid components in avocado pulp, the comparative analysis showed that bagging had no significant effects on the fatty acid components of avocado fruit, but could improve the relative content of unsaturated fatty acid in avocado fruits. Through principal component analysis, the score of nine treatments on three principal components was obtained. After calculating the score according to the expression, the fruit quality traits of white fruit bags were the best among the three bagging treatments of each strain. In conclusion, fruit bagging during the growth and development of avocado can provide a new microenvironment for its growth and development, reduce the impact of various external adverse factors on fruit development, and improve the appearance and internal quality of fruit. This study preliminarily screened out the white paper bag suitable for the bagging of avocado, which can provide reference for growers in the production of avocado, so as to better meet the demand for high-grade fruits in the domestic and foreign markets.
Three color leaves of the adult plant of Schefflera odorata cv. Variegata on the same plant including whole green (WGL), variegated leaf (VL) and whole yellow leaf (WYL) were used as the test material, to determine its photosynthetic pigment content, light response curve, gas exchange parameters and chlorophyll fluorescence parameters and to study the photosynthetic characteristics of three different color leaves of Schefflera odorata cv. Variegata so as to provide theoretical guidance for rational planting and plant cultivation and reference basis. The results showed that the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (Chls) and carotenoid (Car) of the three colors of leaves were extremely different (P<0.01), and they were WGL > VL > WYL, and the Chl a/b of the three colors of leaves were 0.556; the net photosynthetic rate (Pn) of whole green leaf increases with the increase of light intensity greater than that of Variegated leaf and whole yellow leaf (Pn) that was always a negative value; the maximum net photosynthetic rate (Pnmax), light saturation point (LSP) and dark respiration rate (Rd) of whole green leaf and variegated leaf are significantly different (P<0.05), and the light compensation points of the two (LCP) the difference was not significant (P>0.05), which showed that whole green leaf> variegated leaf; the initial slope (α) of variegated leaf was higher than that of whole green leaf, and there was a significant difference (P<0.05); stomatal conductance (Gs), transpiration rate (Tr) and water use efficiency (WUE) are shown as whole green leaf > variegated leaf > whole yellow leaf, while the intercellular carbon dioxide concentration (Ci) was the opposite, and showing whole yellow leaf > variegated leaf > whole green leaf; initial fluorescence (Fo) and Actual quantum yield (ΦPS II) value was positively correlated with chlorophyll content, which was represented by whole green leaf > variegated leaf > whole yellow leaf. The Fv/Fm values of the three leaves were less than the stable value 0.832. The qN and qP values increased firstly and then later with the increase of chlorophyll content. The performance is variegated leaf > whole green leaf > whole yellow leaf. Whole green leaf with higher chlorophyll content have higher photosynthesis efficiency, and the growth and development of whole yellow leaf without chlorophyll will be restricted and die; variegated leaf have stronger adaptability and regulation ability than whole green leaf, and can be used at lower chlorophyll concentrations. Under the circumstances, improving photosynthetic efficiency through self-regulation has higher cultivation and cultivation value.
Amomum villosum Lour is a perennial herbaceous plant of Cardamom genus of Zingiberaceae. The ripe and full fruit of Amomum amomum Lour is one of the four southern medicines in China, which has the effects of warming the spleen, invigorating the stomach, regulating qi and calming the fetus, etc., with extremely high medicinal and economic value. Amomum villosum Lour is adapted to grow in a shady and humid environment, and light is an important factor affecting growth and development of Amomum villosum Lour. Therefore, we should be familiar with the photosynthetic characteristics of Amomum villosum Lour to better protect and utilize germplasm resources. In this paper, using SPAD-502 PLUS chlorophyll meter, 3051D photosynthetic meter and WALZ MINI-PAM-Ⅱ chlorophyll fluorescence meter, the seedlings of ‘Zhansha 11’ were used as materials to determine the parameters such as chlorophyll content (SPAD), net photosynthetic rate (Pn), electron transfer rate (ETR) and photosynthetic available quantum yield (Y2), the farmyard cultivation species ‘Reke 1’ and ‘Reke 2’ as the control, analysis of photosynthetic characteristics and environmental adaptability of ‘Zhansha 11’. The results showed that the leaf SPAD value of ‘Zhansha 11’ was 44, which was between the two controls, 12.47% higher than that of ‘Reke 1’ and 18.59% lower than that of ‘Reke 2’; The net photosynthetic rate (Pn) of ‘Zhansha 11’ was 12.19 µmol/(m2·s), which was 1.63 times and 5.11 times higher than that of ‘Reke 1’ and ‘Reke 2’, and was significantly higher than that of the controls; Transpiration rate (Tr) and stomatal conductance (Gs) of ‘Zhansha 11’ were significantly higher than ‘Reke 2’. Electron transfer rate increased with the increase of photosynthetical active radiation at 10-1082 µmol/(m2·s), and the electron transfer rate of ‘Zhansha 11’ was higher than that of ‘Reke 1’ and ‘Reke 2’, indicates that the photosynthetic system II of ‘Zhansha 11’ has high photoelectron transfer capability. In conclusion, ‘Zhansha 11’ has optimal photosynthetic characteristics, strong ability to transform light energy into electron flow and potential photosynthetic capacity, and has a wide range of light radiation; which is suitable for the growth environment with low shade. The results provided a theoretical basis for the production and cultivation of ‘Zhansha 11’. At the same time, the photosynthetic characteristics of ‘Zhansha 11’ were compared with those of farmyard cultivation species, which provided scientific basis for the selection of ‘Zhansha 11’, and guided the breeding of excellent varieties of Amomum villosum Lour.
The effects of grafting cultivation and nitrogen rates on nitrogen and dry matter accumulation and nitrogen metabolism enzymes of wax gourd were studied to elucidate the dynamic rule of dry matter and nitrogen accumulation and provide theoretical basis for the scientific fertilization of wax gourd. In this study, ‘Tiezhu No. 2’ wax gourd and ‘Haizhen No. 1’ rootstock were used as the materials. A split-plot experiment design was used with cultivation patterns worked as the primary plot, including grafting cultivation and self-root cultivation, and nitrogen rate as the secondary plot, including N0 (0 kg/hm 2), N180 (180 kg/hm2), N360 (360 kg/hm2) and N540 (540 kg/hm2). The accumulation of nitrogen and dry matter and activity of nitrogen metabolism enzymes for wax gourd in five different growth stages were studied. The total accumulation amount of nitrogen and dry matter first increased and then decreased with the increase of nitrogen application rate with peak values at N360, whose nitrogen and dry matter accumulation were significantly higher than those of the other three nitrogen rate treatments. The average nitrogen and dry matter accumulation of grafted wax gourd was 16.11% and 11.76% higher than those of self-rooted wax gourd, respectively in the whole growth period. The maximum theoretical value (K), maximum accumulation rate (Vmax), average accumulation rate (Vmean), the beginning period of rapid accumulation period (T1), the ending period of rapid accumulation period (T2) and the occurrence time of maximum accumulation rate (Tmax) of nitrogen and dry matter accumulation of wax gourd increased first and then decreased with the increase of nitrogen rate with peak values at N360. While K, Vmax) and Vmean of nitrogen and dry matter accumulations in grafted wax gourd were higher than those of self-rooted wax gourd. The activity of nitrate reductase (NR) and glutamine synthetase (GS) increased first and then decreased with the growth period proceeded, and reached the maximum at 75 days. The activity of NR and GS increased first and then decreased with the increase of nitrogen application rate, with peak value at N360, the activity of NR and GS was significantly higher than those at N0, N180 and N540. Compared with self-rooted wax gourd, the NR activitiy of grafted wax gourd increased by 33.08%, 28.95%, 34.63%, 36.03% and 67.23% at 30 days (vine extension stage), 55 days (flowering stage), 75 days (early fruit development stage), 95 days (middle and late fruit development stage) and 110 days (mature stage), with GS increased by 29.64%, 22.11%, 21.00%, 28.00% and 35.27%, respectively. In conclusions, grafting can significantly improve the accumulation and the rate of dry matter and nitrogen and the activity of NR and GS. N360 was the optimum nitrogen application rate for wax gourd in production.
Colletotrichum siamense is an important pathogenic fungus, which is the dominant pathogenic species of Colletotrichum leaf disease (CLD) in the field of rubber trees in China. And it is also the main pathogen of anthracnose of many other agricultural and forestry crops in tropical and subtropical regions. Lipid droplets, the neutral lipids of cells, are present in most eukaryotic cells and are the storage form of intracellular triglycerides. Lipid droplets can be involved in lipid metabolism, membrane transport, protein degradation and signaling in the cell. Perilipin is the most abundant lipid-associated protein on the surfaces of lipid droplets, mainly existed in animals and plants, and plays an important role in regulate lipid metabolism. Cap20 is a homologue of perilipin in Colletotrichum genus. It was previously confirmed that Cap20 is a pathogenic associated protein that involved in the formation of turgor pressure of appressorium, the number of lipid droplets and the pathogenicity of Colletotrichum genus. Understanding the interaction proteins of Cap20 and the biological significance of the interactions may be important for gaining insight into how Cap20 is involved in the molecular mechanisms of pathogenesis. In the previous stage, a candidate protein, acetate kinase, which interacts with Cap20, was screened from the cDNA library of C. siamense by yeast two-hybrid technology. In this study, the full length of acetate kinase coding gene was cloned, and the protein structure and phylogenetic analysis were analyzed firstly. The results showed that the acetate kinase coding gene had a DNA size of 1312 bp including an intron, a cDNA of 1248 bp encoding 415 amino acids and containing an acetate kinase structural domain, which was named as CsAck. Then, the prokaryotic expression vector PET32a-CsAck with 6×His tag was constructed, and the fusion protein CsAck with a 6×His tag and Cap20 with a GST tag was obtained by using it and the GST-tag-containing pGEX6p-1-Cap20 (previously constructed) for protein expression and purification. The in vitro interaction between the Cap20 and CsAck proteins was verified by Pull down expriments. Finally, the expression vectors PXY203-CsAck-S containing the hygromycin transferase gene HPH and the pCB1532-GFP-Cap20 with the sulfonylurea resistant gene ILV1 were constructed and co-transformed into C. siamense wild-type strain to obtain transformants. The results of Co-IP (Co-Immunoprecipitation) confirmed that Cap20 and CsAck could interact with each other in C. siamense. The results confirm that the pathogenesis-related protein Cap20 interacts with the acetate kinase CsAck both in vitro and in vivo, which could lay the foundation for further studies on the pathogenic function and regulatory mechanism of Cap20 in C. siamense.
A Burkholderia sp. DF3-1 strain was isolated and identified previously from contaminated soil in heavy metal mining areas. The strain was preserved in the laboratory of Research Group for Banana Industry Technology. The stain has a strong ability to adsorb cadmium ion. Its adsorption characteristics to cadmium ion (Cd2+) of the environment were analyzed further in this study. The adsorption characteristics for environmental cadmium ion (Cd2+) were further analyzed by multiple ways. The removal efficiency of cadmium ion by the strain was measured under different initial concentrations, pH and culture time. Effects of cadmium on the internal and external morphology of bacterial cells were detected using a scanning electron microscopy (SEM) and a transmission electron microscopy (TEM). The infrared spectroscopy (Fourier transform infrared spectroscopy, FTIR) was used to determine the changes of different groups on bacterial surface. Location of cadmium tolerance genes was also predicted by plasmid elimination. The results showed that the growth of this strain was not almost affected under the initial Cd2+ concentration less than10 mg/L. On the contrary, the initial concentrations of 50 mg/L and 100 mg/L inhibited the growth of Burkholderia sp. DF3-1. Maximum biomass was achieved within 24 hours of culture and decreased in the following tested time points. By analyzing the effects of different pH on the removal efficiency of Burkholderia sp. DF3-1, it was found that the highest removal efficiency was observed at pH 5. The highest removal efficiency was 83.64%. The results of SEM showed that Cd 2+ treatment caused the surface of the bacterial cells to be rough, deformed and shrunken. The results of TME demonstrated that Cd2+ thickened the cell wall and dissolved the bacterial cell membrane. The boundary between cell membrane and cytoplasm was not obvious. The electron microscopy showed that cadmium had a toxic effect on cells. In addition, the results of FTIR showed that the -CH2-, amide I, -NO2, -COOH, -C-OH and -CO- groups were involved in the adsorption process of Cd2+ on the bacterial surface. After using SDS and sodium benzoate to eliminate plasmids, there was little difference in cadmium resistance of Burkholderia sp. DF3-1, so it was preliminarily speculated that the cadmium resistance gene of Burkholderia sp. DF3-1 was located in genomic DNA and not in the plasmid. Therefore, the adsorption of cadmium ions by Burkholderia sp. DF3-1 occurs in both intracellular accumulation and extracellular adsorption. The initial concentration and pH have a greater impact on its adsorption capacity. The above research results would provide an important reference for the later soil bioremediation.
Deep vertically rotary tillage is a new type of deep ploughing and deep loosening farming technology. Different from conventional farming methods, deep vertically rotary tillage could increase the depth of the cultivated layer soil, thereby better improving the soil structure. In order to reveal the influence of different farming methods on the bacterial and fungal communities of sugarcane planting soil, this study took deep vertically rotary tillage farming and conventional farming treated soil as the research object, and analyzed different ecological niches through 16S rRNA and 18S rRNA high-throughput sequencing technologies (bulk soil, rhizosphere, and rhizoplane) soil bacterial and fungal community composition, structure and diversity changes, combined with soil chemical properties to carry out correlation analysis. The results showed that compared with conventional tillage treatments, the proportion of small and medium aggregates (<1 mm) increased after deep vertically rotary tillage treatment and the proportion of larger aggregates (>1 mm) was lower than that of the conventional tillage treatment. The content of available phosphorus and soil organic matter in the soil treated by deep vertically rotary tillage increased significantly, while the content of total nitrogen and total phosphorus decreased. The relative abundance of phyla Firmicutes and Chloroflexi significantly increased after deep vertically rotary tillage treatment, and the alpha diversity of bacterial and fungal communities after deep vertically rotary tillage treatment increased obviously, while there was no significance observed in the microbial alpha diversity among different niches of soils. In terms of the differences in microbial beta diversity, compared with the conventional tillage treatment, the soil microbial community structure in the deep vertically rotary tillage treatment altered significantly. At the same time, the bacterial community structures varied from different niches, while no obvious difference was observed in the fungal community structure among different niches. Combined with the correlation analysis of environmental factors, the changes in total nitrogen, available phosphorus, and organic matter content could significantly affect the soil microbial characteristics, and the available phosphorus and soil organic matter were the main factors affecting the composition of soil microbial communities. In summary, the composition of sugarcane soil nutrients and aggregates altered under different soil tillage treatments, and there were differences in the structure and diversity of soil bacterial and fungal communities. The diversity and structure of microbial communities under different tillage methods were affected by soil available phosphorus and organic matter. These research results can provide a theoretical basis for analyzing the response mechanism of sugarcane soil microbial communities to different tillage methods.
Coffee is one of the three major beverages in the world, which has a wide range of nutritional, economic and medicinal value. Coffee industry has become one of the advantageous industries of cash crops in hot areas in China. Coffee is a perennial crop. With the increase of planting years and the renewal of old coffee plantations, coffee has produced serious replantation diseases, which has become a serious problem affecting the income increase of coffee farmers and restricting the sustainable development of industry. So it needs to be solved urgently. In order to reveal the mechanism of coffee replantation diseases, the healthy and diseased coffee rhizosphere soils were collected from the representative coffee plantations in Chengmai, Qionghai and Wanning, respectively, and CK was collected from adjacent areas. The main physical and chemical indexes, enzyme activity, and the quantity and metabolic characteristics of culturable microorganisms were determined. In the three areas, pH of the rhizosphere soil of healthy and diseased plants was significantly lower than that of CK (P<0.01), and the contents of total potassium, available potassium and total nitrogen in the rhizosphere soil of diseased plants were lower than those of healthy plants, and the difference of total nitrogen between healthy and diseased plants was significant in Chengmai (P<0.05); the content of rhizosphere soil of diseased plants alkali hydrolyzable nitrogen in the three regions was significantly higher than that of healthy plants (P<0.05), and the contents of the rhizosphere soil of diseased plants available phosphorus and organic matter in Qionghai were significantly higher than those of healthyplants (P<0.05). The activity of CAT, POD, cellulase and acid phosphatase in the rhizosphere soil of diseased plants was higher than that of healthy plants, while urease activity was on the contrary. The number and metabolic activity of culturable bacteria, fungi and actinomycetes in the rhizosphere of diseased plants were lower than those in healthy coffee plants, and the microbial diversity decreased. Correlation analysis showed that in Wanning, urease activity was significantly positively correlated with culturable bacteria and actinomycetes, in Chengmai and Qionghai POD activity was significantly positively correlated with culturable bacteria, and in the three areas cellulase activity was significantly positively correlated with culturable fungi. In conclusion, the rhizosphere soil of diseased coffee plants appeared acidification, nutrient imbalance and decreased microbial diversity, and the occurrence of coffee replantation diseases was closely related to the rhizosphere environmental disaster. The conclusion of this study would provide a theoretical basis for overcoming coffee replantation diseases.
Litchi fruit, with the high nutritive value and commercial potential, is an important tropical and subtropical fruit in south China. However, the harvested litchi fruit is prone to quality deterioration such as rot occurrence, pericarp browning and pathogen infection in high temperature and high humidity seasons. Thus, litchi fruit is often processed into different commercial products in addition to fresh-eating, especially dried litchi. Due to the long drying processing time, the dried litchi is highly susceptible to serious pericarp browning development, resulting in the loss of appearance color, product quality and fruit flavor of the dried litchi, and eventually limit the sustainable development of litchi processing industry. Therefore, it is necessary to develop a safe and effective drying processing technology for the dried litchi fruit to delay pericarp browning, stabilize appearance color and improve quality of the dried litchi fruit. Some studies reported that the technologies of blanching and sulfur-free color protection liquids could effectively control the occurrence of enzymatic browning during drying processing by inactivating the activity of polyphenol oxidase (PPO), and thus improve the product quality of the dried horticultural products. However, there are few reports on delaying pericarp browning development and maintaining appearance quality in the dried litchi during hot-air drying processing by using the safe color-protecting technology. Therefore, in this study, in order to control the pericarp browning of litchi fruit during hot-air drying, the technologies of blanching and sulfur-free color-protecting in litchi fruit during drying processing were studied. An L9(34) orthogonal experimental design was used to investigate the effects of different blanching conditions such as blanching temperature and blanching time with different concentration of citric acid, and different sulfur-free color protection liquids including citric acid, sodium chloride (NaCl) and ascorbic acid, on the inactivation of PPO in the pericarp of litchi fruit, which would help to obtain the optimal parameters of blanching and sulfur-free color-protecting in litchi fruit during drying processing. The results displayed that the blanching temperature 95℃, blanching time 3 min and 0.3% citric acid were the optimum blanching conditions for dried litchi fruit during processing. Additionally, the application of the sulfur-free color protection liquids including 0.2% citric acid, 2.0% NaCl and 0.3% ascorbic acid for litchis revealed the best color-protecting of dried litchis. The data indicated that the above optimized parameters could effectively suppress the occurrence of pericarp browning in litchi fruit during drying processing, and thus maintain the better quality of dried litchi fruit.
Yunnan small seed coffee was used to explore the characteristics and optimal mathematical model of hot air drying. The hot air drying characteristics of the coffee beans under different circumstances were studied by an orthogonal experiment with wind speed, pavement thickness and stirring speed, and the applicability of 10 mathematical models in hot air drying characteristics was compared. The results showed hot air velocity could promote the heat and mass transfer in the drying process. The mixing process not only could accelerate heat transfer, but also improve heat efficiency and reduce drying time. Coffee bean pavement thickness mainly affected the drying time. The pavement thickness was inversely proportional to the change of drying rate. Data analysis revealed the drying was mainly characterized by decreasing drying speed, and there was no obvious constant speed drying stage, and the hot air temperature had the greatest influence on the drying characteristics of hot air drying. According to the range and variance analysis of the orthogonal experiment, the optimal hot air drying scheme was temperature 40℃, wind speed 1 m/s and stirring speed 2 r/min. The optimal mathematical model was an logarithmic equation. The range values of the influence of hot air temperature, hot air speed and stirring speed on the total hot air drying time was 19, 6.67, 5.5, respectively. P value of the three factors under 95% confidence interval was 0.011, 0.082 and 0.391 respectively. The primary and secondary order was hot air temperature A> hot air speed B> stirring speed C. Logarithmic model was analyzed by the nonlinear regression fitting of evaluation index R2, SSE, mean square of residual and comparison experimental data with commonly used drying model. According to the date analysis, the results showed that the best-fitting degree of Logarithmic model, R2 is 0.986444, SSE is 0.021734, mean square of residual is 0.002075. Taking all the factors into conclusion the mathematical model not only can predict the hot air drying characteristic curve of Yunnan arabica wet beans, but also provide a theoretical basis and reference for the actual production and processing of small seed coffee.
Cultivation substrate is one of the main factors affecting the growth of Dendrobium officinale. In this study, the substrates of pine bark (PB), coconut coir (CC) and the mixture of pine bark∶coconut coir in 1∶1 ratio (PC) were used to plant D. officinale. On the basis of metabolomics, the effects of the properties of the three cultivation substrates on the metabolism of D. officinale stems were investigated. Cutting ring method was used to determine the physical properties of the three cultivation substrates, phenol-sulphuric acid method was used to detect the content of polysaccharides, and gas chromatography mass spectrometry (GC-MS) method was performed to detect the primary metabolites. The results indicated that the aeration porosity and water holding capacity in the group of coconut coir and complex substrate were higher than that of the substrate of pine bark, the content of polysaccharides were ranked as coconut coir > complex substrate > pine bark. Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) for multivariate statistical analysis of 42 identified metabolites showed that the samples cultivated in different substrates clustered into one group, and the separation between groups was obvious, indicating the metabolites of the samples from different groups existed a significant difference. Combined with fold change (FC), the VIP values calculated based on the OPLS-DA model were used to screen the metabolites with significant difference. When the conditions (VIP>1 and FC≥2 or FC≤0.5) were set, there were a total of fourteen, eleven and five metabolites, with significant differences in content across groups being screened in the comparison of pine bark and complex substrate, pine bark and coconut coir, complex substrate and coconut coir, respectively. A large part of metabolites with significant difference in the group of coconut coir and complex substrate performed a lower accumulation in the pine bark group. The results gained by combined analysis between the physical properties of the three cultivation substrates and the metabolites with significant difference revealed that if the cultivation substrate had the weaker aeration and capacity of water supply, there would be a stronger expression in crassulacean acid metabolism (CAM). Thus, C3 pathway would become weaker, and carbohydrates accumulate less. In other words, the substrate with the properties of high aeration porosity and good water holding capacity was beneficial to the accumulation of carbohydrate in D. officinale. Therefore, this study could provide data and theoretical guidance for the cultivation of D. officinale.
To improve the utilization of coconut processing byproduct, four protein fractions including albumin, globulin, gluten-1 and gluten-2 were separately extracted from coconut cake. The amino acid composition of the protein fractions was analyzed. The subunit composition and molecular weight of the fractions were analyzed by SDS-PAGE. The hydrophobicity of coconut cake albumin, globulin, gluten-1 and gluten-2 were determined. The effects of different ionic strength on emulsifying capacity and emulsion stability of the protein fractions were studied, too. The content of coconut cake albumin, globulin, glutelin-1 and glutelin-2 was (8.84±1.16), (54.35±3.69), (20.49±1.11) and (6.14±1.42) g/100 g, respectively. The protein fractions are all rich in arginine and glutamic acid. In addition, the essential amino acid content of the protein fractions was relatively high than that recommended by FAO/WHO. The essential amino acid composition was reasonable. The albumin showed the highest biological value (58.63) among the four coconut cake protein fractions. This indicates that albumin has the highest nutritional value. Results of the SDS-PAGE analysis showed that there was significant difference in subunit composition among coconut cake albumin, globulin and gluten-1. The molecular weight was ranged from 17.7 to 53.3 kDa. Acid subunit composition of glutlelin-1 was 46.1%, in contrast the content of basic subunits in glutelin-2 was 27.5%, which was one of the main reasons why glutelin-1 and glutelin-2 has different solution. Glutelin-2 was composed of ten subunits with a molecular weight range of 18.8-119.2 kDa, which is the widest range among coconut cake protein fractions. The hydrophobicity of globulin and glutelin-2 was higher than that of albumin and glutelin-1 (P<0.05). In addition, at the same ionic strength, the globulin showed the highest emulsifying capacity (127.3 m2/g) and emulsion stability (98.13%) among the fractions, following which was the glutelin-2 and albumin, whereas glutelin-1 exhibited the lowest emulsifying capacity (11.79 m2/g). There was no significant difference on emulsion stability among coconut cake globulin, glutelin-2 and albumin. But the emulsion stability of globulin, glutelin-2 and albumin was higher than that of soybean protein isolate (P<0.05). When ionic strength was from 0 to 0.4 mol/L, the emulsifying capacity and emulsion stability of globulin were improved as ionic strength increased. In contrast, the emulsifying capacity of albumin and glugelin-2 was reduced as ionic strength increased. This study would provide a theory basis for the further application of coconut cake protein.
