Cloning and Bioinformatics Analysis of Pepper PnCAD Gene by Race

doi:10.3969/j.issn.1000-2561.2022.12.004

Abstract

Abstract:

Lignin has some functions in plants, such as transporting water, supporting plants and strengthening plants against damage, etc. It is one of the important products of phenylpropane metabolism. Among them, cinnamyl alcohol dehydrogenase (CAD) is an important rate-limiting enzyme in the lignin synthesis pathway. In this experiment, on the basis of pepper transcriptome sequencing, the RACE method was used for gene cloning, bioinformatics analysis of the full-length PnCAD gene, and many analyses of its protein such as physicochemical properties, subcellular localization and phylogenetic tree. It was analyzed by qPCR. The cinnamyl alcohol dehydrogenase gene was cloned by the race method. Finally, a full-length cDNA with a length of 1364 bp was obtained, including 1071 bp open reading frame (ORF), encoding 356 amino acids. The predicted relative molecular weight was 3.879 kDa and the isoelectric point was 6.27. It belonged to hydrophilic protein. It contained three N-glycosylation characteristic sequences and nine phosphorylation sites, which were likely to be in the cytoplasm. Domain analysis showed that CAD protein contained NAD (P) binding sites, multiple catalytic zinc and structural zinc binding sites. Phylogenetic tree analysis showed that pepper CAD was closely related to Asarum CAD 6, and the highest homology was 76%, both of which belonged to primitive dicotyledons. Through fluorescence quantitative analysis, it could be found that the expression of P. flaviflorum under the infection of Phytophthora capsici increased, reached the highest value at 8 h, about 10 times that of the control, then increased slightly at 24 h, about 6 times that of the control group, and then decreased. Generally speaking, the gene expression level of P. flaviflorum at all time was higher than that of P. nigrum cv. Reyin-1 and was significantly different. The experimental data could provide reference data for the future study of the anti abiotic stress function of black pepper, and also provide a theoretical basis for the functional study of gene PnCAD.

Key words: Piper nigrum, PnCAD, gene cloning, bioinformatics analysis, qPCR

CLC Number:

S961.6

SUN Yeqiao, WANG Jue, HU Lisong, WU Baoduo, HAO Chaoyun, FAN Rui. Cloning and Bioinformatics Analysis of Pepper PnCAD Gene by Race[J]. Chinese Journal of Tropical Crops, 2022, 43(12): 2422-2430.

Figures/Tables 9

Tab. 1 RACE primer name and sequence

引物名称 Primer name	序列（5′-3′） Sequence (5′-3′)	功能 Function
B497-1(GSP1)	GGTTATTCTGAGTGAG	RACE引物
B497-2(GSP2)	TCATCACTACCAGTAGGCCTC	RACE引物
B497-3(GSP3)	GTACGGTGATAGGATCCCAGA	RACE引物
C357-1	GTTACAGGCGGCACTGGTCTTACG	RACE引物
C357-2	GGAGATGCTCAATTTTTGTGCTGC	RACE引物
PUB1-F	TTACCAGGACTCAGCAGCGAATG	实时荧光定量PCR引物
PUB1-R	AAGCCAATGACTTTACATCCTCCAG	实时荧光定量PCR引物
CAD-F	AACCTGGAAAATGCCTTGGG	实时荧光定量PCR引物
CAD-R	TCTGTCCGCTCCAAGTTCTT	实时荧光定量PCR引物

Fig. 1 RACE cloning and PCR amplification of PnCAD in P. nigrum M: DL2000 marker; 1: PCR product; A: The product of 5°-RACE; B: The product of 3°-RACE.

Fig. 2 cDNA sequence and deduced amino acid sequence of PnCAD gene First black box represents start codon; * represents stopcodon; the single underscore represents the end of Poly(A) and the double underscore represents the Poly(A) sequence. Second black box represents the possible Acyl-activating enzyme (AAE) consensus motifs, and third black box represents the ATP substrate binding regions.

Fig. 3 Signal peptide and transmembrane structure analysis

Fig. 4 Conservative domain prediction of PnCAD protein

Fig. 5 Prediction results of tertiary structure of pepper PnCAD protein

Fig. 6 Comparison of amino acid sequence of pepper PnCAD protein with that of other plant CAD

Fig. 7 Phylogenetic tree analysis of pepper PnCAD protein

Fig. 8 PnCAD gene expression results

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