Cloning and Expression Analysis of EfGRAS Gene of Relative Sugarcane Wild Species Erianthus fluvus

doi:10.3969/j.issn.1000-2561.2020.10.017

Abstract

Abstract:

In order to make deeper research into the drought resistance of sugarcane varieties, the genes of GRAS which playing an important role in the regulation of drought resistance mechanisms in sugarcane varieties for expression analysis were studied. In this experiment, we firstly retrieved the GRAS transcription factor gene sequence information from the transcriptome database, and used RACE-PCR and qRT-PCR techniques to clone a GRAS gene from ‘Sucrose 99-2’ and namde it EfGRAS (GenBank registration No. MT499789), then the bioinformatics analysis and expression analysis under drought stress were conducted on the EfGRAS gene. The results showed that the EfGRAS gene could encode 547 amino acids with a CDS 1644 bp in length, the molecular formula of the protein was C₂₆₄₅H₄₁₄₉N₇₄₇O₈₁₆S₂₁, the relative molecular mass was 60.14 kDa, the instability coefficient was 54.85, the fat coefficient was 84.37, and the GRAVY value was ?0.293, which was a class unstable and hydrophilic protein. The subcellular location indicated that it was located in the nucleus. The main secondary structure of EfGRAS protein was α-helix and random coils, which were closely related to sorghum; qRT-PCR analysis showed that after drought stress, the gene expression in ‘Sugarcane 99-2’ was up-regulated by about 57.6 times compared to the control group, and the gene expression in ‘DZ 01-58’ was up-regulated by about 27.4 times compared to the control group. Compared with the control group, the gene expression level in ‘YC 89-9’ had a lower fold of up-regulation. The results would provide a theoretical basis for the further study on the function of EfGRAS in sugarcane.

Key words: Erianthus fluvus, drought stress, EfGRAS gene, clone, expression analysis

CLC Number:

S566.1
Q78

SHEN Xianyue,GU Shujie,XIE Linyan,QIAN Zhenfeng,ZENG Dan,HE Lilian,LI Fusheng. Cloning and Expression Analysis of EfGRAS Gene of Relative Sugarcane Wild Species Erianthus fluvus[J]. Chinese Journal of Tropical Crops, 2020, 41(10): 2113-2119.

Figures/Tables 12

Tab. 1 qRT-PCR primers

基因名称 Gene name	正向引物（5°-3°） Forward primer (5°-3°)	反向引物（5°-3°） Reverse primer (5°-3°)
EfGRRAS	CGAAGCACGCACA GACG	GGAGAAGACGAG AAGAGGC
25S rRNA	CTGGAATGGTCAA GGCTGGT	TCCTTCTGTCCCAT CCCTACC

Tab. 2 Gene amplification primer sequences

基因名称 Gene name	正向引物（5°-3°） Forward primer (5°-3°)	反向引物（5°-3°） Forward primer (5°-3°)
EfGRAS	AGTCTATCGGACTCTCTGCTG	GTTTTAGTGCTCAGCCCAACATA

Fig. 1 Gel electrophoresis detection results of RNA M: DL2000 Marker; 1: Drought treatment ‘DZ 01-58’ RNA; 2: CK ‘DZ 01-58’ RNA; 3: Drought treatment ‘YC 89-9’ RNA; 4: CK ‘YC 89-9’ RNA; 5: Drought treatment ‘ZM 99-2’ RNA; 6: CK ‘ZM 99-2’ RNA.

Fig. 2 PCR clone of EfGRAS M: DL2000 Marker; 1: The annealing temperature is 56 ℃; 2: The annealing temperature is 58 ℃; 3: The annealing temperature is 60 ℃.

Fig. 3 Nucleotide sequence and derived amino acid sequence of EfGRAS gene

Fig. 4 Hydrophobic/hydrophilic analysis of EfGRAS amino acid sequence

Fig. 5 EfGRAS protein conserved domain prediction

Tab. 3 Subcellular localization of EfGRAS protein

亚细胞位置 Subcellular location	分值Score
亚细胞位置 Subcellular location	LocDB	PotLocDB	Neural Nets	Pentamers	Integral
细胞核	5.8	3.0	0.00	0.00	5.93
细胞膜	0.0	0.0	0.96	0.04	0.10
细胞外	0.0	0.0	0.96	0.14	0.00
细胞质	4.2	0.0	0.00	1.80	2.78
线粒体	0.0	0.0	0.00	2.00	0.03
内质网	0.0	0.0	0.00	0.61	0.12
过氧化氢酶体	0.0	0.0	0.96	0.00	0.00
高尔基体	0.0	0.0	0.11	0.28	0.00
叶绿体	0.0	0.0	0.00	0.31	0.71
液泡	0.0	0.0	0.00	0.01	0.33

Fig. 6 Multiple sequences alignment of EfGRAS

Fig. 7 Phylogenetic tree analysis of EfGRAS

Fig. 8 Prediction of tertiary structure of EfGRAS protein

Fig. 9 EfGRAS gene in different materials at seedling stage Different lowercase letters indicate significant difference between different materials (P <0.05).

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