Cloning and sequence analysis of VaEXO11 and its promoter in Vitis amurensis

Abstract: 【Objective】Low temperature is one of the important climatic factors affecting crop yield and quality. It is urgent to study the mechanisms of low temperature at the molecular and genetic levels and to find ways to improve plant cold resistance. According to the previous transcriptome analysis, it was found that six members of the VvEXO family in Muscat were up-regulated under low temperature conditions, among which VvEXO11 was found to be the most significantly up-regulated. The analysis of the expression profile of VvEXO showed that VvEXO11 responded to low temperature stress in grapevines at early stage, and its role in the response to low temperature stress needed to be further studied. Vitis amurensis has been widely studied in cold resistance breeding. In this study, VvEXO11 and its promoter sequence in V. amurensis were cloned and analyzed by bioinformatics methods, and the function of VaEXO11 was preliminarily predicted, thus providing clues for further gene function verification, and revealing the new cold- resistant grape varieties.【Methods】Using V. amurensis as the test material, DNA and total RNA were extracted using DNAsecure novel plant genomic DNA extraction kit and RNAprep Pure polysaccharide and polyphenol plant total RNA extraction kit produced by Tiangen Biochemical Technology Co. The total RNA and DNA were extracted according to the product instructions. Using qualified total RNA as template, TransScript one- step gDNA removal and cDNA synthesis kit was used to reverse-transcribe the RNA into cDNA according to the instructions. The cDNA obtained by reverse transcription of V. amurensis RNA and extracted V. amurensis DNA were used as templates,respectively, based on the EXORDIUM sequence fragments obtained by sequencing the V. amurensis transcriptome and combined with the design of primers VaEXO11-F/VaEXO11-R, PVaEXO11-F/PVaEXO11 -R for the amplification of ORF and promoter of the target gene. The PCR amplification products were detected by 1.2% agarose gel electrophoresis and DNA Marker DL2000. The amplified DNA fragments were recovered and ligated to the cloning vector, and then the connected products were transformed into the receptor state of DH5α Escherichia coli by heat shock method. After antibiotic screening and colony PCR detection, positive clones were selected for sequencing. The promoter sequence of VaEXO11 gene in grape was firstly cloned and then analyzed by the Plant CARE (http://bioinformatics.psb.ugent. be/webtools/plantcare/html/) to predict promoter cis element. The relationship between promoter PVaEXO11 and low temperature stress was investigated by transient transformation of tobacco.【Results】The cDNA sequence of VaEXO11 gene cloned from the grapevine was 957 bp, and its open reading frame was 957 bp, encoding 318 amino acids. DNA sequence analysis of VaEXO11 revealed that VaEXO11 consisted of only one exon, which had only 6 nucleotides different from VvEXO11. The conserved domain analysis of VaEXO11 protein showed that VaEXO11 protein contained only one conserved domain, Phi_1 domain. VaEXO11 and VvEXO11 protein sequence comparison revealed that there were only 3 amino acid differences between them. The basic physical and chemical properties of VaEXO11 protein were analyzed by Protparam. The molecular weight of VAEXO11 protein was 33855.58 Da and the theoretical isoelectric point (pI) was 9.15. In amino acid composition, serine (Ser) content was the highest, reaching 12.3%, followed by leucine (Leu) and glycine (Gly), which were 11.3% and 8.8%, respectively. The instability index (Ⅱ) was 31.75, indicating that the protein was stable. The Grand average of hydropathicity (GRAVY) is -0.082 and the Aliphatic index is 85.03, indicating that it was a hydrophobic fat-soluble protein. Analysis of grape genome data showed that EXL11 was present on the 18th chromosome of grape. Grape PVaEXO11 promoter contained multiple action elements. In addition to core promoters like CAAT-box and TATA-box, some functional response elements were also found, including hormone response elements, action elements involved in drought stress, circadian rhythm regulation and trauma response elements. Phylogenetic analysis showed that besides V. vinifera and V. riparia, Tripterygium wilfordii was closely related to VaEXO11. Transient transformation result in tobacco showed that low-temperature activation of promoter activity induced a rapid increase in the relative expression of VaEXO11, directly or indirectly promoting the production of intracellular antioxidant enzymes, thereby reducing the damage of low- temperature to cells and improving cold resistance.【Conclusion】In this study, VaEXO11 gene was cloned from V. amurensis. The similarity of VaEXO11 sequence with VvEXO11 sequence in Pinot Noir was 99.37%, and the similarity of protein sequence with VvEXO11 sequence was 98.75%. The protein contained a highly conserved Phi_1 domain and a signal peptide, which was predicted to be a hydrophobic fat-soluble protein. The cis-acting elements was predicted in cloned promoter sequence, and it was found that the promoter region contained a variety of elements related to stress, indicating that the gene may participate in response to a variety of biological and abiotic stresses. Then, the relationship between VaEXO11 and low temperature was explored, and it was preliminarily speculated that low temperature could activate the promoter activity and induce the relative expression of VaEXO11 to increase rapidly, directly or indirectly promoting the production of antioxidant enzymes in cells, and thereby reducing the damage of low temperature to cells and improving cold resistance.