Functional Identification Of Differential Genes And VvINVA Genes During Hypothermic Exercise Based On Whole Transcriptome Analysis Of Vitis Amurensis

In the process of grapes responding to biological stress and abiotic stress,there have been more extensive studies focusing on mRNA,whlie little research has been done on CircRNA,lncRNA and miRNA.In this study,Vitis amurensis,which is more resistant to cold,were used as the test material,and treated with natural low-temperature growth exercises in the north.We set the growth period(A,28±1oC)and the initial cold resistance(B,4 ±1oC),intermediate cold resistance(C,0±1oC),late cold resistance(D,-5±1oC),and deep dormant period(E,-10±1oC).And the comparison of the differences between these five periods is divided into ten comparisons(I-X).The following results were obtained by RNA-seq,miRNA-seq and analysis:1.In this study,in five different cold-tempered Vitis amurensis samples,Clean Data obtained a total of 87.95 Gb in mRNA,lncRNA and CircRNA.The four types of RNA obtained were analyzed by KEGG and GO to determine the specific changes in BP,CC,and MF in the ten comparisons of I-X.One of the significant pathways related to cold resistance,starch and sucrose pathways,through the analysis of the differential gene expression of this pathway,using venn diagrams and heat maps and pathway diagrams,respectively,and finally obtained their phase specificity during the cold resistance exercise period,and specific involved gene expression.2.We obtained 21 miRNAs corresponding to the 21 differentially expressed genes related to cold resistance from the whole transcriptome data,most of which were unknown miRNAs.The only known miRNAs were miR3624-5P,vvi-miR396 b,vvi-miR397 a.Taking 21 key miRNAs as routines,its co-expression analysis with CircRNA,lncRNA,and mRNA showed that there were 1,603 mRNAs,191 lncRNAs,and 875 CircRNAs were identified and decomposed with miRNAs.15 differential genes were used for fluorescence quantitative verification,and 13 genes have consistent expression trends in qRT-PCR and RNA-seq in the second-generation sequencing.3.The VvINVA gene was cloned,and the total length of the gene was 2465 bp,the length of the CDS fragment was 1614 bp,the isoelectric point was 5.17,and the molecular weight was 60080.59 D.The entire polypeptide chain of VvINVA was hydrophilic and does not contain signal peptides and transit peptides;the protein was mainly composed of irregular coils and extended chains;subcellular localiza tio n prediction revealed that it may be localized in vacuoles.After overexpression of the gene in tobacco,it was found that the sucrose and fructose contents of the transgenic lines increased with the decrease of the environmental temperature;in the transgenic plants,the MDA content was significantly lower than that of the control;the electrica l conductivity gradually increased with the decrease of temperature,Wild-type leaf cells were more seriously injured than transgenic ones.The activities of SOD,POD and CAT in the leaves of transgenic plants were significantly higher than those of wild type.The temperature of 4oC was lower than that of 8oC.Cell membrane damage may occur,indicating that the transgenic plants have certain low temperature resistance characteristics.