Transcriptome Analysis Of Chinese Wild Vitis Amurensis And Vitis Vinifera Under Cold Stress

As one of the most important economic fruit trees in the world,grape is also one of the most widely cultivated fruit trees in China.Most cultivated grapevines belong to Vitis vinifera L.,which have a good berries quality but a poor low cold-resistance.Northwest China is suitable for planting grape for its unique climatic and soil conditions.However,under winter low temperature,European cultivars need to be buried to prevent cold damage,which is inconvenient in grape production.Therefore,breeding new cultivars with cold resistance is the key to solve this problem.Among the various Chinese wild grapevines,Vitis amurensis is the most cold-resistant species which can tolerate low temperatures at-40?.Therefore,it is a significant gene resource of cold resistance.Exploring the cold-resistant gene in V.amurensis has a great significance for revealing molecular mechanism of cold-resistance and breeding cold-resistant grapevine.V.amurensis acc.‘Shuangyou'(cold-resistant),a Chinese wild grape,and V.vinifera cv.‘Red Globe'(cold-sensitive)were chosen as the study materials.Large amount of differentially expressed genes(DEGs)from the two grapevines were screened after low temperature stress treatment and RNA-seq analysis.Further more,cold-related candidate genes were found via bioinformatic analysis.Finally,expression analysis of the candidate genes were performed by qRT-PCR.Results obtained were as follows:1.The comparisons of physiochemical indices were made between ‘Shuangyou' and ‘Red Globe' under 0? stress at 3,12,48 and 72 h.Untreated samples at room temperature were harvested as controls(0h).The results showed that the electrolyte leakage(EL)and malondialdehyde(MDA)contents increased with duration of cold treatment in the two grapevines.At the same treatment point,EL and MDA contents in ‘Shuangyou' were lower than ‘Red Globe';the proline contents in ‘Shuangyou' and ‘Red Globe' declined and then rised and finally went down with the extension of cold stress time;the proline contents in ‘Shuangyou' was higher than ‘Red Globe' at 0 and 48 h,while lower than ‘Red Globe' at 3,12 and 72h;the changes of catalase(CAT)activity was in a fluctuation and ascorbate peroxidase(APX),superoxide dismutase(SOD),peroxidase(POD)activity were all increased and then decreased;CAT activity in ‘Shuangyou' was higher than ‘Red Globe' at other time points except 48h;SOD activity in ‘Shuangyou' was higher than ‘Red Globe' at other time points except 72h;APX activity in ‘Shuangyou' was lower than ‘Red Globe' at 0,3,12,48 and 72 h;POD activity in ‘Shuangyou' was higher than ‘Red Globe' at 0,3,12,48 and 72 h.All the results indicated that ‘Shuangyou' is better than ‘Red Globe' in terms of the cold resistance.2.Total RNA was extracted from ‘Shuangyou' and ‘Red Globe' leaves trained under cold stress 0,3,12,48 and 72 h.The cDNA library was constructed and approximately 3?g of total RNA from each sample was used to sequence.A total of 5992 DEGs were obtained,including 796 new genes.Transcriptome analysis revealed that 32,1161,1894 and 3290 DEGs were found in ‘Shuangyou' at 3,12,48,and 72 h,respectively;while 240,310,1072,and 1107 DEGs were detected in ‘Red Globe' at 3,12,48,and 72 h,respectively.By contrast of the two grapevines at the same treatment,1059,519,1713,1414 and 1215 genes were differentially expressed,respectively.3.The analysis of GO richment showed that the DEGs of ‘Shuangyou' mainly concentrated on response to cold,response to chitin,jasmonic acid mediated signaling pathway,regulation of plant-type hypersensitive response,protein targeting to membrane,abscisic acid-activated signaling pathway,response to jasmonic acid;the DEGs of ‘Red Globe' mainly focus on response to cold,response to chitin,response to mechanical stimulus,intracellular signal transduction,abscisic acid-activated signaling pathway,response to wounding,response to gravity and chalcone biosynthetic process.The KEGG pathway analysis showed that the DEGs of ‘Shuangyou' were mainly involved in glutathione metabolism,circadian rhythm-plant,photosynthesis,plant hormone signal transduction and other special pathways;the DEGs of ‘Red Globe' were mainly involved in plant hormone signal transduction,plant-pathogen interaction,circadian rhythm-plant,flavonoid biosynthesis,phenylpropanoid biosynthesis and other special pathways.4.Twelve candidate genes with different expression levels were tested by qRT-PCR.Eight and other four DEGs had the same and the similar expression trends with the RNA-seq data,which proved the reliability of RNA-seq.