Preliminary Study Of Chilling Tolerance Molecular Mechanism And Mining Functional Gene At Seedling Stage In Upland Cotton Based On Transcriptome

Upland cotton originated from subtropics and is fond of warm and afraid of cold.With the large-scale adjustment of cotton-planting area,Xinjiang has become the largest cotton-planting area in China.However,the climate in Xinjiang is cold,and the“late spring cold" often leads to the replay of cotton fields,which seriously affects the yield and quality of cotton fields.Seedling stage is the key period of chilling injury of cotton.Screening of cold-tolerant varieties in cotton seedling stage is of great significance and value to resist "late spring cold" and ensure the survival and quality and yield of cotton.This study was mainly carried out from the following two aspects:1.Through the cold resistance identification and variety screening of cotton germplasm resources in the Yellow River Basin,the Yangtze River Basin,the Northwest Inland Basin,the Northern Precocious Maturity and Foreign Countries five ecological areas,and 50 representative varieties were identified by multiple indexes;2.This study based on RNA-seq technology found six chilling candidate genes;3.The chilling-tolerant gene GhZAT10(Gh_D05G2011)was cloned and its function was verified.The main findings are as follows:1.After 4? low temperature treatment for 4.5 days and 5 days recovery,the survival seedling rate is used as the main index of chilling tolerance in cotton seedling stage.The chilling tolerance of 315 germplasms from the cotton region of the Yangtze River basin,the cotton region of the Yellow River basin,the Northwest Inland Basin,Northern Precocious Maturity and Foreign Countries were preliminarily identified.The result showed that the cotton varieties in the Northwest Inland Basin had the strongest chilling tolerance,followed by the foreign varieties.Cotton germplasms in other ecological areas had no chilling tolerance,and the difference of chilling tolerance was not significant.According to the difference of survival seedling rates,10 cotton germplasms were selected from each ecological area,and the cold tolerance was evaluated by 4 indexes,such as survival rate,cold damage grade,morphology index and relative conductivity.The final evaluation results are as follows:there have one high cold-resistant variety in seedling stage,which is Xinluzhong16,and the high chilling-resistant varieties were Xinluzao28 and Xinluzhongl.There are three chilling-resistant varieties,named STS458,Shan70 and KN27-3.There are four low chilling-tolerance varieties,named Shan79,Chaoyangmian2,Kyrgyzstan and FM1735.In addition,the remaining forty cultivars are sensitive to low temperature.2.The transcriptome sequencing of two significantly different chilling-tolerance germplasms(Xinluzhong16 and Xinluzhong32)were further performed for 4? low temperature treatment of 0 h,1 h,3 h,6 h,9 h and 12 h.A total of 67982 genes were detected.We found that transcription factors were highly up-regulated in early response to low temperature,and ZAT10 accounted for the highest proportion of the "first wave"transcription factors,reaching almost half.During 3-9 hours of chilling stress,a large number of differentially expressed genes were up-regulated between and within the groups of two germplasms.Six genes significantly enriched in GO pathways responding to low temperature and temperature stimuli were selected based on GO and KEGG enrichment analysis:Gh_D05G2011?Gh_D10G2455?Gh_D13G0160?Gh_D13G2112?Gh_A03G0210?Gh_D03G137 1.Among the six candidate genes,GhZAT10(Gh_D05G2011)belongs to the "first wave" transcription factor.And the expression of GhZAT10 in chilling resistant varieties is significantly higher than that in chilling sensitive varieties,so we cloned the gene and verified its function of chilling tolerance.3.The full length of GhZAT10 gene is 813 bp and GhZAT10 encodes 270 amino acids.GhZAT10 protein does not possess signal peptide.Further more,it was found to be localized on the nucleus by subcellular localization technology.The expression of GhZAT10 in root tissue is higher than that in stem tissue and leaf tissue,and up-regulating after chilling stress in these issues.The GhZAT10 silencing cotton plants were more sensitive to low temperature.In summary,the following conclusions are drawn from this experiment:the cotton varieties in the Northwest Inland Basin have the strongest chilling tolerance.Through the multi-index identification,we obtained one high cold-resistant variety,two high chilling-resistant varieties,three chilling-resistant varieties,four low chilling-tolerance varieties and forty chilling-sensitive cultivars.GhZAT10(Gh_D05G2011)is one of the"first wave" transcription factors by RNA-seq analysis,and its expression level in cold-resistant germplasm is significantly higher than that in chilling-sensitive germplasm.GhZAT10 silencing cotton plants showed obvious leaf wilt.So GhZAT10 posiitive regulated the chilling tolerance of cotton.This study not only provid materials and theoretical basis for cotton variety improvement and agricultural production,but also lay a theoretical foundation for cultivating chilling-resistant cotton varieties.