The Analysis Of The Characteristics Of Gene Expression And Their Responding To High Temperature Stress During Cotton Anther Development

Cotton(Gossypium hirsutum L.)is one of the most important economic crops in the world,and its fiber is the main natural source of the world's textile industry.The suitable temperature range for cotton growth and development is 20-30?.When the day/night temperature lasts more than 30?/20? for 13 hours,it will cause the bud and boll shedding,and the longer the duration of high temperature(HT),the higher the shedding rate,which seriously affects the yield of seed cotton.With population growth and the process of industrialization,global warming has become an irreversible trend,and extreme weather such as periodic high temperatures and droughts will occur more frequently.Therefore,it is urgent to deeply understand the effect of HT on cotton growth and development and the mechanism of HT tolerance,and to develop new HT-tolerant cotton cultivars.In the present study,high-throughput deep sequencing technique was used to carry out transcriptome and smallRNA sequencing on the different development stages of anthers between HT-tolerant cotton line and HT-sensitive cotton line in order to explore the different responding of developing anthers to HT stress,dissect the molecular adaptation mechanism of anther/pollen tolerance to HT,and further to discover new HT-tolerant genes.The results will provide theoretical basis and gene resources for the improvement of cotton tolerance to HT.1.The gene expression characteristics of cotton anther during the process of development were deeply analyzed.Plant anther development is a systematic and complex process precisely controlled by a lot of genes.The genes and their regulatory mechanisms for this process remain elusive.In contrast to some researches on anther development with respect to mRNAs or miRNAs in many crops,the association analysis combining both omics has not been reported on cotton anther.Here the molecular mechanism of cotton anther development was investigated with the employment of association analysis of transcriptome and smallRNA sequencing during the predefined four stages of anther development,sporogenuous cell proliferation(SCP),meiotic phase(MP),microspore release period(MRP)and pollen maturity(PM).Analysis revealed that the differentially expressed genes are increasingly recruited along with the developmental progress.Expression of functional genes differed significantly among developmental stages.The genes related with cell cycle,progesterone-mediated oocyte maturation,and meiosis are predominantly expressed at the early stage of anther development(SCP and MP),and the expression of genes involved in energy metabolism,flavonoid biosynthesis,axon guidance and phospholipase D signaling pathways is mainly enriched at the late stage of anther development(MRP and PM).Analysis of expression patterns revealed that there was the largest number of differentially expressed genes in the MP and the expression profiles of differentially expressed genes were significantly increased,which implied the importance of MP in the entire anther development cycle.In addition,prediction and analysis of miRNA targeted genes suggested that miRNAs play important roles in anther development.The miRNAs ghr-miR393,Dt?chr12?6065 and At?chr9?3080 participated in cell cycle,carbohydrate metabolism and auxin anabolism through the target genes,respectively,to achieve the regulation of anther development.Anyway,Through the association analysis of mRNA and miRNA,our work gives a better understanding of the preferentially expressed genes and regulation in different developmental stages of cotton anther and the importance of meiotic phase,and also the involvement of miRNAs in precise regulation for this process,which would be valuable for clarifying the mechanism of plant anther development in response to internal and external environments.2.The HT-responsive miRNAs in cotton anther varied with different development stages.We performed miRNA sequencing during the four stages of cotton anther development under normal and high temperature(NT and HT,respectively)conditions to investigate the response of miRNA profiling to HT.A total of 77 miRNAs,including 33 known miRNAs and 44 novel miRNAs,were identified,and41 and 28 miRNAs were differentially expressed under NT and HT stress conditions,respectively.The sporogenous cell proliferation(SCP),meiotic phase(MP),microspore release period(MRP),and pollen maturity(PM)stages had 10(including12 miRNAs),4(including 6 miRNAs),4(including 5 miRNAs),and 7(including 11 miRNAs)HT stress-responsive miRNA families,respectively,which were identified after removing the changes in genotype-specific miRNAs under NT condition.Seven miRNA families(miR2949,miR167,and miR160 at the SCP stage;miR156 and miR172 at the MP stage;miR156 at the MRP stage;and miR393 and miR3476 at the PM stage),which had expression abundance of more than 10% of the total expression abundance,served as the main regulators responding to HT stress with positive or negative regulation patterns.These miRNAs orchestrated the expression of the corresponding target genes and led to different responses in the HT-tolerant and the HT-sensitive lines.3.Effects of HT stress on anther transcriptome and fatty acid content were characterized.The effect of HT stress on gene expression in cotton anther at different developmental stages was analyzed by transcriptomic sequencing technology using HT-tolerant and HT-sensitive lines as materials.KEGG enrichment showed that glycosaminoglycan degradation,protein processing,biofilm formation and fatty acid biosynthesis were the four mutual signal pathways in the early stages of anther development,while unsaturated fatty acid biosynthesis,?-linolenic acid metabolism and flavonoid biosynthesis pathway were the mutual signal pathways in the later stages of anther development.The target genes of the main HT-responsive miRNA families in anther at different developmental stages were predicted from the differentially expressed genes at the same stages.KEGG enrichment of these taget genes showed that miRNAs were mainly involved in the regulation of sugar metabolism(starch and sucrose metabolism,carbohydrate metabolism,pentose and glucuronic acid transformation)and fatty acid metabolism(fatty acid degradation,fatty acid biosynthesis,fatty acid metabolism and ABC transport carrier)in cotton anther under HT stress.Further analysis of fatty acids by targeted metabolome detection method showed that content of the most unsaturated fatty acids,including Arachidic acid,Linoleic acid,Linolenic acid,Oleic acid and ?-Linoenic acid in the anthers of the HT-tolerant line was significantly lower than that of the HT sensitive line,while Docosadienoic acid,Dodecanoic acid,Eicosadienoic acid and Pentadecenoic acid contents in the anthers of the HT-tolerant line were significantly higher than those of the HT-sensitive line,especially in the anthers at the MRP stage.4.Identification and preliminary functional verification of candidate genes in response to HT stress.According to the above studies,Gh?D10G2577(Acx),Gh?A06G0328(DPBF3),Gh?D10G2551(WRI1)and Gh?A08G1739(FATA)were identified as important candidate genes involved in responding to HT stress in anther.The plant overexpression vectors of the four genes were constructed and transformed into Arabidopsis thaliana.The homozygotes of transgenic Arabidopsis thaliana were selected and used for HT stress treatments.The results showed that tehe transgenic plants with overexpression of Gh?D10G2577 gene showed obvious characteristics of HT sensitivity and all died under HT stress.The other transgenic plants with overexpression of Gh?A06G0328,Gh?D10G2551 and Gh?A08G1739 genes showed characteristics of HT sensitivity with a part of transgenic lines,while the other part of transgenic lines showed HT stress phenotype similar to the wild type.The siRNAs near the coding region of Gh?D10G2577 gene showed significantly differential expression in the later stage of anther development between HT-tolerant and HT-sensitive lines under HT stress.The Cp G island methylation in the promoter region of Gh?D10G2577 gene in anther under HT stress was detected,and the results showed that the methylation frequency of CHG site was significantly higher in HT-tolerant line than HT-sensitive line.Accordingly,the siRNA-mediated epigenetic regulation might affect Gh?D10G2577 gene expression in the later stages of anther development under HT stress,and the different regulation of the gene expression between HT-tolerant and HT-sensitive cotton lines may be one of the important reasons for different tolerance of anthers to HT.