A Preliminary Study On Alternative Splicing Of Specific Genes In Upland Cotton

In eukaryotes,alternative splicing(AS)is a process of splicing of mRNA precursors(pre-mRNA)with different modes,resulting in diverse transcript isoforms,and ultimately producing multiple proteins from a single gene.As an important means of post-transcriptional regulation,the AS process greatly enhances the diversity of eukaryotic genetic levels.In order to further understand the status of this AS-induced diversity in plants,we selected a cultivar accession(Maxxa)and a wild variety of Gossypium hirsutum(Tx2094)as research materials,taking five different types of AS events as the examples.The occurrence and expression patterns of the isoforms of five genes in leaves and fibers of the upland cottons were qualitatively and quantitatively compared.The functional differentiation of different isoforms of each gene was also discussed.The main results are as follows:1.Based on our previous fiber transcriptome data of upland cotton,TUBA1,CSFS50,GBE1,Myosin2 and DAD genes were selected from the genes that possessed five basic types of alternative splicing events(retained intron,RI;skipped exon,SE;alternative 5? splice sites,A5SS;alternative 3? splices site,A3SS;mutually exclusive exons,MXE).The five genes produce 2-3 splicing isoforms,respectively.The isoforms of TUBA1 and DAD are expressed in both leaves and fibers,but the expression level is much higher in fiber than that of in leaves;the isoforms of the other three genes are mainly expressed in the leaves.A significant differential expression was observed between the different isoforms of each of the five genes.In addition,the homologous isoforms of the five genes also showed significant expression differentiation between the cultivator and wild variety of upland cottons.2.The DAD was used as an example to show the expression pattern of multiple transcripts(parental duplicated homoeologs and isoforms derived by AS events)within same polyploid plant.By cloning and sequencing the mRNA of DAD gene,it was found that in the G.hirsutum with AADD genome,the AS regulation of DAD gene was only acted on the homoeolog of D genome,which generating a total of three transcripts Dt1,Dt2 and At.Quantitative RT-PCR analysis showed that the three transcripts had obvious spatial and temporal differentiation expressions,in which At was mainly expressed in leaves;Dt1 was expressed in both leaves and fibers,but the expression level in fibers was higher,and the expression level was significant up-regulated during fiber development;while Dt2 has a lower expression only in the young leaves and the late fiber development stage in cultivated cotton.3.To investigate whether the differential expressions of three transcripts of DAD gene in upland cotton are related to their functions,three pBI121 overexpression vectors were constructed and introduced into ecotype Col-0 of Arabidopsis thaliana using Agrobacterium-mediated transformation.The phenotypic comparison of the transgenic plants and wild type of Col-0 showed that the Dt1-overexprssion transgenic plants had the strongest vitality,and vigorous growth with the largest leaves and the highest plants;the Dt2-overexprssion plants were the next;At-overexprssion plants were similar to wild type in terms of phenotype and viability except for having less fruits.