Cloning And Analysis Of Fiber-superiority GhRACK1Gene And Promoter From Cotton

Cotton is an important cash crops in the world. With the progress of the times and the developmentof technology, people have become increasingly demanding of fiber quality, cotton fiber in ourtraditional breeding can not meet the demand for high-end textiles. Cloning of fiber-specific gene andpromoter is an important way on improving cotton fiber of plant molec ular biology. Now most ofpromoters of controlling cotton gene are constitutive promoters. For example the CaMV35S, althoughit can drive stable expression of the exogenous gene, as a constitutive promoter, it has defects caused bytheir own properties, aid gene is expressed in the various stages of the development of cotton, variousperiods, it will not only consumethe cotton body a lot of energy substances, but also will affect plantgrowth and development. Using upland cotton sumian12as material we clone a fiber-superiority fromgene GhRACK1cotton. We continue to clone the5’ upstream of the gene promoter sequence of1987bp,and the promoter sequence for the deletion analysis. The main results are as follows:1、 Analys is of our laboratory cotton fibers obtained the differentially expressed cDNA sequecesfragment, found a cotton fiber predominantly expressed gene sequences.2、 Using3’ RACE and5’ RACE cloning of the full length cDNA of the gene, and cloning the gene onthe genome, finding the gene containing a929bp intron, analysing the cDNA sequence of the genewe found that the open reading frame of981bp encoding326amino acids. Comparing andanalys ing the gene on the NCBI, we found it gene homology of up to78%with kidney beanRACK1, expressed protein homology up to89%. Plant RACK1gene is first found in tobacco, It isan auxin-mediated gene which plays a very important role on the control of flowering andformation of meristem, so we named the clone gene GhRACK1.3、 We use inverse PCR with touchdown PCR to amplify the5’ upstream sequence of the GhRACK1gene. First amplified514bp upstream sequence, the second amplified1473bp upstream sequence.Finally we get1987bp by splicing the flanking regions.4、 With promoter prediction element analys is of the sequence, we found it has TATA box, CAAT box,GATA box cis-acting elements, as well as with trichomes, seeds, pollen development specific actingelements.5、 Delete promoter according to the distribution of specific elements, then construct5plant expressionvectors with gus at the downstream of deleted promoters as well as a full-length promoter. Wetransformation the5vectors into tobacco by Agrobacterium tumefaciens, which the prGhRACK1-0,the prGhRACK1-1, the prGhRACK1-2plant expression obtained transgenic tobacco plants.6、 Gus assay of the positive tobacco plants, we found that the prGhRACK1-0plant expression vectordriven gus gene expressing in leaf trichomes, and apical parts; the prGhRACK1-1plant expressionvector driven the gus gene highly expressed in the entire blade root; the prGhRACK1-2plantexpression vector driven as the gus gene expression only in root hairs.