The Cloning Of Epsps Gene From Cotton And Analysis Of Expression Character

The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase is essential for the biosynthesis of aromatic compounds in plants and microbes. It is the unique target of the herbicide glyphosate and catalyzes the reaction between phosphoenolpyruvate (PEP) and shikimate-3-phosphate (S3P) to synthesize 5-enolpyruvylshikimate 3-phosphate (EPSP).Glyphosate is a broad-spectrum herbicide, which is obsorbed and transfered to other parts of the plants. It acts on plants and microorganisms by inhibiting synthesis of 5-enolpyruvyl-3-phosphoshikimate.the precursor or aromatic amino acid, which leads the plants lacking of aromatic amimo acid and finally died。Cotton was planted in valleies of the Changjiang River , Huanghe River ,Huaihe River and area of xinjiang on a large scale, and it was a very important economic crop in our courtry. The output and quality of cotton impressed by the herbicides, Therefore, it is extrememly important for the agriculture development to study the glyphosate-insensitive gene aiming at the cultivation of the transgenic cotton.In this study, a new RNA isolation method for the cotton tissue was investigated. The cotton tissues contain a plenty of gossypol, saccharide, tannin and other secondary metabolites, which increase the difficulty of RNA isolation. This new method was easy to operate, less time using and high-efficacy. In this research, cotton line Y18 was investigated. Two homologous epsps genes, epspsA and epspsB, were discovered in Y18. The expression character analysis of this two genes indicated that epspsA was expressed invariably and the expression of epspsB was induced.in cotton. It was a basic to cultivate the transgenic glyphosate-resistant cotton. Meanwhile, epspsB was cloned successfully by using RACE method. The full legnth of epspsB contained 1928bp, coding sequence 1563bp and 522 amino acids coded, 5'UTR 117bp, 3'UTR 249bp, and poly (A) tail in 3'end. The content of G+C% was 42.32%. epspsB had homologous with the epsps genes from other spices. epspsB was ligated with pPIC3.5k vector by using the Pichia pastoris expression system.