Preliminary Study On The Cotton Transformation Conditions Of 3GT Gene Driven By GhCesA4

Gossypium hirsutum L. is an important economic crop in our country and even in the world. Cotton fiber is a kind of main raw material of textile industry. With the improvement of people’s claim to the colour of natural textile, using genetic engineering technology and modern molecular biology methods to improve the colour of cotton fiber has becoming an important direction of the molecular breeding of cotton. Using Agrobacterium mediated transformation of plant genetic variations of cotton hypocotyl explant, started by cotton fiber tissue specific promoter Gh Ces A4, the key enzyme of anthocyanin synthesis 3GT gene was conversed into cotton hypocotyls. Resistant callus with exogenous genes were obtained. Lay a foundation for the obtain of transgenic cotton which have key enzyme gene 3GT started by cotton fiber tissue specific promoter Gh Ces A4. And then lay a foundation for the improvement of cotton fiber colour by molecular biology techniques. The main results were as follows:1. The induction of callus from cotton hypocotylRegard MSB as basic medium, explore the influence of different 2,4-D and KT combinations on the production of cotton hypocotyl callus. The results showed that 0.1 mg/L 2,4-D+ 0.1 mg/L KT is conducive to the production of cotton hypocotyl callus. The callus induction was 80%.2. The construction of plant expression vector1503 bp Gh Ces A4 cotton fiber cell-specific promoter was obtained from the cotton genomic DNA by PCR amplification. Connect the received promoter with the p CAMBIA1305.1 plasmid which has the key enzyme of anthocyanin synthesis 3GT gene. Obtain the plant expression vector p CAMBIA1305.1-3GT-Gh Ces A4 which has the cotton fiber tissue specific promoter Gh Ces A4 and 3GT gene.3. The antibiotic sensitivity testThe effects of different concentrations of Hygromycin(Hyg) and Cefotaxime(Cef) on cotton hypocotyl callus induction and the inhibitory effect of Cefotaxime to Agrobacterium were studied to determine the optimum concentration of screening agent and antimicrobial agent of cotton hypocotyl in Agrobacterium-mediated genetic transformation. The results showed that when the concentration of Hygromycin is 100 mg/L, Hygromycin won’t induce big effect on hypocotyl callus, and will effectively transformed explant selection. When the concentration of Cefotaxime is 600 mg/L, the induce of cotton explant callus won’t impacted largely, at the same time the Cefotaxime can effectively inhibit the grow and reproduction of Agrobacterium tumefaciens and prevent contamination caused by Agrobacterium tumefaciens.4. The Agrobacterium-mediated genetic transformation of cotton hypocotylWell-built plant expression vector p CAMBIA1305.1-3GT-Gh Ces A4 was conversed into Agrobacterium(LBA4404) cells. The cotton hypocotyls were infected by the prepared Agrobacterium. In Agrobacterium-mediated genetic transformation of cotton hypocotyl, the time of Agrobacterium infection and co-culture can affect the resistance rate of callus formation to a certain extent. The results showed that when the infection time of Agrobacterium was 15 min, the resistant callus rate was 53.33%, and when the co-culture time was 24 h, the resistant callus rate was 56.66%, the conversion efficiency reached the expection. Therefore, 15 min of Agrobacterium infection and 24 h of co-culture were good for the production of resistant callus. The cotton hypocotyl callus which have cotton fiber tissue specific promoter Gh Ces A4 and 3GT gene were obtained by Hygromycin screening and 3GT gene PCR detection.