Cloning Of AtSUS3 Gene And Genetic Transformation Of Cotton

Cotton (Gossypium spp.) is one of the most important economic crops in the world. Cotton fibers are developed from a part of epidermal cells of the ovules, and they involve in the regulation for many gene expression. UDPG (uridine 5’-diphosphoglucose) is a substrate of cellulose synthesis. Sucrose synthase (SuSy) plays an important role in cell elongation and cellulose synthesis by producing UDPG, decreasing UDP content and improving turgor pressure in epidermal cells. Comparison of ovules between fiber-free mutant and wild type showed that ovular epidermal cells in the mutant lacked the typical bud-like projections, and the expression of SuSy was undetectable in the fiber-free seed mutant ovules at the day of anthesis (0 DPA). Furthermore, low expression SUS3 levels in the cotton ovular epidermis led to the reduced typical bud-like projections at 0 DPA and a shortened fiber phenotype at 3 DPA.Previous studies in our lab confirmed that knock-down AtSUS3 gene in Arabidopsis may cause compensation increase in other members of SUS3 family. This could promote bolting, higher rate fruit-ripening and yields of siliques in transgenic Arabidopsis. Based on the results, AtSUS3 gene was cloned from Arabidopsis and was subsequently constructed into the plant-specific vector pBI121, which was then transformed into Agrobacterium LBA4404. In order to further study the AtSUS3 in cotton, we used two different transformation methods to obtain the transgenic cotton. Results are as follows:1. The cloning of AtSUS3. AtSUS3 gene was cloned from Arabidopsis. The full length of AtSUS3 is 2430bp, and it encodes 809 amino acids. The molecular weight of AtSUS3 is 92.00kDa, theoretical isoelectric point is 5.85, the average hydrophobicity is-0.276, and the most abundant amino acids are Leu, Glu, Val and Gly. Structural prediction of proteins showed that the AtSUS3 protein belongs to the glycosyl transferase family and contains two functional domains, sucrose-synth in the N terminal as well as glycos-transf-1 domain in the C terminal. The secondary structure of AtSUS3 protein contains 34.49% of alpha helix,48.95% of random coil, and 16.56% extended-chain. Alpha helix and random coil are major elements in AtSUS3 protein.2. The constructing of expression vector. The AtSUS3 gene was constructed to the vector pB1121, named as pB1121-AtSUS3, and the pB1121-AtSUS3 was subsequently transferred into Agrobacterium strain LBA4404. By using Agrobacterium to infect cotton hypocotyl callus regeneration, the AtSUS3 was transfected into cotton cells. Under sterile conditions, we cultured full and lint-free cotton seeds to culture for 7 days. Then cotton hypocotyl segments were transfected 3 minutes with the Agrobacterium strain, washed and then co-culture with Agrobacterium for 48 hours. We acquired kanamycin-resistant callus effectively. Its conversion process included callus, embryogenic callus and embryogensis.3. Analyzing grafting methods. Three different grafting methods, which are cleft graft, conjoint graft and approach graft respectively, were tested. The results revealed that for the soil-culture seedings, the survival rate of conjoint graft was the highest, while the growth state of cotton seedings using conjoint graft was the best. For sterile seedings, the survival rate of cleft graft was the highest, and the cotton seedlings grew well.4. Acquiring AtSUS3 cotton lines. Ninetheen transgenic AtSUS3 cottons were acquired by cleft grafting regenerative cotton lines that contain AtSUS3 gene. Using ELISA, we tested the contents and activities of SuSy, INV and SPS in transgenic cotton lines. The results showed that transgenic cotton lines were generally higher than the wild type ones, but all positive lines had differences in the contents and activities of SuSy, INV and SPS.5. Exploring the method of cotton embryo transformation of exogenous gene. We developed a cotton transformation system based on direct Agrobacterium inoculation of mechanically isolated living embryos of cotton. The semi cotyledon embryos were infected by Agrobacterium containing GFP at a concentration of OD6000.4～0.6 for 3 hours, and were co-incubated for 48 hours. Then the embryos were transferred to selective medium containing 100 mg/L kanamycin for screening. The resistant plants were obtained 30 days later. The GFP was detected at DNA and RNA level, and the green fluorescence was observed in cotton leaves. However, the expression of exogenous gene was abated 40 days later, and even lost after 60 days. It indicated the transgenic cotton lines mostly were chimeras.