Cloning, Identification And Function Analysis Of One Sucrose Synthase Gene In Cotton

Cotton(Gossypium spp.) is an important economic crop and the largest source of textile fiber in the world. As a single cell without the complication of cell division and multicellular development, cotton fiber was considered to a unique experimental system for investigating the regulation of fiber-specific genes. Cotton fiber was operated by a lot of genes expressed specifically during the ovule and fiber development. At the same time, improvement of the cotton transformation efficiency and isolation of fiber-specific genes provided a platform for production of transgenic cotton and function analysis of cotton key genes. Sucrose synthase (SuSy, EC2.4.1.13), as one of the key enzymes impelling sucrose into various metabolic paths, exists in prokaryote and eukaryote universally. Its product, UDP-glucose, is known as the most directly substrate of cellulose synthesis in fiber secondary wall formation.In this study, we obtained a fragment of sucrose synthase from the library of7235, and cloned its full length sequence of genome using TAIL-PCR technology, which was5582bp, included15exons and14introns. Meanwhile, we amplified its2790bp cDNA sequence, which has a2430bp ORF, coding809amino acids, and named GhSUSAl. This gene belonged GT-1family, which contained typical glycosyl transfer domain, and more than one phosphorylation site. We amplified the genome sequences from two diploid cottons, and confirmed at least one copy of the A sub-genome and the D sub-genome in tetraploid cotton. This gene had steady expression in different tissues of cotton, high in the root and stamen. It expressed differently in fiber with various DPAs, predominantly in the initial time and cell wall deposition time of fiber development. The expression level of GhSUSA1was higher in ovule than fiber during elongation time. Analysis of expression in different genetic cotton materials showed GhSUSAl expressed low in0DPA of two lintless-fuzzless mutants, and high in7235during elongation time compared with TM-1. Although had similarity in amino acids, the gene of GhSUSA1had significant differences in molecular characteristics and evolutional relationship as compared to other two cotton SuSy genes. Three SuSy genes not only had differences in gene constructions, but also in expressions.Functional roles of cotton sucrose synthase gene (GhSUSA1) in cotton development were analyzed using the anti-genetic technology. In vitro enzyme assays indicated that GhSUSA1exhibited activity with existence of sucrose and UDP by prokaryotic expression system. To further functional analysis, the antisense constructs of GhSUSAl driven by the E6(E6AS-SA) and the constitutive CaMV35S (35AS-SA) promoter were developed using pBI121as the fragment, respectively. Then, this plant-expressed vector was transformed into the cotton genome by the Agrobacterium-medlated transformation. All of TO, T1and T2transgenic plants were detected by the PCR analysis using the NPTII and promoter-gene specific primers. Finally,12antisense transgenic pure lines were obtained after three rounds selections. All of the12pure lines with one to four copies transgene insertion tested by southern blot analysis of the NPTII were selected to further analyze. We found that all of the transgenic lines containing the antisense GhSUSA1demonstrated the various changes in the fiber lengthen and part of them difference in fiber strengthen and fineness. We selected7lines for further analysis, in which had no suppressed in other two SuSys. The GhSUSA1expression in5DPA of the four E6AS-SA transgenic cotton lines were suppressed with different degree of inhibition varied. Although the reduction of SuSy enzymatic activity by decreasing GhSUSA1transcripts didn’t affect the amount of cellulose, the content of fructose and glucose decreased severely, and the sucrose decreased except one line, which was consistent with activity. In the lines of35AS-SA, there was more obvious exchange in seeds compared with fibers. Both ball size and100-seed weigth were decreased and the seedling biomass of pure lines was lower than WO, too. Q-PCR analysis of three35AS-SA lines in10-20DPA seeds showed significantly decreasing of GhSUSAl, especially in10DPA seeds. Subsequently, the reduction of SuSy enzymatic activity by decreasing GhSUSA1transcripts severely affected the sugar content and finally leaded to shrinking of starch. Ultimately, the seeds size and weigth was reduced, especially in Line45-49. These results indicated that suppression of GhSUSA1in fiber could change differences of turgor between seed.and fibers then leaded to reduction of fiber length. While the consistive suppression of GhSUSA1had more significant effection in sink strength and seed development.In addition, a sense-expression construct of GhSUSA1driven by the35S promoter (35S-SA) was introduced into the WO embryogenic calli for further research of GhSUSA1function. There were co-suppression of sense GhSUSA1, but some plants showed higher level in expression than WO. Plants with over-expression of GhSUSA1had corresponding high content of sugar and T2seeding biomass. Some of TO and T1plants with over-expression of GhSUSA1showed longer fiber than WO, but segregation in T1. These need further research by pure lines.Alos, we found and selected a mutant whose phenotypes could be stably inherited into the next generation:a cotton long internode mutant. We analysesed phynotype of this mutant and had a preliminary identification of the T-DNA insertions. Moreover, a light yellow sprout mutant and a curled leaf mutant appeared in the prosess of transgene. These cotton mutants were purified, which could be the valuable materials using for cotton biology analysis.