Cloning And Expression Characterization Of GhRGP1, GhRGP2, GhUPTG1 And GhUPTG2 In Cotton

The process of fiber development is involved in a series of reactions of polysaccharide biosynthesis. Many glycosyltransferases genes isolated in plants conform to glycosyltransfering reactions. These glycosyltransferases, such as CesA, Csl, RGP, UPTG etc, are important in fiber cells. It is important to isolate and characterize the functional expression of these genes. We isolated GhRGPl, GHRGP2, GhUPTGl, GhUPTG2 genes in previous work, and these genes may related to the growths of cotton fibers. The experiment results as follows:1. Sequence analysisDatabank searches revealed that GhRGP shared more than 79% homology at the amino acid level with the known RGP cDNAs from other plants, such as rice, wheat, pea , cowpea, Arabidopsis, maize andpotato( 90%, 87%, 90%, 86%, 88%, 87% and 89%, respectively). GhRGPl and GhRGP2 genes belong to the RGP gene. Compareing the predicted polypeptide sequence of the cotton GhUPTGl gene with those known UDP-transglucosylase family ami no acid sequence, we found that their homology is 30% - 43%, whereas the GhUPTG2gene is 32% - 46%, suggesting that GhUPTGl and GhUPTG2 gene may be members of UDP- transglucosylase family.2. Expression of the genes€otton total RNA was isolated from different tissues of immature plants and expression of these genes was analyzed by RT-PCR. The experimental results showed that the GhRGPl gene is expressed at the low level in the four tissues, and in cotyledon slightly higher. GhRGP2 gene is expressed high in cotyledon, hypocotyl and root, but is not expressed in leaf; The GhUPTGl gene is not expressed in all four tissues; but GhUPTG2 gene is expressed only in leaf, and is not expressed in the other three tissues. This experiments showed a differential expression pattern of mRNA levels in different cotton tissues. Expression of gene in other tissues and each tissue from different growth period will be further studied.3. Vector construction and transformationBy molecular biology technique, the ORF of the four cDNAs was inserted pBI121 vector to construct the overexpression vector, and simultaneity to construct suppression vector. The results of PCR reaction and double digestion showed that the target gene has been already successfully fused with vector. At present, these recombinant vectors were transforraated into Agrobacterium (LBA4404). We are transformating tobacco and cotton. This experiments make the foundation on studying the function of the related enzymes to cotton cellulose biosynthesis.