Cloning And Expression Analysis Of Two Cotton Fiber Advantage Express Gene AGP

Cotton fiber as the main products of cotton production, whether or not advantages of its quality directdecides the quality and efficiency of textiles. How to improve the quality of cotton fiber has always been amain objective of cotton breeding research. There are a large number of active expression genes in cottonfiber tissues. These genes strictly control the growth and development of the fiber. Isolation andidentification of cotton fiber development related genes can provide an important theoretical significancefor using genetic engineering means to improve quality of cotton fiber.Arabinogalactan-proteins(AGPs) is a kind of glycoprotein family and it is rich in hydroxyproline,fasciclin-like AGPs(FLAs) is a subfamily of AGPs proteins. Study show that AGPs can promote expansionof cells, extension of root’s epidermal cells, elongation of pollen tubes and signal transductions,and so on.This study we isolate GhFLA1and GhFLA4genes from cotton fiber, and preliminary analysis the sequenceof genes, Space-time characterization and function of this sequences in cotton tissues. The results of thestudy are as follows:1.GhFLA1gene’s overall length has1013bp, it can encode244amino acids. GhFLA4gene’s overall lengthhas878bp, it can encode245amino acids. Both of them are not contain introns.2.We utilize real-time fluorescence quantification PCR for analysing expression characteristics of twogenes in cotton tissues and different development days’s fiber. The results show that: GhFLA1and GhFLA4genes hardly expression in cotyledon. They have a small amount of expression in roots and stems, butperformance preferential expression in cotton fiber and is regulated by the development of cotton fiber. Inanother word, with the development of cotton fiber, expression quantity performance unimodal curve. Themaximum appeared in18DPA, and it suggests that this gene is associated with cotton fiber development.3.To study the function of GhFLA1and GhFLA4genes, we utilize “Gateway” technology for building plantexpression vector, and transform BY-2tobacco suspension cells, tobacco and cotton YZ-1by genetictransformation which is mediated by agrobacterium. Compared transgene BY-2cells with wild type cells,transgene BY-2cells’ length is obviously longer than wild type, the leaf epidermal hair of tobacco issignificantly more than the wild type; We build plant expression vector which is mixed of GFP, theinstantaneous expression by injecting a tobacco leaf, and we find by confocal microscopy that, thefluorescence signal of GhFLA1protein appearance in cytoplasm, but GhFLA4main exist in cytoplasm andnucleus; We build yeast expression vector of GhFLA1and GhFLA4, and get a genetically modified yeast.Compare to the synchronous culture wild type, we find that transform gene cells’ length has increase. Andlast, we successfully build GhFLA1and GhFLA4’s protein expression vector, and transform EscherichiaColi DE3bacterial strains. The microbial is induced by IPTG, and then analysed by SDS polyacrylamidegel electrophoresis, we find that the target proteins all appearance at29KD finaly.