Identification And Functional Analysis Of Cotton Aquaporin Gene And Vesicle Membrane Protein Gene

In cotton breeding practices, in order to improve the quality of cotton fiber, cultivate high-yield, high-quality and stress resistance new varieties, so we master the law of heredity and variation of cotton fiber are necessary. With the progress made in cotton molecular biology and functional genomics, especially for the large scale EST (expressed sequence tag) sequencing projects, more and more fiber development-associated genes have been cloned and characterized. These data pave the way for gene expression and discovering regulation mechanisms involved in cotton fiber development.In this experiment, we separated a gene from cotton fiber library which is constructed from G. hirsutum super-fiber quality line 7235. The gene fragment related to vesicle trafficking. By 5’RACE technique, we obtained a 1215bp length cDNA which open reading frame is 660bp, encode 219 amino acids. Blastx analysis indicated this gene had a 90% identity with homogentisate Vesicle-associated membrane protein of Ricinus communis. In G. hirsutum TM-1, GhVAMP genomic sequence is 2880bp that have 4 introns and 5 exons. GhAQP genomic sequence is 1111bp in G. hirsutum TM-1 and contains 3 introns and 4 exons.We use qRT-PCR and RT-PCR study GhVAMP and GhAQP genes in cotton (G hirsutum TM-1 and G. hirsutum 7235) root, stem, leaf and different development stage fibers. The results showed that GhVAMP gene predominantly expressed in roots, GhVAMP gene between the two varieties express extremely significance different. In 1DPA, GhVAMP gene expression was also higher, speculated that the gene may be related to cotton fiber development and the formation of the ovule. Because in this period, GhVAMP gene participate in a lot of carbohydrates and lipids synthesis and transport, and fiber cytoskeleton establish.GhAQP gene has the highest expression in stem, and has a significance difference in roots and 21DPA. The root is the important parts of plants obtain water; stem is the only transport channel of water from the ground. Some aquaporin can also transport small molecules hydrophilic neutral solutes, which plays a very important role in metabolism of the water and material metabolism. From 8DPA to 21DPA, aquaporin gene expression gradually increased, speculated that the aquaporin gene is closely related to the cotton fiber elongation and fiber maturity.Plant expression vector pCAMBIA1304 which contains GFP reporter gene, we use it making up GhAQP gene subcellular localization vector pCAMBIA1304-GhAQP. The results showed that GhAQP gene is located at the onion cell’s membrane.In order to clarify the genes function, we constructed three plant expression vectors and transformed into cotton plant cells via Agrobacterium-mediated genetic transformation. By PCR text, now we obtained GhAQP gene sense vector 5 clones positive regenerated plants,3 clones had been get seeds of To generation; obtained GhAQP gene antisense vector 7 clones positive regenerated plants,6 clones had been get seeds of To generation; obtained Gh VAMP gene antisense vector 10 clones positive regenerated plants,8 clones had been get seeds of To generation; Some of the characters changed of the regenerated plants. For example, flowers malformation, leaves become bigger and so on. Southern blot showed that, GhAQP gene and GhVAMP gene had been inserted to the cotton genome.Using transform GhAQP gene cotton’s leaves did protoplast permeability experiment. The result show that sense vector regenerated plants aquaporin expression increase and translation more aquaporin gene, contribute to plant absorb and transport water. Enhance of plant tolerance drought and salt ability.