Preliminary Function Analysis Of Two Annexins In Cotton (Gossypium Hirsutum L.)

Cotton is an important raw material of textile industry. Cotton fiber grows out of a single epidermal cell. Cotton fiber development can be divided into four different development processes, in each process there are many kinds of different genes participating jointly in fiber morphology. So to illustrate cotton fiber development related genes function not only has the production significance, but also can help to elucidate the molecular mechanism of single-celled development.Cotton often suffers all sorts of harm, such as drought, salt, low temperature, diseases and pests, and is seriously affected in its quality and yield; therefore to foster high resistant cotton varieties of cotton breeding work is one of the important tasks.Annexins constitute an evolutionary conserved multigene family of Ca2+-dependent membrane binding proteins, which can be found in eukaryotes, except the yeast. Annexins play an impotant role in the process of plant growth and respond to environmental stresses and stimulation.Our lab cloned two annexins from cotton material 7235. On this basis, we studied the expression of genes under abiotic stresses, and constructed different expression vectors. Now, we have got several transgenic pure lines, providing a platform for further functions analysis of annexins.The material is jinmianl9, abiotic stresses consist of low temperature (4℃), PEG6000, ABA, NaCl and Salicylic acid (SA). Q-PCR was used to detect the expression of GhFAnnl and GhFAnnx under various abiotic stresses. Compared with control, GhFAnn1 expression was induced strongly after drought,4℃, ABA and Salicylic acid (SA) treaments, whereas it seems to be unaffected by NaCl treatment. The level of GhFAnnx expression increased after the treatment with drought and SA, down-regulated after NaCl treatment. After treatment with ABA, the expression level of GhFAnnx did not clearly change. The results indicated that GhFAnn1 and GhFAnnx were the resistance genes in respond to abiotic stress。We constructed 6 expression vectors of GhFAnn1 and GhFAnnx, including sense expression vector of GhFAnn1 gene (SAnn1), anti-sense expression vector of full length of GhFAnn1 gene (AsAnn1), anti-sense expression vector of 3'-upstream fragment of GhFAnn1 gene (3AsAnn1), sense expression vector of GhFAnnx gene (SAnn2), anti-sense expression vector of full length of GhFAnnx gene (AsAnn2), interfered expressed vector (IAnn2). Then, all of the 6 plant-expressed vectors were transformed into the cotton genome. On the basis of abtained partial T1 and T2 regeneration plants, we continued to create and identificate different transgenic clone lines. Using PCR and Kanamycin resistance, we obtained many transgenic pure lines. Q-PCR was used to study the genes expression of transgenic pure lines. Among all the pure lines of SAnn1, the level of expression about 4 pure lines (2-2,110-2,84-3 and 80-1-1) were abundant, and the others (57-1,77-1-2,96-2,110-4-2,110-6-2,110-7-3,107-4-1,107-5-1 and 107-7-1) were low, in all the developmental stages of the fiber compared to the non-trangenic line(W0). The level of expression of all pure lines from AsAnn1 (67-1,140-1 and 157-1) was similar to W0. Three pure lines from 3AsAnn1 were lower than control in all the developmental stages of the fiber. In all pure lines of SAnn2, the level of expression of 32-1 increased, while the others declined. The level of expression of pure line(2-1) was up-regulated at 0DPA, and down-regulated at 5DPA,10DPA,15DPA and 20DPA,2-1 was a clone of anti-sense vector (AsAnn2). The clones of 2-1-1,83-1-3 and 18-1 came from interfered expressed vector (IAnn2), GhFAnnx expression was significantly lower in 2-1-1, and higher in 83-1-3 than W0. The level of expression of pure line (18-1) increased at 0DPA and 5DPA, and reduced at 10DPA,15DPA and 20DPA. In different transgenic pure lines, expression pattern of target genes remain to be verified.Two years of test results of the determination of cellulose indicated that among transgenic pure lines of SAnn1, the fiber length of two pure lines (107-7-1 and 80-1-1) were significantly higher than the control, but micronaire and strength did not significantly change. The fiber length of four pure lines(2-2,57-1,110-5-3 and 110-7-3) got shorter, fiber strength did not change significantly, and other genetically modified material of the fiber quality indicators with no significantly change compared to control. All of the AsAnn1 trangenic pure lines did not change significantly in fiber length. Among them, clone 67-1 and 157-1, their strength and micronaire significant difference and very significant compared to control. Among all of 3AsAnnl trangenic pure lines, a pure line (63-2), its fiber length was significantly shorter than control, fiber quality parameters of the other trangenic pure lines did not change significantly. Among all of SAnn2 genetically modified material, the colne 42-1, its fiber length was significantly shorter than control, the others'fiber quality indicators did not change clearly. There is only one pure line (2-1) of all the AsAnn2 trangenic cotton, its fiber length is significant shoter than control, micronaire and strength did not change. Three pure lines of IAnn2 genetically modified material were significantly or very significantly shorter in fiber length than control. The relationship of fiber quality and gene expression needs to further study.