Cloning And Functional Analysis Of Gossypium Barbadense Rar1, Sgt1, Rac1, KTN1 Genes And Gossypium Arboreum Cab Promoter

Cotton, as one of the most important economic crops, has played an important role in our national economy development. Currently two key problems are facing in cotton production in China that is the prevention from infection of Verticllium wilt and the upgrade of fiber quality. Targeting to solve these two problems at a gene level, three parts of studies have been carried out in this thesis as following:(1) Cloning of GbRar1, GbSgt1, and GbRacl genes from Gossypium barbadense and analysis of disease resistance of transgenic tobacco plantsThe coding sequence of the three defense signaling components, Rar1, Sgt1 and Racl proteins, known act at a position between downstream of R gene and oxidative burst, was cloned respectively from G. barbadense. Southern blot analysis indicated GbRarl and GbRacl genes were existed in G barbadense as a single copy. Northern-blot showed the level of mRNAs of GbRarl, GbSgtl or GbRac1 was dramatically increased upon induction with Verticllium dehliae inoculation. It suggests that the GbRar1, GbSgt1 or GbRacl may involve in the defense response of G. barbadense to Verticllium wilt infection.Three plant expression vectors pSgt, pRar and pRac, harboring GbRar1, GbSgtl or GbRacl gene driven by CaMV 35S promoter, were constructed and used for transformation of Nicotiana tabacum var. NC89, respectively. Southern-blot demonstrated that all these three genes were integrated into the genome of tobacco. Disease challenge test of leaves of transgenic plants in vitro by inoculation of Alternaria alternata showed that the resistance was enhanced compared with the non-transgenic plants. This implicates that they may have a potential application in genetic engineering of plants with enhanced disease resistance.(2) Cloning of GbKTN1 gene from G. barbadense and effect of GbKTNl over-expression on yeast cell elongationKatanin is a protein with a function of coupled ATP hydrolysis and microtubule severing. It is well known that the homologous gene coding for katanin-like protein in Arabidopsis has a significant role in cell elongation and cell wall biosynthesis. The cDNA of GbKTNl gene was isolated from 10 DPA fiber cells of G. barbadense using 5'RACE/3'RACE techniques. Southern-blot hybridization indicates it is a single copygene in G.barbadense. Semi-quantitative RT-PCR combined with southern-blot analysis revealed that GbKTN1 was expressed in roots, hypocotyls, leaves and fibers. However, the transcripts mRNA of GbKTNl was most abundant in fiber cells, while it was lowest in leaves. It is likelihood that GbKTN1 gene is preferentially expressed in the sclerenchyma cells than that in other types of cells.The GbKTNl cDNA was transformed into S. pombe to confirm its function on cell elongation. Results showed that most yeast cells over-expressing GbKTNl gene were elongated dramatically with a cell length of 2-3 fold increase than that of non-transformed cells. Its implication is the GbKTN1 gene may correlate with the cell elongation in G.barbadense.(3) Cloning and functional analysis of a light-inducible Gacab promoter from Gossypium arboreumA 1009 bp promoter sequence of cab gene encodes chlorophyll a/b binding protein belonging to a class of light-inducible proteins was cloned from G. arboreum. Sequence analysis showed no obvious homology to the published cab promoters. The full-length Gacab P and 5' end deletions with a length of 197 bp, 504 bp or 779 bp was fused with gus (uid A) gene respectively and plant expression vectors constructed. Construct with Gacab P:: gus was used for transformation of Nicotiana tabacum var. NC89 and transgenic tobacco plants generated. GUS histochemical assay showed that GUS was specifically expressed in leaves and young green tissues. GUS was not detected in leaves of transgenic plants grown in the dark for 6d, whereas GUS was highly expressed in leaves of these plants further induced with light for 6d, demonstrating Gacab P was a light-inducible promoter. Transit GUS expression in rice calli indicated that the expression level of 504 bp...