Resistance Mechanism Of GbVe Gene From Gossypium Barbadense To Verticillium Wilt

Verticillium wilt caused by Verticillium dahliae is a soilborne vascular diseasewhich is difficult to control and harmful to cotton production severely in China. In theformer research of our team, we had cloned a Verticillium wilt resistance gene (GbVe)from cotton (Gossypium barbadense) and verified GbVe transgenic Arabidopsis thalianahad greater levels of resistance to V. dahliae. In this research, the resistance mechanismto Verticillium wilt was investigated of transgenic Arabidopsis thaliana with Gossypiumbarbadense Ve from molecular biology, physiology and biochemistry, and phenotype.And the relations between GbVe and root, lignin, H2O2, POD, disease resistance relatedgenes EDS1, NDR1, BAK1, PR4, PR5were researched. Meanwhile, Ve genes and theirpromoters from different cotton varieties were cloned and compared. GbVe transgeniccotton was obtained via the pollentube pathway method. The research results wouldprovide important theoretical basis for revealing the resistant mechanism of Ve. Theresults were as follows:1. Compared to wild type Arabidopsis, the transgenic Arabidopsis obviously had lesshyphaes in the stem at21d post inoculation. It was shown that the proliferation ofVerticillium wilt fungus was suppressed in transgenic Arabidopsis. Identification ofdisease resistance showed that the transgenic Arabidopsis of GbVe had greater levels ofresistance to V. dahliae. The roots of GbVe transgenic Arabidopsis developed more rapidlythan the wide type Arabidopsis.2. After inoculated with V. dahliae17days, lignin content of transgenic Arabidopsisand wild type all increased. The lignin content of transgenic Arabidopsis was more thanthat of wild type Arabidopsis before inoculation or after inoculation. In the wild type, H2O2content reached the peak at30hpi post inoculation, then began to decline, but was higherthan that before inoculation. But in the transgenic Arabidopsis H2O2content reached thepeak at12hpi after inoculation, then began to decline and was lower than that beforeinoculation. H2O2content in wild type was about2times of transgenic Arabidopsis at72hpi. After inoculated, the POD activity of transgenic Arabidopsis increased more than thatof the wild type. In the wild type POD activity reached the top at18hpi, but fell rapidly.But in the transgenic Arabidopsis POD activity was the largest at12hpi, then reducedslightly, obviously dropped after42hpi.3. After inoculation, the expression quantity of GbVe was increased obviously in transgenic Arabidopsis compared to wild type. And expression quantity of EDS1was alsohigher than wild type. But the expression quantity of NDR1was lower than wild type inwhich it was increased, and it would increase later. It proved that the signal transductionpathway of GbVe-induced resistance against the Verticillium dahliae depeneded on EDS1and NDR1. The expression quantity of PR4and PR5all increased suggested that PR4andPR5involved in the GbVe-mediated resistance to Verticillium wilt. So GbVe-mediatedresistance signaling conducts mainly through the SA-mediated signal transductionpathways, and the JA-mediated signal transduction pathways is also involved in. Theexpression quantity of BAK1had no change in transgenic Arabidopsis, but substantiallyincreased in wild type. The results showed that GbVe mediated signaling cascade did notdepend on BAK1.4. The length of each Ve gene was3387bp from different kinds of cottons includingJimian14, Jimian20, CRI8, Han208, Hai7124. The ORFs were completely the same.The ORFs of Hai7124and Pima90-53just had one different site, which had no differencein the amino acid sequence. Four sites on GhVe and GbVe were different, and they had onedifferent amino acid, which wasn’t in the conservative domains.5. The promoter sequence of Ve cloned from Jimian11and CRI8were2067bp andquite the same. The promoter sequence of Ve cloned from Pima90-53was2083bp.Compare to GbP, GhP had16bp missing and4mutation sites. Bioinformatics analysisshowed that both GbP and GhP had regulatory elements related to disease resistance andstress, such as GT1CONSENSUS, GT1GMSCAM4, WRKY71OS and so on. GbP had anARR1AT related to cytokinin at134bp, a TAAAGSTKST1related to guard cell-specificgene expression at138bp; but GhP had no. One of DOFCOREZM elements was atdifferent locations compared GbP with GhP, which had relation to stress response. GbPhad a S1FBOXSORPS1L21and a S1FSORPL21at1552bp which play a role in theexpression of the plastid ribosomal protein S1and L21, but GhP had no one.6. GbVe was transformed into cotton through the pollen-tube pathway. Transgenicplants of T0generation were sceened by PCR, and16of Han208plants with GbVe and17of Jimian11cottons with GbVe were obtained. The positive ratios were33.33%and3.47%, respectively. At present, parts of T1generation are growing in the greenhouse.