| Cotton production was serious destroy with soil-borne fungal Verticillium dahliea and difficult to prevent. Although many research works about the Verticillium dahliea was carried out, there were no effective research achievements for disease control, due to lack of disease resistance gene resources in cotton and complexity pathogenicity of Verticillium dahliae. Therefore, cloning and identification the function of disease resistance genes to Verticillium dahliae is the urgent task to disease resistance transgenic breeding.In this research, leucine-rich repeats transmembrane (LRR-TM) genes of GbaVd1 and GbaVd2 were cloned from the disease resistance cultivar Gossypium barbadense cv. Hai7124 by TAIL-CPR and RACE methods, which homologus to anti-Verticillium genes Ve1 and Ve2 in tomato. The full length cDNA of GbaVd1 is 3262 bp in which a 3063 bp ORF encoding a 1020 aa protein, a 3′UTR and 5′UTR of 130 bp and 69 bp, respectively. The full length cDNA of GbaVd2 comprised 3466 bp and included an ORF of 3249 bp, encoding a 1082 aa protein, a 68 bp and 149 bp long 5′UTR and 3′UTR, respectively. The flanking sequences of GbaVd1 and GbaVd2 were cloned by TAIL-PCR method and 900 bp and 1107 bp fragment were obtained, respectively, and there was no intron in GbaVd1 and GbaVd2. Although the identity of the nucleotides between GbaVd1/GbaVd2 and Ve1/Ve2 is less than 60% and code proteins is lower than 50%, their structures was similar to the LRR-TM disease resistance gene, which contains signal peptide, N terminus of the mature protein, leucine-rich repeats (LRRs), extracytoplasmic domain, transmembrane domain and cytoplasmic domain. In GbaVd1 and GbaVd2, there are 32 and 33 LRRs, also include 22 and 26 potential glycosylation sites, respectively. The subcellular localization of GbaVd1 and GbaVd2 was predicted in plasma membrane by Psort. Morever, the flanking sequences were possible contain many cis-elements, such as fungal elicitor responsive element of Box-W1, cis-acting regulatory element involved in the MeJA-responsiveness of TGACG motif and CGTCA motif, cis-acting element involved in the abscisic acid responsiveness of ABRE, cis-acting element involved in defense and stress responsiveness of TC-rich repeats and so on.Vd1 and Vd2 genes and theirs flanking sequences were cloned from different wild cottons base on GbaVd1 and GbaVd2 sequences, and the relation between genes difference and cottons sensitivity to Verticillium dahliae was analysis. The difference of Vd1 and Vd2 genes were mainly shown single nucleotide polymorphisms which lead to many sense mutations. The lack of short nucleotide fragment was also exist in Vd1 and Vd2 genes, such as GneVd1 from Gossypium nelsonii, GdaVd2 from Gossypium davidsonii, GstVd2 from Gossypium sturtianum and so on, which result in the deletion of signal peptide, change the N terminus of mature protein, add or delete the glycosylation sites, lose of cytoplasmic domain and so on. However, there are no obvious correlation between the genes difference and the cottons sensitivity to Verticillium dahliae.Overexpressin of GbaVd1 and GbaVd2 were shown to enhance the tolerance to Verticilliun dahliae toxin (VD-toxin) in transgenic Arabidopsis, and did not found the symptoms of leaf chlorosis and root depression. Morever, the resistance of GbaVd1 and GbaVd2 transgenic plants to V. dahliae was obvious improved. For example, compared to the infected of wild type Col-0, the tillers of transgenic plants were returned to normal, the growth retardants was improved, the influence of inflorescence height was reduced and the seeded was shown normal, but the leaves of transgenic plant were also shown chlorosis as the wild type. Therefore, the results were shown that GbaVd1 and GbaVd2 genes possess anti-Verticillium function in V. dahliae invade progress.Analysis the gene expression profile were shown that 44 genes were co-upregulated and 61 genes co-downregulated more than 1.5 folds in both GbaVd1 and GbaVd2 transgenic plants, and many differential expression genes were related to response to stimulus which contained biotic stress, abiotic stress, immune response, regulation of response to stimulus and so on. The research was found that some signal factors of disease resistance were regulated in transgenic plants, such as SERK2, SERK4, SGT1A, SGT1B, NDR1, EDS1, kinase genes and so on. The ethylene and jasmonic acid signal transduction pathway were also effected in transgenic plants, the expression of EBF1, EBF2, EIN3, ABA1, AOS, AOC1, JMT, JAR1, JAZ7, etc were induced. The biosynthesis pathway of flavonoid and terpenoid were changed too, their related genes of DFR, TT5, LDOX, SQP2, GGPS1 and SQE3 were regulated. Overexpression of GbaVd1 and GbaVd2 were also promoted the expression of cell wall related protein genes, such as nodulin, lectin, proline-rich protein, hydroxyproline-rich glycoprotein, glycine-rich protein, arabinogalactan-protein and so on, and the activity ofβ-1,3-glucanase andβ-D-xylosidase were enhanced, which were related to cell wall synthesis or degradation.Base on these results, the mechanism of GbaVd1 and GbaVd2 resistance model was supposed, two genes encode LRR-TM receptor like protein could directly or indirectly combine with the V. dahliae elicitors, this action would regulate the expression of signal factor of disease resistance, then active the ethylene and jasmonic acid signal transduction pathway which lead to change the synthesis of phytoalexin and expression of cell wall related proteins, form the antifungus activity compound and enhance the cell wall structure obstacle, finally increase the host resistance to V. dahliae. |
