| Verticillium wilt is one of the major cotton diseases and has significantly affected both the yield and quality of cotton. Because of the lack of understanding of the mechanism of cotton disease resistance, the lack of existing cultivars to serve as sources of resistance, the breeding of Verticillium wilt resistant cottons has been unsuccessful. To reveal the mechanism of disease resistance in uplandcotton and wild cotton and identify key genes related to resistance, SSH,2-DE and MALDI-TOF-MS were employed to identify the differential expressed ESTs/proteins related to the disease resistance in cotton inoculated with Verticillium dahliae. Seven full-length cDNAs of genes related to the disease resistance were cloned.(1) To analyze the differential gene expressions related to Verticillium wilt resistance, forward and reverse subtractive cDNA libraries were constructed by suppression subtractive hybridization (SSH). The Yumian21, designated as standard cultivar resistant to Verticillium wilt in China, was used as material, in which the cDNA from roots inoculated with medium pathogenicity W strain of Verticillium dahliae was used as tester and drive, and roots without inoculation as corresponding driver and tester. A total of169unigenes of early induced expression related to disease-resistance were enriched, including68up-regulated genes and91down-regulated genes. By means of Blast and cluster analysis in the six databases of Nr, Nt, Swissprot, GO,COG&KEGG,152genes were annotated successfully, and the other16unclear genes couldn't be annotated, which may represent the new genes. Results were as follows,(i) These genes which play19biological functions were divided into18biological processes and53metabolic pathways, and the coding proteins of which were the subunits of eight different cellular components. The results indicated that the response to Verticillium dahliae in upland cotton is complex.(ii) These gene expressions mainly involved in metabolic process, cellular process, response to stimulus, biological regulation and regulation of biological process. The percentages of the genes were26.9%,24.7%,14%and12.9%, respectively. The genes involved in growth and immune system process were only3.7%equally. From the perspective of cellular components, the genes involved in the cells or cell structures accounted for65.4%, and followed by organelles and organelle parts, accounting for28.0%.The percentages of the genes involved in Macrocomplexes and membrane-enclosed lumens were the least, accounting for3.9%and1.9%, respectively.For the molecular functions, the genes involved in catalytic activity and binding activity accounted for50%and42.5%, respectively. Others involved in molecular transduction, transport and receptor activity accounted for7.5%. For the biological functions, in addition to the genes involved in general function prediction accounting for13.2%, the genes involved in signal transduction and translation, post-translational modification and protein folding interactions occupied up to 11.4%equally. For the metabolic pathways, these genes mainly acted in basal metabolism and biosynthesis of secondary metabolites, accounting for33.33%and27.03%, respectively. The other genes were less than10%.(iii) Three up-regulated genes, including FH11(calmodulin-related protein), FH42(Elongation factor Tu), FH67(heat shock protein90) were involved in the plant-pathogen interaction. The result suggested that induced structural resistance manipulated by calcium signal, the disease-resistance pathway mediated by ethylene signal and the unknown R-mediated disease-resistance are likely to the three important pathways in upland cotton.(2) Fifty-seven spotes of differentially expressed protein were identified in Gossipium thurberi. They represented51known proteins in protein database,which cover11%of the total protein spots in the2-DE gel. These proteins involved in disease and stress resistance, transcriptional regulation, protein processing and degradation, photosynthesis, energy production and basic metabolic processes. The results showed that resistance to Verticillium wilt in G. thurberi was the expression of multiple proteins combined effect of synergy. More importantly, expression of five disease resistance proteins were all up-regulated, suggesting that TIR-NBS-LRR like R-genes in Gossipium thurberi may play a leading role in resistance to Verticillium wilt.(3) Using an EST of a dirigent-like gene from the SSH as the query probe to blast cotton EST database, a full-length768bp cDNA sequence of the dirigent-like gene, named GhDIR,was obtained and cloned by PCR. Sequence analysis indicated that it contained a complete open reading fragment (ORF), from70bp to594bp, encoding175amino acids, and had no intron. Sequence comparison and phylogenetic analysis showed the GhDIR cDNA was highly identical with its ortholog from Sea Island cotton, and the next most similar orthologs coming from other dicotyledons, but sharing low homology with monocotyledons. With the above technics, a SUMO gene, named GhSUMO,with a complete open reading fragment (ORF)396bp encoding a protein of95amino acids, was successfully cloned from Yumian21(uplandcotton). The GhSUMO was verified by PCR amplifications using the cDNA and DNA templates from the cotton root, and sequencing results showed that GhSUMO gene had not intron. Amino acid sequence analysis showed that the protein had a conserved ubiqitin domain, the C-terminal double Gly fracture/Connection site, a conservative hydrophobic surface and an Ulpl-Smt3interaction site. Sequence comparison and phylogenetic analysis showed the GhSUMO protein was highly identical with its ortholog from Castor (Ricinus communis), and the next most similar orthologs coming from other dicotyledons. Quantitative RT-PCR analysis showed that GhDIR and GhSUMO were activated in roots of Upland cotton inoculated with Verticillium dahliae. It meant that the two genes may play a role in V. wilt resistance.(4) The PGIP gene of G.thurberi, named ThPGIP was cloned. Its full lenth was1165pb, ORF1095pb, encoding364amino acids. Sequence analysis showed that the PGIP belonged to a LRR super-family and shared a singal peptide and a transmembrane region. It should belong to the secretory protein. This gene had no intron. Sequence comparison and phylogenetic analysis showed the ThPGIP protein was highly identical with its ortholog from Panax ginseng, but sharing low homology with upland cotton and sea island cotton. Quantitative RT-PCR analysis showed that the ThPGIP was induced in roots inoculated with Verticillium dahliae post of20h in G.thurberi.(5) The four full-length cDNA of TIR-NBS-LRR gene analogs, respectively named ThTNLRl, ThTNLR2, ThTNLR3, and ThTNLR4, were cloned from roots of G.Thurberi. The length of cDNAs were3149bp,2011bp,1379bp and4683bp respectively. The analysis of those sequences showed that they all had the conserved domains related to disease resistance, such as TIR, P-loop, Kinase-2, and LRR et al. The phylogenetic tree with others known resistance genes (R genes) suggested that there was the nearest distance with the N gene of tobacco, and there were further distance with the RPP series genes and CS1gene of Arabidopsis thaliana. The results of quantitative RT-PCR showed that their expressions were indused by V. dahliae in roots of G.Thurberi. The uprelated expressions of the two former genes were the observed at20h after inoculation with V. dahliae, however, the two latters were exactly the opposite. The cloning of the four genes established the foundation for researching the interaction of the genes and their function related to disease resistance further. |
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