Cloning And Functional Study Of CKX And PG Family Genes From Gossypium Barbadense

Cotton fiber quality in our country are mostly low, which can’t meet the market needs; while the cotton fiber quality is mainly decided by plant genotype, so the study of fiber development related genes expression and regulation is greatly significant to the improvement of fiber quality.Verticillium wilt is one of the most destructive diseases of cotton, which has serious influence to the cotton production. The effective prevention and control measure is screening resistance genes and breeding transgenic disease-resistant varieties. Therefore, a deep understanding of cotton verticillium wilt resistance mechanism, cloning related genes and validating gene functions, are greatly significant for breeding disease-resistant varieties.Cytokinin(CTK), an important plant hormone, plays a significant role in the regulation of plant growth and disease resistance. Cytokinin dehydrogenase(CKX), the only known plant enzyme that irreversibly degrades natural cytokinin, is the key factor of the endogenous CTK degradation. Studies have shown that CKX can affect the development and production of cotton fiber, and is involved in the regulation of stress.Polygalacturonase(PG) is associated with not only cell development, pollen tube elongation, pollen grain maturation, xylem cell formation, but also pathogen defense, plant host interaction. Studies have shown that PG plays a role in cotton pollen and fiber development, as well as plant disease-resistance process.Two cytokinin dehydrogenase family genes Gb CKX1, Gb CKX2 and five polygalacturonase family genes Gb PG2, Gb PG3, Gb PG4, Gb PG5, Gb PG6 were selected from a full-length c DNA library of Gossypium barbadense Pima90-53 under Verticillium dahliae stress. Gene cloning, bioinformatics analysis, differential expression analysis and primary functional study after genes silencing were performed in this study. The results were as follows:1. Cloning, expression analysis and primary functional study of Gb CKX1, Gb CKX2(1) The open reading frame(ORF) length of Gb CKX1 was 1560 bp, which encoded a protein of 519 amino acids. The ORF length of Gb CKX2 was 1539 bp and encoded a protein of 512 amino acids. Multiple sequence analysis showed that the homology of the protein coding by Gb CKX1 and Gb CKX2 was 80%. They both contain a highly conserved FAD binding domain and a cytokinin-binding domain like other CKX family genes. But they are different in the structure. Only Gb CKX2 contains L-galactose lactone dehydrogenase domain(PLN02465), D-lactate dehydrogenase domain(PLN02805) and protein-protein interaction motifs site PAS. Bioinformatics analysis showed that Gb CKX1 and Gb CKX2 had no signal peptide and the transmembrane domains, which indicates that both of them belong to non-secreted proteins. Phylogenetic tree analysis showed that Gb CKX1 and Gb CKX2 were close to Tc CKX、Pt CKX1、At CKX7.(2) The expression patterns of CKX1 and CKX2 in differential fiber cell stages were analyzed using real-time PCR. The results showed each gene had different expression pattern between Pima 90-53 and CCRI8. CKX1 had a peak in the secondary wall thickening stage, and expression level was significantly higher in Pima90-53 than that of CCRI8; while CKX1 had a peak in the fiber elongation stage and expression level was significantly higher in CCRI8 than that of Pima90-53. Therefore,it predicted that CKX1 may affect the form of cotton fiber strength by participating in the secondary wall thickening; CKX2 may affect the fiber length by participating in the cotton fiber elongation.(3) The expression level of CKX1 and CKX2 both increased post inoculated with V. dahliae compared with control. The expression level of CKX1 in Pima90-53 was significantly higher than control at 12 hpi, while in CCRI8 the peak appeared at 24 hpi. The expression level of CKX2 in Pima90-53 was significantly higher than control from the beginning to 24 hpi, but no significant difference in CCRI8.(4) The eukaryotic overexpression vectors p BI121-FBP7-Gb CKX1 and p BI121-FBP7-Gb CKX2 were successfully constructed using an expression vector p BI121-FBP7 with a cotton seed coat-specific promoter FBP7.(5) Silencing of CKX1 and CKX2 by VIGS enhanced cotton plants’ susceptibility to V. dahliae, and plants exhibited higher disease index than the vector control plants at 20 dpi. 2. Cloning and expression analysis of polygalacturonase family genes(1)The ORF length of Gb PG2, Gb PG3, Gb PG4 was 1425 bp, 1449 bp, 1449 bp respectively, each encoded a protein of 474, 482, 482 amino acids. It predicted that the ORF length of Gb PG5, Gb PG6 was 1449 bp, 1185 bp respectively, each encoding a protein of 482, 394 amino acids.(2) Bioinformatics analysis showed that, Gb PG2 contains no signal peptide and the transmembrane domain, which make it belong to non-secreted proteins; Gb PG3, Gb PG5 belong to signal anchor protein due to the transmembrane domain of these two proteins; containing both signal peptide and the transmembrane domain, Gb PG4, Gb PG6 belong to transmembrane protein.(3) Conserved domain analysis indicated that Gb PG2, Gb PG3, Gb PG4, Gb PG5, Gb PG6 all belong to PG gene family. Plant PG gene family have four structural domains,I(SPNTDG), II(GDDC), III(CGPGHGISIGSLG), IV(RIK). Multiple sequence analysis showed that, Gb PG2, Gb PG3, Gb PG4, Gb PG5 contained I, II, IV domains, while the location of III was replaced by other amino acids; Gb PG6 contained I, II, III, IV domains, Phylogenetic tree analysis showed that Gb PG5 was close to cotton PG which have been reported, but Gb PG2, Gb PG3, Gb PG4, Gb PG6 were clustered into different subfamily with Gb PG5.(4) The expression patterns of PGs in differential fiber cell stages were analysed by real-time PCR. The expression levels of PG4, PG6 in the fiber elongation stage were significantly different between Pima90-53 and CCRI8; PG2, PG5, PG6 expression were significantly higher in the secondary wall thickening stage in Pima90-53 than that of CCRI8; The expression level of PG3 was different between Pima90-53 and CCRI8 in the secondary wall thickening stage. Therefore, PG4 may affect the fiber length by participating in the cotton fiber elongation; PG2, PG3, PG5 may affect the form of cotton fiber strength by participating in the secondary wall thickening; while PG6 may affect cotton fiber length and strength by participating both in the cotton fiber elongation and the secondary wall thickening.(5) The expression patterns of PGs were different between Pima90-53 and CCRI8 post inoculated with V. dahliae. The expression levels of PG2, PG4, PG6 were significantly induced both in Pima90-53 and CCRI8; PG3 was only significantly induced in Pima90-53; PG5 was only significantly induced in CCRI8.