Functional Analysis Of GbSTK And GhLBD100 From Cotton In Verticillium Wilt Resistance

Verticillium dahliae is one of the most destructive fungal diseases in cotton.At present,there are no effective fungicides to control it.Therefore,it is of great significance to study the molecular mechanism of Verticillium wilt resistance,which could help to improve cotton resistance and yield.Serine/threonine protein kinase(S/TK)is an important protein kinase in the process of protein phosphorylation.STK is involved in intercellular signal transduction and plays an important role in regulating development and disease/stress resistance.A cotton serine/threonine protein kinase gene(GbSTK)was identified in our previous study.The gene was induced by Verticillium dahliae Kleb and a variety of signal molecules and the GbSTK overexpression Arabidopsis significantly improved the resistance to Verticillium wilt.In this study,we further carried out studies including investigating the expression of GbSTK gene in different resistant cotton varieties,the function of GbSTK gene in the regulation of resistance and development,and its possible interaction protein.The results would provide the basis for revealing the mechanism of STK gene involved in disease resistance and development.LBD gene family is a plant-specific transcription factor.It contains a conserved LOB domain,which plays an important role in plant growth and metabolism.At present,the function of LBD genes in G.hirsutum has not been reported.In this study,we identified and analyzed the gene family of LBD genes in upland cotton genime.Based on the transcriptome data and promoter motif analysis,we screened the gene of GhLBD100,which was induced by Verticillium wilt and different signal moleculars,and the gene function was studied.The main results are as follows:1.The expression pattern of GbSTK gene in different resistant varieties was analyzed by quantitative real-time PCR.The expression level of GbSTK gene in resistant varieties was higher than that in susceptible varieties(Han208),among which the expression of STK in Nongda Mian 8 was the highest,indicating that the transcription level of STK gene was positively correlated with cotton resistance to Verticillium wilt.Virus induced gene silencing(VIGS)was used to effectively silence the GbSTK gene.The silencing of GbSTK gene reduced the plant resistance to Verticillium wilt,indicating that GbSTK gene palys positive role in cotton resistance to V.dahliae.2.The pGBKT7-GbSTK vector of yeast two hybrid was successfully constructed,which was non-toxic and has no self-activated.According to screening a cDNA library of Pima 90-53 induced by Verticillium wilt based on yeast two hybrid,30 proteins with interacting with GbSTK were screened.The selected SAMS proteins have been reported to be involved in plant disease resistance.3.The average number of rosette leaves of transgenic GbSTK Arabidopsis of was 7.5,which was significantly lower than that of empty vector control plants(11.9).The flowering time of transgenic plants was 5-7 days earlier than that of control plants.Furthermore,the transcription level of FT,SOC1 and LFY genes,which are markers of Arabidopsis flowering pathway,was detected.The results showed that the expression level of FT and SOC1 genes in different transgenic lines was significantly higher than in the control plants,while the expression of LFY gene was less affected,indicating that STK gene can affect the expression level of FT and SOC1 genes,and then regulated the early flowering of transgenic plants.4.Atotal of 128 LBD genes were identified in upland cotton genome.All the members of LBD gene family contain conservative LOB domains,which distributed on 13 chromosomes.By phylogenetic tree analysis,LBD genes in upland cotton were classified into two classes:Class I and Class II,which were divided into eight subgroups including Ⅰa、Ⅰb、Ⅰc、Ⅰd、Ⅰe、Ⅰf、Ⅱa and Ⅱb.Combined with the transcriptome data of Nongda 601 inoculated with V.dahliae,we found that four members in class Ⅱa were up-regulated in the root when induced by V.dahlia.Based on the investigation of the promoter motif sequence,we found that GhLBD100 gene was rich in salicylic acid(SA),jasmonic acid(JA),gibberellin(GA)and other regulatory elements related to stress.5.The GhLBD100 gene was cloned from Nongda 601 and its function was analyzed.qRT-PCR showed that the expression of GhLBD100 was higher than in the control during 2 to 36h.We also detected it expression in different tissues and found that GhLBD100 gene expressed with the highest level in the root,then in the stem,and with the lowest level in the leaf.Besides,GhLBD100 could also responsed to SA,GA,ET,MeJA signals.Virus induced gene silencing(VIGS)was used to silence GhLBD100 gene effectively,the resistance of cotton seedlings to V.dahliae was reduced.The content of H2O2 at 0,6,12,24,36 and 48 h was significantly higher than that of the control.There was no significant change in POD activity of the silence plants at 0 h compared with the mock,but it was significantly higher at 6 h than that of the control,and the POD activity of the silenced plants was significantly higher than the mock at 6 and 12 h,then POD activity decreased during 24 to 48 h,significantly lower than the control plants.At the same time,the CAT activity was inhibited in the silenced cottons,resulting H2O2 over-accumulation,which lead to cells toxicosis.The results showed that GhLBD100 gene was involved in the regulation of ROS balance in plants.6.The yeast two hybrid vector GhLBD100 was successfully constructed,and 70 proteins interacting with GhLBD100,including SPL protein,were screened from the yeast two hybrid cDNA library induced by Verticillium wilt.It was reported that SPL transcription factors participate in adverse environmental changes such as plant stress and hormone response.In summary,differences in expression patterns of GbSTK gene among upland cotton varieties with different resistance and sensitivity in this study.GbSTK gene has the biological function of improving Verticillium wilt resistance and flowering earlier.128 LBD genes in upland cotton were identified in the whole genome.GhLBD100 gene was identified as a potential Verticillium wilt resistance gene by promoter sequence analysis.GhLBD100 gene affects H2O2 content by regulating CAT activity,and regulates POD content,which is involved in regulating ROS balance in plants and improving cotton resistance to Verticillium wilt.The above results show that GhLBD100 gene is a functional gene involving in the Verticillium wilt resistance in cotton.