Cloning And Preliminary Verification Of Genes Related To Fusarium Wilt Resistance In Gossypium Barbadense L.

Fusarium wilt seriously affects the quality and yield of Gossypium barbadense L.exploring the genes related to the resistance of sea-island cotton to Fusarium wilt by molecular means and analyzing the mechanism of sea island cotton defense against Fusarium oxysporum have important theoretical and practical value for cultivating sea island cotton varieties resistant to fusarium wilt and solving the impact and harm of Fusarium wilt on Gossypium barbadense L.In the early stage,the resistant strain Hai 06-146 and susceptible variety Xinhai 14 of Gossypium barbadense L.were infected with Fusarium wilt for 40 h,respectively,and the stems were taken for transcriptome sequencing.The down regulated genes in resistant and susceptible materials were screened,and 34 differentially expressed genes were obtained.Ten target genes were cloned by NCBI function annotation.Real time fluorescence quantitative(q RT-PCR)expression analysis was performed on these 10 genes to verify the transcriptome results,and the function of candidate genes was preliminarily verified by virus-mediated gene silencing technology.The results are as follows :(1)The differentially expressed target genes were cloned and 10 genes were successfully obtained as follows: Gb LTP(366 bp),Gb SDR(795 bp),Gb CYCLA(1170 bp),Gb NCAPG(1062 bp),Gb DNAJ6(861 bp),Gb FAM179B(918 bp),Gb ATX1(1122 bp),Gb IQ1(1440 bp)、Gb DNAP(1349 bp),Gb MCM4(2520 bp).Bioinformatics analysis showed that Gb LTP contained signal peptide at the N-terminal and sub-cellular localization outside the cell,Gb FAM179 B,Gb SDR,Gb DNAPri2 sub-cellular localization in the cytoplasm,and its localization in the nucleus.Gb CYCLA,Gb DNAP and Gb MCM4 are related to DNA replication signaling pathway;Gb LTP has lipid transport function domain(ns LTP family),Gb SDR has NADPH conserved domain(SDR family),Gbcyclin A has cyclin domain,Gb DNAPri2 has Pril family domain,Gb MCM4 has MCM family domain.(2)QRT-PCR was used to analyze the expression of the cloned genes in roots,stems and leaves at 0,8,16,24,32 and 40 h after fusarium infection.The relative expression levels of these 10 genes in Hai 06-146 were lower than those in Xinhai 14,and there was tissue specificity for expression in roots,stems and leaves of plants.(3)Silencing fragments were designed,and 10 target gene silencing vectors were successfully constructed.Virus-mediated gene silencing technology was used to silence related genes in resistant strain Hai 06-146 and susceptible variety Xinhai 14,q RT-PCR was used to confirm the success of 10 target gene silencing.Fusarium wilt was inoculated in Hai06-146 and Xinhai 14,respectively.The disease index was counted 15 days later.In this experiment,the VIGS technique was used to preliminarily verify that Gb SDR and Gb LTP play an important role in the negative regulation of Fusarium wilt in Gossypium barbadense L.