| Kenaf(Hibiscus cannabinus L.)is an annual phloem fiber crop with high salinity tolerance and can be used for phytoremediation in salinealkali land.However,the mechanism of kenaf in response to salt stress remains unclear.Therefore,it is very important to study the molecular mechanism of kenaf salt tolerance and identify the genes responding to salt stress for breeding kenaf salt tolerance varieties and soil improvement.Previous studies have shown that WRKY transcription factors actively participate in the process of plant salt tolerance response.In this study,we first analyzed the physiological characteristics of kenaf seedlings in response to salt stress,then identified the members of kenaf WRKY gene family and analyzed their expression under salt stress.Finally,we obtained the gene HcWRKY75 that positively responds to salt stress,and verified the function of HcWRKY75 by virus-induced gene silencing VIGS technique.To provide genetic resources for the analysis of kenaf HcWRKY75 salt tolerance molecular mechanism and breeding of kenaf salt tolerance varieties.The main results are as follows:1.In order to explore the physiological characteristics of kenaf seedlings in response to salt stress,the salt-sensitive variety "Zanyin No.1" and the salt-tolerant variety "Fuhong 952" were taken as materials and treated with 250 m M Na Cl for 8 days,and 11 indexes including growth morphology,antioxidant enzyme activity and content of osmotic regulatory substances were measured respectively.The results showed that the salt damage of "Zanyin 1" variety was more obvious than that of "Fuhong 952" after salt stress.The plant height and relative water content of "Zanyin 1" were decreased by 41.9% and 20.7%,respectively.The plant height of "Fuhong 952" decreased by 39.8% compared with the control group,and the relative water content decreased by 16.6%.After salt stress,the peroxidase and catalase activities of "Fuhong 952" were significantly enhanced compared with "Zanyin No.1".The content of osmotic regulating substances such as proline in Fuhong 952 was higher than that in Zanyin 1.It showed that "Fuhong 952" was more salt-tolerant than "Zanyin 1".2.In order to analyze the expression pattern of the WRKY gene family in kenaf under salt stress,the members of the WRKY gene family in the whole genome of kenaf were identified by bioinformatics method based on the existing reference genome and transcriptome data in our research group.A total of 82 WRKY gene family members were identified.All of them had the typical conserved WRKYGQK sequence of WRKY gene family members.By phylogenetic tree analysis,the kenaf WRKY gene could be divided into three subfamilies,Group I,II and III,each containing 9,63 and 10 members,respectively.Analysis of WRKY gene expression patterns in kenaf under salt stress showed that the expression of six WRKY genes in "Fuhong 952" and "Zanyin 1" was significantly different.Among them,HcWRKY75 gene was more significantly expressed than other differential genes.Therefore,the HcWRKY75 gene was used as a candidate gene for subsequent analysis of salt tolerance.3.In order to further explore the function of HcWRKY75 gene in kenaf response to salt stress,the cloned gene sequence analysis revealed that HcWRKY75 gene belongs to Group III subgroup,the full length of CDS is807 bp,encoding 269 amino acids.We constructed VIGS silencing vector of HcWRKY75 gene and transformed Fuhong 952.Salt stress treatment showed that compared with the control plants,HcWRKY75 gene silenced plants were more sensitive to salt stress,the plant height of silenced plants decreased by 36.9%,the content of malondialdehyde increased by 2.3times,and the catalase activity decreased by 43.7%.These results indicate that the silencing of HcWRKY75 gene reduces the salt tolerance of "Fuhong952".It is suggested that HcWRKY75 gene plays a positive role in response to salt stress in kenaf seedling stage. |
