苦荞耐盐基因FtGST2的克隆及功能初步分析

Cloning and Preliminary Functional Analysis of Salt Tolerance Gene FtGST2 in Tartary Buckwheat

课题组前期通过全基因组关联分析获得1个苦荞耐盐相关基因FtGST2(FtPinG0707941400.01)。通过同源比对和保守序列分析发现,该基因属于谷胱甘肽-S-转移酶(glutathione-S-transferases)中的四氯代氢醌脱卤素酶亚族(TCHQD)。从中苦3号中克隆出FtGST2基因,对该基因上游2000 bp的启动子序列进行分析发现,34个元件中含有2个脱落酸响应元件与6个茉莉酸甲酯响应元件,而脱落酸与茉莉酸正是植物调控盐胁迫途径的重要激素。为进一步验证FtGST2的功能,对FtGST2在苦荞不同器官中的表达量和不同浓度NaCl处理下的表达量差异进行检测。结果显示,FtGST2在根中的表达量最高,并且FtGST2在100 mmol/L NaCl处理下的表达量最高。同时构建了FtGST2的过表达拟南芥与过表达毛状根,并检测过表达植株的种子发芽率、主根长、鲜重及其生理指标。结果显示,盐处理下FtGST2的过表达拟南芥发芽率和根长均高于野生型,盐处理下FtGST2的过表达毛状根鲜重显著高于对照组A4毛状根。此外,对毛状根的超氧化物歧化酶、过氧化物酶、过氧化氢酶及丙二醛4个生理指标进行检测后发现过表达FtGST2基因的确能有效提高苦荞毛状根过表达株系的耐盐性。以上试验对FtGST2基因的抗盐功能进行了初步验证,为后续苦荞耐盐品种的选育奠定了一定基础。

In the previous study,a salt tolerance related gene FtGST2(FtPinG0707941400.01)in tartary buckwheat was identified through the whole genome association analysis.The homologous alignment and conserved sequence analysis revealed that the gene belongs to the tetrachlorohydroquinone dehalogenase subfamily of glutathione-S-transferases.We cloned the FtGST2 gene from the cultivar‘Zhongku-3’,and identified 34 responsive elements including 2 abscisic acid responsive elements and 6 methyl jasmonate responsive elements,in the 2000 bp upstream promoter sequence.Abscisic acid and jasmonic acid are important stimuli for regulating salt stress pathways in plants.Through analyzing the expression levels of FtGST2 in different organs of tartary buckwheat and the differences in expression levels under different concentrations of NaCl treatment,the highest transcripts of FtGST2 were detected in roots,and also under 100 mmol/L NaCl treatment.In addition,we over-expressed FtGST2 in Arabidopsis thaliana and hairy roots,and detected the seed germination rate,main root length,fresh weight,and physiological indicators.The germination rate and root length of Arabidopsis thaliana overexpressing FtGST2 under salt treatment were higher than those of wild-type.The fresh weight of hairy roots overexpressing FtGST2 under salt treatment was significantly higher than that of the control group A4 hairy roots.After testing the four physiological indicators of SOD,POD,CAT,and MDA in hairy roots,it was found that overexpression of FtGST2 gene can effectively improve the salt tolerance of tartary buckwheat hairy root overexpression strains.Collectively,these experiments provided preliminary evidence on the salt resistance function of the FtGST2 gene,laying a certain foundation for the breeding of salt tolerant tartary buckwheat varieties in the future.