RNA沉默技术作为探索基因功能的实验手段应用于多种生物.以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象,检测酿酒酵母双链RNA介导的基因沉默效应.以pESC-LEU为骨架,构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499.用RT-PCR...RNA沉默技术作为探索基因功能的实验手段应用于多种生物.以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象,检测酿酒酵母双链RNA介导的基因沉默效应.以pESC-LEU为骨架,构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499.用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调.结果表明,双链RNA介导的基因沉默技术,能够用作降低酿酒酵母某一特定基因表达水平的工具.并有助于理解芽殖酵母的RNA干扰现象.展开更多
Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous comp...Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous compo un ds.Studies on Cyt-b5 are more con centrated in mammals,but are relatively rare in in sects.The characteristics and functi on of Cyt-b5 from Locusta migratoria have not been described yet.We sequeneed the full-length cDNA sequenee of Cyt-b5 from L.migratoria(LmCyt-b5)by reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea.LmCyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of LmCyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of L.mCYP6FD1 with cytochrome P450 reductase(LmCPR)or LmCyt-b5 separately.These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L.migratoria.展开更多
文摘RNA沉默技术作为探索基因功能的实验手段应用于多种生物.以编码酿酒酵母NADPH依赖型醛糖还原酶的GRE3基因为对象,检测酿酒酵母双链RNA介导的基因沉默效应.以pESC-LEU为骨架,构建重组质粒psiLENT-GRE3并用于转化酿酒酵母YPH499.用RT-PCR检测到诱导1 kb RNA双螺旋和136 bp loop结构引起的GRE3基因表达下调.结果表明,双链RNA介导的基因沉默技术,能够用作降低酿酒酵母某一特定基因表达水平的工具.并有助于理解芽殖酵母的RNA干扰现象.
基金This research was supported by the National Natural Science Foundation of China(31320103921 and 31872010)and the Graduate Outstanding Irinovation Project of Shanxi Province,China(2017 BY011).
文摘Cytochrome b5(Cyt-b5)is a small heme protein and known to be involved in a wide range of biochemical transformations,in eluding cytochrome P450 monooxyge nase(CYP)-mediated metabolism of endoge nous and exogenous compo un ds.Studies on Cyt-b5 are more con centrated in mammals,but are relatively rare in in sects.The characteristics and functi on of Cyt-b5 from Locusta migratoria have not been described yet.We sequeneed the full-length cDNA sequenee of Cyt-b5 from L.migratoria(LmCyt-b5)by reverse transcription-PCR(RT-PCR)based on locust transcriptome database.The phylogenetic analysis showed that LmCyt-b5 was closely related to the Cyt-b5 from Blattodea.LmCyt-b5 was highly expressed in ovary,Malpighian tubules,midgut,gastric caeca,and fat bodies.Silencing of LmCyt-b5 had no effect on the susceptibility of L.migratoria to four different insecticides.Suppression of LmCyt-b5 or silencing of both LmCyt-b5 and LmCPR did not significantly change the total CYP activity toward the substrate 7-ethoxycoumarin(7-EC).However,coexpression of LmCYP6FD1 with LmCPR and LmCyt-b5 together in Sf9 cells by using Bac-to-Bac baculovirus expression system significantly increased the catalytic activity of LmCYP6FD1 toward 7-EC as compared with the coexpression of L.mCYP6FD1 with cytochrome P450 reductase(LmCPR)or LmCyt-b5 separately.These results suggest that LmCyt-b5 plays an important role in the catalytic reaction of LmCYP6FD1 toward 7-EC in our in vitro experiments.Further study is needed to clarify the role of LmCyt-b5 in CYP-mediated catalytic reactions in L.migratoria.
