VIGS技术鉴定药用菊花皇菊中的DFR基因功能研究  

Study on the Function of DFR Gene in Medicinal Chrysanthemum by VIGS Technology

作　　者：王自布[1,2] 邓可[1] 曹剑锋[1,2] Wang Zibu;Deng Ke;Cao Jianfeng(Guizhou Normal College,Guiyang,550018;Institute of Medicinal Plant Biotechnology,Guizhou Normal College,Guiyang,550018)

机构地区：[1]贵州师范学院,贵阳550018 [2]贵州师范学院药用植物生物技术研究所,贵阳550018

出　　处：《基因组学与应用生物学》2020年第5期2141-2147,共7页Genomics and Applied Biology

基　　金：贵州省一流大学重点建设项目“中学生物实验教学研究”一流课程(黔教高发[2017]158号)资助。

摘　　要：从药用菊花皇菊中克隆二氢黄酮醇还原酶(dihydroflavonol 4-reductase,DFR)基因片段,插入病毒诱导载体中来抑制菊花内源性基因的表达,用于其基因功能分析。根据已报道的菊花DFR基因序列(GenBank登录号GU324979)设计引物,克隆部分基因序列,应用生物信息学方法对其氨基酸序列进行分析。采用病毒诱导基因沉默技术(VIGS),以菊花的扦插苗生长到第3~4片叶期的菊花植株为实验材料,沉默菊花的DFR基因,用半定量和荧光定量PCR检测病毒诱导沉默后DFR基因的表达。克隆得到黄菊DFR基因的最大开放阅读框为1029 bp,共编码342个氨基酸,等电点是6.03,分子量是41089.36,与同一科属植物之间同源性达到80%以上,说明DFR蛋白氨基酸序列在同一科属间具有高度保守性。根据DFR基因全长设计构建病毒诱导载体的引物得到453 bp的目的基因,命名为NbDFR。构建病毒载体(TRV)并利用携带目的基因的TRV重组载体病毒感染菊花幼苗,10 d后可以看出菊花出现发育迟缓且叶片皱缩干枯的现象,检测DFR基因发现基因表达下调约20%,但未达到显著性。DFR基因是否被成功沉默还进一步验证。病毒诱导的基因沉默技术可在药用菊花中初步快速鉴定相关基因的功能。The dihydroflavonol 4-reductase(dihydroflavonol 4-reductase,DFR)gene fragment was cloned from chrysanthemum,which was inserted into the virus inducible vector to inhibit the expression of the endogenous gene of chrysanthemum,and used for its gene function analysis.Based on the primers of the reported DFR gene sequence of chrysanthemum,some gene sequences were cloned and virus induced gene silencing was used.Its aminoacid sequence was analyzed by bioinformatics.The chrysanthemum plants grown to the 3~4 leaf stage of chrysanthemum were used as experimental materials.The DFR gene of chrysanthemum was silenced.The expression of DFR gene was detected by semi quantitative and fluorescence quantitative PCR,and the function of the gene in the plant was verified.It was 1029 bp in length,encoding 324 amino acids.The molecular weight of DFR protein was about 41089.36,with a theoretical isoelectricpoint(pI)of 6.03.The similarity of protein DFR amino acid sequences between the same families of plants were over 80%.Amino acid sequence of DFR protein was highly conservative.The target gene with a fragment length of 453 bp was cloned,named NbDFR.The virus vector(TRV)was constructed and the TRV recombinant vector carrying the target gene was used to infect chrysanthemum seedlings.After 10 days,the growth retardation and leaf shrinkage of the chrysanthemum could be seen.The gene expression of the DFR gene was down regulated about 20%,but not significant and the DFR gene was successfully silenced.

关 键 词：病毒诱导基因沉默(VIGS) 菊花皇菊 DFR基因 

分 类 号：S567.239[农业科学—中草药栽培]