红麻TIR1基因克隆及其表达载体构建

摘要

[目的]分析红麻运输抑制剂响应蛋白1(TIR1)基因在不同组织和不同发育时期花药中的表达情况,并构建其超量表达载体和干扰载体,为研究该基因在雄蕊发育中的调控功能打下基础.[方法]根据红麻花药转录组数据,利用生物信息学方法,克隆TIR1基因cDNA序列,实时荧光定量PCR(qPCR)分析TIR1基因在红麻保持系722B和不育系722A根、茎、叶及不同发育时期花药中的表达情况,并构建超量表达载体和干扰载体.[结果]TIR1基因的开放阅读框(ORF)为1761 bp,编码586个氨基酸(GenBank登录号KY613992).qPCR检测结果显示,与不育系722B相比,不育系722A TIR1基因在四分体期花药中呈显著下调表达(P<0.05,下同),在茎和双核期花药中呈显著上调表达,在单核期花药中极显著上调表达(P<0.01);而在根和叶中,保持系722B和不育系722A中TIR1基因表达水平差异均不显著(P>0.05).超量表达载体pBI121-GFP-TIR1和干扰载体pART27-pK-Tz-Tf构建成功.[结论]红麻TIR1基因编码一个富含亮氨酸重复的F-box蛋白.红麻不育系722A的单核期花药TIR1基因表达较保持系722B极显著上调表达,可能与花药败育有关.构建的超量表达载体和干扰载体,可用于红麻TIR1基因功能及其与雄蕊发育的关系研究.%[Objective]Expressions of transport inhibitor response protein1(TIR1)gene in different tissues and anther at various developmental stages of Hibiscus cannabinus L. were studied,and its over-expression vector and interference vector were constructed in order to investigate the regulation function of TIR1 gene in stamen development.[Method]cD-NA sequence of TIR1 gene was cloned using bioinformatics method based on transcriptome data of H. cannabinus anthers. The expression patterns of TIR1 in root,stem,leaf and anther at different developmental stages of sterile lines 722A and maintainer line 722B were analyzed by real time fluorescence quantitative qPCR. And its over-expression vector and inter-ference vector were constructed.[Result]The open reading frame(ORF)of TIR1 was 1761 bp,encoding 586 amino acids (GenBank accession number KY613992). qPCR analysis results showed that,compared with maintainer line 722B,in terile line 722A,TIR1 expression significantly down-regulated in anther at tetrad stage(P<0.05,the same below),signifi-cantly up-regulated in stem and anther at binucleate stage,and extremely significantly up-regulated in anther at singlenu-cleus stage(P<0.01). In root and stem,the expression of TIR1 in maintainer line 722B and terile line 722A were not sig-nificantly different(P>0.05). Over-expression vector pBI121-GFP-TIR1 and interference vector pART27-pK-Tz-Tf were constructed.[Conclusion]The cloned TIR1 gene encodes a F-box protein which is featured with leucine-rich repeats. Expression of TIR1 in anther at singlenucleus stage significantly up-regulates in sterile lines 722A compared with main-tainer line 722B,which may be related to anther abortion. Over-expression vector and interference vector are constructed successfully and can be used for studying the relationship between function of TIR1 gene in H. cannabinus and stamen development.