Cloning of TIR1 gene in Hibiscus cannabinus L. and construction of its expression vector

ObjectiveExpressions 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.MethodcD-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.ResultThe 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.ConclusionThe 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.