Changes Of Endogenous Hormones Of Sterile Line Which Is Reduced By Partial-HcPDIL5-2a And The Cloning And Transformation Of HcTIR1

Endogenous hormones regulates almost every aspect of plant growth and development,and the role of endogenous hormones in stamen is very important for stamen development.The synthesis,transport and signal transduction of endogenous hormones in anther development will lead to male sterility.As a member of the signal transduction process,the induction of auxin by TIR1 protein plays an important role in the anther development related genes dependent on auxin signaling.The normal development of anther is related to the fertility of the plants.The study of the role of auxin related genes in the development of Kenaf anthers provides a theoretical basis for the study of the mechanism of male sterility in kenaf.The partial-HcPDIL5-2a gene was used to transform the 722B maintainer line of kenaf by the pollen tube pathway,and the male sterile mutant HMS was obtained.On the basis of previous research,the partial-HcPDIL5-2a tansgenic male sterile line and 722B maintainer line were used as materials in this study.The high performance liquid chromatography was used to analyze the changes of content in the HMS and 722B.Auxin receptor gene(HcTIR1)was cloned by homology cloning and molecular biology methods,and the RT-qPCR method was used to analyze relative expression between male sterile lines and maintainer lines.In order to further confirm the role of HcTIR1 gene in the male sterile lines,the HcTIR1 gene was used to construct the over-expression vector and interference vector,and the gene function was verifed by Agrobacterium mediated transformation of tobacco.The main results of this experiment are as follows:1.The content of IAA and GA in three stages of HMS male sterile line anther arc both lower then 722B.By contrast,the content of ABA in three stages of HMS male sterile are higher then 722B.The change trend of IAA and GA content was decreased.The change trend of IAA content in 722B is also the same as HMS male sterile line,instead,the change trend of GA content elevate.The change of ABA content in two lines have the same trend,however,the content of ABA in three stages of HMS male sterile line were significantly higher than the corresponding period of 722B.So we concluded that partial-HcPDIL5-2a gene did affect the hormone metabolism of HMS male sterile line.2.We obtained cDNA and gDNA sequence of HcTIR1 gene based on transcriptome data of kenaf anthers and bioinformatic methods.HcTIR1 gene has 2 introns.Their length are 688 bp and 277 bp.The cDNA sequence of HcTIRl gene has been submitted to NCBI database,and GenBank accession number is KY613992.We used bioinformatic methods to analyze protein sequence of HcTIR1 gene,and found that HcTIR1 gene encode 586 amino acids and TIR1 protein is a F-box protein which consist of several leucine-rich repeats(LRR).3.We used RT-qPCR technique to analyze the expression of HcTIR1 gene in root?stem?leave and different developmental stages of anther,and the results show that there are not significant difference in roots stems and leaves between two lines,while there are quite difference in the different developmental stages of anther.HcTIRl gene show down-regulation in all three stages of HMS mutant anther.The relative expression of HcTIR1 gene in HMS sterile line is only 0.088 fold in tetrad stage,0.116 fold in mononucleus phase,and 0.018 fold in binucleate stage.The results show that partial-HcPDIL5-2a gene have an impact on The relative expression of HcTIR1 gene in HMS sterile.The over-expression vector and RNA interference vector of HcTIRl gene were constructed successfully.They can be used to study the function of the gene.The over-expression vector and RNA interference vector of HcTIRl gene were transferred into tobacco by the method of leaf disc transformation,and the function of HcTIR1 gene will be studied.At present,a number of positive seedlings were identified by PCR.