红麻转非全长HcPDIL5-2a诱导的雄性不育系内源激素的变化及TIR1基因的克隆和遗传转化

发布时间：2019-02-16 08:50

【摘要】：植物激素几乎调节着植物发育的每一方面,而其在雄蕊发育中的调节作用,对于雄蕊育性显得尤为重要。花药发育中植物激素的合成、运输、信号转导任何一步出现问题都将会导致雄性不育。TIR1蛋白作为信号转导过程中的一员,它对生长素的感应对于其下游依赖生长素信号的相关花药发育基因的表达举足轻重。花药发育的正常与否往往关系着植物的育性。对生长素相关基因在红麻花药发育中作用的研究,为红麻雄性不育机理研究提供了理论依据。前人通过花粉管通道法,利用红麻非全长HcPDIL5-2a基因转化红麻722B保持系材料,从而获得雄性不育突变体HMS。本研究在这基础上以红麻转非全长HcPDIL5-2a基因雄性不育系HMS和722B保持系为材料,通过高效液相测定内源激素含量,分析HMS和722B内源激素的变化;采用同源克隆和分子生物学方法克隆了红麻生长素受体基因HcTIR1基因,并利用RT-qPCR的方法分析了该基因在不育系和保持系中的相对表达量;为了进一步确认HcTIR1基因在雄性不育中的作用,将HcTIR1基因构建植物超量表达载体和干扰载体,通过农杆菌介导转化烟草,验证其基因功能。获得的主要结果如下:1.内源激素测定结果显示,HMS不育系中三个时期花药的IAA、GA均低于722B,而ABA的情况却与之相反。HMS不育系中IAA和GA含量的变化趋势为下降,而722B中IAA含量变化也是呈下降趋势,但其GA变化趋势为上升。两系的ABA含量变化趋势一致,但HMS不育系每个时期的ABA含量明显高于722B的相应时期。可见,非全长HcPDIL5-2a基因确实在HMS雄性不育系中对激素代谢产生了影响。2.根据红麻花药转录组数据和生物信息学方法克隆了HcTIR1基因的cDNA和gDNA,它们的长度分别为1761bp和2726bp;HcTIR1基因有2个内含子,它们的长度分别为688bp、277bp,将HcT1R1基因cDNA序列提交至NCBI数据库,GenBank登陆号:KY613992;HcTIR1基因编码586个氨基酸,是富含LRR的F-box蛋白。3.利用RT-qPCR技术分析了HcTIR1基因在根、茎、叶及花药不同发育时期的表达情况,结果表明,HMS系和保持系722B中HcTIR1基因在根、茎、叶表达差异不显著,而花药不同发育时期表达差异显著。在花药三个时期均显著下调,在四分体时期表达量仅为722B的0.088倍,单核期为0.116倍,双核期为0.018倍。说明非全长HcPDIL5-2a基因影响了HTIR1基因的表达。成功构建了HTIR1基因的超量表达载体和RNAi干扰载体,它们可以用于研究该基因的功能。通过浓杆菌介导法,将超量表达载体和干扰载体转入烟草,从而研究HcTIR1基因的功能。目前通过普通PCR鉴定出了一批阳性苗。
[Abstract]:Plant hormones regulate almost every aspect of plant development, and their regulatory role in stamen development is particularly important for stamen fertility. Any problem in plant hormone synthesis, transport and signal transduction in anther development will lead to male sterility. TIR1 protein is a member of signal transduction. Its sensitivity to auxin plays an important role in the expression of auxin signaling related anther development genes. Whether anther development is normal or not is often related to plant fertility. The study on the role of auxin related genes in the development of kenaf anther provides a theoretical basis for the study of the mechanism of kenaf male sterility. By pollen-tube pathway method, the maintainer line of kenaf 722B was transformed by using kenaf non-full-length HcPDIL5-2a gene, thus the male sterile mutant HMS. was obtained. In this study, the content of endogenous hormones in kenaf transformed non-full-length HcPDIL5-2a male sterile lines HMS and 722B were determined by HPLC, and the changes of HMS and 722B endogenous hormones were analyzed. The HcTIR1 gene of kenaf growth hormone receptor gene was cloned by homologous cloning and molecular biology, and the relative expression of the gene in male sterile line and maintainer line was analyzed by RT-qPCR method. In order to further confirm the role of HcTIR1 gene in male sterility, HcTIR1 gene was constructed into plant overexpression vector and interference vector, and its gene function was verified by Agrobacterium tumefaciens mediated transformation of tobacco. The main results are as follows: 1. The results of endogenous hormone determination showed that the IAA,GA of anthers was lower than 722B in the three stages of HMS sterile line, but the situation of ABA was opposite. The content of IAA and GA decreased in HMS sterile line, and the content of IAA decreased in 722B sterile line. But its GA change trend is rising. The change trend of ABA content in two lines was the same, but the ABA content of HMS male sterile line was obviously higher than that of 722B in each stage. It can be seen that the nonfull length HcPDIL5-2a gene does affect hormone metabolism in HMS male sterile lines. 2. 2. The cDNA and gDNA, of HcTIR1 gene were cloned from anther transcriptome data and bioinformatics method of kenaf. The length of cDNA and gDNA, were 1761bp and 2726bprespectively. There are two introns in HcTIR1 gene, the length of which is 688bpn277bp. the cDNA sequence of HcT1R1 gene is submitted to NCBI database. GenBank accession number: KY613992;HcTIR1 gene encodes 586 amino acids, which is a LRR rich F-box protein. The expression of HcTIR1 gene in root, stem, leaf and anther was analyzed by RT-qPCR technique. The results showed that there was no significant difference in the expression of HcTIR1 gene in root, stem and leaf between HMS line and maintainer line 722B. There were significant differences in anther expression at different developmental stages. The expression level was only 0.088 times of that of 722B at tetrad stage, 0.116 times at mononuclear stage and 0.018 times at binuclear stage. The results showed that the non-full length HcPDIL5-2a gene affected the expression of HTIR1 gene. The overexpression vector and RNAi interference vector of HTIR1 gene were successfully constructed, which can be used to study the function of the gene. In order to study the function of HcTIR1 gene, the overexpression vector and interference vector were transferred into tobacco by concentrated bacillus mediated method. At present, a number of positive seedlings have been identified by ordinary PCR.
【学位授予单位】：广西大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S563.5

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