紅麻轉(zhuǎn)非全長HcPDIL5-2a誘導的雄性不育系內(nèi)源激素的變化及TIR1基因的克隆和遺傳轉(zhuǎn)化
發(fā)布時間：2019-02-16 08:50
【摘要】：植物激素幾乎調(diào)節(jié)著植物發(fā)育的每一方面,而其在雄蕊發(fā)育中的調(diào)節(jié)作用,對于雄蕊育性顯得尤為重要�；ㄋ幇l(fā)育中植物激素的合成、運輸、信號轉(zhuǎn)導任何一步出現(xiàn)問題都將會導致雄性不育。TIR1蛋白作為信號轉(zhuǎn)導過程中的一員,它對生長素的感應對于其下游依賴生長素信號的相關(guān)花藥發(fā)育基因的表達舉足輕重。花藥發(fā)育的正常與否往往關(guān)系著植物的育性。對生長素相關(guān)基因在紅麻花藥發(fā)育中作用的研究,為紅麻雄性不育機理研究提供了理論依據(jù)。前人通過花粉管通道法,利用紅麻非全長HcPDIL5-2a基因轉(zhuǎn)化紅麻722B保持系材料,從而獲得雄性不育突變體HMS。本研究在這基礎(chǔ)上以紅麻轉(zhuǎn)非全長HcPDIL5-2a基因雄性不育系HMS和722B保持系為材料,通過高效液相測定內(nèi)源激素含量,分析HMS和722B內(nèi)源激素的變化;采用同源克隆和分子生物學方法克隆了紅麻生長素受體基因HcTIR1基因,并利用RT-qPCR的方法分析了該基因在不育系和保持系中的相對表達量;為了進一步確認HcTIR1基因在雄性不育中的作用,將HcTIR1基因構(gòu)建植物超量表達載體和干擾載體,通過農(nóng)桿菌介導轉(zhuǎn)化煙草,驗證其基因功能。獲得的主要結(jié)果如下:1.內(nèi)源激素測定結(jié)果顯示,HMS不育系中三個時期花藥的IAA、GA均低于722B,而ABA的情況卻與之相反。HMS不育系中IAA和GA含量的變化趨勢為下降,而722B中IAA含量變化也是呈下降趨勢,但其GA變化趨勢為上升。兩系的ABA含量變化趨勢一致,但HMS不育系每個時期的ABA含量明顯高于722B的相應時期。可見,非全長HcPDIL5-2a基因確實在HMS雄性不育系中對激素代謝產(chǎn)生了影響。2.根據(jù)紅麻花藥轉(zhuǎn)錄組數(shù)據(jù)和生物信息學方法克隆了HcTIR1基因的cDNA和gDNA,它們的長度分別為1761bp和2726bp;HcTIR1基因有2個內(nèi)含子,它們的長度分別為688bp、277bp,將HcT1R1基因cDNA序列提交至NCBI數(shù)據(jù)庫,GenBank登陸號:KY613992;HcTIR1基因編碼586個氨基酸,是富含LRR的F-box蛋白。3.利用RT-qPCR技術(shù)分析了HcTIR1基因在根、莖、葉及花藥不同發(fā)育時期的表達情況,結(jié)果表明,HMS系和保持系722B中HcTIR1基因在根、莖、葉表達差異不顯著,而花藥不同發(fā)育時期表達差異顯著。在花藥三個時期均顯著下調(diào),在四分體時期表達量僅為722B的0.088倍,單核期為0.116倍,雙核期為0.018倍。說明非全長HcPDIL5-2a基因影響了HTIR1基因的表達。成功構(gòu)建了HTIR1基因的超量表達載體和RNAi干擾載體,它們可以用于研究該基因的功能。通過濃桿菌介導法,將超量表達載體和干擾載體轉(zhuǎn)入煙草,從而研究HcTIR1基因的功能。目前通過普通PCR鑒定出了一批陽性苗。
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S563.5
本文編號：2424272
[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.
【學位授予單位】：廣西大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S563.5
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