本文選題：煙草 + orf25基因��； 參考：《江西農業(yè)大學》2017年碩士論文

【摘要】：煙草是我國乃至世界上重要的經濟作物,同時也是植物基因工程常用的模式植物。ATP合酶是一種線粒體復合酶,主要由F0和F1兩部分組成,異常編碼的ATP合酶單個基因或多個基因產物常伴隨植物雄性不育的形成。orf25基因是F0部分的亞基基因,其異常表達可能會導致煙草雄性不育的產生。本研究以atp9基因為對照基因,前期已構建orf25和atp9基因的RNA干擾表達載體,通過農桿菌介導法分別轉化保持系煙草中煙90(zy90)和K326,并均獲得轉基因陽性植株。通過分析轉RNAi-orf25和轉RNAi-atp9煙草在形態(tài)學、ATP酶活性、ATP含量、orf25基因相對表達量以及花粉活力上與對照組之間的差異,來探究orf25基因與煙草雄性不育的關系。主要研究結果如下:1、共獲得轉RNAi-orf25煙草12株,其中1株在苗期死亡,1株未開花;轉RNAi-atp9煙草14株,其中有2株未開花。對轉基因煙草的形態(tài)學特征研究表明:干擾orf25或atp9基因的表達后,植株明顯變矮、生長緩慢、分枝增加、葉片數少、葉片變小、易染病。2、生殖生長期非轉基因煙草葉、花、莖和根四個器官中,保持系zy90中orf25基因相對表達量顯著高于不育系MSzy90,且花中orf25基因相對表達量始終保持最高;保持系K326中atp9基因相對表達量顯著高于不育系MSK326,且atp9基因相對表達量在花器官中為最高。RT-PCR分析結果表明,轉RNAi-orf25煙草葉、花、莖和根中orf25基因相對表達量明顯低于對照組,且花中為最低;同樣地,與對照組一致的是,轉RNAi-atp9煙草atp9基因相對表達量在花中為最低,并且在四個器官中均明顯低于對照組。3、比較生殖生長期非轉基因煙草zy90和MSzy90以及MSK326和K326葉、花、莖和根四個器官的ATP酶活性,結果表明均是保持系中的ATP酶活性顯著高于不育系。兩系皆是花器官中的ATP酶活性最大。兩種轉基因煙草轉RNAi-orf25和轉RNAi-atp9中的ATP酶活性和ATP含量均顯著低于對照煙草,且花中均明顯高于其他三個器官。表明兩種煙草RNAi表達載體皆具有干擾效果。4、相比于對照煙草,轉基因煙草的花粉活力明顯下降。其中有一株轉RNAi-atp9煙草育性發(fā)生改變,形成雄性不育煙草。5、干擾煙草orf25基因和atp9基因的正常表達均能特異性地減弱目的基因的表達,降低ATP酶活性和ATP含量以及煙草的花粉活力,推測ATP合酶的這兩個亞基基因可能具有相似的功能。orf25基因的異常表達雖不能直接引起煙草雄性不育,但是否與其他煙草CMS相關基因共同調控煙草雄性不育性有待于進一步研究。
[Abstract]:Tobacco is an important cash crop in China and even the world. It is also a common model plant. ATP synthase is a mitochondrial complex enzyme, which is mainly composed of F0 and F1. The abnormal encoding of ATP synthase single gene or multiple gene products is often associated with the formation of plant male sterility. Orf25 gene is the F0 subunit gene, and its abnormal expression may lead to the production of tobacco male sterility. In this study, atp9 gene was used as control gene to construct RNAi expression vectors of orf25 and atp9 genes. The vector was transformed into tobacco maintainer line zy90 (zy90) and K326 by Agrobacterium tumefaciens, respectively, and transgenic plants were obtained. In order to explore the relationship between orf25 gene and male sterility of tobacco, the relative expression of orf25 gene and the relative expression of orf25 gene in morphological ATPase activity were analyzed by analyzing the difference between transgenic RNAi-orf25 and transRNAi-atp9 tobacco. The main results were as follows: 1 of 12 tobacco plants transferred to RNAi-orf25, 1 of which died at seedling stage did not bloom, and 14 of them were transferred to RNAi-atp9, 2 of which did not bloom. The morphological characteristics of transgenic tobacco were studied. The results showed that after interfering with the expression of orf25 or atp9 gene, the plants became shorter, grew slowly, increased branches, decreased the number of leaves, reduced the leaves, became susceptible to disease. The relative expression of orf25 gene in the maintainer line zy90 was significantly higher than that in the sterile line MSzy90 in four organs of stem and root, and the relative expression of orf25 gene in flower was always the highest. The relative expression of atp9 gene in the maintainer line K326 was significantly higher than that in the sterile line MSK326, and the relative expression of atp9 gene was the highest in flower organs. RT-PCR analysis showed that the relative expression of orf25 gene in the leaves, flowers, stems and roots of transgenic tobacco was significantly lower than that in the control group. The relative expression of RNAi-atp9 gene was the lowest in the flower, and the lowest in the control group. The activities of zy90 and MSzy90 in non-transgenic tobacco, leaves, flowers, stems and roots of MSK326 and K326 in reproductive period were compared. The results showed that the ATPase activity in both maintainer lines was significantly higher than that in male sterile lines. Both strains had the highest ATPase activity in floral organs. The ATPase activity and ATP content in two transgenic tobacco transRNAi-orf25 and RNAi-atp9 were significantly lower than those in the control, and the flowers were significantly higher than the other three organs. The results showed that both RNAi expression vectors had interference effect. Compared with control tobacco, the pollen viability of transgenic tobacco decreased significantly. One of the transgenic RNAi-atp9 plants changed its fertility to form male sterile tobacco. 5, which interfered with the normal expression of orf25 gene and atp9 gene in tobacco, which could specifically weaken the expression of the target gene, decrease the activity of ATPase, the content of ATP and the pollen vigor of tobacco. It is speculated that these two subunits of ATP synthase may have similar function. The abnormal expression of orf25 gene can not directly cause tobacco male sterility, but whether the two subunits can regulate tobacco male sterility together with other tobacco CMS genes needs further study.
【學位授予單位】：江西農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S572

【參考文獻】

相關期刊論文 前10條

1 吳凌敏;陶瑤;謝麗娟;聶亞平;鐘思榮;朱肖文;劉齊元;;生殖生長期煙草orf25基因的表達和ATP酶活性分析[J];江西農業(yè)大學學報;2016年04期

2 陶瑤;王瑜;鐘思榮;吳凌敏;謝麗娟;聶亞平;周瑋;王建革;劉齊元;;煙草ATP合酶F_0部分4個亞基基因轉錄本編輯位點分析[J];作物學報;2016年12期

3 陶瑤;王瑜;吳凌敏;謝麗娟;聶亞平;鐘思榮;周瑋;王建革;劉齊元;;細胞質雄性不育煙草orfB基因的PCR擴增與序列分析[J];基因組學與應用生物學;2016年06期

4 李可琪;曾新華;袁榮;閆曉紅;吳剛;;甘藍型油菜溫敏細胞核雄性不育系TE5A花藥發(fā)育的細胞學研究[J];中國農業(yè)科學;2016年12期

5 孫麗芳;鄧杰;王霞;趙偉;楊克軍;苗興芬;高樹仁;;玉米細胞質雄性不育系胞質類型鑒定及花粉敗育研究[J];作物雜志;2016年03期

6 陳慧芳;王鑫;謝康;李懿;趙萍;;家蠶V型ATP酶B亞基的克隆及表達特征[J];生物工程學報;2016年04期

7 馬蓉麗;成妍;吳海濤;焦彥生;喬寧;;洋蔥T型細胞質雄性不育與花蕾ATP含量的關系[J];中國蔬菜;2016年02期

8 周伯;沙偉;張梅娟;張春蕾;索荔;;砂蘚ATP合酶Ⅱ亞基基因的克隆及表達分析[J];基因組學與應用生物學;2015年07期

9 王開芳;張詠梅;張金文;楊磊;王鵬;王志偉;;甘藍型油菜細胞質雄性不育系105A花藥敗育的細胞學觀察[J];中國農學通報;2015年13期

10 黃謙心;信曉陽;龐紅喜;胡勝武;;白菜型油菜雄性不育材料的花藥結構及遺傳研究[J];西北農業(yè)學報;2015年03期

相關博士學位論文 前2條

1 楊鵬;棉花晉A細胞質雄性不育系與保持系差異轉錄組和蛋白質組研究[D];河南農業(yè)大學;2013年

2 孫新菊;蘿卜CMS育性相關基因分離鑒定與表達特征分析[D];南京農業(yè)大學;2012年

相關碩士學位論文 前4條

1 林娜;苧麻atp2基因RNA干擾表達載體的構建及其干擾效果初步研究[D];云南大學;2015年

2 趙彥朋;同步抑制FAD2-1與FatB基因對棉籽油品質影響的初步研究[D];石河子大學;2014年

3 蔡一鳴;MSH1 RNAi干擾載體的構建及轉基因芥菜的獲得[D];西南大學;2014年

4 馮忠梅;大白菜胞質雄性不育生理生化機制的研究[D];山東農業(yè)大學;2004年

，

本文編號：2110377