本文選題：甘藍型油菜　切入點：Bnams4~b　出處：《華中農業(yè)大學》2017年碩士論文

【摘要】：甘藍型油菜核不育系統(tǒng)7365AB(Bnams3ms3ms4~bms4~b/BnaMs3ms3ms4~bms4~b)由于不育性穩(wěn)定、敗育徹底,具有廣泛的恢復源等特點,成為目前油菜雜種優(yōu)勢利用的重要途徑之一。不育基因Bnams4~b能夠引起油菜和擬南芥花藥功能性缺失和葉片黃化,導致雄性不育。BnaMs3是Bnams4~b的恢復基因,能夠恢復Bnams4~b的破壞效應,但BnaMs3在其它作物中的同源基因與Bnams4~b的關系以及對Bnams4~b的功能有什么影響,目前還沒有相關研究。本研究將不育基因Bnams4~b和恢復基因BnaMs3轉化到普通煙草和栽培番茄A57中,對Bnams4~b在其它作物中的功能以及不同作物Tic40對Bnams4~b功能的影響進行研究。主要研究結果如下:1.煙草的遺傳轉化和陽性鑒定將不育基因Bnams4~b轉化到煙草中,獲得陽性株。取不同黃化程度的轉基因植株葉片進行qRT-PCR。結果顯示,Bnams4~b基因的表達量隨轉基因煙草葉片黃化程度的增加而升高。2.轉基因煙草的表型分析轉基因煙草葉片出現(xiàn)了不同程度的黃化,對其黃化葉片進行透射電鏡觀察和葉綠素含量檢測,發(fā)現(xiàn)葉綠素含量隨葉片由黃到綠呈逐漸升高的趨勢,黃化葉片中的葉綠體結構出現(xiàn)異常。醋酸洋紅染色結果顯示,轉基因黃化煙草的花藥能夠產生有活力的花粉粒,并未出現(xiàn)預期的雄性不育表型。3.轉基因煙草未出現(xiàn)不育的原因分析對轉基因煙草未出現(xiàn)不育的原因進行分析,首先更換花藥特異啟動子A9和TA29后轉化煙草,依然沒有出現(xiàn)雄性不育表型;其次對煙草不同Tic40拷貝的功能進行分析,結果證實煙草的各Tic40拷貝都不能恢復Bnams4~b引起的擬南芥雄性不育表型。4.轉基因番茄的陽性鑒定和表達量分析將番茄A57的Tic40拷貝轉化擬南芥,與包含Bnams4~b的擬南芥雜交,結果顯示番茄的Tic40拷貝不能恢復Bnams4~b引起的擬南芥雄性不育。將不育基因Bnams4~b和恢復基因BnaMs3導入番茄中,出現(xiàn)了黃化表型,qRT-PCR顯示葉色黃化程度越重,Bnams4~b的表達量越高,花藥中也有一定程度的表達。5.轉基因番茄的表型分析和育性觀察共得到19株Bnams4~b轉基因番茄陽性苗,有6株葉片黃化,相同的生長環(huán)境下黃化株比正常的栽培材料生長矮小。其中兩株花藥中沒有花粉粒,部分轉化株的花藥中有少量可育花粉粒和大量干癟的、沒有活力的花粉粒。黃化程度最嚴重的單株出現(xiàn)死蕾現(xiàn)象,不能正常結實。6.煙草和番茄Tic40中的功能變異位點分析將擬南芥、甘藍型油菜、煙草、番茄的Tic40進行氨基酸序列比對分析。結果顯示,番茄Tic40中的六個功能位點與Bnams3完全一致,推測番茄Tic40與Bnams3有類似功能,番茄中只有存在類似于Bnams3的Tic40拷貝時,Bnams4~b才能表現(xiàn)出較強的破壞功能。煙草Tic40序列中的第一個功能位點與BnaMs3和Bnams3的都不相同,且缺失了第六個功能位點,結合轉基因煙草沒有得到不育表型,推測與煙草Tic40的兩個變異位點有關。
[Abstract]:The genetic male sterile system of Brassica napus, 7365ABN Bnams3ms3ms4bms4nbm4nbm4bm4b) has become one of the important ways of utilization of heterosis because of its stable sterility, complete abortion and extensive recovery source, which is one of the most important methods for the utilization of heterosis in Brassica napus (Brassica napus L.).The sterile gene Bnams4~b can cause anther functional loss and leaf yellowing in rape and Arabidopsis thaliana, and cause male sterility. BnaMs3 is the restoring gene of Bnams4~b, which can restore the destructive effect of Bnams4~b.However, the relationship between Bnams4~b and homologous genes of BnaMs3 in other crops and their effects on the function of Bnams4~b have not been studied.In this study, the sterile gene Bnams4~b and restorer gene BnaMs3 were transformed into common tobacco and cultivated tomato A57 to study the function of Bnams4~b in other crops and the effect of Tic40 on Bnams4~b function.The main results are as follows: 1.Genetic transformation and positive identification of tobacco transformed sterile gene Bnams4~b into tobacco to obtain positive plants.The leaves of transgenic plants with different yellowing degree were used for qRT-PCR.The results showed that the expression of Bnams4B gene increased with the increase of yellowing degree of transgenic tobacco leaves.Phenotypic Analysis of transgenic Tobacco leaves the yellow leaves of transgenic tobacco were observed with transmission electron microscope and chlorophyll content was detected. It was found that the chlorophyll content increased gradually with the leaves from yellow to green.The chloroplast structure in yellow leaves was abnormal.The results of acetic acid magenta staining showed that the anthers of transgenic yellow tobacco could produce active pollen grains, and no expected male sterility phenotype. 3.Analysis of the reasons for the absence of sterility in transgenic tobacco. First, the anther specific promoters A9 and TA29 were replaced and then transformed into tobacco, and the phenotype of male sterility was still not found in transgenic tobacco.The results showed that none of the Tic40 copies of tobacco could recover the male sterility phenotype of Arabidopsis thaliana induced by Bnams4~b.The Tic40 copy of transgenic tomato A57 was transformed into Arabidopsis thaliana and crossed with Arabidopsis thaliana containing Bnams4~b. The results showed that the Tic40 copy of tomato could not restore the male sterility induced by Bnams4~b in Arabidopsis thaliana.When the sterile gene Bnams4~b and restorer gene BnaMs3 were introduced into tomato, the yellow phenotype was detected by qRT-PCR. The higher the degree of leaf color yellowing, the higher the expression of Bnams4B, and the higher the expression of Bnams4B in anthers.The phenotypic analysis and fertility observation of transgenic tomato obtained 19 Bnams4~b transgenic tomato positive seedlings, 6 of which were yellowing in leaves, and the growth of yellow plants in the same growing environment was smaller than that of normal cultivated materials.There were no pollen grains in two anthers, a small number of fertile pollen grains and a large number of dry, inactive pollen grains in the anthers of some transformed plants.The most serious degree of yellowing appeared in the dead bud of single plant and could not bear fruit normally. 6.Amino acid sequence alignment analysis of Tic40 in Arabidopsis thaliana, Brassica napus, tobacco and tomato was carried out in tobacco and tomato Tic40.The results showed that the six functional loci in tomato Tic40 were completely consistent with Bnams3. It was inferred that tomato Tic40 and Bnams3 had similar functions. Only when there was a Tic40 copy similar to Bnams3 in tomato could Bnams4B show strong damage function.The first functional locus of tobacco Tic40 sequence was different from that of BnaMs3 and Bnams3, and the sixth functional locus was missing. The combination of transgenic tobacco did not get sterile phenotype, which was related to the two mutation sites of Tic40 in tobacco.
【學位授予單位】：華中農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.4

【參考文獻】

相關期刊論文 前10條

1 陳晉瑩;姜友軍;劉洋;;淺談農業(yè)生物技術及轉基因農作物[J];糧油倉儲科技通訊;2016年04期

2 任志強;肖建紅;楊慧珍;卜華虎;;轉基因植物在農業(yè)上的應用[J];安徽農業(yè)科學;2016年03期

3 俎峰;夏勝前;頓小玲;周正富;曾芳琴;易斌;文靜;馬朝芝;沈金雄;涂金星;傅廷棟;;基于分子標記的油菜隱性核不育7-7365AB遺傳模式探究[J];中國農業(yè)科學;2010年15期

4 崔欣,陳慶山,楊慶凱,曹越平;植物轉基因沉默與消除[J];植物學通報;2002年03期

5 夏蘭芹,王遠,郭三堆;外源基因在轉基因植物中的表達與穩(wěn)定性[J];生物技術通報;2000年03期

6 傅廷棟;中國油菜生產和品種改良的現(xiàn)狀與前景[J];安徽農學通報;2000年01期

7 肖國櫻,李德清,唐傳道,朱列書;煙草雜種優(yōu)勢利用的途徑和方法[J];作物研究;1999年04期

8 陳梁鴻,王新望,張文俊,張曉東,胡道芬,劉廣田;抗除草劑草甘膦EPSPs基因在小麥中的轉化[J];遺傳學報;1999年03期

9 孫玉合,丁昌敏,張歷歷,卜鍋章,龔明良;煙草新胞質雄性不育系86-6的創(chuàng)造及其利用[J];中國煙草學報;1999年01期

10 胡日華,黃英金,何寬信,肖文俊;烤煙新質源雄性不育系的選育及應用研究初報[J];江西農業(yè)大學學報;1999年01期

相關博士學位論文 前4條

1 頓小玲;甘藍型油菜核不育系7365A恢復基因克隆和進化分析[D];華中農業(yè)大學;2013年

2 周正富;甘藍型油菜隱性核不育分子機理及相關基因功能分析[D];華中農業(yè)大學;2011年

3 黃鎮(zhèn);分子標記輔助選擇培育新型甘藍型油菜隱性細胞核雄性不育系[D];華中農業(yè)大學;2009年

4 易斌;甘藍型油菜隱性核不育基因Bnms1的精細定位和克隆[D];華中農業(yè)大學;2008年

相關碩士學位論文 前2條

1 謝瑩;番茄功能雄性不育系形態(tài)與生理特性分析及ps基因的精細定位[D];東北農業(yè)大學;2014年

2 朱云;甘藍型油菜隱性上位核不育的分子機理研究[D];華中農業(yè)大學;2008年

，

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