本文選題：甘藍(lán)型油菜　切入點(diǎn)：Bnams4~b　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：甘藍(lán)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(fù)源等特點(diǎn),成為目前油菜雜種優(yōu)勢(shì)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(dǎo)致雄性不育。BnaMs3是Bnams4~b的恢復(fù)基因,能夠恢復(fù)Bnams4~b的破壞效應(yīng),但BnaMs3在其它作物中的同源基因與Bnams4~b的關(guān)系以及對(duì)Bnams4~b的功能有什么影響,目前還沒(méi)有相關(guān)研究。本研究將不育基因Bnams4~b和恢復(fù)基因BnaMs3轉(zhuǎn)化到普通煙草和栽培番茄A57中,對(duì)Bnams4~b在其它作物中的功能以及不同作物Tic40對(duì)Bnams4~b功能的影響進(jìn)行研究。主要研究結(jié)果如下:1.煙草的遺傳轉(zhuǎn)化和陽(yáng)性鑒定將不育基因Bnams4~b轉(zhuǎn)化到煙草中,獲得陽(yáng)性株。取不同黃化程度的轉(zhuǎn)基因植株葉片進(jìn)行qRT-PCR。結(jié)果顯示,Bnams4~b基因的表達(dá)量隨轉(zhuǎn)基因煙草葉片黃化程度的增加而升高。2.轉(zhuǎn)基因煙草的表型分析轉(zhuǎn)基因煙草葉片出現(xiàn)了不同程度的黃化,對(duì)其黃化葉片進(jìn)行透射電鏡觀察和葉綠素含量檢測(cè),發(fā)現(xiàn)葉綠素含量隨葉片由黃到綠呈逐漸升高的趨勢(shì),黃化葉片中的葉綠體結(jié)構(gòu)出現(xiàn)異常。醋酸洋紅染色結(jié)果顯示,轉(zhuǎn)基因黃化煙草的花藥能夠產(chǎn)生有活力的花粉粒,并未出現(xiàn)預(yù)期的雄性不育表型。3.轉(zhuǎn)基因煙草未出現(xiàn)不育的原因分析對(duì)轉(zhuǎn)基因煙草未出現(xiàn)不育的原因進(jìn)行分析,首先更換花藥特異啟動(dòng)子A9和TA29后轉(zhuǎn)化煙草,依然沒(méi)有出現(xiàn)雄性不育表型;其次對(duì)煙草不同Tic40拷貝的功能進(jìn)行分析,結(jié)果證實(shí)煙草的各Tic40拷貝都不能恢復(fù)Bnams4~b引起的擬南芥雄性不育表型。4.轉(zhuǎn)基因番茄的陽(yáng)性鑒定和表達(dá)量分析將番茄A57的Tic40拷貝轉(zhuǎn)化擬南芥,與包含Bnams4~b的擬南芥雜交,結(jié)果顯示番茄的Tic40拷貝不能恢復(fù)Bnams4~b引起的擬南芥雄性不育。將不育基因Bnams4~b和恢復(fù)基因BnaMs3導(dǎo)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(dá)量越高,花藥中也有一定程度的表達(dá)。5.轉(zhuǎn)基因番茄的表型分析和育性觀察共得到19株Bnams4~b轉(zhuǎn)基因番茄陽(yáng)性苗,有6株葉片黃化,相同的生長(zhǎng)環(huán)境下黃化株比正常的栽培材料生長(zhǎng)矮小。其中兩株花藥中沒(méi)有花粉粒,部分轉(zhuǎn)化株的花藥中有少量可育花粉粒和大量干癟的、沒(méi)有活力的花粉粒。黃化程度最嚴(yán)重的單株出現(xiàn)死蕾現(xiàn)象,不能正常結(jié)實(shí)。6.煙草和番茄Tic40中的功能變異位點(diǎn)分析將擬南芥、甘藍(lán)型油菜、煙草、番茄的Tic40進(jìn)行氨基酸序列比對(duì)分析。結(jié)果顯示,番茄Tic40中的六個(gè)功能位點(diǎn)與Bnams3完全一致,推測(cè)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(shí),Bnams4~b才能表現(xiàn)出較強(qiáng)的破壞功能。煙草Tic40序列中的第一個(gè)功能位點(diǎn)與BnaMs3和Bnams3的都不相同,且缺失了第六個(gè)功能位點(diǎn),結(jié)合轉(zhuǎn)基因煙草沒(méi)有得到不育表型,推測(cè)與煙草Tic40的兩個(gè)變異位點(diǎn)有關(guān)。
[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(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S565.4

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