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不同作物Tic40对新型嵌合基因Bnams4~b功能的影响

发布时间:2018-04-09 16:23

  本文选题:甘蓝型油菜 切入点:Bnams4~b 出处:《华中农业大学》2017年硕士论文


【摘要】:甘蓝型油菜核不育系统7365AB(Bnams3ms3ms4~bms4~b/BnaMs3ms3ms4~bms4~b)由于不育性稳定、败育彻底,具有广泛的恢复源等特点,成为目前油菜杂种优势利用的重要途径之一。不育基因Bnams4~b能够引起油菜和拟南芥花药功能性缺失和叶片黄化,导致雄性不育。BnaMs3是Bnams4~b的恢复基因,能够恢复Bnams4~b的破坏效应,但BnaMs3在其它作物中的同源基因与Bnams4~b的关系以及对Bnams4~b的功能有什么影响,目前还没有相关研究。本研究将不育基因Bnams4~b和恢复基因BnaMs3转化到普通烟草和栽培番茄A57中,对Bnams4~b在其它作物中的功能以及不同作物Tic40对Bnams4~b功能的影响进行研究。主要研究结果如下:1.烟草的遗传转化和阳性鉴定将不育基因Bnams4~b转化到烟草中,获得阳性株。取不同黄化程度的转基因植株叶片进行qRT-PCR。结果显示,Bnams4~b基因的表达量随转基因烟草叶片黄化程度的增加而升高。2.转基因烟草的表型分析转基因烟草叶片出现了不同程度的黄化,对其黄化叶片进行透射电镜观察和叶绿素含量检测,发现叶绿素含量随叶片由黄到绿呈逐渐升高的趋势,黄化叶片中的叶绿体结构出现异常。醋酸洋红染色结果显示,转基因黄化烟草的花药能够产生有活力的花粉粒,并未出现预期的雄性不育表型。3.转基因烟草未出现不育的原因分析对转基因烟草未出现不育的原因进行分析,首先更换花药特异启动子A9和TA29后转化烟草,依然没有出现雄性不育表型;其次对烟草不同Tic40拷贝的功能进行分析,结果证实烟草的各Tic40拷贝都不能恢复Bnams4~b引起的拟南芥雄性不育表型。4.转基因番茄的阳性鉴定和表达量分析将番茄A57的Tic40拷贝转化拟南芥,与包含Bnams4~b的拟南芥杂交,结果显示番茄的Tic40拷贝不能恢复Bnams4~b引起的拟南芥雄性不育。将不育基因Bnams4~b和恢复基因BnaMs3导入番茄中,出现了黄化表型,qRT-PCR显示叶色黄化程度越重,Bnams4~b的表达量越高,花药中也有一定程度的表达。5.转基因番茄的表型分析和育性观察共得到19株Bnams4~b转基因番茄阳性苗,有6株叶片黄化,相同的生长环境下黄化株比正常的栽培材料生长矮小。其中两株花药中没有花粉粒,部分转化株的花药中有少量可育花粉粒和大量干瘪的、没有活力的花粉粒。黄化程度最严重的单株出现死蕾现象,不能正常结实。6.烟草和番茄Tic40中的功能变异位点分析将拟南芥、甘蓝型油菜、烟草、番茄的Tic40进行氨基酸序列比对分析。结果显示,番茄Tic40中的六个功能位点与Bnams3完全一致,推测番茄Tic40与Bnams3有类似功能,番茄中只有存在类似于Bnams3的Tic40拷贝时,Bnams4~b才能表现出较强的破坏功能。烟草Tic40序列中的第一个功能位点与BnaMs3和Bnams3的都不相同,且缺失了第六个功能位点,结合转基因烟草没有得到不育表型,推测与烟草Tic40的两个变异位点有关。
[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.
【学位授予单位】:华中农业大学
【学位级别】:硕士
【学位授予年份】:2017
【分类号】:S565.4

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