大丽轮枝菌微菌核发育异常突变体的筛选及其相关基因的鉴定

发布时间：2018-04-20 11:40

本文选题：大丽轮枝菌 + T-DNA插入突变体库　；参考：《南京师范大学》2015年硕士论文

【摘要】：为了更加深入的分析研究大丽轮枝菌微菌核发育相关基因的功能,利用农杆菌介导的遗传转化(ATMT)方法,成功构建了大丽轮枝菌菌核型菌株V08DF1、中间型菌株VBp2的突变体库,其中产生大量黑色微菌核的V08DF1库中包含2000个转化子,产生少量黑色微菌核的VBp2库中包含1000个转化子。从菌核型菌株V08DF1库中筛选出14株不产微菌核的突变体,分别是1C2、2H3、5G4、617、 7E6、9G4、10B5、10E1、11A6、11B7、11C3、11G3、12C8、1213。从中间型菌株VBp2库中筛选到9株微菌核发育异常的突变体,包括4株在查氏平板上产紫黑色素的突变体,分别为7C8、7H4、9B1、9C10,与5株在查氏培养基上不生长的突变体,分别为7F4、10B10、10C6、10E12、IOG8。对上述23株微菌核发育异常的突变体进行T-DNA插入的PCR验证,均能扩增到潮霉素抗性基因。通过Southern杂交对V08DF1库中筛选出的14株微菌核发育异常突变体进行检测,结果表明7个为单拷贝插入,5个为双拷贝插入,2个为三拷贝插入。7株单拷贝插入的微菌核发育异常的突变菌株,分别是6I7、10B5、10E1、12C8、1213、1C2和2H3。Southern杂交检测VBp2库中筛选出的微菌核发育异常的突变体,仅有突变体7F4、9B1、10E12获得结果,这3株突变体均为双拷贝插入。这些微菌核异常突变体的获得为进一步克隆参与调控微菌核形成发育的基因奠定了基础。由于单拷贝突变体易于找出它们的T-DNA插入位点,可进一步找出被破坏的微菌核发育相关基因,所以重点研究单拷贝插入的突变体。随机选择2H3和1C2这2个单拷贝插入的突变体进行微菌核发育相关基因的研究。首先利用TAIL-PCR方法找到T-DNA区的插入位点,并通过与美国BROAD研究所公布的大丽轮枝菌的全基因组数据库以及NCBI网站上的序列进行BLAST,找到并分析T-DNA区插入的侧翼序列与微菌核发育相关基因。结果表明突变体2H3被T-DNA区破坏的基因编码conidial yellow pigment biosynthesis polyketide synthase,基因编号为VDAG_00190.1,对其基因功能成功进行了回补与敲除验证,其中,2H3的回补转化子共得到7个,并且这些转化子的表型也都得到了恢复,敲除转化子共得到5个,经过了Southern杂交验证,确定是定点敲除,得到的敲除转化子均为单拷贝,初步证实了这个基因确实与微菌核发育有关。突变体1C2被破坏的微菌核发育相关基因为一个假设蛋白,基因编号为VDAG_01680.1,对其基因功能进行了回补验证,共得到1个阳性回补转化子。另外,对双拷贝突变体5G4和9G4用Tail-PCR方法与质粒拯救方法寻找其T-DNA区插入的位点,最终只获得其中一个插入位点,所以后续的基因功能验证工作很难开展。推测突变体5G4可能破坏下游的G931P828RE17.T0基因表达序列标签；突变体9G4可能被破坏的基因也编码假设蛋白,基因编号为VDAG_09865.1。
[Abstract]:In order to further analyze the function of the genes related to the development of microsclerotia, the library of the mutant V08DF1, the intermediate strain VBp2, was successfully constructed by means of Agrobacterium tumefaciens mediated genetic transformation (ATMTT). The V08DF1 library which produced a large number of black microsclerotia contained 2000 transformants and the VBp2 library which produced a small amount of black microsclerotia contained 1000 transformants. Fourteen non-microsclerotia mutants were screened from the sclerotia strain V08DF1 library. They were 1C2O2H3H3O5G4617, 7E6O9G4G4G4O10E1A11A11A11B711C3O11C3O12C8101313. The results showed that the microsclerotia could not produce microsclerotia from the Sclerotinia sclerotiorum strain V08DF1 library. Nine mutants with abnormal microsclerotia development were screened from the VBp2 library of intermediate strains, including four mutants producing purple melanin on Charing plate, respectively, 7C8H4H4B1B1C10 and 5 mutants that did not grow on Charing's medium, respectively, 7F410B10C610E12IOG8. The hygromycin resistant genes were amplified from 23 microsclerotia mutants with T-DNA insertion by PCR. Fourteen microsclerotia abnormal mutants screened from V08DF1 library were detected by Southern hybridization. The results showed that 7 were single copy inserts, 5 were double copy inserts, and 2 were three copies inserted into 7 strains of mutant microsclerotia with single copy insertion. The results were obtained from the microsclerotia dysplasia selected from the VBp2 library by 6I7H7, 10B5, 10E1, 10E1, 12C8O1213131C2 and 2H3.Southern hybridization. Only the mutant 7F4O9B1O10E12 was obtained, and the three mutants were all double-copy inserted. The acquisition of these abnormal microsclerotia mutants laid a foundation for further cloning of genes involved in the regulation of microsclerotia formation and development. Because single copy mutants are easy to find their T-DNA insertion sites, they can further find out the genes related to microsclerotia development, so we focus on single copy inserted mutants. Two single copy inserts, 2H3 and 1C2, were randomly selected to study the genes associated with microsclerotia development. First, the insertion site of T-DNA region was found by TAIL-PCR method. The genes related to the development of microsclerotia and flanking sequences inserted in T-DNA region were identified and analyzed by BLAST with the whole genome database and NCBI website published by the BROAD Institute of the United States. The results showed that the mutant 2H3 was encoded by conidial yellow pigment biosynthesis polyketide synthase (VDAG00190.1), which was destroyed by T-DNA region. The gene function of 2H3 was successfully mended and knocked out. Moreover, the phenotypes of these transformants were also recovered. Five transformants were obtained from the knockout transformants. After Southern hybridization, the knockout transformants were confirmed to be site-specific knockout, and all the knockout transformants were single-copy. It is preliminarily confirmed that this gene is related to the development of microsclerotia. The gene associated with microsclerotia development of the mutant 1C2 was a hypothetical protein, and the gene number was VDAG _ (01680.1). The function of the gene was verified and a positive complement transformant was obtained. In addition, Tail-PCR method and plasmid rescue method were used to search for the insertion site of the T-DNA region of the double copy mutants 5G4 and 9G4, and only one of the insertion sites was obtained, so it is difficult to carry out the following gene function verification work. It is speculated that the mutant 5G4 may destroy the downstream G931P828RE17.T0 gene expression sequence tag, and the gene that the mutant 9G4 may be destroyed also encodes the hypothetical protein, the gene number is VDAGV 09865.1.
【学位授予单位】：南京师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S432.4

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