稻曲病菌T-DNA插入突变体B1812、B1241侧翼基因克隆及Uvt-726互作因子的筛选

发布时间：2018-07-31 11:17

【摘要】：稻曲病是发生在水稻穗部的一种真菌病害,其病原菌稻曲病菌(Ustilaginoidea virens)可侵染水稻小花,形成的稻曲球严重危及水稻的产量和稻米的品质,进而危害人类和家畜的健康。基于对稻曲病菌的形态、分离技术、孢子萌发、侵染机制等方面的研究,目前对于水稻稻曲病的防治已经取得了一些进展,然而由于稻曲病菌的致病机制仍不明确,尚不能从根本本上防治该病害。研究稻曲病菌的致病机制,有助于深入认识和有效防控稻曲病的发生,同时对于水稻抗稻曲病品种的选育具有一定的指导意义。利用农杆菌介导的转化技术(ATMT,Agrobacterium-mediated transformation technology)对病原菌进行遗传改造,再通过表型筛选进行基因功能的研究已经成为一种简便有效的方法。本实验室前期通过ATMT构建了稻曲病菌P1的突变体库,本研究以从中筛选得到致病力减弱菌株B1812和B1241为研究材料,观察它们的生物学特性及致病情况,通过基因克隆的手段获得了 T-DNA插入位点侧翼基因,并初步研究了基因的功能,为研究稻曲病菌的致病机制提供了一定的理论基础。1.稻曲病菌P1的T-DNA插入突变菌株B1812,其致病力、液体摇培产分生孢子能力与野生菌株P1相比明显下降,且孢子明显小于P1。而在固体培养基PSA和TB3菌落形态和生长速率与P1相比无显著差异,在MM上的生长速率下降。突变菌株B1812在不含潮霉素的PSA平板上转接5代之后,仍能扩增到HPH基因,说明T-DNA已经稳定地插入到B1812的基因组中。Southern杂交表明T-DNA在该突变菌株的基因组中为单拷贝。序列比对显示,侧翼基因UvHac1与UV-8b菌株中的UV8b_1075同源。UvHac1全长共2196bp,编码蛋白为转录激活因子Hac1,其中ORF为1690bp,含有一个70 bp的内含子,编码539个氨基酸。5'非编码区长为360 bp,3'非编码区长为146 bp。分析插入位点侧翼序列发现,突变菌株B1812的基因组在T-DNA插入位点处缺失了 42 bp。T-DNA插入在UvHac1的5'UTR区,距起始密码子46 bp。qRT-PCR分析显示该基因表达量显著下降。经NCBI同源性比对,UvHac1编码蛋白Hac1含有一个高度保守的结构域bZIP。2.致病力减弱突变菌株B1241在固体培养基MM、PSA和TB3上的菌落形态、生长速率,以及液体摇培产生的分生孢子形态等与P1相比无显著差异,但分生孢子能力呈极显著下降。T-DNA已经稳定地插入到B1241的基因组中且为单拷贝。经序列比对发现,突变体中T-DNA插入位点处少了 28 bp的稻曲病菌基因组序列,有37bp的序列在T-DNA及稻曲病菌基因组中都没有比对到。侧翼基因UvGH18与UV-8b菌株的UV8b-7878同源,开放阅读框长2317 bp,包含81 bp和106 bp的两个内含子,编码709个氨基酸。UvGH18基因全长2650bp,5·非编码区长度为14bp,3·非编码区长度为319 bp。T-DNA插入在UvGH18的启动子区域,位于起始密码子之前516 bp处。qRT-PCR得知,突变体中UvGH18的表达量下降。UvGH18编码的糖基水解酶18家族蛋白(GH18)含有一个保守结构域D××D×D×E。酵母双杂交筛选稻曲病菌P1的cDNA文库,得到4个可能与GH18互作的蛋白,分别为泛素类、小泛素相关修饰蛋白连接酶、海藻糖-6-磷酸合成酶/磷酸酶和细胞形态蛋白Sog2。3.Uvt-726是致病力减弱的T-DNA插入突变菌株B726通过基因克隆得到的假定基因。为进一步探究致病相关基因Uvt-726的功能,本研究通过酵母双杂交技术筛选其互作蛋白。将目的基因Uvt-726连接至质粒pGBKT7构建诱饵质粒,并对诱饵蛋白进行了毒性及自激活检测。诱饵质粒和文库质粒分别转化进酵母菌株Y2H和Y187中进行杂交。诱饵蛋白与BD蛋白在二倍体酵母中融合表达,与稻曲病菌P1的cDNA文库酵母中的捕获蛋白结合,从而激活下游报告基因的表达。通过DDO/X/A和QDO/X/A进行筛选,测序后与稻曲病菌全基因组进行比对,筛选得到4个Uvt-726的互作蛋白,包括AP-3复合物β亚基、RNA聚合酶II亚基A磷酸酶、寡糖转移酶STT3亚基和ADP/ATP载体蛋白。
[Abstract]:Rice koji is a fungal disease occurring at the ear of rice. The pathogen of Oryza sativa (Ustilaginoidea virens) can infect small rice flowers. The formation of rice curl seriously endangers the yield of rice and the quality of rice, and then endangers the health of human and domestic animals. Based on the morphology, separation technique, spore germination, and infection mechanism of the strains of Oryza sativa At present, some progress has been made in the prevention and control of rice rice curd disease. However, the disease mechanism of Oryza is still not clear because the pathogenic mechanism of Oryza is still unclear. It is helpful to understand and effectively prevent and control the occurrence of rice curd disease. The breeding of ATMT, Agrobacterium-mediated transformation technology, is a simple and effective method to study the genetic function of the pathogenic bacteria by using the transformation of Agrobacterium mediated transformation (Agrobacterium-mediated transformation), and it has been constructed by ATMT in the early stage of the laboratory. In this study, we screened the pathogenic strain B1812 and B1241 as the research materials, observed their biological characteristics and pathogenicity, obtained the flanking gene of the T-DNA insertion site by means of gene cloning, and preliminarily studied the function of the gene, which provided a certain mechanism for the study of the pathogenic mechanism of Oryza Oryza. On the basis of the theoretical basis, the T-DNA insertion mutant strain B1812 of.1., the pathogen of Oryza Oryza P1, its pathogenicity was significantly lower than that of the wild strain P1, and the spores were obviously less than P1., but the morphology and growth rate of PSA and TB3 in the solid medium were not significantly different from P1, and the growth rate on MM decreased. After transferring to 5 generations on the PSA plate without hygromycin, the HPH gene was still amplified, indicating that T-DNA had been inserted into the genome of B1812 steadily and.Southern hybridization showed that T-DNA was a single copy in the mutant genome. Sequence alignment showed that the full length of UV8b_1075 homologous.UvHac1 in the flanking gene UvHac1 and UV-8b strain was 2196bp, The encoding protein is a transcription activating factor Hac1, in which ORF is 1690bp, contains a 70 BP intron, and the non coding region length of the 539 amino acid.5'is 360 BP, the 3' non coding region length is 146 bp. analysis insertion site flanking sequence, and the mutant strain B1812's genome is missing at the T-DNA insertion site and inserted in the UvHac1 region. 46 bp.qRT-PCR analysis from the initial codon showed that the gene expression decreased significantly. After NCBI homology, the UvHac1 encoded protein Hac1 contained a highly conserved domain bZIP.2. pathogenicity strain B1241 in the solid culture medium MM, PSA and TB3, the colony morphology, the growth rate, and the conidia form produced by the liquid shake culture. There was no significant difference compared with P1, but the ability of conidia was significantly reduced by.T-DNA, which had been steadily inserted into the genome of B1241 and was a single copy. By sequence alignment, it was found that the T-DNA insertion site in the mutant was less than 28 BP of the genome sequence of Oryza Oryza, and there was no comparison of 37bp sequence in the genome of T-DNA and Oryza. Right. The flanking gene UvGH18 is homologous to the UV8b-7878 of the UV-8b strain. The open reading frame is 2317 BP, containing two introns of 81 BP and 106 BP, encoding 709 amino acid.UvGH18 gene full length 2650bp, 5. The length of non coding region is 14bp, and 3. The length of the non coding region is 319 bp.T-DNA is inserted in the UvGH18 promoter region, located before the starting codon 516. BP.QRT-PCR found that the expression of UvGH18 in the mutant was reduced by.UvGH18 encoded glycosyl hydrolase 18 family protein (GH18) containing a cDNA Library of D x D x D x E. yeast two hybrid screening of P1 of the pathogen of Oryza in a conservative domain, and 4 proteins that might interact with the GH18 were obtained, which were ubiquitin, small ubiquitin related protein ligase and algae. Sugar -6- phosphate synthetase / phosphatase and cell morphogenetic protein Sog2.3.Uvt-726 are the hypothetical genes obtained by the T-DNA insertion mutant strain B726, which have weakened the pathogenicity. In order to further explore the function of the pathogenic related gene Uvt-726, this study screened the interaction protein by yeast two hybrid technique. The target gene Uvt-726 was connected to the quality of the gene. The decoy plasmid was constructed by grain pGBKT7, and the bait protein was tested for toxicity and self activation. The bait plasmid and library plasmid were transformed into yeast strain Y2H and Y187 respectively. The bait protein and BD protein were fused in diploid yeast and combined with the capture protein in the cDNA Library of P1 of Oryza Oryza, thus activating the downstream newspaper. It was screened by DDO/X/A and QDO/X/A, and compared with the whole genome of Oryza Oryza after sequencing, 4 Uvt-726 intercrop proteins were screened, including AP-3 complex beta subunit, RNA polymerase II subunit A phosphatase, oligosaccharide transferase STT3 subunit and ADP/ATP carrier protein.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.111.4

【参考文献】