稻曲病菌T-DNA插入突變體B1812、B1241側翼基因克隆及Uvt-726互作因子的篩選
發(fā)布時間：2018-07-31 11:17
【摘要】：稻曲病是發(fā)生在水稻穗部的一種真菌病害,其病原菌稻曲病菌(Ustilaginoidea virens)可侵染水稻小花,形成的稻曲球嚴重危及水稻的產量和稻米的品質,進而危害人類和家畜的健康。基于對稻曲病菌的形態(tài)、分離技術、孢子萌發(fā)、侵染機制等方面的研究,目前對于水稻稻曲病的防治已經(jīng)取得了一些進展,然而由于稻曲病菌的致病機制仍不明確,尚不能從根本本上防治該病害。研究稻曲病菌的致病機制,有助于深入認識和有效防控稻曲病的發(fā)生,同時對于水稻抗稻曲病品種的選育具有一定的指導意義。利用農桿菌介導的轉化技術(ATMT,Agrobacterium-mediated transformation technology)對病原菌進行遺傳改造,再通過表型篩選進行基因功能的研究已經(jīng)成為一種簡便有效的方法。本實驗室前期通過ATMT構建了稻曲病菌P1的突變體庫,本研究以從中篩選得到致病力減弱菌株B1812和B1241為研究材料,觀察它們的生物學特性及致病情況,通過基因克隆的手段獲得了 T-DNA插入位點側翼基因,并初步研究了基因的功能,為研究稻曲病菌的致病機制提供了一定的理論基礎。1.稻曲病菌P1的T-DNA插入突變菌株B1812,其致病力、液體搖培產分生孢子能力與野生菌株P1相比明顯下降,且孢子明顯小于P1。而在固體培養(yǎng)基PSA和TB3菌落形態(tài)和生長速率與P1相比無顯著差異,在MM上的生長速率下降。突變菌株B1812在不含潮霉素的PSA平板上轉接5代之后,仍能擴增到HPH基因,說明T-DNA已經(jīng)穩(wěn)定地插入到B1812的基因組中。Southern雜交表明T-DNA在該突變菌株的基因組中為單拷貝。序列比對顯示,側翼基因UvHac1與UV-8b菌株中的UV8b_1075同源。UvHac1全長共2196bp,編碼蛋白為轉錄激活因子Hac1,其中ORF為1690bp,含有一個70 bp的內含子,編碼539個氨基酸。5'非編碼區(qū)長為360 bp,3'非編碼區(qū)長為146 bp。分析插入位點側翼序列發(fā)現(xiàn),突變菌株B1812的基因組在T-DNA插入位點處缺失了 42 bp。T-DNA插入在UvHac1的5'UTR區(qū),距起始密碼子46 bp。qRT-PCR分析顯示該基因表達量顯著下降。經(jīng)NCBI同源性比對,UvHac1編碼蛋白Hac1含有一個高度保守的結構域bZIP。2.致病力減弱突變菌株B1241在固體培養(yǎng)基MM、PSA和TB3上的菌落形態(tài)、生長速率,以及液體搖培產生的分生孢子形態(tài)等與P1相比無顯著差異,但分生孢子能力呈極顯著下降。T-DNA已經(jīng)穩(wěn)定地插入到B1241的基因組中且為單拷貝。經(jīng)序列比對發(fā)現(xiàn),突變體中T-DNA插入位點處少了 28 bp的稻曲病菌基因組序列,有37bp的序列在T-DNA及稻曲病菌基因組中都沒有比對到。側翼基因UvGH18與UV-8b菌株的UV8b-7878同源,開放閱讀框長2317 bp,包含81 bp和106 bp的兩個內含子,編碼709個氨基酸。UvGH18基因全長2650bp,5·非編碼區(qū)長度為14bp,3·非編碼區(qū)長度為319 bp。T-DNA插入在UvGH18的啟動子區(qū)域,位于起始密碼子之前516 bp處。qRT-PCR得知,突變體中UvGH18的表達量下降。UvGH18編碼的糖基水解酶18家族蛋白(GH18)含有一個保守結構域D××D×D×E。酵母雙雜交篩選稻曲病菌P1的cDNA文庫,得到4個可能與GH18互作的蛋白,分別為泛素類、小泛素相關修飾蛋白連接酶、海藻糖-6-磷酸合成酶/磷酸酶和細胞形態(tài)蛋白Sog2。3.Uvt-726是致病力減弱的T-DNA插入突變菌株B726通過基因克隆得到的假定基因。為進一步探究致病相關基因Uvt-726的功能,本研究通過酵母雙雜交技術篩選其互作蛋白。將目的基因Uvt-726連接至質粒pGBKT7構建誘餌質粒,并對誘餌蛋白進行了毒性及自激活檢測。誘餌質粒和文庫質粒分別轉化進酵母菌株Y2H和Y187中進行雜交。誘餌蛋白與BD蛋白在二倍體酵母中融合表達,與稻曲病菌P1的cDNA文庫酵母中的捕獲蛋白結合,從而激活下游報告基因的表達。通過DDO/X/A和QDO/X/A進行篩選,測序后與稻曲病菌全基因組進行比對,篩選得到4個Uvt-726的互作蛋白,包括AP-3復合物β亞基、RNA聚合酶II亞基A磷酸酶、寡糖轉移酶STT3亞基和ADP/ATP載體蛋白。
[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.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S435.111.4
本文編號：2155416
[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.
【學位授予單位】：南京農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S435.111.4
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