立枯丝核菌第45家族糖苷水解酶PAMP活性区段及位点的研究
发布时间：2018-11-26 15:19
【摘要】：玉米在我国国民经济和农业生产中发挥着重要的作用,在我国粮食和饲料作物及工业原料方面占有举足轻重的地位。玉米纹枯病已成为生产中的主要病害,随着高密度栽培和高氮的推广应用,该病害快速蔓延,在全国各地不同玉米产区产生不同程度的减产,严重影响我国农业生产的产量和质量。植物细胞壁是抵抗外界病原物入侵的天然屏障。植物病原真菌和细菌都能产生细胞壁降解酶,成为在植物细胞内和细胞之间蔓延的主要致病因子,如纤维素酶和果胶酶等。近年来,人们越来越注意到在真菌与植物互作机制方面起作用的细胞壁降解酶。PAMP分子是病原微生物表面存在的一些保守分子,广泛存在于微生物中。植物模式识别受体通过识别病原物模式分子来激活体内信号途径,诱导防卫反应从而限制病原物的入侵。前期研究发现,R.solani AG-1-IA融合群中具有PAMP活性的酶中有β-1,4-内切纤维素酶EG1,EG1是一个PAMP分子,并且有研究证明了PAMP活性与催化活性相互独立。为了进一步确定其发挥作用的区段及具体位点,我们通过一系列实验来探究。我们将EG1的野生型命名为WT,通过基因定点突变将EG1氨基酸序列第32位的天冬氨酸(Aspartic acid,Asp)突变为丙氨酸(Alanine,Ala),导致其催化活性的丧失并命名为D32A。为了确定EG1发挥PAMP活性的区段,我们对EG1的六个相对保守的区段进行了突变,结果发现野生型WT突变区段C6后仍然具有较高的催化活力,能够引起玉米、烟草等植物的叶片坏死,但是不能诱导PAL、POD等防卫反应基因的过量表达;D32A突变区段C6后几乎没有催化活性,既不能引起玉米、烟草等植物的叶片坏死,也不能诱导PAL、POD等防卫反应基因的过量表达。然后利用PVX表达系统进行实验。首先利用PCR扩增法将信号肽序列插入基因5’末端,然后将片段插入pGR106空载体构建PVX表达载体,而后经过冻融法转化农杆菌(Agrobacterium tumefaciens)GV3101感受态细胞。将携带重组质粒的农杆菌接种烟草叶片,以转化空pGR106载体的农杆菌作为对照(CK),结果发现WT-C6可以使烟草叶片产生病斑,而D32A-C6则不可以。对于WT-C6和D32A-C6的不同表现,我们认为是其对细胞壁纤维素的降解所产生的效果。这些结果证明,C6保守区(序列为GCNWRFDWF)是EG1发挥PAMP活性的关键区段。接着,为了进一步探究EG1的具体活性位点,我们将C6区段内的所有氨基酸进行定点突变,并获得相应的突变工程菌和蛋白,将调整好浓度的纯酶接种玉米后发现,其中一个蛋白RA不能引起玉米叶片的坏死。然后,用调整好浓度的RA的纯酶接种玉米和烟草叶片,同时测定RA接种对玉米和烟草叶片活性氧产生的影响,以及对防卫反应基因表达的影响。结果发现,RA不能使玉米和烟草叶片坏死,也不能使活性氧大量产生,同时不能引起防卫反应基因的过量表达。同时利用PVX表达载体对d32a-ra进行表达,发现携带pGR106/pd32a-ra的重组质粒不能引起烟草叶片的坏死。这些实验结果初步确定了EG1的具体活性位点。本研究证实了R.solani第45家族糖苷水解酶β-1,4-内切纤维素酶EG1发挥PAMP活性的关键区段,同时对其具体位点进行了初步探究。
[Abstract]:The corn plays an important role in our national economy and agricultural production, and plays a very important role in our country's grain and feed crops and industrial raw materials. The corn sheath blight has become the main disease in the production, and with the popularization and application of high-density cultivation and high-nitrogen, the disease is rapidly spreading, and the yield and the quality of the agricultural production in China are seriously affected. The plant cell wall is a natural barrier against the invasion of the environment. Plant pathogenic fungi and bacteria can produce cell wall degrading enzymes, and become the main pathogenic factors that spread among plant cells and cells, such as cellulase and pectase. In recent years, more and more attention has been paid to cell wall degrading enzymes that function in the interaction of fungi with plants. The PAMP molecules are some of the conserved molecules in the surface of the pathogenic micro-organisms and are widely present in the micro-organisms. The plant pattern recognition receptor activates the in vivo signaling pathway by identifying the pathogen pattern molecules, inducing a defense response to limit the invasion of the pathogen. It was found that EG1 and EG1 in the enzyme with PAMP activity in the R. solani AG-1-IA fusion group were a PAMP molecule, and the study demonstrated that the activity of PAMP and the catalytic activity were independent. In order to further determine the sections and specific sites for which they function, we are exploring through a series of experiments. The wild-type of EG1 was named WT, and the amino acid (Asp) at position 32 of the EG1 amino acid sequence was mutated to alanine (Alanine, Ala) by a gene-directed mutation, resulting in a loss of catalytic activity and a designation of D32A. In order to determine the extent of the activity of EG1 as a PAMP, we have mutated the six relatively conserved segments of EG1, and the results show that the wild-type WT mutant segment C6 still has a high catalytic activity, which can cause leaf necrosis of plants such as corn, tobacco and the like, but can not induce PAL, The excessive expression of the defense reaction gene such as POD and the like is almost no catalytic activity after the D32A mutant segment C6, so that the leaf necrosis of the plants such as corn, tobacco and the like can not be caused, and the excessive expression of the defense reaction genes such as PAL, POD and the like can not be induced. The experiment was then carried out using a PVX expression system. The signal peptide sequence was first inserted into the 5 'end of the gene by the PCR amplification method, then the fragment was inserted into the pGR106 empty vector to construct the PVX expression vector, and then the Agrobacterium tumefaciens GV3101 competent cell was transformed by a freeze-thaw method. The agrobacterium carrying the recombinant plasmid was inoculated with the tobacco leaf to transform the Agrobacterium as the control (CK) of the empty pGR106 vector, and it was found that WT-C6 could cause the tobacco leaf to produce a disease spot, while D32A-C6 could not. For the different manifestations of WT-C6 and D32A-C6, we believe that it has an effect on the degradation of the cell wall cellulose. These results demonstrate that the C6 conserved region (sequence GCNWRDWF) is the key section of the EG1 to play the PAMP activity. Then, in order to further explore the specific active site of EG1, all the amino acids in the C6 segment are mutated, and the corresponding mutant engineering bacteria and proteins are obtained, and the adjusted concentration of the pure enzyme is inoculated with the corn, so that the necrosis of the corn leaves can not be caused by one of the proteins RA. Then, corn and tobacco leaves were inoculated with the pure enzyme of RA with good concentration, and the effect of RA on the active oxygen production of corn and tobacco leaves was also determined, and the effect of RA on the expression of the defense response gene was also determined. The results showed that RA could not cause necrosis of corn and tobacco leaves, and could not produce a large amount of active oxygen, and could not cause excessive expression of defense response gene. At the same time, the expression of d32a-ra was carried out by using the PVX expression vector, and it was found that the recombinant plasmid carrying the pGR106/ pd32a-ra could not cause necrosis of the tobacco leaf. The results of these experiments initially determined the specific active site of EG1. In this study, R. solani's 45 family of glycosidase-1, 4-endoglucanase EG1 was used to play the key part of the activity of PAMP, and the specific site was explored.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.131.4
本文编号：2358921
[Abstract]:The corn plays an important role in our national economy and agricultural production, and plays a very important role in our country's grain and feed crops and industrial raw materials. The corn sheath blight has become the main disease in the production, and with the popularization and application of high-density cultivation and high-nitrogen, the disease is rapidly spreading, and the yield and the quality of the agricultural production in China are seriously affected. The plant cell wall is a natural barrier against the invasion of the environment. Plant pathogenic fungi and bacteria can produce cell wall degrading enzymes, and become the main pathogenic factors that spread among plant cells and cells, such as cellulase and pectase. In recent years, more and more attention has been paid to cell wall degrading enzymes that function in the interaction of fungi with plants. The PAMP molecules are some of the conserved molecules in the surface of the pathogenic micro-organisms and are widely present in the micro-organisms. The plant pattern recognition receptor activates the in vivo signaling pathway by identifying the pathogen pattern molecules, inducing a defense response to limit the invasion of the pathogen. It was found that EG1 and EG1 in the enzyme with PAMP activity in the R. solani AG-1-IA fusion group were a PAMP molecule, and the study demonstrated that the activity of PAMP and the catalytic activity were independent. In order to further determine the sections and specific sites for which they function, we are exploring through a series of experiments. The wild-type of EG1 was named WT, and the amino acid (Asp) at position 32 of the EG1 amino acid sequence was mutated to alanine (Alanine, Ala) by a gene-directed mutation, resulting in a loss of catalytic activity and a designation of D32A. In order to determine the extent of the activity of EG1 as a PAMP, we have mutated the six relatively conserved segments of EG1, and the results show that the wild-type WT mutant segment C6 still has a high catalytic activity, which can cause leaf necrosis of plants such as corn, tobacco and the like, but can not induce PAL, The excessive expression of the defense reaction gene such as POD and the like is almost no catalytic activity after the D32A mutant segment C6, so that the leaf necrosis of the plants such as corn, tobacco and the like can not be caused, and the excessive expression of the defense reaction genes such as PAL, POD and the like can not be induced. The experiment was then carried out using a PVX expression system. The signal peptide sequence was first inserted into the 5 'end of the gene by the PCR amplification method, then the fragment was inserted into the pGR106 empty vector to construct the PVX expression vector, and then the Agrobacterium tumefaciens GV3101 competent cell was transformed by a freeze-thaw method. The agrobacterium carrying the recombinant plasmid was inoculated with the tobacco leaf to transform the Agrobacterium as the control (CK) of the empty pGR106 vector, and it was found that WT-C6 could cause the tobacco leaf to produce a disease spot, while D32A-C6 could not. For the different manifestations of WT-C6 and D32A-C6, we believe that it has an effect on the degradation of the cell wall cellulose. These results demonstrate that the C6 conserved region (sequence GCNWRDWF) is the key section of the EG1 to play the PAMP activity. Then, in order to further explore the specific active site of EG1, all the amino acids in the C6 segment are mutated, and the corresponding mutant engineering bacteria and proteins are obtained, and the adjusted concentration of the pure enzyme is inoculated with the corn, so that the necrosis of the corn leaves can not be caused by one of the proteins RA. Then, corn and tobacco leaves were inoculated with the pure enzyme of RA with good concentration, and the effect of RA on the active oxygen production of corn and tobacco leaves was also determined, and the effect of RA on the expression of the defense response gene was also determined. The results showed that RA could not cause necrosis of corn and tobacco leaves, and could not produce a large amount of active oxygen, and could not cause excessive expression of defense response gene. At the same time, the expression of d32a-ra was carried out by using the PVX expression vector, and it was found that the recombinant plasmid carrying the pGR106/ pd32a-ra could not cause necrosis of the tobacco leaf. The results of these experiments initially determined the specific active site of EG1. In this study, R. solani's 45 family of glycosidase-1, 4-endoglucanase EG1 was used to play the key part of the activity of PAMP, and the specific site was explored.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.131.4
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