立枯絲核菌第45家族糖苷水解酶PAMP活性區(qū)段及位點(diǎn)的研究
發(fā)布時(shí)間：2018-11-26 15:19
【摘要】：玉米在我國(guó)國(guó)民經(jīng)濟(jì)和農(nóng)業(yè)生產(chǎn)中發(fā)揮著重要的作用,在我國(guó)糧食和飼料作物及工業(yè)原料方面占有舉足輕重的地位。玉米紋枯病已成為生產(chǎn)中的主要病害,隨著高密度栽培和高氮的推廣應(yīng)用,該病害快速蔓延,在全國(guó)各地不同玉米產(chǎn)區(qū)產(chǎn)生不同程度的減產(chǎn),嚴(yán)重影響我國(guó)農(nóng)業(yè)生產(chǎn)的產(chǎn)量和質(zhì)量。植物細(xì)胞壁是抵抗外界病原物入侵的天然屏障。植物病原真菌和細(xì)菌都能產(chǎn)生細(xì)胞壁降解酶,成為在植物細(xì)胞內(nèi)和細(xì)胞之間蔓延的主要致病因子,如纖維素酶和果膠酶等。近年來(lái),人們?cè)絹?lái)越注意到在真菌與植物互作機(jī)制方面起作用的細(xì)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,廣泛存在于微生物中。植物模式識(shí)別受體通過(guò)識(shí)別病原物模式分子來(lái)激活體內(nèi)信號(hào)途徑,誘導(dǎo)防衛(wèi)反應(yīng)從而限制病原物的入侵。前期研究發(fā)現(xiàn),R.solani AG-1-IA融合群中具有PAMP活性的酶中有β-1,4-內(nèi)切纖維素酶EG1,EG1是一個(gè)PAMP分子,并且有研究證明了PAMP活性與催化活性相互獨(dú)立。為了進(jìn)一步確定其發(fā)揮作用的區(qū)段及具體位點(diǎn),我們通過(guò)一系列實(shí)驗(yàn)來(lái)探究。我們將EG1的野生型命名為WT,通過(guò)基因定點(diǎn)突變將EG1氨基酸序列第32位的天冬氨酸(Aspartic acid,Asp)突變?yōu)楸彼?Alanine,Ala),導(dǎo)致其催化活性的喪失并命名為D32A。為了確定EG1發(fā)揮PAMP活性的區(qū)段,我們對(duì)EG1的六個(gè)相對(duì)保守的區(qū)段進(jìn)行了突變,結(jié)果發(fā)現(xiàn)野生型WT突變區(qū)段C6后仍然具有較高的催化活力,能夠引起玉米、煙草等植物的葉片壞死,但是不能誘導(dǎo)PAL、POD等防衛(wèi)反應(yīng)基因的過(guò)量表達(dá);D32A突變區(qū)段C6后幾乎沒(méi)有催化活性,既不能引起玉米、煙草等植物的葉片壞死,也不能誘導(dǎo)PAL、POD等防衛(wèi)反應(yīng)基因的過(guò)量表達(dá)。然后利用PVX表達(dá)系統(tǒng)進(jìn)行實(shí)驗(yàn)。首先利用PCR擴(kuò)增法將信號(hào)肽序列插入基因5’末端,然后將片段插入pGR106空載體構(gòu)建PVX表達(dá)載體,而后經(jīng)過(guò)凍融法轉(zhuǎn)化農(nóng)桿菌(Agrobacterium tumefaciens)GV3101感受態(tài)細(xì)胞。將攜帶重組質(zhì)粒的農(nóng)桿菌接種煙草葉片,以轉(zhuǎn)化空pGR106載體的農(nóng)桿菌作為對(duì)照(CK),結(jié)果發(fā)現(xiàn)WT-C6可以使煙草葉片產(chǎn)生病斑,而D32A-C6則不可以。對(duì)于WT-C6和D32A-C6的不同表現(xiàn),我們認(rèn)為是其對(duì)細(xì)胞壁纖維素的降解所產(chǎn)生的效果。這些結(jié)果證明,C6保守區(qū)(序列為GCNWRFDWF)是EG1發(fā)揮PAMP活性的關(guān)鍵區(qū)段。接著,為了進(jìn)一步探究EG1的具體活性位點(diǎn),我們將C6區(qū)段內(nèi)的所有氨基酸進(jìn)行定點(diǎn)突變,并獲得相應(yīng)的突變工程菌和蛋白,將調(diào)整好濃度的純酶接種玉米后發(fā)現(xiàn),其中一個(gè)蛋白R(shí)A不能引起玉米葉片的壞死。然后,用調(diào)整好濃度的RA的純酶接種玉米和煙草葉片,同時(shí)測(cè)定RA接種對(duì)玉米和煙草葉片活性氧產(chǎn)生的影響,以及對(duì)防衛(wèi)反應(yīng)基因表達(dá)的影響。結(jié)果發(fā)現(xiàn),RA不能使玉米和煙草葉片壞死,也不能使活性氧大量產(chǎn)生,同時(shí)不能引起防衛(wèi)反應(yīng)基因的過(guò)量表達(dá)。同時(shí)利用PVX表達(dá)載體對(duì)d32a-ra進(jìn)行表達(dá),發(fā)現(xiàn)攜帶pGR106/pd32a-ra的重組質(zhì)粒不能引起煙草葉片的壞死。這些實(shí)驗(yàn)結(jié)果初步確定了EG1的具體活性位點(diǎn)。本研究證實(shí)了R.solani第45家族糖苷水解酶β-1,4-內(nèi)切纖維素酶EG1發(fā)揮PAMP活性的關(guān)鍵區(qū)段,同時(shí)對(duì)其具體位點(diǎn)進(jìn)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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.131.4
本文編號(hào)：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.
【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.131.4
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 姜婷;蘇喬;安利佳;;多重脅迫下玉米實(shí)時(shí)定量PCR內(nèi)參基因的篩選與驗(yàn)證[J];植物生理學(xué)報(bào);2015年09期
2 李蘆江;陳文生;張敏;蘭海;潘光堂;楊克誠(chéng);;240份玉米自交系紋枯病抗性鑒定與評(píng)價(jià)[J];植物遺傳資源學(xué)報(bào);2014年05期
3 董章勇;王琪;秦世雯;王振中;;香蕉枯萎病菌1號(hào)和4號(hào)生理小種細(xì)胞壁降解酶的比較[J];植物病理學(xué)報(bào);2010年05期
4 楊俊品,唐海濤,楊家秀,李曉,陳德全,張彪,石永剛,黃宜祥;抗玉米紋枯病材料的鑒定及抗性遺傳研究[J];植物病理學(xué)報(bào);2005年02期
5 黃澤軍,黃榮峰,黃大f ;ERF轉(zhuǎn)錄因子及其在植物防衛(wèi)反應(yīng)中的作用[J];植物病理學(xué)報(bào);2004年03期
6 唐海濤,榮延昭,楊俊品;玉米紋枯病研究進(jìn)展[J];玉米科學(xué);2004年01期
7 陳其軍,肖玉梅,王學(xué)臣,周海夢(mèng);植物功能基因組研究中的基因敲除技術(shù)[J];植物生理學(xué)通訊;2004年01期
8 陳捷,劉艷舞,辛彥軍,高增貴,紀(jì)明山,薛春生,付波,陳剛,王作英,時(shí)俊光,牛小帆;生物農(nóng)藥防治玉米土傳性病害藥效的測(cè)定[J];沈陽(yáng)農(nóng)業(yè)大學(xué)學(xué)報(bào);2000年05期
9 陳捷,唐朝榮,高增貴,薛春生,牛小帆,宋佐衡;玉米紋枯病病菌侵染過(guò)程研究[J];沈陽(yáng)農(nóng)業(yè)大學(xué)學(xué)報(bào);2000年05期
10 陳捷;玉米紋枯病菌致病因子的研究[J];沈陽(yáng)農(nóng)業(yè)大學(xué)學(xué)報(bào);1999年03期
，本文編號(hào)：2358921
本文鏈接：http://sikaile.net/shoufeilunwen/zaizhiyanjiusheng/2358921.html
最近更新
教材專著
