本文主要研究内容
作者薛媛（2019）在《拟南芥SNARE蛋白VAMP711通过调控质膜H~+-ATPase响应干旱胁迫的机制研究》一文中研究指出：干旱胁迫是植物生长发育过程中遇到的主要非生物胁迫之一,它严重抑制植物的生长和作物的产量。植物激素脱落酸(Abscisic Acid,ABA)在植物干旱胁迫响应过程中具有非常重要的作用。研究表明,干旱胁迫来临时,植物可以通过ABA信号转导途径抑制质膜H+-ATPase活性,从而促进气孔的关闭以防止体内过多的水分损耗。质膜H+-ATPase持续激活的突变体ost2-2D(H+-ATPase家族成员AHA1基因的突变)具有明显的干旱胁迫敏感表型。表明质膜H+-ATPase活性在响应干旱胁迫方面具有非常重要的作用。但是目前ABA调节质膜H+-ATPase活性的机制还不是很清楚。为了寻找参与ABA通过质膜H+-ATPase促进气孔关闭过程中的调控因子,本研究利用ABA处理的稳定表达AHA1的转基因材料,通过免疫共沉淀(Co-immunoprecipitation,Co-IP)和液相色谱-串联质谱(Liquid Chromatography-tandem Mass Spectrometry,LC-MS/MS)的方法筛选到与 AHA 1互作的蛋白 VAMP711(Vesicle-Associated Membrane Protein711)。拟南芥VAMP711 属于 SNARE(Soluble NSF-Attachment Protein Receptor)蛋白家族,在介导囊泡与液泡的融合过程具有重要功能。通过荧光素酶互补成像(Firefly Luciferase Complementation Imaging,LCI)实验发现,VAMP711 与 AHA1 之间的互作受 ABA 诱导。双分子荧光互补(Bimolecular Fluorescence Complementation,BiFC)实验,发现VAMP711与AHA1的互作部分存在于细胞膜上。酵母双杂交(Yeast Two-hybrid)实验表明,AHA1/AHA2的C端887至897之间的11个氨基酸残基对VAMP711与AHA1/AHA2的互作是必需的。拟南芥质膜H+-ATPase活性分析发现,VAMP711基因的缺失会造成质膜H+-ATPase活性的升高;在体外,原核表达的VAMP711蛋白能够直接抑制质膜H+-ATPase活性;酵母系统中,VAMP711能够抑制依赖拟南芥质膜H+-ATPase AHA2活性的酵母在葡萄糖培养基上生长。这些结果表明,植物VAMP711为质膜H+-ATPase的负调控因子。为了进一步探究植物中VAMP711通过质膜H+-ATPase响应干旱胁迫的分子机制,本研究检测了 VAMP711对质膜H+-ATPase在细胞膜上蛋白量的影响。蛋白免疫印迹(Western Blotting,WB)实验结果表明,VAMP711不影响质膜H+-ATPase在细胞膜上的含量。对GFP-VAMP711的亚细胞定位分析发现,在ABA处理下VAMP711向细胞膜靠近;非损伤微测实验(Non-invasive Micro-test Technique,NMT)和质膜H+-ATPase活性实验结果表明,ABA虽然对野生型和vamp711的质膜H+-ATPase活性都有抑制,但是对vamp711的抑制程度低于野生型,表明ABA对质膜H+-ATPase的活性的抑制部分由VAMP711介导。在拟南芥ost2-2D突变体中过表达VAMP711能够部分的抑制ost2-2D的干旱胁迫敏感表型,表明在拟南芥中VAMP711通过抑制质膜H+-ATPase的活性响应干旱胁迫。综上所述,本研究发现拟南芥SNARE蛋白VAMP711参与调节ABA介导的质膜H+-ATPase活性抑制以促进气孔关闭过程。在干旱胁迫下,植物中ABA的积累促进VAMP711与AHA1/AHA2在细胞膜上互作,抑制了质膜H+-ATPase的活性,从而促进气孔关闭,减少植物体内水分的损耗,提高植物的耐旱性。
Abstract
gan han xie pai shi zhi wu sheng chang fa yo guo cheng zhong yu dao de zhu yao fei sheng wu xie pai zhi yi ,ta yan chong yi zhi zhi wu de sheng chang he zuo wu de chan liang 。zhi wu ji su tuo la suan (Abscisic Acid,ABA)zai zhi wu gan han xie pai xiang ying guo cheng zhong ju you fei chang chong yao de zuo yong 。yan jiu biao ming ,gan han xie pai lai lin shi ,zhi wu ke yi tong guo ABAxin hao zhuai dao tu jing yi zhi zhi mo H+-ATPasehuo xing ,cong er cu jin qi kong de guan bi yi fang zhi ti nei guo duo de shui fen sun hao 。zhi mo H+-ATPasechi xu ji huo de tu bian ti ost2-2D(H+-ATPasejia zu cheng yuan AHA1ji yin de tu bian )ju you ming xian de gan han xie pai min gan biao xing 。biao ming zhi mo H+-ATPasehuo xing zai xiang ying gan han xie pai fang mian ju you fei chang chong yao de zuo yong 。dan shi mu qian ABAdiao jie zhi mo H+-ATPasehuo xing de ji zhi hai bu shi hen qing chu 。wei le xun zhao can yu ABAtong guo zhi mo H+-ATPasecu jin qi kong guan bi guo cheng zhong de diao kong yin zi ,ben yan jiu li yong ABAchu li de wen ding biao da AHA1de zhuai ji yin cai liao ,tong guo mian yi gong chen dian (Co-immunoprecipitation,Co-IP)he ye xiang se pu -chuan lian zhi pu (Liquid Chromatography-tandem Mass Spectrometry,LC-MS/MS)de fang fa shai shua dao yu AHA 1hu zuo de dan bai VAMP711(Vesicle-Associated Membrane Protein711)。ni na gai VAMP711 shu yu SNARE(Soluble NSF-Attachment Protein Receptor)dan bai jia zu ,zai jie dao nang pao yu ye pao de rong ge guo cheng ju you chong yao gong neng 。tong guo ying guang su mei hu bu cheng xiang (Firefly Luciferase Complementation Imaging,LCI)shi yan fa xian ,VAMP711 yu AHA1 zhi jian de hu zuo shou ABA you dao 。shuang fen zi ying guang hu bu (Bimolecular Fluorescence Complementation,BiFC)shi yan ,fa xian VAMP711yu AHA1de hu zuo bu fen cun zai yu xi bao mo shang 。jiao mu shuang za jiao (Yeast Two-hybrid)shi yan biao ming ,AHA1/AHA2de Cduan 887zhi 897zhi jian de 11ge an ji suan can ji dui VAMP711yu AHA1/AHA2de hu zuo shi bi xu de 。ni na gai zhi mo H+-ATPasehuo xing fen xi fa xian ,VAMP711ji yin de que shi hui zao cheng zhi mo H+-ATPasehuo xing de sheng gao ;zai ti wai ,yuan he biao da de VAMP711dan bai neng gou zhi jie yi zhi zhi mo H+-ATPasehuo xing ;jiao mu ji tong zhong ,VAMP711neng gou yi zhi yi lai ni na gai zhi mo H+-ATPase AHA2huo xing de jiao mu zai pu tao tang pei yang ji shang sheng chang 。zhe xie jie guo biao ming ,zhi wu VAMP711wei zhi mo H+-ATPasede fu diao kong yin zi 。wei le jin yi bu tan jiu zhi wu zhong VAMP711tong guo zhi mo H+-ATPasexiang ying gan han xie pai de fen zi ji zhi ,ben yan jiu jian ce le VAMP711dui zhi mo H+-ATPasezai xi bao mo shang dan bai liang de ying xiang 。dan bai mian yi yin ji (Western Blotting,WB)shi yan jie guo biao ming ,VAMP711bu ying xiang zhi mo H+-ATPasezai xi bao mo shang de han liang 。dui GFP-VAMP711de ya xi bao ding wei fen xi fa xian ,zai ABAchu li xia VAMP711xiang xi bao mo kao jin ;fei sun shang wei ce shi yan (Non-invasive Micro-test Technique,NMT)he zhi mo H+-ATPasehuo xing shi yan jie guo biao ming ,ABAsui ran dui ye sheng xing he vamp711de zhi mo H+-ATPasehuo xing dou you yi zhi ,dan shi dui vamp711de yi zhi cheng du di yu ye sheng xing ,biao ming ABAdui zhi mo H+-ATPasede huo xing de yi zhi bu fen you VAMP711jie dao 。zai ni na gai ost2-2Dtu bian ti zhong guo biao da VAMP711neng gou bu fen de yi zhi ost2-2Dde gan han xie pai min gan biao xing ,biao ming zai ni na gai zhong VAMP711tong guo yi zhi zhi mo H+-ATPasede huo xing xiang ying gan han xie pai 。zeng shang suo shu ,ben yan jiu fa xian ni na gai SNAREdan bai VAMP711can yu diao jie ABAjie dao de zhi mo H+-ATPasehuo xing yi zhi yi cu jin qi kong guan bi guo cheng 。zai gan han xie pai xia ,zhi wu zhong ABAde ji lei cu jin VAMP711yu AHA1/AHA2zai xi bao mo shang hu zuo ,yi zhi le zhi mo H+-ATPasede huo xing ,cong er cu jin qi kong guan bi ,jian shao zhi wu ti nei shui fen de sun hao ,di gao zhi wu de nai han xing 。
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论文作者分别是来自中国农业大学的薛媛，发表于刊物中国农业大学2019-01-17论文，是一篇关于拟南芥论文,质膜论文,干旱胁迫响应论文,中国农业大学2019-01-17论文的文章。本文可供学术参考使用，各位学者可以免费参考阅读下载，文章观点不代表本站观点，资料来自中国农业大学2019-01-17论文网站，若本站收录的文献无意侵犯了您的著作版权，请联系我们删除。
标签：拟南芥论文; 质膜论文; 干旱胁迫响应论文; 中国农业大学2019-01-17论文;