本文主要研究内容
作者刘明明（2019）在《水稻类病斑半显性突变体als1的基因定位与互补验证》一文中研究指出：植物在长期的进化过程中已经具备了相对完善的防御系统来抵御病原菌的侵入与生长。植物病害影响植株的养分吸收、光合作用、生长发育等代谢活动,因而植物产量、品质也会受到影响。类病斑突变体往往能够增强植株对病原菌的抗性,这对于植物的育种以及抗病机制的研究极为重要。本研究经甲基磺酸乙酯（ethylmethane sulfonate,EMS）诱变水稻籼型三系恢复系缙恢10号,获得了一个叶片和叶鞘均产生类病斑的突变体als1（apoptosis leaf and sheath 1）,对als1突变体进行了表型分析、细胞学观察、防御反应相关基因的表达分析、组织化学分析、遗传分析、生理指标测定、基因的表达部位分析、光诱导分析、光合色素含量测定等研究,并利用分子标记对该突变体进行遗传连锁分析和精细定位,同时进行了候选基因的功能互补验证。本研究为水稻抗病机制的研究奠定了基础。主要研究结果如下:1突变体als1表型分析突变体als1在苗期表型与野生保持一致,从四叶期开始在倒四叶叶尖和叶鞘出现褐色的斑点,随着植株生长斑点数逐渐增多直至布满所有叶片及叶鞘,进入成熟期叶片逐渐从叶尖开始至叶片中上部呈现枯萎。在野生型作对照情况下,突变体als1的株高、一次枝梗数、二次枝梗数、穗长以及千粒重显著降低,另外,有效穗数、每穗粒数以及每穗实粒数都极显著降低,并且结实率极显著降低。2突变体als1的光诱导反应分蘖期,将野生型叶片和突变体als1未出现锈斑的叶片用锡箔纸进行遮光,观察锈斑的出现是否受光的诱导。结果显示,遮光处理的叶片未出现锈斑,再将野生型和als1叶片遮光部位恢复光照后,als1叶片锈斑重新产生,而野生型并无变化。3光合色素含量分析光合色素含量测定表明:在突变体中,类胡萝卜素（car）、总的叶绿素（Total Chl）、叶绿素a（Chl a）、叶绿素b（Chl b）与野生型相比,都表现出显著降低。4细胞学观察利用荧光显微镜观察叶片横切面,在白光下,野生型叶肉细胞呈现嫩绿色,在紫外光下,细胞排列整齐几乎没有胼胝质的积累,而突变体als1在白光下叶肉细胞呈现棕色,在紫外光下,细胞排列松弛破坏严重,并且有大量的胼胝质沉积。说明突变体中叶肉细胞的结构遭受破坏。5组织化学分析对分蘖期野生型和突变体als1的叶片进行二氨基联苯胺（3,3,-Diaminobenzidine,DAB）和台盼蓝染色。DAB染色结果显示,在突变体als1叶片锈斑部位及其周围有大量深褐色聚集物沉淀,而在野生型叶片几乎没有出现深褐色聚集物。台盼蓝染色后,突变体als1叶片锈斑部位及其周围呈现出深蓝色,野生型叶片表现均匀的浅蓝色分布。表明突变体als1锈斑的周围有大量活性氧（ROS）积累,并且als1叶片出现细胞死亡现象。6生理指标的测定与野生型相比,突变体als1中保护酶系统过氧化氢酶（CAT）、超氧化物歧化酶（SOD）、过氧化物酶（POD）的酶活分别在倒一叶、倒二叶以及倒三叶均呈现极显著降低。除H2O2在倒三叶中几乎没有上升,·OH、O2-在倒一叶、倒二叶以及倒三叶中均显著上升。同时,H2O2在倒一叶和倒二叶也明显上升。表明突变体als1存在大量活性氧的爆发。7防御反应相关基因表达据报道,类病斑突变体中植物的防御反应往往被激活。在野生型与突变体als1中,参与防御反应的相关基因定量表达分析表明:氧胁迫相关蛋白基因POX22.3和POC1、植物次级代谢产物途径相关蛋白基因OsPAL4、获得性免疫相关蛋白基因NH1都明显上调。在水稻的叶片和叶鞘中,病原相关蛋白基因PR1a、PR1b、PR2、PR10表达量均明显上调。表明在突变体als1中抗性反应被激活。8遗传分析利用EMS诱变缙恢10号得到稳定遗传的突变体als1,并与西大1A进行杂交,F1代与突变体als1表型一致,F2代发生性状分离,其中类病斑突变体植株2198株,正常植株688株。其遗传分离比符合孟德尔遗传定律3:1的比例。表明突变体als1受一对显性核基因控制。9基因定位及目的基因互补验证利用F2代群体中正常植株进行基因定位。最终将目的基因定位于第7染色体标记DD-1和DD2-1之间,物理物理距离242kb,此区间内有11个候选基因。通过基因克隆并测序,发现只有Os07g0488400的编码框发生了单碱基突变,导致编码氨基酸的改变。为了进一步确定候选基因Os07g0488400是否为我们的目的基因:ALS1。通过基因工程技术将在野生型中超量表达突变体Os07g0488400的编码框,得到的转基因T0代植株出现了突变体als1的表型,且基因测序突变位点为双峰。这一结果证实了Os07g0488400是目的基因ALS1。10 ALS1基因表达分析取突变体als1孕穗期根、茎、叶、鞘、穗,进行定量分析发现,ALS1基因主要在叶片和叶鞘中表达。另外,对不同病变程度的突变体叶片进行定量发现,病变程度越深ALS1表达水平越高。11稻瘟病抗性分析与激素测定利用HPLC法,测定野生型和突变体中总水杨酸（SA）和茉莉酸（JA）含量。与野生型相比,突变体als1中SA含量显著升高,JA含量极显著升高。说明ALS1基因参与SA和JA介导的防卫反应。为了进一步说明突变体als1对稻瘟病的抗性,在五叶期,对突变体als1和野生型均接种ZA31和ZB15两个稻瘟病菌。发现突变体als1对两种菌株均表现出抗病性,稻瘟病病斑总面积明显少于野生型。
Abstract
zhi wu zai chang ji de jin hua guo cheng zhong yi jing ju bei le xiang dui wan shan de fang yu ji tong lai di yu bing yuan jun de qin ru yu sheng chang 。zhi wu bing hai ying xiang zhi zhu de yang fen xi shou 、guang ge zuo yong 、sheng chang fa yo deng dai xie huo dong ,yin er zhi wu chan liang 、pin zhi ye hui shou dao ying xiang 。lei bing ban tu bian ti wang wang neng gou zeng jiang zhi zhu dui bing yuan jun de kang xing ,zhe dui yu zhi wu de yo chong yi ji kang bing ji zhi de yan jiu ji wei chong yao 。ben yan jiu jing jia ji huang suan yi zhi （ethylmethane sulfonate,EMS）you bian shui dao xian xing san ji hui fu ji jin hui 10hao ,huo de le yi ge xie pian he xie qiao jun chan sheng lei bing ban de tu bian ti als1（apoptosis leaf and sheath 1）,dui als1tu bian ti jin hang le biao xing fen xi 、xi bao xue guan cha 、fang yu fan ying xiang guan ji yin de biao da fen xi 、zu zhi hua xue fen xi 、wei chuan fen xi 、sheng li zhi biao ce ding 、ji yin de biao da bu wei fen xi 、guang you dao fen xi 、guang ge se su han liang ce ding deng yan jiu ,bing li yong fen zi biao ji dui gai tu bian ti jin hang wei chuan lian suo fen xi he jing xi ding wei ,tong shi jin hang le hou shua ji yin de gong neng hu bu yan zheng 。ben yan jiu wei shui dao kang bing ji zhi de yan jiu dian ding le ji chu 。zhu yao yan jiu jie guo ru xia :1tu bian ti als1biao xing fen xi tu bian ti als1zai miao ji biao xing yu ye sheng bao chi yi zhi ,cong si xie ji kai shi zai dao si xie xie jian he xie qiao chu xian he se de ban dian ,sui zhao zhi zhu sheng chang ban dian shu zhu jian zeng duo zhi zhi bu man suo you xie pian ji xie qiao ,jin ru cheng shou ji xie pian zhu jian cong xie jian kai shi zhi xie pian zhong shang bu cheng xian ku wei 。zai ye sheng xing zuo dui zhao qing kuang xia ,tu bian ti als1de zhu gao 、yi ci zhi geng shu 、er ci zhi geng shu 、sui chang yi ji qian li chong xian zhe jiang di ,ling wai ,you xiao sui shu 、mei sui li shu yi ji mei sui shi li shu dou ji xian zhe jiang di ,bing ju jie shi lv ji xian zhe jiang di 。2tu bian ti als1de guang you dao fan ying fen nie ji ,jiang ye sheng xing xie pian he tu bian ti als1wei chu xian xiu ban de xie pian yong xi bo zhi jin hang zhe guang ,guan cha xiu ban de chu xian shi fou shou guang de you dao 。jie guo xian shi ,zhe guang chu li de xie pian wei chu xian xiu ban ,zai jiang ye sheng xing he als1xie pian zhe guang bu wei hui fu guang zhao hou ,als1xie pian xiu ban chong xin chan sheng ,er ye sheng xing bing mo bian hua 。3guang ge se su han liang fen xi guang ge se su han liang ce ding biao ming :zai tu bian ti zhong ,lei hu luo bo su （car）、zong de xie lu su （Total Chl）、xie lu su a（Chl a）、xie lu su b（Chl b）yu ye sheng xing xiang bi ,dou biao xian chu xian zhe jiang di 。4xi bao xue guan cha li yong ying guang xian wei jing guan cha xie pian heng qie mian ,zai bai guang xia ,ye sheng xing xie rou xi bao cheng xian nen lu se ,zai zi wai guang xia ,xi bao pai lie zheng ji ji hu mei you pian zhi zhi de ji lei ,er tu bian ti als1zai bai guang xia xie rou xi bao cheng xian zong se ,zai zi wai guang xia ,xi bao pai lie song chi po huai yan chong ,bing ju you da liang de pian zhi zhi chen ji 。shui ming tu bian ti zhong xie rou xi bao de jie gou zao shou po huai 。5zu zhi hua xue fen xi dui fen nie ji ye sheng xing he tu bian ti als1de xie pian jin hang er an ji lian ben an （3,3,-Diaminobenzidine,DAB）he tai pan lan ran se 。DABran se jie guo xian shi ,zai tu bian ti als1xie pian xiu ban bu wei ji ji zhou wei you da liang shen he se ju ji wu chen dian ,er zai ye sheng xing xie pian ji hu mei you chu xian shen he se ju ji wu 。tai pan lan ran se hou ,tu bian ti als1xie pian xiu ban bu wei ji ji zhou wei cheng xian chu shen lan se ,ye sheng xing xie pian biao xian jun yun de jian lan se fen bu 。biao ming tu bian ti als1xiu ban de zhou wei you da liang huo xing yang （ROS）ji lei ,bing ju als1xie pian chu xian xi bao si wang xian xiang 。6sheng li zhi biao de ce ding yu ye sheng xing xiang bi ,tu bian ti als1zhong bao hu mei ji tong guo yang hua qing mei （CAT）、chao yang hua wu qi hua mei （SOD）、guo yang hua wu mei （POD）de mei huo fen bie zai dao yi xie 、dao er xie yi ji dao san xie jun cheng xian ji xian zhe jiang di 。chu H2O2zai dao san xie zhong ji hu mei you shang sheng ,·OH、O2-zai dao yi xie 、dao er xie yi ji dao san xie zhong jun xian zhe shang sheng 。tong shi ,H2O2zai dao yi xie he dao er xie ye ming xian shang sheng 。biao ming tu bian ti als1cun zai da liang huo xing yang de bao fa 。7fang yu fan ying xiang guan ji yin biao da ju bao dao ,lei bing ban tu bian ti zhong zhi wu de fang yu fan ying wang wang bei ji huo 。zai ye sheng xing yu tu bian ti als1zhong ,can yu fang yu fan ying de xiang guan ji yin ding liang biao da fen xi biao ming :yang xie pai xiang guan dan bai ji yin POX22.3he POC1、zhi wu ci ji dai xie chan wu tu jing xiang guan dan bai ji yin OsPAL4、huo de xing mian yi xiang guan dan bai ji yin NH1dou ming xian shang diao 。zai shui dao de xie pian he xie qiao zhong ,bing yuan xiang guan dan bai ji yin PR1a、PR1b、PR2、PR10biao da liang jun ming xian shang diao 。biao ming zai tu bian ti als1zhong kang xing fan ying bei ji huo 。8wei chuan fen xi li yong EMSyou bian jin hui 10hao de dao wen ding wei chuan de tu bian ti als1,bing yu xi da 1Ajin hang za jiao ,F1dai yu tu bian ti als1biao xing yi zhi ,F2dai fa sheng xing zhuang fen li ,ji zhong lei bing ban tu bian ti zhi zhu 2198zhu ,zheng chang zhi zhu 688zhu 。ji wei chuan fen li bi fu ge meng de er wei chuan ding lv 3:1de bi li 。biao ming tu bian ti als1shou yi dui xian xing he ji yin kong zhi 。9ji yin ding wei ji mu de ji yin hu bu yan zheng li yong F2dai qun ti zhong zheng chang zhi zhu jin hang ji yin ding wei 。zui zhong jiang mu de ji yin ding wei yu di 7ran se ti biao ji DD-1he DD2-1zhi jian ,wu li wu li ju li 242kb,ci ou jian nei you 11ge hou shua ji yin 。tong guo ji yin ke long bing ce xu ,fa xian zhi you Os07g0488400de bian ma kuang fa sheng le chan jian ji tu bian ,dao zhi bian ma an ji suan de gai bian 。wei le jin yi bu que ding hou shua ji yin Os07g0488400shi fou wei wo men de mu de ji yin :ALS1。tong guo ji yin gong cheng ji shu jiang zai ye sheng xing zhong chao liang biao da tu bian ti Os07g0488400de bian ma kuang ,de dao de zhuai ji yin T0dai zhi zhu chu xian le tu bian ti als1de biao xing ,ju ji yin ce xu tu bian wei dian wei shuang feng 。zhe yi jie guo zheng shi le Os07g0488400shi mu de ji yin ALS1。10 ALS1ji yin biao da fen xi qu tu bian ti als1yun sui ji gen 、jing 、xie 、qiao 、sui ,jin hang ding liang fen xi fa xian ,ALS1ji yin zhu yao zai xie pian he xie qiao zhong biao da 。ling wai ,dui bu tong bing bian cheng du de tu bian ti xie pian jin hang ding liang fa xian ,bing bian cheng du yue shen ALS1biao da shui ping yue gao 。11dao wen bing kang xing fen xi yu ji su ce ding li yong HPLCfa ,ce ding ye sheng xing he tu bian ti zhong zong shui yang suan （SA）he mo li suan （JA）han liang 。yu ye sheng xing xiang bi ,tu bian ti als1zhong SAhan liang xian zhe sheng gao ,JAhan liang ji xian zhe sheng gao 。shui ming ALS1ji yin can yu SAhe JAjie dao de fang wei fan ying 。wei le jin yi bu shui ming tu bian ti als1dui dao wen bing de kang xing ,zai wu xie ji ,dui tu bian ti als1he ye sheng xing jun jie chong ZA31he ZB15liang ge dao wen bing jun 。fa xian tu bian ti als1dui liang chong jun zhu jun biao xian chu kang bing xing ,dao wen bing bing ban zong mian ji ming xian shao yu ye sheng xing 。
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论文作者分别是来自西南大学的刘明明，发表于刊物西南大学2019-09-24论文，是一篇关于水稻论文,类病斑论文,基因定位论文,互补验证论文,西南大学2019-09-24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，资料来自西南大学2019-09-24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：水稻论文; 类病斑论文; 基因定位论文; 互补验证论文; 西南大学2019-09-24论文;