茭白抗病基因Zl-RPM1和Zl-ADR的克隆及表达分析

发布时间：2018-06-17 19:00

本文选题：茭白 + 菰黑粉菌　；参考：《中国计量大学》2016年硕士论文

【摘要】：茭白肉质茎的形成是茭白植株与菰黑粉菌互作的结果。分析本实验室前期开展的茭白茎部发育中的蛋白质组发现:茎部膨大发育期间存在与抗病及胁迫响应相关蛋白的表达变化,推测茭白植株对菰黑粉菌侵染产生的防卫反应可能对维持茭白植株的生长发育及孕茭具有重要作用,相关基因的克隆及其在茭白孕茭中的表达模式分析将有助于探讨茭白的孕茭机制。本研究以双季茭白“龙茭2号”为试验材料,克隆获得了茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因的全长并进行了生物信息学分析,比较分析了三个基因在茭白茎部发育期间、不同表型及不同部位的表达变化;通过茭白茎部组织切片观察,比较分析了茭白3种表型茎部菰黑粉菌的分布特点,结合杀菌剂三唑酮处理对茭白菰黑粉菌生长分布的抑制作用,分析了三个抗病基因的表达变化与菰黑粉菌生长分布的关系,并通过菰黑粉菌与愈伤组织的体外互作培养对相关表达变化进行了验证分析。结果如下:一、茭白中菰黑粉菌分布的显微观察菰黑粉菌在不同表型茭白茎部的生长分布存在明显差异,正常茭的老茭茎部存在少量的菰黑粉菌孢子,与灰茭茎部中灰孢子类似。正常茭茎部膨大至长度为10 cm和15 cm时,茎部菰黑粉菌菌丝簇数量较多,但在茎部膨大至长度为20 cm时,茎部菰黑粉菌菌丝簇数量明显减少,可能与茭白茎部老化时茎部营养物质供应不足相关。二、茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因的克隆及序列分析通过RACE法克隆获得了茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR 3个基因的全长序列,同源性分析发现:3个基因均与水稻中抗病相关基因具有较高相似性,相似性分别为61%、79%和95%,均具有NBS-LRR类抗病基因的保守结构域CC、NBS、LRR等。其中Zl-RPM1.1 cDNA全长为1665 bp,含有一个1593 bp的开放阅读框,编码531个氨基酸,预测的分子量约为61.25 kDa,理论等电点为5.75;Zl-RPM1.2 cDNA全长为3480 bp,含有一个3195 bp的开放阅读框,编码1028个氨基酸,预测的分子量约为117.8 kDa,理论等电点为6.30;Zl-ADR cDNA全长为1164 bp,含有一个完整1161 bp的ORF,编码387个氨基酸,预测的分子量约为44.10 kDa,理论等电点为6.43。通过NCBI中的Spidey程序分析发现:Zl-RPM1.1和Zl-RPM1.2基因中没有内含子,但Zl-ADR基因含两个内含子,分别在其第629-718 bp、931-1315 bp之间包含一个大小为90 bp和185 bp的内含子。三、茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因在茭白孕茭中的表达模式分析正常茭白茎部发育期间3个抗病相关基因的响应时期不同:Zl-RPM1.1和Zl-RPM1.2基因的表达量显著上调时期主要在8叶期和茎部膨大至15 cm,而Zl-ADR因的表达量上调时期在8叶期,但在膨大至15 cm时表达量下调;从基因表达水平上表明8叶期可能是茭白防卫反应的重要时期,相关基因的表达变化可能与正常茭白茎部菰黑粉菌的生长分布及对茎部细胞的侵染相关。茭白中3个基因在不同部位的表达存在差异:正常茭白中Zl-RPM1.1在叶片表达量显著高于茎部;而Zl-RPM1.2和Zl-ADR在茎部的表达量均显著高于叶片;此外,3个基因在不同茭白表型间存在表达差异:膨大初期茎部中,Zl-RPM1.1和Zl-RPM1.2在灰茭中的表达量显著高于同时期的雄茭和正常茭茎部;而Zl-ADR基因在正常茭茎部中的表达量显著高于雄茭和灰茭茎部,可能与不同表型茭白植株中茎部菰黑粉菌的侵染能力及活体营养方式的变化相关。三唑酮喷施8叶期茭白植株后能够明显延迟茭白茎部的膨大时间(1-2周),并在一定范围内具有剂量效应;茎部组织切片观察发现:三唑酮能够明显抑制茎部菰黑粉菌菌丝的生长,茭白茎部的膨大与菰黑粉菌菌丝的生长分布密切相关;荧光定量PCR结果表明:低浓度(20 mg/L)的三唑酮喷施早期,能够促进茎部Zl-RPM1.1、Zl-RPM1.2和Zl-ADR的表达;但高浓度(80 mg/L)的三唑酮喷施能够明显抑制三个基因的表达。茭白茎部的膨大及三个抗病基因的表达变化与菰黑粉菌的侵染及生长密切相关。分析菰黑粉菌与茭白愈伤组织的体外互作结果发现:MT型菰黑粉菌和T型菰黑粉菌侵染茭白愈伤组织后均可诱导Zl-RPM1.1、Zl-RPM1.2和Zl-ADR的表达变化,T型菰黑粉菌侵染后三个抗病基因的响应早于MT型菰黑粉菌。病原菌(胡麻斑病菌和锈病菌)分别侵染茭白叶片后,Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因在叶片和茎部中的表达量与对照相比出现了显著下调。
[Abstract]:The formation of the fleshy stem of Zizania Zizania is the result of the interaction between the Zizania Zizania plant and the rhizome of Zizania Zizania. The cloning of the related genes and the analysis of the expression pattern in the Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania Zizania gestation mechanism. This study took the double cropping water bamboo "long Zizania 2" as the experimental material, cloned to obtain the full length of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR gene of Zizania Zizania. Bioinformatics analysis was used to compare and analyze the expression changes of three genes in the stem development of Zizania Zizania, different phenotypes and different parts. Through the observation of stem tissue section of Zizania Zizania, the distribution characteristics of Zizania Zizania in 3 phenotypic stems were compared and analyzed, and the growth and distribution of Zizania Zizania Zizania Zizania was inhibited by the treatment of three zizanone. The relationship between the changes of the expression of three disease resistance genes and the growth distribution of Zizania black fungus was analyzed, and the related expression changes were verified by the culture of the callus and the callus in vitro. The results are as follows: 1. The distribution of the wild rice in Zizania Zizania and the distribution of the growth and distribution of the fungus in the stem of different phenotypes. There was a small amount of Zizania spore spores in the old Zizania stem of normal Zizania Zizania, which was similar to the ash spore in the ash stem. The number of mycelium in the stem was more than that in the length of 10 cm and 15 cm, but the number of mycelium in the stem was significantly reduced when the stem expanded to 20 cm, and it might be with the Zizania Zizania. The stem nutrient supply was insufficient in the stem aging. Two, the cloning and sequence analysis of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR genes of Zizania Zizania were cloned by RACE method to obtain the full length sequence of 3 genes of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR in Zizania Zizania. The homology analysis showed that the 3 genes were all similar to the resistance related genes in rice. The similarity is 61%, 79% and 95%, respectively, which have the conservative domain CC, NBS, LRR, etc. of the NBS-LRR resistance gene. The Zl-RPM1.1 cDNA full length is 1665 BP, contains a 1593 BP open reading frame, encodes 531 amino acids, the predicted molecular weight is about 61.25 kDa, the theoretical isoelectric point is 5.75; Zl-RPM1.2 cDNA is 3480 BP, and contains a 3195 The open reading frame, which encodes 1028 amino acids, predicts the molecular weight of about 117.8 kDa, the theoretical isoelectric point is 6.30, the Zl-ADR cDNA is 1164 BP, contains a complete 1161 BP ORF, encodes 387 amino acids, and the predicted molecular weight is about 44.10 kDa, and the theoretical isoelectric point is found in 6.43. through Spidey program analysis in NCBI: Zl-RPM1.1 and Zl-RPM1.2 There is no intron in the gene, but the Zl-ADR gene contains two introns and contains an intron of 90 BP and 185 BP respectively in its 629-718 BP and 931-1315 BP. Three, the expression pattern of the Zizania Zizania, Zl-RPM1.2 and Zl-ADR gene in the Zizania Zizania Zizania Zizania, the response of 3 disease related genes during the development of the normal stem. The expression of Zl-RPM1.1 and Zl-RPM1.2 genes was significantly up-regulated during the 8 leaf period and the stem expansion to 15 cm, while the expression of Zl-ADR was up to the 8 leaf stage, but the expression decreased at 15 cm. From the gene expression level, the 8 leaf stage may be an important period for the defense response of Zizania Zizania, and the expression of related genes changes. The expression of the 3 genes in Zizania Zizania was significantly higher than that in the stem, while the expression of Zl-RPM1.1 in the leaves of the normal Zizania Zizania was significantly higher than that in the stem, while the expression of Zl-RPM1.2 and Zl-ADR in the stem was significantly higher than that in the leaves; in addition, the 3 genes were different. The expression difference was found in the phenotypes of Zizania Zizania: the expression of Zl-RPM1.1 and Zl-RPM1.2 in the stem was significantly higher than that of the male and normal stem of the same period, while the expression of Zl-ADR gene in the normal stem was significantly higher than that of the male and gray Zizania Zizania. Three Zizania Zizania can obviously delay the expansion time of the stem of Zizania Zizania (1-2 weeks) and have a dose effect in a certain range after spraying the 8 leaf stage of Zizania Zizania. It is found that three zizanone can obviously inhibit the growth of the mycelium of the stalks of Zizania Zizania, the expansion of the stem of Zizania Zizania and the mycelium of Zizania latifolia. The growth distribution of mycelium was closely related; the fluorescence quantitative PCR results showed that the expression of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR in the stem could be promoted by the early spraying of three zolone with low concentration (20 mg/L). But the high concentration (80 mg/L) of three zazolone spraying could obviously inhibit the expression of three genes. The expansion of stem and the expression of three resistance genes in the stem of Zizania Zizania It was closely related to the infection and growth of Zizania black fungus. The in vitro interaction of Zizania black fungus and Zizania Zizania callus found that the expression of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR could induce the changes of the expression of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR after the infection of the callus of Zizania Zizania, and the response of the three resistance genes of the T Zizania black fungus was earlier than that of the MT type Zizania Zizania. The expression of Zl-RPM1.1, Zl-RPM1.2 and Zl-ADR genes in the leaves and stems of the pathogens (Hu Maban and rust fungi) were significantly down regulated compared with the control.
【学位授予单位】：中国计量大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S645.2

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