本文選題：茭白 + 菰黑粉菌　； 參考：《中國計(jì)量大學(xué)》2016年碩士論文

【摘要】：茭白肉質(zhì)莖的形成是茭白植株與菰黑粉菌互作的結(jié)果。分析本實(shí)驗(yàn)室前期開展的茭白莖部發(fā)育中的蛋白質(zhì)組發(fā)現(xiàn):莖部膨大發(fā)育期間存在與抗病及脅迫響應(yīng)相關(guān)蛋白的表達(dá)變化,推測茭白植株對菰黑粉菌侵染產(chǎn)生的防衛(wèi)反應(yīng)可能對維持茭白植株的生長發(fā)育及孕茭具有重要作用,相關(guān)基因的克隆及其在茭白孕茭中的表達(dá)模式分析將有助于探討茭白的孕茭機(jī)制。本研究以雙季茭白“龍茭2號”為試驗(yàn)材料,克隆獲得了茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因的全長并進(jìn)行了生物信息學(xué)分析,比較分析了三個基因在茭白莖部發(fā)育期間、不同表型及不同部位的表達(dá)變化;通過茭白莖部組織切片觀察,比較分析了茭白3種表型莖部菰黑粉菌的分布特點(diǎn),結(jié)合殺菌劑三唑酮處理對茭白菰黑粉菌生長分布的抑制作用,分析了三個抗病基因的表達(dá)變化與菰黑粉菌生長分布的關(guān)系,并通過菰黑粉菌與愈傷組織的體外互作培養(yǎng)對相關(guān)表達(dá)變化進(jìn)行了驗(yàn)證分析。結(jié)果如下:一、茭白中菰黑粉菌分布的顯微觀察菰黑粉菌在不同表型茭白莖部的生長分布存在明顯差異,正常茭的老茭莖部存在少量的菰黑粉菌孢子,與灰茭莖部中灰孢子類似。正常茭莖部膨大至長度為10 cm和15 cm時,莖部菰黑粉菌菌絲簇?cái)?shù)量較多,但在莖部膨大至長度為20 cm時,莖部菰黑粉菌菌絲簇?cái)?shù)量明顯減少,可能與茭白莖部老化時莖部營養(yǎng)物質(zhì)供應(yīng)不足相關(guān)。二、茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因的克隆及序列分析通過RACE法克隆獲得了茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR 3個基因的全長序列,同源性分析發(fā)現(xiàn):3個基因均與水稻中抗病相關(guān)基因具有較高相似性,相似性分別為61%、79%和95%,均具有NBS-LRR類抗病基因的保守結(jié)構(gòu)域CC、NBS、LRR等。其中Zl-RPM1.1 cDNA全長為1665 bp,含有一個1593 bp的開放閱讀框,編碼531個氨基酸,預(yù)測的分子量約為61.25 kDa,理論等電點(diǎn)為5.75;Zl-RPM1.2 cDNA全長為3480 bp,含有一個3195 bp的開放閱讀框,編碼1028個氨基酸,預(yù)測的分子量約為117.8 kDa,理論等電點(diǎn)為6.30;Zl-ADR cDNA全長為1164 bp,含有一個完整1161 bp的ORF,編碼387個氨基酸,預(yù)測的分子量約為44.10 kDa,理論等電點(diǎn)為6.43。通過NCBI中的Spidey程序分析發(fā)現(xiàn):Zl-RPM1.1和Zl-RPM1.2基因中沒有內(nèi)含子,但Zl-ADR基因含兩個內(nèi)含子,分別在其第629-718 bp、931-1315 bp之間包含一個大小為90 bp和185 bp的內(nèi)含子。三、茭白Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因在茭白孕茭中的表達(dá)模式分析正常茭白莖部發(fā)育期間3個抗病相關(guān)基因的響應(yīng)時期不同:Zl-RPM1.1和Zl-RPM1.2基因的表達(dá)量顯著上調(diào)時期主要在8葉期和莖部膨大至15 cm,而Zl-ADR因的表達(dá)量上調(diào)時期在8葉期,但在膨大至15 cm時表達(dá)量下調(diào);從基因表達(dá)水平上表明8葉期可能是茭白防衛(wèi)反應(yīng)的重要時期,相關(guān)基因的表達(dá)變化可能與正常茭白莖部菰黑粉菌的生長分布及對莖部細(xì)胞的侵染相關(guān)。茭白中3個基因在不同部位的表達(dá)存在差異:正常茭白中Zl-RPM1.1在葉片表達(dá)量顯著高于莖部;而Zl-RPM1.2和Zl-ADR在莖部的表達(dá)量均顯著高于葉片;此外,3個基因在不同茭白表型間存在表達(dá)差異:膨大初期莖部中,Zl-RPM1.1和Zl-RPM1.2在灰茭中的表達(dá)量顯著高于同時期的雄茭和正常茭莖部;而Zl-ADR基因在正常茭莖部中的表達(dá)量顯著高于雄茭和灰茭莖部,可能與不同表型茭白植株中莖部菰黑粉菌的侵染能力及活體營養(yǎng)方式的變化相關(guān)。三唑酮噴施8葉期茭白植株后能夠明顯延遲茭白莖部的膨大時間(1-2周),并在一定范圍內(nèi)具有劑量效應(yīng);莖部組織切片觀察發(fā)現(xiàn):三唑酮能夠明顯抑制莖部菰黑粉菌菌絲的生長,茭白莖部的膨大與菰黑粉菌菌絲的生長分布密切相關(guān);熒光定量PCR結(jié)果表明:低濃度(20 mg/L)的三唑酮噴施早期,能夠促進(jìn)莖部Zl-RPM1.1、Zl-RPM1.2和Zl-ADR的表達(dá);但高濃度(80 mg/L)的三唑酮噴施能夠明顯抑制三個基因的表達(dá)。茭白莖部的膨大及三個抗病基因的表達(dá)變化與菰黑粉菌的侵染及生長密切相關(guān)。分析菰黑粉菌與茭白愈傷組織的體外互作結(jié)果發(fā)現(xiàn):MT型菰黑粉菌和T型菰黑粉菌侵染茭白愈傷組織后均可誘導(dǎo)Zl-RPM1.1、Zl-RPM1.2和Zl-ADR的表達(dá)變化,T型菰黑粉菌侵染后三個抗病基因的響應(yīng)早于MT型菰黑粉菌。病原菌(胡麻斑病菌和銹病菌)分別侵染茭白葉片后,Zl-RPM1.1、Zl-RPM1.2和Zl-ADR基因在葉片和莖部中的表達(dá)量與對照相比出現(xiàn)了顯著下調(diào)。
[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.
【學(xué)位授予單位】：中國計(jì)量大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S645.2

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1 李帥;茭白抗病基因Zl-RPM1和Zl-ADR的克隆及表達(dá)分析[D];中國計(jì)量大學(xué);2016年

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本文編號：2032117