本文選題：玉米 + 赤霉素�。� 參考：《四川農(nóng)業(yè)大學(xué)》2016年碩士論文

【摘要】：玉米(Zea mays L.)是重要糧食作物,但病蟲害導(dǎo)致其產(chǎn)量和品質(zhì)下降,威脅糧食安全。植保素是植物受病害真菌侵染產(chǎn)生的小分子次生代謝化合物。Kauralexins是玉米中首次發(fā)現(xiàn)的一種二萜植保素,具有良好的抗病原真菌活性,且對玉米螟幼蟲有拒食作用。Kauralexin具有(異)貝殼杉烯骨架,其生物合成途徑是由柯巴基焦磷酸合酶CPS和(異)貝殼杉烯合酶KS(L)連續(xù)催化前體GGPP形成貝殼杉烯或異貝殼杉烯,進(jìn)一步由細(xì)胞色素P450氧化酶氧化修飾形成。參與kauralexin生物合成的CPS已經(jīng)被鑒定為An2,但其貝殼杉烯合酶還未鑒定。另外植物激素赤霉素的生物合成也需要貝殼杉烯合酶的參與,有報(bào)道稱玉米矮化突變體彷是由于貝殼杉烯合酶基因突變導(dǎo)致赤霉素合成受阻而產(chǎn)生的矮化,但相關(guān)基因還未被鑒定。并且到目前為止,還未有報(bào)道鑒定玉米貝殼杉烯合酶基因,這限制了玉米植保素kauralexin的相關(guān)研究及其應(yīng)用。同時,植保素積累與其代謝關(guān)鍵基因表達(dá)密切相關(guān),分析kauralexin代謝關(guān)鍵基因An2的啟動子活性并鑒定其順式元件,能為kauralexin的代謝調(diào)控研究奠定基礎(chǔ)。本研究首先在玉米全基因組中搜索貝殼杉烯合酶,并通過系統(tǒng)進(jìn)化對其進(jìn)行功能預(yù)測。其中ZmKSL3、ZmKSL5和ZmTPS1與禾本科植物的貝殼杉烯合酶具有較近的進(jìn)化關(guān)系,即作為候選基因進(jìn)行功能研究。通過大腸桿菌重組表達(dá)和代謝工程分析,并結(jié)合本氏煙瞬時表達(dá),對產(chǎn)物進(jìn)行GC-MS檢測確定其生化功能,即催化ent-CPP形成貝殼杉烯。進(jìn)一步通過亞細(xì)胞定位分析確認(rèn)其功能。同時分析了這三個基因在病原菌侵染和激素處理下的表達(dá)模式,并結(jié)合玉米d5矮化突變體確定了這三個基因的生物學(xué)功能。另外,還利用基因槍瞬時表達(dá)體系,分析了An2基因啟動子的活性區(qū)域。主要結(jié)果如下：1.對從玉米基因組中搜索出的貝殼杉烯合酶基因,通過氨基酸序列比對,與其他禾本科植物的KS(L)s等進(jìn)行系統(tǒng)進(jìn)化分析,發(fā)現(xiàn)ZmKSL3與多種植物中參與赤霉素合成的KS的親緣關(guān)系十分相近,而ZmKSL5與ZmTPS1除了本身相近之外,還與小麥中的TaKSL5-1/2相近,它們都不含有γ結(jié)構(gòu)域。進(jìn)一步分析發(fā)現(xiàn)ZmKSL3、 ZmKSL5和ZmTPS1在玉米二號染色體上呈現(xiàn)串聯(lián)排列,預(yù)示著這三個基因在進(jìn)化上可能是串聯(lián)復(fù)制而來。2.在大腸桿菌重組表達(dá)這三個基因并通過代謝工程的方法與玉米中的ent-CPP合酶An2共表達(dá),GC-MS檢測產(chǎn)物發(fā)現(xiàn)ZmKSL3、ZmKSL5和ZmTPS1都能催化ent-CPP生成貝殼杉烯(ent-kaurene)。前人研究認(rèn)為ZmTPS1為倍半萜合酶,本研究通過分析發(fā)現(xiàn)ZmTPS1確實(shí)具有一定的倍半萜合酶活性,能催化FPP產(chǎn)生一系列倍半萜,而ZmKSL3和ZmKSL5則不具有該功能。但進(jìn)一步在本氏煙草中瞬時表達(dá)這三個基因,結(jié)果證明它們在植物體內(nèi)也能產(chǎn)生貝殼杉烯。亞細(xì)胞定位分析顯示,這三個蛋白都定位與質(zhì)體中,符合其貝殼杉烯合酶的功能。因此結(jié)合生物化功能鑒定和亞細(xì)胞定位分析確定這三個基因均編碼貝殼杉烯合酶。3.對玉米d5突變體進(jìn)行半定量PCR分析發(fā)現(xiàn)這三個基因中只有ZmKSLS在d5突變體中不表達(dá),并且以往轉(zhuǎn)錄組數(shù)據(jù)顯示其在生長旺盛的區(qū)域有高表達(dá),表明ZmKSL3參與赤霉素生物合成。而對該突變體中ZmKSL3基因組序列分析,發(fā)現(xiàn)與其野生型母本和B73自交系中的序列相比,突變體中的ZmKSL3在5’-UTR部分起始密碼子之前缺失了100bp的片段,其中包含與轉(zhuǎn)錄相關(guān)的CAAT元件,這可能就是引起其不表達(dá)的原因。而ZmTPS1或ZmKSL5的突變體均并未出現(xiàn)矮化的表現(xiàn)型,表明不參與赤霉素生物合成。前人研究發(fā)現(xiàn)d5突變體中仍然可檢測到少量的貝殼杉烯,對d5突變體中ZmKSLS(?)ZmTPS1序列進(jìn)行分析未發(fā)現(xiàn)明顯突變,進(jìn)一步進(jìn)行體外生化功能分析發(fā)現(xiàn)這兩個基因的d5克隆仍然具有貝殼杉烯合酶功能,但可能具有嚴(yán)格的調(diào)控機(jī)制使其不能對d5突變進(jìn)行補(bǔ)償。4.利用熒光定量PCR,分析了ZmKSL3、 ZmKSL5和ZmTPS1在不同處理下的表達(dá)情況,并與kauralexin代謝關(guān)鍵基因An2進(jìn)行比較。研究表明,ZmKSL3在禾谷鐮刀菌(Fusarium graminearum)孢子侵染的葉片和ABA處理的根部有少量誘導(dǎo),這可能與其啟動子中所含有的相關(guān)元件有關(guān)。而ZmKSL5和ZmT PS1在禾谷鐮刀菌孢子侵染的葉片、ABA處理的地下部分、MeJA和ETH聯(lián)合處理的葉片和MeJA單獨(dú)處理的葉片中都明顯上調(diào),并且與An2在這四種處理中的表現(xiàn)呈現(xiàn)正相關(guān)。對玉米生長發(fā)育各時期的轉(zhuǎn)錄組數(shù)據(jù)分析發(fā)現(xiàn)ZmTPS1和ZmKSL5與An2之間也有相似的表達(dá)模式,而ZmKSL3則不同,這表明ZmTPS1與ZmKSL5參與植保素kauralexin的合成。5.通過在線預(yù)測發(fā)現(xiàn)An2啟動子中含有2個W-box,可能與其參與植保素kauralexin的誘導(dǎo)表達(dá)有關(guān)。利用基因槍轟擊介導(dǎo)的瞬時轉(zhuǎn)化體系,在玉米愈傷組織中進(jìn)行了An2基因啟動子活性的分析,結(jié)果發(fā)現(xiàn),不含有第一個W-box元件的啟動子片段A2F2與突變掉第一個W-box元件的A2F1M都無法檢測的啟動子活性,而含有第一個元件的A2F1則能檢測到活性。這證明能夠介導(dǎo)An2啟動子的關(guān)鍵順式元件為其遠(yuǎn)離起始密碼子的第一個-box元件,這為研究kaurlexins代謝調(diào)控機(jī)制提供了基礎(chǔ)。結(jié)論：本研究鑒定了三個玉米貝殼杉烯合酶基因,其中一個參與赤霉素生物合成,為玉米矮化突變體d5的突變基因,而另外兩個能夠被病原菌侵染和激素處理所誘導(dǎo),可能參與植保素kauralexin生物合成,同時這三個基因在染色體上形成串聯(lián)排列,應(yīng)起源為基因復(fù)制,之后發(fā)生功能異化。對kauralexin代謝關(guān)鍵酶基因An2以啟動子活性進(jìn)行了分析,確定了其關(guān)鍵順式元件W-box。本研究鑒定的An2以啟動子順式元件為后續(xù)玉米植保素kauralexin的代謝調(diào)控研究提供了基礎(chǔ)。
[Abstract]:Maize (Zea mays L.) is an important grain crop, but disease and insect pests cause its yield and quality to decrease and threaten food safety. The plant protection element is a small molecular secondary metabolite of plant infected by plant diseases,.Kauralexins is a kind of two terpenoids found in maize for the first time. It has good antifungal activity and is young to corn borer. The insect antifeedant.Kauralexin has the cytoskeleton of (hetero) beetle, its biosynthesis pathway is the continuous catalysis of cochbyl pyrophosphate synthase CPS and (ISO) beetle synthase KS (L) to catalyze the formation of beetle or ISO beetene, which is further oxidized by cytochrome P450 oxidizing enzyme, and participates in the CPS of kauralexin biosynthesis. The biosynthesis of the plant hormone gibberellin also needs to participate in the biosynthesis of the plant hormone gibberellin, and it is reported that the maize dwarf mutant seems to be dwarf caused by the inhibition of the synthesis of gibberellin by the mutation of the An2, but the related genes have not been identified. So far, there has not been a report on the identification of the maize beta - tetalene synthase gene, which restricts the related research and application of kauralexin. At the same time, the accumulation of plant protection hormone is closely related to the expression of key genes of metabolism. It analyses the promoter activity of the key gene of kauralexin metabolism and identification of its cis element, which can be used for the metabolism of kauralexin. In this study, we first search for the whole genome of Maize to search for the beta - chitosan synthase and predict its function through phylogenetic evolution. ZmKSL3, ZmKSL5 and ZmTPS1 have a close relationship with the phylogeny of the gramineous plants, that is, the functional study of the candidate genes. And metabolic engineering analysis, combined with the instantaneous expression of Benedict smoke, GC-MS detection of the product to determine its biochemical function, that is, catalyzing ent-CPP to form beta fume. Further by subcellular location analysis to confirm its function. At the same time, the expression patterns of the three genes in the pathogen infection and hormone location are analyzed, and the D5 dwarf mutation of maize is combined. The body has identified the biological functions of the three genes. In addition, the active region of the An2 gene promoter is analyzed by the instantaneous expression system of the gene gun. The main results are as follows: 1. the phyllogenase gene of the maize genome, through the alignment of amino acid sequences, was systematically evolved with the KS (L) s of other gramineous plants. It was found that ZmKSL3 was closely related to KS in a variety of plants that were involved in gibberellin synthesis, while ZmKSL5 and ZmTPS1 were similar to TaKSL5-1/2 in wheat, and they all did not contain gamma domain. Further analysis found that ZmKSL3, ZmKSL5 and ZmTPS1 were arranged in series on chromosome two. These three genes may have evolved in tandem to reproduce the three genes in Escherichia coli and co express the ent-CPP synthase An2 in Maize by metabolic engineering. The GC-MS detection products found that ZmKSL3, ZmKSL5 and ZmTPS1 can catalyze the formation of ent-CPP (ent-kaurene). The previous study considered ZmTPS1 to be half a half. Terpene synthase, in this study, found that ZmTPS1 did have a certain number of sesquiterpene synthase activity, which can catalyze FPP production of a series of sesquiterpene, while ZmKSL3 and ZmKSL5 do not have the function. However, these three genes are expressed instantaneously in benotiana tobacco, and the results show that they can also produce pteroterene. Subcellular location within the plant. The analysis showed that the three proteins were located in the plastids and in conformity with the function of its beta contienase. Therefore, the three genes were identified by biologic functional identification and subcellular localization analysis, and all of the three genes were encoded by the beta conetene synthase.3. for semi quantitative PCR analysis of maize mutants, and only ZmKSLS was not expressed in the D5 mutants, And the previous transcriptional data showed that it was highly expressed in the growing region, indicating that ZmKSL3 was involved in gibberellin biosynthesis, and that the ZmKSL3 genome sequence analysis of the mutant found that the ZmKSL3 in the mutant was missing 100bp before the 5 '-UTR partial starting codon compared with the sequence in the wild type mother and the B73 inbred line. The fragment, which contains the CAAT element associated with the transcription, may be the cause of its non expression, and the mutant of ZmTPS1 or ZmKSL5 does not appear to be dwarf phenotype, indicating that it does not participate in gibberellin biosynthesis. Previous studies have found that a small amount of beta fir still can be detected in the D5 mutants, and ZmKSLS (?) ZmTPS1 sequence in the D5 mutant. No obvious mutation was found in the column analysis. Further biochemical analysis in vitro found that the D5 clones of the two genes still had the function of the beta clenpene synthase, but it might have a strict regulatory mechanism that could not compensate the D5 mutation by.4. using fluorescence quantitative PCR, and analyzed the tables of ZmKSL3, ZmKSL5 and ZmTPS1 under different treatments. The results were compared with An2, a key gene of kauralexin metabolism. The study showed that a small amount of ZmKSL3 was induced in the leaves of the spores infected by Fusarium graminearum (Fusarium graminearum) and the root of ABA treatment, which may be related to the related components contained in the promoter. The ZmKSL5 and ZmT PS1 were infected with the Fusarium graminearum spores, ABA, and ABA. The MeJA and ETH combined leaves and the leaves treated by the MeJA and ETH were significantly up-regulated in the leaves treated separately, and there was a positive correlation with the performance of An2 in these four treatments. The synthesis.5. of ZmTPS1 and ZmKSL5 involved in the synthetic.5. of BF kauralexin found that the An2 promoter contains 2 W-box in the An2 promoter, which may be related to its participation in the induced expression of the PbO kauralexin. The activity of An2 gene promoter in the callus of maize was analyzed by bombarding the transient transformation system in the maize callus. The results were found, The promoter fragment A2F2 that does not contain the first W-box element and the A2F1M that abrupt the first W-box element cannot detect the promoter activity, and the A2F1 with the first element can detect the activity. This proves that the key cis element that can mediate the An2 promoter is the first -box element far away from the beginning of the secret code, this is the study of kaurl The metabolic regulation mechanism of exins provides a basis. Conclusion: Three Maize biosynthesis genes are identified, one of which participates in the biosynthesis of gibberellin, the mutant gene of maize dwarf mutant D5, and the other two can be induced by pathogenic bacteria infection and hormone treatment, and may be involved in the biosynthesis of flocking kauralexin. These three genes are arranged in series on chromosomes, which should be derived from gene replication and then function dissimilation. The promoter activity of kauralexin metabolism key enzyme gene An2 is analyzed, and its key cis element, W-box., is identified as the promoter of the promoter cis element as the subsequent metabolic regulation of the subsequent maize plant protection element kauralexin. Control research provides the basis.

【學(xué)位授予單位】：四川農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S513

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