玉米贝壳杉烯合酶功能鉴定及Kauralexins关键酶基因An2启动子分析

发布时间：2018-05-10 06:27

  本文选题：玉米 + 赤霉素　； 参考：《四川农业大学》2016年硕士论文

【摘要】：玉米(Zea mays L.)是重要粮食作物,但病虫害导致其产量和品质下降,威胁粮食安全。植保素是植物受病害真菌侵染产生的小分子次生代谢化合物。Kauralexins是玉米中首次发现的一种二萜植保素,具有良好的抗病原真菌活性,且对玉米螟幼虫有拒食作用。Kauralexin具有(异)贝壳杉烯骨架,其生物合成途径是由柯巴基焦磷酸合酶CPS和(异)贝壳杉烯合酶KS(L)连续催化前体GGPP形成贝壳杉烯或异贝壳杉烯,进一步由细胞色素P450氧化酶氧化修饰形成。参与kauralexin生物合成的CPS已经被鉴定为An2,但其贝壳杉烯合酶还未鉴定。另外植物激素赤霉素的生物合成也需要贝壳杉烯合酶的参与,有报道称玉米矮化突变体彷是由于贝壳杉烯合酶基因突变导致赤霉素合成受阻而产生的矮化,但相关基因还未被鉴定。并且到目前为止,还未有报道鉴定玉米贝壳杉烯合酶基因,这限制了玉米植保素kauralexin的相关研究及其应用。同时,植保素积累与其代谢关键基因表达密切相关,分析kauralexin代谢关键基因An2的启动子活性并鉴定其顺式元件,能为kauralexin的代谢调控研究奠定基础。本研究首先在玉米全基因组中搜索贝壳杉烯合酶,并通过系统进化对其进行功能预测。其中ZmKSL3、ZmKSL5和ZmTPS1与禾本科植物的贝壳杉烯合酶具有较近的进化关系,即作为候选基因进行功能研究。通过大肠杆菌重组表达和代谢工程分析,并结合本氏烟瞬时表达,对产物进行GC-MS检测确定其生化功能,即催化ent-CPP形成贝壳杉烯。进一步通过亚细胞定位分析确认其功能。同时分析了这三个基因在病原菌侵染和激素处理下的表达模式,并结合玉米d5矮化突变体确定了这三个基因的生物学功能。另外,还利用基因枪瞬时表达体系,分析了An2基因启动子的活性区域。主要结果如下：1.对从玉米基因组中搜索出的贝壳杉烯合酶基因,通过氨基酸序列比对,与其他禾本科植物的KS(L)s等进行系统进化分析,发现ZmKSL3与多种植物中参与赤霉素合成的KS的亲缘关系十分相近,而ZmKSL5与ZmTPS1除了本身相近之外,还与小麦中的TaKSL5-1/2相近,它们都不含有γ结构域。进一步分析发现ZmKSL3、 ZmKSL5和ZmTPS1在玉米二号染色体上呈现串联排列,预示着这三个基因在进化上可能是串联复制而来。2.在大肠杆菌重组表达这三个基因并通过代谢工程的方法与玉米中的ent-CPP合酶An2共表达,GC-MS检测产物发现ZmKSL3、ZmKSL5和ZmTPS1都能催化ent-CPP生成贝壳杉烯(ent-kaurene)。前人研究认为ZmTPS1为倍半萜合酶,本研究通过分析发现ZmTPS1确实具有一定的倍半萜合酶活性,能催化FPP产生一系列倍半萜,而ZmKSL3和ZmKSL5则不具有该功能。但进一步在本氏烟草中瞬时表达这三个基因,结果证明它们在植物体内也能产生贝壳杉烯。亚细胞定位分析显示,这三个蛋白都定位与质体中,符合其贝壳杉烯合酶的功能。因此结合生物化功能鉴定和亚细胞定位分析确定这三个基因均编码贝壳杉烯合酶。3.对玉米d5突变体进行半定量PCR分析发现这三个基因中只有ZmKSLS在d5突变体中不表达,并且以往转录组数据显示其在生长旺盛的区域有高表达,表明ZmKSL3参与赤霉素生物合成。而对该突变体中ZmKSL3基因组序列分析,发现与其野生型母本和B73自交系中的序列相比,突变体中的ZmKSL3在5’-UTR部分起始密码子之前缺失了100bp的片段,其中包含与转录相关的CAAT元件,这可能就是引起其不表达的原因。而ZmTPS1或ZmKSL5的突变体均并未出现矮化的表现型,表明不参与赤霉素生物合成。前人研究发现d5突变体中仍然可检测到少量的贝壳杉烯,对d5突变体中ZmKSLS(?)ZmTPS1序列进行分析未发现明显突变,进一步进行体外生化功能分析发现这两个基因的d5克隆仍然具有贝壳杉烯合酶功能,但可能具有严格的调控机制使其不能对d5突变进行补偿。4.利用荧光定量PCR,分析了ZmKSL3、 ZmKSL5和ZmTPS1在不同处理下的表达情况,并与kauralexin代谢关键基因An2进行比较。研究表明,ZmKSL3在禾谷镰刀菌(Fusarium graminearum)孢子侵染的叶片和ABA处理的根部有少量诱导,这可能与其启动子中所含有的相关元件有关。而ZmKSL5和ZmT PS1在禾谷镰刀菌孢子侵染的叶片、ABA处理的地下部分、MeJA和ETH联合处理的叶片和MeJA单独处理的叶片中都明显上调,并且与An2在这四种处理中的表现呈现正相关。对玉米生长发育各时期的转录组数据分析发现ZmTPS1和ZmKSL5与An2之间也有相似的表达模式,而ZmKSL3则不同,这表明ZmTPS1与ZmKSL5参与植保素kauralexin的合成。5.通过在线预测发现An2启动子中含有2个W-box,可能与其参与植保素kauralexin的诱导表达有关。利用基因枪轰击介导的瞬时转化体系,在玉米愈伤组织中进行了An2基因启动子活性的分析,结果发现,不含有第一个W-box元件的启动子片段A2F2与突变掉第一个W-box元件的A2F1M都无法检测的启动子活性,而含有第一个元件的A2F1则能检测到活性。这证明能够介导An2启动子的关键顺式元件为其远离起始密码子的第一个-box元件,这为研究kaurlexins代谢调控机制提供了基础。结论：本研究鉴定了三个玉米贝壳杉烯合酶基因,其中一个参与赤霉素生物合成,为玉米矮化突变体d5的突变基因,而另外两个能够被病原菌侵染和激素处理所诱导,可能参与植保素kauralexin生物合成,同时这三个基因在染色体上形成串联排列,应起源为基因复制,之后发生功能异化。对kauralexin代谢关键酶基因An2以启动子活性进行了分析,确定了其关键顺式元件W-box。本研究鉴定的An2以启动子顺式元件为后续玉米植保素kauralexin的代谢调控研究提供了基础。
[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.

【学位授予单位】：四川农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S513

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