玉米萜类合成酶基因TPS10表达调控的研究

发布时间：2018-04-08 17:47

本文选题：玉米　切入点：虫害　出处：《中国农业大学》2015年博士论文

【摘要】：玉米(Zea mays L.)作为重要的粮食作物和工业原料在国民经济中占据重要地位。虫害是制约玉米产量的关键因素之一。已有的研究结果表明玉米萜类合成酶在玉米的间接防御中起到非常重要的作用。玉米受到鳞翅目幼虫为害后,叶片会释放一系列的萜类化合物吸引害虫的天敌。虽然在过去的二十年中玉米已经成为研究植物间接防御的一个模式系统,但是对于萜类合成酶基因的表达调控机制在玉米中还未见报道。本研究以玉米萜类合成酶基因TPS10启动子为主要研究对象,目的是阐明玉米萜类合成酶基因TPS10的表达调控机制。qPCR结果表明TPS10基因受虫害和MeJA诱导表达,表达部位在叶片并且其表达水平在一定程度上受光合作用的影响。PLACE预测结果显示1727 bp的TPS10启动子序列中存在多个与植物防御相关的顺式作用元件,如GCC-box,G-box,W-box。转基因拟南芥的GUS组织化学染色及酶活性测定结果表明TPS10启动子的-300至-200区域为启动子保持虫害诱导活性的功能序列,其中的GCC-box为TPS10基因表达的一个关键元件。同时189份玉米自交系品种的检测结果表明GCC-box在不同玉米自交系的TPS10启动子中是保守的。以TPS10启动子的-300至-200区域为诱饵序列,酵母单杂交高通量筛选拟南芥转录因子文库获得7个拟南芥AP2/ERF转录因子。通过系统进化树分析得到与7个拟南芥AP2/ERF转录因子进化关系较近的17个玉米ERF转录因子。qPCR结果表明17个玉米ERF转录因子中只有EREB58受虫害和MeJA诱导表达,并且它的表达模式和TPS10的非常类似。亚细胞定位结果显示EREB58特异定位在细胞核中。酵母单杂交实验,凝胶阻滞实验结果表明EREB58能够与TPS10启动子中的GCC-box特异结合。拟南芥原生质体瞬时表达分析结果表明EREB58通过与GCC-box的结合激活基因的表达,具有转录激活活性。转基因玉米中过表达EREB58能够激活TPS10基因的转录及其倍半萜化合物(E)-β-farnesene和(E)-a-bergamotene的释放。相比之下,将EREB58通过RNAi敲除掉后在MeJA处理条件下TPS10基因不转录也无倍半萜化合物的释放。这表明在玉米中EREB58对于TPS10的诱导表达以及倍半萜挥发物的合成都是充分必要的。此外,我们对玉米萜类合成酶基因TPS6的启动子也进行了初步的研究,研究结果表明TPS6启动子的-100至-1区段具有启动子活性,下一步我们将对该区段进行详细研究。以上实验结果表明,玉米中EREB58通过与TPS10启动子中的顺式作用元件GCC-box结合,激活TPS10基因的表达及其倍半萜化合物的释放,阐明了玉米萜类合成酶基因TPS10的表达调控机制,对于解析玉米防御体系的分子调控网络及培育抗虫玉米品种具有重要意义,同时也为其他植物的防御体系的分子调控网络的研究提供新的思路。
[Abstract]:Zea mays L.As an important food crop and industrial raw material, it occupies an important position in the national economy.Pest is one of the key factors restricting maize yield.Previous studies have shown that terpene synthase plays an important role in indirect defense of maize.When maize is damaged by Lepidoptera larvae, the leaves release a series of terpenoids to attract the natural enemies of pests.Although maize has become a model system for the study of plant indirect defense in the past two decades, the regulation of terpene synthase gene expression has not been reported in maize.In this study, the TPS10 promoter of maize terpenoid synthase gene was used as the main research object. The aim of this study was to elucidate the regulation mechanism of TPS10 expression of terpene synthase gene in maize. The results showed that TPS10 gene was induced by insect pests and MeJA.The expression site was located in the leaves and its expression level was affected by photosynthesis to some extent. PLACE prediction results showed that there were many cis-acting elements related to plant defense in the 1727 BP TPS10 promoter sequence, such as GCC-boxG-box W-box.The results of GUS histochemical staining and enzyme activity determination of transgenic Arabidopsis thaliana showed that the regions of -300 to -200 of the TPS10 promoter were the functional sequences of the promoter to maintain the insect-inducing activity, and the GCC-box was a key element of TPS10 gene expression.The results showed that GCC-box was conserved in TPS10 promoter of different maize inbred lines.Using the -300 to -200 regions of TPS10 promoter as bait sequences, seven Arabidopsis AP2/ERF transcription factors were obtained by yeast single hybrid high-throughput screening of Arabidopsis transcription factor library.Through phylogenetic tree analysis, 17 maize ERF transcription factors, which were closely related to the evolution of 7 AP2/ERF transcription factors in Arabidopsis thaliana, were obtained. The results showed that only EREB58 was induced by insect pests and MeJA among the 17 ERF transcription factors.And its expression pattern is very similar to that of TPS10.Subcellular localization showed that EREB58 was specifically located in the nucleus.The results of yeast single hybridization and gel block assay showed that EREB58 could specifically bind to GCC-box in TPS10 promoter.Transient expression analysis of Arabidopsis protoplasts showed that EREB58 had transcriptional activation activity by binding to GCC-box.Overexpression of EREB58 in transgenic maize can activate the transcription of TPS10 gene and the release of sesquiterpene compounds such as 尾 -farnesene and Ela-a-bergamotene.In contrast, the TPS10 gene was not transcribed and no sesquiterpene compounds were released under MeJA treatment after EREB58 was knocked out by RNAi.This indicated that EREB58 was necessary for the induction of TPS10 expression and the synthesis of sesquiterpene volatiles in maize.In addition, we also studied the promoter of maize terpene synthase gene TPS6. The results showed that the -100 to -1 region of TPS6 promoter had promoter activity.The results show that EREB58 activates the expression of TPS10 gene and the release of sesquiterpene compounds by binding to GCC-box, a cis-acting element in TPS10 promoter, and elucidates the mechanism of TPS10 expression in maize.It is of great significance to analyze the molecular regulatory network of maize defense system and to develop insect-resistant maize varieties. At the same time, it also provides a new idea for the study of molecular control network of other plant defense systems.
【学位授予单位】：中国农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S513

【共引文献】