小麦TaGW2-6A等位变异近系籽粒发育过程中GA相关基因的表达及作用分析

发布时间：2018-03-12 22:48

本文选题：小麦　切入点：Ta　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：赤霉素(GA)在种子发育过程中发挥着非常重要的作用,植物通过对GA信号感知和转导从而调控种子发育过程。E3泛素连接酶广泛参与到GA信号感知和转导,从而调控GA信号途径介导籽粒发育过程。TaGW2-6A负调控籽粒的大小,研究发现TaGW2-6A编码RING E3泛素连接酶。本课题前期研究发现TaGW2-6A编码区中第八外显子977bp处“T”碱基的插入导致小麦的粒宽和粒重显著增加。本研究利用中国春CS(野生型)及其近等基因系材料NIL31(T碱基插入突变型)研究了GA相关基因在花后3,6,9,12,15,20,25天的转录水平和表达模式。通过外源GA处理和种子萌发实验验证TaGW2-6A不同等位变异对GA的反应,从而解析TaGW2-6A等位变异促进籽粒变大原因。得到以下主要结果:1.花后3-25天,NIL31中籽粒胚乳鲜重和干重均高于CS。同时我们发现,9-20天,NIL31中干物质积累速率显著高于CS。表明,TaGW2-6A等位变异通过影响籽粒灌浆及干物质积累速率进而控制籽粒的大小。2.实时定量PCR结果表明,GA合成基因(GA 3-oxidase)和GA响应基因(GASA4)在NIL31具有比CS更高的表达水平。此外,NIL31中内源GA含量水平显著高于CS。表明,NIL31中高表达的GA 3-oxidase促进GA合成,使得GASA4表达量升高,进而促进胚乳细胞分裂与发育,从而影响种子的大小。此外,GA合成和响应相关基因与TaGW2-6A表达模式分析发现,随着TaGW2-6A表达量的降低,GA合成和响应相关基因表达量逐渐升高,表明TaGW2-6A与GA相关基因具有负相关的关系。3.外源GA处理实验结果发现,CS中,和未处理相比,胚乳细胞增大,数目增多,最终籽粒变大。此外,GA 3-oxidase和GASA4基因表达水平升高,内源GA含量水平升高。表明,外源GA处理促进了CS中GA 3-oxidase基因的表达,促进内源GA含量增加,使得GASA4表达量升高,进而影响胚乳细胞的发育最终导致籽粒变大。然而,在NIL31中,和未处理相比,胚乳细胞变小、数目减少、最终籽粒变小、内源GA含量水平降低。此外,GA 3-oxidase水平显著升高,但GASA4表达水平降低。由于GASA4基因的低表达,进而影响胚乳细胞的发育最终导致籽粒的变小。其原因是NIL31本身就有高含量的内源GA,当受到外源GA刺激时,GA响应蛋白作为GA信号转导的分子开关从而负调节GA信号,GA 3-oxidase受到负反馈调节,从而调控植物体内GA平衡,使GASA4低水平表达,从而引起籽粒的变化。4.种子萌发实验结果表明,GA处理前,NIL31具有比CS高的萌发率和发芽势,此外,NIL31茎的生长速度也明显高于CS。此外,NIL31籽粒中内源GA含量水平显著高于CS。表明,NIL31中GA合成基因、响应基因较高表达水平,使其体内含有较高水平的GA,进而促进种子萌发和植物整个生长过程。GA处理后,种子萌发实验发现,CS和NIL31表现出截然相反的萌发效果。可见,植物体内GA水平较高时,外源GA的使用,由于负反馈调节反而抑制GASA4的表达,进而影响了种子发育。我们推测,TaGW2-6A与GA信号协同作用来调节小麦籽粒的发育。
[Abstract]:Gibberellin (GA) plays a very important role in the process of seed development in plants, the GA signal perception and transduction to regulate seed development process of.E3 ubiquitin ligase GA is widely involved in signal perception and transduction by regulating GA signaling pathway mediated negative regulation of grain development process of.TaGW2-6A grain size, found that TaGW2-6A encoding RING E3 ubiquitin ligase. Our previous study found that eighth of the TaGW2-6A encoding region of exon 977bp of "T" base insertion resulted in wheat grain width and grain weight increased significantly. This study uses Chinese spring CS (wild type) and its near isogenic lines (NIL31 T insertion mutant) of GA gene in the days after flowering 3,6,9,12,15,20,25 transcription and expression pattern. Germination response experimental verification TaGW2-6A different alleles of GA by exogenous GA and TaGW2-6A seeds, and analysis A variation to promote larger grain. The main results are as follows: 3-25 1. days after anthesis, NIL31 in grain endosperm of fresh weight and dry weight were significantly higher than that of CS. at the same time, we found that 9-20 days, dry matter accumulation rate of NIL31 was significantly higher than that of CS. showed that the size of.2. real-time quantitative PCR results TaGW2-6A alleles by grain filling and influence the rate of dry matter accumulation and grain control showed that the synthesis of GA gene (GA 3-oxidase) and GA (GASA4) with responsive gene expression level higher than CS in NIL31. In addition, the level of endogenous GA content in NIL31 was significantly higher than that of CS., high expression of NIL31 in GA 3-oxidase to promote GA synthesis, the GASA4 expression increased, and then promote the endosperm cell division and development, thus affecting seed size. In addition, GA synthesis and response related genes and TaGW2-6A expression pattern analysis showed that the expression of TaGW2-6A with decreased GA synthesis and response related genes Because the expression level increased gradually, showed that TaGW2-6A and GA related genes have a negative correlation between the.3. results showed that exogenous GA treatment, CS, compared with the untreated, the endosperm cells increased, the increase of the number of final grain size. In addition, increased the expression level of GA 3-oxidase and GASA4 gene, increased the content of endogenous GA level shows. That exogenous GA treatment promoted the expression of CS in GA of 3-oxidase gene, promote endogenous GA content increased, the expression of GASA4 increased, thereby affecting endosperm cell development leads to larger grain. However, in NIL31, and the untreated than endosperm cells become smaller, the number of final grain becomes small, endogenous the content of GA decreased. In addition, GA 3-oxidase increased significantly, but the expression level of GASA4 decreased. Due to the low expression of GASA4 gene, thereby affecting endosperm cell development resulting in grain becomes smaller. The reason is that NIL31 itself High content of endogenous GA, when subjected to exogenous GA stimulation, GA response protein as a molecular switch and signal transduction of GA negatively regulates GA signaling, GA 3-oxidase by negative feedback regulation, and regulation of plant GA balance, the low expression of GASA4, the experiment result showed that the changes of.4. seeds germination, before GA treatment NIL31 is higher than CS, the germination rate and germination potential, in addition, the growth rate of NIL31 stem was significantly higher than CS. in addition, the level of endogenous GA content in grains was higher than that of CS. NIL31 showed that the GA synthetic gene NIL31, gene expression in response to the higher level, the body contains higher levels of GA, and then promote the seed germination and the whole plant growth process after.GA treatment, the seed germination experiment showed that CS and NIL31 showed the opposite germination effect. Therefore, the level of GA in higher plants, the use of exogenous GA, but due to the negative feedback regulation Inhibiting the expression of GASA4 and further affecting the development of seeds, we speculate that TaGW2-6A and GA signals are synergistic to regulate the development of wheat grain.

【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.1

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