小麦谷醇溶蛋白盒结合因子基因TaPBF-D在小麦中过表达促进了籽粒谷蛋白的积累

发布时间：2019-04-24 08:48

【摘要】：小麦是世界上三大粮食作物之一,因为其种子中富含谷醇溶蛋白而可以加工成丰富多样的食品。谷醇溶蛋白由醇溶蛋白和谷蛋白组成,谷蛋白又可根据分子量的大小分为高分子谷蛋白亚基和低分子谷蛋白亚基,决定了面粉的粘弹性。尽管已有的报道表明谷蛋白的表达主要受到相关转录因子的控制,但未见转化小麦的报道。本论文首先将课题组前期克隆的小麦转录因子PBF-D的全长cDNA编码序列构建到表达载体pGA3626上,利用小麦茎尖转化技术侵染济麦22,经PCR和qRT-PCR检测后得到10个T0代阳性株系,4个T1代株系和2个T2株系。qRT-PCR结果表明,TaPBF-D的过表达促进了谷蛋白亚基1By8,1Bx7和1Dyl2的表达,同时我们发现胚乳特异转录因子TaAPA在胚乳不同发育时期表达量都有所提高,而TaGAMYB没有变化。通过软件SPSS2.0对转录因子和谷蛋白亚基进行了相关性分析,结果发现TaPBF-D的过表达与TaSPA,1By8,1Bx7和1Dyl2的表达之间存在显著的正相关。谷蛋白定量实验表明在T1代和T2代种子中谷蛋白的含量与对照组相比显著提高。以上结果表明TaPBF-D的过表达促进了谷蛋白的积累,结果同样表明转录水平的调控对小麦谷蛋白的表达起着十分重要的调控作用,而这种作用也体现在胚乳发育相关转录因子的互作上。为了探讨转录水平的变化是否影响了表观遗传上的调控,我们检测了在胚乳中表达的DNA甲基化酶(MET2a,MET2b,MET3,CMT,Dnmt和DRM)和去甲基化酶(DME,DML)的表达水平,发现在T1代和T2代中,TaPBF-D表达水平的改变并未引起甲基化酶和去甲基化酶的表达水平出现规律性的变化。在小麦不同HMWv-GS亚基突变体中,我们同样分析了胚乳发育不同时期相关转录因子TaPBFs(-A,-B,-D),TaSPAs(-A,-B,-D),TaGAMYB,DNA甲基化酶(MET2a,MET2b,MET3,CMT,Dnmt和DRM)和去甲基化酶(DME,DML)的表达水平,结果发现在野生型小麦L03-227(HMW-GS亚基组成5+10,1,17+18)中,转录因子TaPBFs,TaSPAs,TaGAMYB的表达水平最高；在突变体L03-221(HMW-GS亚基组成Null)中,转录因子TaPBFs,TaSPAs,TaGAMYB的表达水平最低；在剩余几种突变体中随着HMW-GS亚基表达沉默数量的增多,TaPBFs和TaSPAs的表达水平逐渐减少,而TaGAMYB并没有发现类似TaPBFs,TaSPAs那样规律性的变化。分析还发现DNA甲基化酶和去甲基化酶的表达水平也没有规律性的变化。这些结果表明HMW-GS的表达与相关转录因子TaPBF和TaSPA的表达之间可能存在表达关联现象。此外,我们克隆了高冰草中PBF基因序列,序列比对分析发现小麦体细胞杂种渐渗系SR3中PBF序列与其亲本JN177高度同源,结果表明小麦渐渗系中的PBF序列来自受体亲本JN177而不是供体亲本高冰草。
[Abstract]:Wheat is one of the three major food crops in the world, because its seeds are rich in gliadin and can be processed into a variety of foods. Glutenin is composed of gliadin and glutenin. Glutenin can be divided into high molecular weight glutenin subunits and low molecular weight glutenin subunits according to the molecular weight, which determines the viscoelasticity of flour. Although it has been reported that the expression of glutenin is mainly controlled by transcription factors, there are no reports of transformation of wheat. In this thesis, the full-length cDNA coding sequence of wheat transcription factor PBF-D cloned in our research group was first constructed into the expression vector pGA3626. The wheat stem tip transformation technique was used to infect Jimai 22. Ten T0 generation positive lines were obtained after detection by PCR and qRT-PCR. The results of qRT-PCR showed that the overexpression of TaPBF-D promoted the expression of glutenin subunit 1By8, 1Bx7 and 1Dyl2. At the same time, we found that the expression of endosperm-specific transcription factor TaAPA was increased at different stages of endosperm development. And TaGAMYB hasn't changed. The correlation between transcription factor and glutenin subunit was analyzed by software SPSS2.0. The results showed that there was a significant positive correlation between the overexpression of TaPBF-D and the expression of TaSPA,1By8,1Bx7 and 1Dyl2. The glutenin content in T _ 1 and T _ 2 generation seeds was significantly higher than that in the control group. The results indicated that the overexpression of TaPBF-D promoted the accumulation of glutenin, and the regulation of transcription level also played an important role in the regulation of glutenin expression in wheat. This effect is also reflected in the interaction of transcription factors associated with endosperm development. In order to investigate whether changes in transcription level affect epigenetic regulation, we examined the expression of DNA methylase (MET2a,MET2b,MET3,CMT,Dnmt, DRM) and demethylase (DME,DML) expressed in endosperm. It was found that the changes of TaPBF-D expression in T1 and T2 generations did not result in regular changes in the expression levels of methylase and demethylase. In different HMWv-GS subunit mutants of wheat, we also analyzed the transcription factors TaPBFs (- A, B, D), TaSPAs (- A, TaGAMYB,DNA methylase), TaGAMYB,DNA methylase (MET2a,MET2b,MET3,CMT,) at different stages of endosperm development. The expression levels of Dnmt and DRM) and demethylase (DME,DML) were detected. The results showed that the expression level of transcription factor TaPBFs,TaSPAs,TaGAMYB was the highest in wild type wheat L03A227 (HMW-GS subunit composed of 510, 1, 17 18). The expression level of transcription factor TaPBFs,TaSPAs,TaGAMYB was the lowest in the mutant L03 HMW-GS 221 (composed of Null subunit). Among the remaining mutants, the expression levels of TaPBFs and TaSPAs decreased gradually with the increase of silencing quantity of HMW-GS subunits, but no regular changes were found in TaGAMYB as that of TaPBFs,TaSPAs. It was also found that the expression level of DNA methylase and demethylase did not change regularly. These results suggest that there may be an expression association between the expression of HMW-GS and the expression of transcription factors TaPBF and TaSPA. In addition, we cloned the sequence of PBF gene from Hippophae aestivum. Sequence alignment analysis showed that the PBF sequence of wheat somatic hybrid SR3 was highly homologous to its parent JN177. The results showed that the PBF sequence in wheat impermeable lines came from the recipient parent JN177 rather than the donor parent Hippophora albicans.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S512.1;Q943.2

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