【摘要】：小麥?zhǔn)鞘澜缟先蠹Z食作物之一,因為其種子中富含谷醇溶蛋白而可以加工成豐富多樣的食品。谷醇溶蛋白由醇溶蛋白和谷蛋白組成,谷蛋白又可根據(jù)分子量的大小分為高分子谷蛋白亞基和低分子谷蛋白亞基,決定了面粉的粘彈性。盡管已有的報道表明谷蛋白的表達主要受到相關(guān)轉(zhuǎn)錄因子的控制,但未見轉(zhuǎn)化小麥的報道。本論文首先將課題組前期克隆的小麥轉(zhuǎn)錄因子PBF-D的全長cDNA編碼序列構(gòu)建到表達載體pGA3626上,利用小麥莖尖轉(zhuǎn)化技術(shù)侵染濟麥22,經(jīng)PCR和qRT-PCR檢測后得到10個T0代陽性株系,4個T1代株系和2個T2株系。qRT-PCR結(jié)果表明,TaPBF-D的過表達促進了谷蛋白亞基1By8,1Bx7和1Dyl2的表達,同時我們發(fā)現(xiàn)胚乳特異轉(zhuǎn)錄因子TaAPA在胚乳不同發(fā)育時期表達量都有所提高,而TaGAMYB沒有變化。通過軟件SPSS2.0對轉(zhuǎn)錄因子和谷蛋白亞基進行了相關(guān)性分析,結(jié)果發(fā)現(xiàn)TaPBF-D的過表達與TaSPA,1By8,1Bx7和1Dyl2的表達之間存在顯著的正相關(guān)。谷蛋白定量實驗表明在T1代和T2代種子中谷蛋白的含量與對照組相比顯著提高。以上結(jié)果表明TaPBF-D的過表達促進了谷蛋白的積累,結(jié)果同樣表明轉(zhuǎn)錄水平的調(diào)控對小麥谷蛋白的表達起著十分重要的調(diào)控作用,而這種作用也體現(xiàn)在胚乳發(fā)育相關(guān)轉(zhuǎn)錄因子的互作上。為了探討轉(zhuǎn)錄水平的變化是否影響了表觀遺傳上的調(diào)控,我們檢測了在胚乳中表達的DNA甲基化酶(MET2a,MET2b,MET3,CMT,Dnmt和DRM)和去甲基化酶(DME,DML)的表達水平,發(fā)現(xiàn)在T1代和T2代中,TaPBF-D表達水平的改變并未引起甲基化酶和去甲基化酶的表達水平出現(xiàn)規(guī)律性的變化。在小麥不同HMWv-GS亞基突變體中,我們同樣分析了胚乳發(fā)育不同時期相關(guān)轉(zhuǎn)錄因子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)的表達水平,結(jié)果發(fā)現(xiàn)在野生型小麥L03-227(HMW-GS亞基組成5+10,1,17+18)中,轉(zhuǎn)錄因子TaPBFs,TaSPAs,TaGAMYB的表達水平最高；在突變體L03-221(HMW-GS亞基組成Null)中,轉(zhuǎn)錄因子TaPBFs,TaSPAs,TaGAMYB的表達水平最低；在剩余幾種突變體中隨著HMW-GS亞基表達沉默數(shù)量的增多,TaPBFs和TaSPAs的表達水平逐漸減少,而TaGAMYB并沒有發(fā)現(xiàn)類似TaPBFs,TaSPAs那樣規(guī)律性的變化。分析還發(fā)現(xiàn)DNA甲基化酶和去甲基化酶的表達水平也沒有規(guī)律性的變化。這些結(jié)果表明HMW-GS的表達與相關(guān)轉(zhuǎn)錄因子TaPBF和TaSPA的表達之間可能存在表達關(guān)聯(lián)現(xiàn)象。此外,我們克隆了高冰草中PBF基因序列,序列比對分析發(fā)現(xiàn)小麥體細(xì)胞雜種漸滲系SR3中PBF序列與其親本JN177高度同源,結(jié)果表明小麥漸滲系中的PBF序列來自受體親本JN177而不是供體親本高冰草。
[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.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S512.1;Q943.2

【相似文獻】

相關(guān)期刊論文 前10條

1 段淑娥;;小麥麥谷蛋白亞基及其基因的研究進展[J];西安文理學(xué)院學(xué)報(自然科學(xué)版);2007年03期

2 趙文明;水稻谷蛋白是類似于豆球蛋白的蛋白質(zhì)[J];實驗生物學(xué)報;1988年02期

3 段淑娥,阮禹松,趙文明,王波;小麥低分子量麥谷蛋白亞基組成研究[J];西北植物學(xué)報;2005年04期

4 沈蕾;龍海;顏澤洪;魏育明;鄭有良;;小麥新品種“川麥42”低分子量谷蛋白亞基新基因的分子克隆[J];遺傳;2006年01期

5 紀(jì)軍;劉冬成;王靜;李俊明;張愛民;;一種小麥高、低分子量麥谷蛋白亞基的提取方法[J];遺傳;2008年01期

6 覃建兵;任燕萍;祝長青;;小麥低分子量麥谷蛋白亞基分離條件優(yōu)化[J];生物技術(shù);2010年04期

7 蔡民華;胡英考;李雅軒;李建國;晏月明;;小麥谷蛋白亞基對品質(zhì)性狀的影響[J];首都師范大學(xué)學(xué)報(自然科學(xué)版);2006年03期

8 張曉科,魏益民,胡新中,鄭建梅;谷蛋白溶漲指數(shù)在小麥早代品質(zhì)選擇中的應(yīng)用價值分析[J];西北植物學(xué)報;2004年06期

9 吳芳;潘志芬;余懋群;;小麥低分子量麥谷蛋白亞基研究進展[J];西北植物學(xué)報;2006年04期

10 忻驊;江瑛;黃偉達;顧其敏;孫崇榮;;小麥高分子量谷蛋白基因的結(jié)構(gòu)分析[J];Journal of Integrative Plant Biology;1992年10期

相關(guān)會議論文 前10條

1 李友軍;朱志勇;黃明;;鎘脅迫對不同抗性小麥品種可溶性糖轉(zhuǎn)運及谷蛋白表達的影響[A];中國作物學(xué)會50周年慶祝會暨2011年學(xué)術(shù)年會論文集[C];2011年

2 馬啟林;李陽生;;水稻儲藏谷蛋白的聚合化特性研究[A];中國植物學(xué)會七十五周年年會論文摘要匯編（1933-2008）[C];2008年

3 晏本菊;任正隆;;四川小麥高分子量谷蛋白亞基分析[A];中國生物化學(xué)與分子生物學(xué)會第八屆會員代表大會暨全國學(xué)術(shù)會議論文摘要集[C];2001年

4 王林海;何中虎;夏先春;;普通小麥低分子量谷蛋白亞基基因克隆與功能標(biāo)記的開發(fā)[A];全國植物分子育種研討會摘要集[C];2009年

5 郎淑平;王海燕;曹愛忠;王蘇玲;張守忠;陳佩度;亓增軍;王秀娥;;分子標(biāo)記輔助選育小麥抗赤霉病、白粉病、谷蛋白5+10亞基聚合體[A];江蘇省遺傳學(xué)會第七屆代表大會暨學(xué)術(shù)研討會論文摘要匯編[C];2006年

6 張宏;吉萬全;任志龍;王長有;王秋英;;染色體工程法聚合小麥優(yōu)質(zhì)麥谷蛋白亞基研究[A];2003年全國作物遺傳育種學(xué)術(shù)研討會論文集[C];2003年

7 陳凡國;趙峰;楊亮;莊倩倩;夏光敏;;小麥及衍生材料/近緣禾草LMW-GS基因的克隆與功能特性[A];植物分子生物學(xué)與現(xiàn)代農(nóng)業(yè)——全國植物生物學(xué)研討會論文摘要集[C];2010年

8 易蓉;黃琳;丁毅;;水稻灌漿期谷蛋白亞基表達的初步分析[A];中國的遺傳學(xué)研究——遺傳學(xué)進步推動中國西部經(jīng)濟與社會發(fā)展——2011年中國遺傳學(xué)會大會論文摘要匯編[C];2011年

9 晏月明;姜怡;孫e鹐，

本文編號：2464314