本文選題：水稻 + 油菜素內(nèi)酯　； 參考：《揚(yáng)州大學(xué)》2017年碩士論文

【摘要】：油菜素內(nèi)酯(Brassinosteroids,BR)是一種廣泛存在于植物體內(nèi)的甾醇類植物激素,被稱為第六大植物激素。BR參與一系列生理生化反應(yīng),在植物的生長(zhǎng)發(fā)育中發(fā)揮著重要作用。目前在雙子葉模式植物擬南芥中已確立一條由細(xì)胞表面BR受體BRI1到細(xì)胞核內(nèi)轉(zhuǎn)錄因子BZR1和BES1的較完整的信號(hào)轉(zhuǎn)導(dǎo)途徑,但在作為單子葉模式植物和重要作物的水稻中BR信號(hào)轉(zhuǎn)導(dǎo)途徑中很多重要元件還未被克隆,部分已克隆基因的生物學(xué)功能仍不明確。本研究以BR合成關(guān)鍵基因DWF4、BR受體蛋白基因D61、以及BR信號(hào)傳導(dǎo)轉(zhuǎn)錄因子(BZR1～BZR4)等6個(gè)重要基因?yàn)閷?duì)象,一方面以相關(guān)基因的突變體為實(shí)驗(yàn)材料,同時(shí)創(chuàng)建BR關(guān)鍵基因的過(guò)表達(dá)和敲除材料,初步分析并探討調(diào)控BR合成或信號(hào)轉(zhuǎn)導(dǎo)對(duì)水稻株型、稻米外觀和食味品質(zhì)等的影響。相關(guān)研究與結(jié)果為深入解析BR功能、以及通過(guò)遺傳操作優(yōu)化BR途徑培育高產(chǎn)優(yōu)質(zhì)水稻新品種提供了重要的材料基礎(chǔ)。主要研究結(jié)果如下:1、對(duì)收集的BR信號(hào)通路關(guān)鍵基因的突變體bzr1、bzr4-1、bzr4-2、d61-1和d61-2進(jìn)行分子鑒定、表達(dá)分析、農(nóng)藝性狀測(cè)定和稻米品質(zhì)分析。結(jié)果顯示bzr1的基本農(nóng)藝性狀和稻米品質(zhì)均無(wú)明顯改變。bzr4-1和bzr4-2株高變矮,劍葉長(zhǎng)寬比和穗粒數(shù)減少,堊白度明顯增加;稻米品質(zhì)中RVA譜的峰值粘度、熱漿粘度、冷膠粘度均下降,且淀粉一級(jí)結(jié)構(gòu)長(zhǎng)B鏈含量顯著降低。d61-1和d61-2株高降低,分蘗數(shù)減少,劍葉長(zhǎng)寬比增加,籽粒變短變寬,堊白度顯著減少;表觀直鏈淀粉含量AAC明顯增加,RVA譜的峰值粘度、熱漿粘度和冷膠粘度均升高,低分子量淀粉顯著減少。2、利用CRISPR/Cas9基因組定點(diǎn)編輯技術(shù),創(chuàng)建了水稻BZR家族4個(gè)基因的基因敲除水稻。為獲得單基因及多基因同時(shí)敲除的遺傳材料,我們?cè)?個(gè)基因外顯子上分別選擇各基因特異性和保守性序列作為基因編輯的靶位點(diǎn)。DNA測(cè)序鑒定結(jié)果表明,BZR家族各成員的單基因和多基因突變體水稻已構(gòu)建成功,且突變種類豐富。具體而言,BZR1～BZR4的單基因突變體分別包括6種(D1、D3、D4、D5、D9和I1)、5種(D3、I1、S1、S2和S3)、5種(D1、D2、D5、D8和I1)和2種(D4和D5)突變類型;雙突變體包括BZR1 BZR2、BZR1 BZR3 和BZR2 BR3三種基因組合,每種組合又分別包括4種、17種和2種突變組合類型;三基因突變體BZR1 BZR2 BZR3包括8種突變類型(D1-S4-D2、D1-I1-D4、D3-D3-D4、D1-D3-D4、D3-D3-I1、I1-D3-I1、I1-S1-I1 和 I1-S1-D3)。這些突變體的創(chuàng)建為后續(xù)系統(tǒng)開(kāi)展水稻BZR基因的功能分析奠定了基礎(chǔ)。3、將BR合成關(guān)鍵基因OsDWF4分別克隆到由胚乳特異性啟動(dòng)子Gt1和組成型表達(dá)啟動(dòng)子Ubi驅(qū)動(dòng)的過(guò)表達(dá)載體pCAMBIA-Gt1和pCAMBIA-Ubi并轉(zhuǎn)入受體品種鹽稻8號(hào)。對(duì)獲得的轉(zhuǎn)基因水稻進(jìn)行分子鑒定、表達(dá)分析、農(nóng)藝性狀鑒定和稻米品質(zhì)分析。分子鑒定結(jié)果顯示已成功獲得46個(gè)pGt1::OsDWF4和13個(gè)pUbi::OsDWF4的獨(dú)立轉(zhuǎn)化子。對(duì)轉(zhuǎn)基因水稻純合系的表達(dá)分析表明目的基因在轉(zhuǎn)基因水稻中有較高表達(dá)。田間農(nóng)藝性狀分析表明pGt1::OsDWF4的株高稍有增加,而pUbi::OsDWF4的株高有所降低。此外,OsDWF4過(guò)表達(dá)材料與對(duì)照相比,其粒長(zhǎng)和千粒重增加,而其他農(nóng)藝性狀變化不明顯。進(jìn)一步稻米理化品質(zhì)分析表明OsDWF4轉(zhuǎn)基因水稻除RVA譜有所改變,其表觀直鏈淀粉含量和淀粉的鏈長(zhǎng)分布均無(wú)明顯變化。
[Abstract]:Brassinosteroids (Brassinosteroids, BR) is a plant sterol hormone widely exists in plants, known as the sixth plant hormone.BR is involved in a series of physiological and biochemical reactions play an important role in plant growth and development. At present in Arabidopsis has established a cell surface by BR BRI1 receptor to transcription factor BZR1 and BES1 in the nucleus of the complete signal transduction pathway, but as in the signal transduction of BR model monocot plants and important crops in rice in the way many important components have not been cloned, has been part of the biological function of gene cloning is still not clear. In this study, the BR synthesis of the key gene DWF4. BR receptor protein gene D61 and BR signal transduction transcription factor (BZR1 ~ BZR4) 6 important genes for the object, on the one hand, related gene mutants as experimental materials, and the creation of the BR based key Overexpression and knockdown of materials, preliminary analysis and study the regulation of BR synthesis or signal transduction of rice plant type, effect of appearance and eating quality. The related research and the analysis of BR function, and the genetic operation optimization of BR provides an important basis for breeding new varieties of high yield and quality of rice. The main research the results are as follows: 1, BR pathway key genes for the collection of the mutant bzr1, bzr4-1, bzr4-2, d61-1 and d61-2 molecular identification, expression analysis, analysis of agronomic traits and grain quality were determined. The results showed that there were no significant changes in.Bzr4-1 and bzr4-2 height of bzr1 based on the agronomic traits and quality of rice, flag leaf the length width ratio and grain number per spike decreased, chalkiness increased significantly; the peak viscosity of RVA spectrum of rice quality, hot viscosity, cool viscosity decreased, and starch level structure of B long chain content significantly decreased.D61-1 and D6 1-2鏍珮闄嶄綆,鍒嗚槚鏁板噺灝，

本文編號(hào)：1739274