油菜素内酯相关突变体和转基因水稻的鉴定及其效应的初步分析

发布时间：2018-04-12 10:04

  本文选题：水稻 + 油菜素内酯　； 参考：《扬州大学》2017年硕士论文

【摘要】：油菜素内酯(Brassinosteroids,BR)是一种广泛存在于植物体内的甾醇类植物激素,被称为第六大植物激素。BR参与一系列生理生化反应,在植物的生长发育中发挥着重要作用。目前在双子叶模式植物拟南芥中已确立一条由细胞表面BR受体BRI1到细胞核内转录因子BZR1和BES1的较完整的信号转导途径,但在作为单子叶模式植物和重要作物的水稻中BR信号转导途径中很多重要元件还未被克隆,部分已克隆基因的生物学功能仍不明确。本研究以BR合成关键基因DWF4、BR受体蛋白基因D61、以及BR信号传导转录因子(BZR1～BZR4)等6个重要基因为对象,一方面以相关基因的突变体为实验材料,同时创建BR关键基因的过表达和敲除材料,初步分析并探讨调控BR合成或信号转导对水稻株型、稻米外观和食味品质等的影响。相关研究与结果为深入解析BR功能、以及通过遗传操作优化BR途径培育高产优质水稻新品种提供了重要的材料基础。主要研究结果如下:1、对收集的BR信号通路关键基因的突变体bzr1、bzr4-1、bzr4-2、d61-1和d61-2进行分子鉴定、表达分析、农艺性状测定和稻米品质分析。结果显示bzr1的基本农艺性状和稻米品质均无明显改变。bzr4-1和bzr4-2株高变矮,剑叶长宽比和穗粒数减少,垩白度明显增加;稻米品质中RVA谱的峰值粘度、热浆粘度、冷胶粘度均下降,且淀粉一级结构长B链含量显著降低。d61-1和d61-2株高降低,分蘖数减少,剑叶长宽比增加,籽粒变短变宽,垩白度显著减少;表观直链淀粉含量AAC明显增加,RVA谱的峰值粘度、热浆粘度和冷胶粘度均升高,低分子量淀粉显著减少。2、利用CRISPR/Cas9基因组定点编辑技术,创建了水稻BZR家族4个基因的基因敲除水稻。为获得单基因及多基因同时敲除的遗传材料,我们在4个基因外显子上分别选择各基因特异性和保守性序列作为基因编辑的靶位点。DNA测序鉴定结果表明,BZR家族各成员的单基因和多基因突变体水稻已构建成功,且突变种类丰富。具体而言,BZR1～BZR4的单基因突变体分别包括6种(D1、D3、D4、D5、D9和I1)、5种(D3、I1、S1、S2和S3)、5种(D1、D2、D5、D8和I1)和2种(D4和D5)突变类型;双突变体包括BZR1 BZR2、BZR1 BZR3 和BZR2 BR3三种基因组合,每种组合又分别包括4种、17种和2种突变组合类型;三基因突变体BZR1 BZR2 BZR3包括8种突变类型(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)。这些突变体的创建为后续系统开展水稻BZR基因的功能分析奠定了基础。3、将BR合成关键基因OsDWF4分别克隆到由胚乳特异性启动子Gt1和组成型表达启动子Ubi驱动的过表达载体pCAMBIA-Gt1和pCAMBIA-Ubi并转入受体品种盐稻8号。对获得的转基因水稻进行分子鉴定、表达分析、农艺性状鉴定和稻米品质分析。分子鉴定结果显示已成功获得46个pGt1::OsDWF4和13个pUbi::OsDWF4的独立转化子。对转基因水稻纯合系的表达分析表明目的基因在转基因水稻中有较高表达。田间农艺性状分析表明pGt1::OsDWF4的株高稍有增加,而pUbi::OsDWF4的株高有所降低。此外,OsDWF4过表达材料与对照相比,其粒长和千粒重增加,而其他农艺性状变化不明显。进一步稻米理化品质分析表明OsDWF4转基因水稻除RVA谱有所改变,其表观直链淀粉含量和淀粉的链长分布均无明显变化。
[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�楂橀檷浣�,鍒嗚槚鏁板噺灏，

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