甘蓝型油菜少分枝突变体sfz基因的克隆和功能分析

发布时间：2018-03-20 07:27

本文选题：油菜　切入点：突变体sfz　出处：《江苏大学》2017年硕士论文　论文类型：学位论文

【摘要】：油菜作为油料作物之一,在全球各地被广泛种植。我国是植物油消费大国,大量植物性食用油需要进口,对外进口率超过65%,因此提高油菜产量以减少植物油进口量刻不容缓。油菜分枝与机械化收获,提高生产效率相关,同时也影响油菜的产量,探究油菜分枝发育机理意义重大。本研究以少分枝突变体sfz作为实验对象,克隆出BnSFZ基因,分析该基因的功能,从而丰富对油菜分枝发育的分子机制的了解。以下是本研究的主要部分:1.油菜突变体sfz的表型分析本实验室已经成功获得含有激活标签载体PXT246的少分枝突变体sfz。本研究对油菜sfz植株进行性状统计得出油菜sfz的一次分枝数较少,一次分枝高度明显高于野生型NY12植株。近红外光谱检测油菜sfz种子中脂肪酸各组成成分的含量与野生型植株无差别。突变体sfz植株表皮蜡质含量较少在菌核病接菌实验中,突变体sfz叶片被较快侵染且菌斑面积较大。2.油菜sfz的蛋白质组差异表达分析蛋白二维电泳技术,分析突变体sfz蛋白表达差异,鉴定得到16个差异蛋白点,将差异蛋白点通过二级质谱分析,最终得到8个差异蛋白。对其进行理化性质、功能等分析发现差异蛋白与能量代谢、抗逆、抗病虫、能量生成、生长发育等相关,在油菜生长发育和蜡质合成过程中可能发挥重要作用。3.油菜突变体sfz突变基因的克隆hiTAIL-PCR、FPNI-PCR技术扩增突变体sfz的T-DNA插入位点的旁侧序列,经过大量改进尝试,最终通过FPNI-PCR方法克隆出侧翼序列。软件分析序列得到插入位点附近基因SFZ1、SFZ2、SFZ3、SFZ4。4.候选基因表达量及生物信息学分析qRT-PCR分析发现在油菜sfz植株中SFZ4基因表达量升高,其余无变化。将SFZ4作为最终候选基因并命名为BnSFZ。生物信息学分析发现BnSFZ蛋白属于AP2大家族,在植物生长发育和蜡质合成中具有重要作用。5.候选基因互补实验验证候选基因BnSFZ的互补实验验证,构建BnSFZ的过表达和抑制表达载体,农杆菌浸花法转化到野生型油菜中。6.目标区域GWAS分析在油菜分枝数全基因组关联分析结果的基础上,选取插入位点前后20Kb序列中寻找SNPs和Indel位点,进而分析这些位点与植物分枝性状的相关性。结果得出SFZ-SNP-18409641位点对分枝性状影响显著(P0.01),该位点位于BnSFZ基因的启动子区域,说明在自然群体关联分析中,BnSFZ基因与分枝数具有相关关系。本研究结果表明,突变体sfz中BnSFZ基因表达量升高,导致植株分枝减少,蜡质含量降低,推测该基因参与植物生长发育和蜡质合成途径。本研究的开展有助于了解植物分枝发育及蜡质合成的分子机理。
[Abstract]:Rape, as one of the oil crops, is widely cultivated around the world. China is a big consumer of vegetable oil, and a large number of vegetable edible oils need to be imported. The foreign import rate is more than 65%, so it is urgent to increase rape yield so as to reduce the import of vegetable oil. Rape branching is related to mechanized harvesting, improving production efficiency, and also affecting rape yield. It is of great significance to explore the mechanism of branching development in rape. In this study, the BnSFZ gene was cloned and the function of the BnSFZ gene was analyzed by using the small branching mutant sfz as the experimental object. The following is the main part of this study: 1. Phenotypic analysis of rapeseed mutant sfz has been successfully obtained in our laboratory with the activation tag vector PXT246, the oligobranched mutant sfz. In this study, the characters of rapeseed sfz plants were analyzed and the results showed that the number of primary branches of sfz in rape was less. The primary branching height was significantly higher than that of wild type NY12 plants. The content of fatty acid components in sfz seeds of rape was not different from that of wild type plants by near infrared spectroscopy. The epidermal wax content of mutant sfz plants was less than that of wild type plants. The mutant sfz leaves were quickly infected and the plaque area was larger. 2.The proteome differential expression analysis of rapeseed sfz by two-dimensional electrophoresis was used to analyze the difference of sfz protein expression in the mutant, and 16 differentially expressed protein spots were identified. Eight differentially expressed proteins were obtained by secondary mass spectrometry. The physical and chemical properties and functions of differential proteins were analyzed. It was found that differential proteins were related to energy metabolism, stress resistance, disease and insect resistance, energy generation, growth and development, etc. It may play an important role in the growth, development and waxy synthesis of rapeseed. 3. Cloning of sfz mutant gene from rapeseed mutant (hiTAIL-PCRN-FPNI-PCR) was used to amplify the flanking sequence of T-DNA insertion site of mutant sfz, and a large number of attempts were made to improve it. Finally, the flanking sequence was cloned by FPNI-PCR method. The gene SFZ1, SFZ2, SFZ3, SFZ4.4near the insertion site was obtained by software analysis. The expression of candidate genes and bioinformatics qRT-PCR analysis showed that the expression of SFZ4 gene was increased in the sfz plants of Brassica napus. SFZ4 was the final candidate gene and named BnSFZ.The bioinformatics analysis found that the BnSFZ protein belonged to the AP2 family. It plays an important role in plant growth, development and waxy synthesis. 5. Candidate gene complementation test verifies candidate gene BnSFZ, constructs BnSFZ overexpression and inhibits expression vector. Transformation of Agrobacterium tumefaciens to wild rapeseed. Based on the results of genome association analysis of branch number of rape, SNPs and Indel loci were selected from 20kb sequence before and after insertion. The results showed that the SFZ-SNP-18409641 locus had a significant effect on the branching traits, which was located in the promoter region of the BnSFZ gene. The results of this study showed that the expression of BnSFZ gene in the mutant sfz increased, resulting in the decrease of plant branching and the decrease of waxy content, and the correlation between the BnSFZ gene and the number of branches was found in the natural population association analysis. It is speculated that this gene is involved in plant growth and waxy synthesis pathway. The development of this study is helpful to understand the molecular mechanism of plant branching and waxy synthesis.
【学位授予单位】：江苏大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S565.4

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