大白菜早抽薹突变基因ebm1的精细定位

发布时间：2018-02-02 22:58

本文关键词： 大白菜抽薹突变体基因定位克隆　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：大白菜[Brassica campestris ssp.pekinensis(Lour)Olsson]是东亚地区重要的蔬菜作物,未熟抽薹会大大降低其商品价值,因此,抽薹性状遗传机理的研究有重要意义。鉴于突变体是研究植物性状遗传机理的理想材料,本研究以大白菜DH系'FT'为材料,利用EMS诱变与游离小孢子培养技术相结合的方法创制出一个早抽薹突变体ebml,构建了突变基因ebml的遗传连锁图谱,并预测出了候选基因。主要研究结果如下:1.突变体表型鉴定结果表明,与野生型'FT'相比,早抽薹突变体ebml表现为极早抽薹,同时伴有叶缘上卷。经流式细胞仪检测,其为二倍体;经TTC染色鉴定,其花粉有正常活性。2.以突变体ebml及其野生型'FT'为亲本,配制F1、F2和BC1。遗传分析结果表明,早抽薹突变性状为单隐性核基因遗传。3.以突变体ebml和青梗菜DH系'13A516'为亲本构建F2分离群体,选取1502株表型为突变性状的单株为定位群体,采用SSR标记方法,将早抽薹突变基因ebwI定位于A04染色体的SSRh1-53和SSRh1-61两个标记之间,物理距离为73.4kb,定位区间内有7个基因。4.通过对目的区域内7个基因的编码序列进行克隆测序,发现在突变体中只有Bra032169基因的cDNA序列与野生型存在差异。根据基因注释信息,Bra032169编码组蛋白甲基转移酶,为拟南芥卷叶基因(CURLYLEAF,CLF)。CLF能够调控与开花相关基因(如AG、FLC、AGL19、FT)的表达。预测Bra032169为ebml的候选基因。5.基因序列结构分析表明,突变体Bra032169基因的第12个外显子中插入了一个53bp片段,可导致翻译提前终止。基因表达模式分析证明,此插入突变并未影响自身的转录,推测可能产生了丧失原有功能的蛋白质,影响其对下游基因的调控作用。
[Abstract]:Chinese cabbage. [Brassica campestris ssp.pekinensis(Lour)Olsson is an important vegetable crop in East Asia. Therefore, it is important to study the genetic mechanism of sprouting characters. In view of the mutant is an ideal material to study the genetic mechanism of plant traits. In this study, an early sprouting mutant ebml was created by combining EMS mutagenesis with free microspore culture with DH strain FT' of Chinese cabbage as the material. The genetic linkage map of mutant gene ebml was constructed and candidate genes were predicted. The main results were as follows: 1. The ebml of early sprouting mutant was very early and accompanied by leaf edge curl, and it was diploid by flow cytometry. The pollen had normal activity by TTC staining. The mutant ebml and its wild type FT 'were used as parents to prepare F _ 1 F _ 2 and BC _ 1.The results of genetic analysis showed that F _ 1 F _ 2 and BC _ 1 were used as parents. The mutant character of early heading and moss was a single recessive nuclear gene. The F _ 2 segregation population was constructed by using the mutant ebml and the DH line of Dioscorea sinensis as parents. 1502 individuals with mutant phenotypes were selected as localizing population and SSR markers were used. The ebwI gene was located between the SSRh1-53 and SSRh1-61 markers of chromosome A04, and the physical distance was 73.4 kb. There were 7 genes. 4. The coding sequences of 7 genes in the target region were cloned and sequenced. Only the cDNA sequence of the Bra032169 gene was different from that of the wild type in the mutant. Bra032169 encodes histone methyltransferase according to the gene annotation information. It is suggested that CURLYLEAF CLFN. CLF can regulate genes related to flowering (such as AGFLCL AGL19) in Arabidopsis thaliana. Bra032169 was predicted to be a candidate gene for ebml. A 53bp fragment was inserted into the 12th exon of the mutant Bra032169 gene, which led to the early termination of translation. The insertion mutation does not affect the transcription of the gene itself, and it may produce a protein that has lost its original function and affect the regulation of the downstream gene.
【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S634.1

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