番茄杂色叶基因vg的遗传定位分析

发布时间：2018-03-14 18:44

本文选题：番茄　切入点：叶色突变体　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：番茄是重要的蔬菜作物,植株光合效率对番茄的产量及品质有着极其重要的影响,而叶片是其光合积累有机物的场所。叶色突变是比较常见并容易观察表型的一类突变体,该类突变体的形成是由于叶绿素含量、叶绿体发育以及光合作用等过程受到影响导致,从而影响作物品质或产量。本研究以在遗传转化过程发现的一株番茄杂色叶突变体vg为研究对象,通过从生理、遗传、及分子水平对突变体进行表型鉴定、突变原因、遗传分析、基因定位、功能验证等研究,了解vg突变体杂色叶产生的机制,为研究番茄光合途径及效率提供理论基础。主要的研究结果如下:1.番茄叶色突变体vg的表型:vg突变体表现为苗期正常,在播种后一个月左右开始出现叶色变化,首先是刚成熟的叶片出现部分泛白,随着幼苗生长有泛白的叶片增多,并且出现包括生长点的幼叶出现黄化,植株长势受到抑制。秋季育苗高温强光照条件下,表型更加明显,生长势更弱。2.番茄叶色突变体vg的生理特性分析:观察叶绿体的超微结构发现与AC相比,vg突变体黄化叶片的叶绿体形态异常,没有类囊体片层形成;vg突变体泛白叶片和正常绿色叶片的叶绿体形态基本正常,但泛白叶片叶绿体的类囊体片层的排列分散、类囊体基粒较少;正常绿色叶片叶绿体的类囊体片层排列不紧密。且在vg突变体的叶片中,没有明显可见的淀粉粒形成。分别测定三种叶色的叶绿素含量发现,黄化叶片的叶绿素a、叶绿素b都显著降低,泛白叶片的叶绿素b降低,正常绿色叶片的叶绿素含量基本不变。且正常绿色叶片的可溶性糖含量以及淀粉含量都明显减少。3.番茄叶色突变体vg的共分离及遗传分析表明:vg突变体的表型与转入的基因没有关系,且不是T-DNA插入导致的突变,而是遗传转化过程引起的突变。以LA1589为母本,vg突变体为父本构建F_2遗传分离群体,分析表明vg突变体的突变表型是单核基因控制的隐性性状。4.利用分离群体基因定位:从F_2群体分离的45株有突变表型的单株,以及在每条染色体上开发并筛选出具有稳定多态性的2个In Del标记,将目的基因定位在第7条染色体。进一步开发筛选了15对In Del分子标记,结果将目的基因定位在7-59980和7-60636两个标记之间物理距离为756 kb的区段。扩大分离F_2群体,分离出了182株有突变表型的单株用于精细定位,利用1个In Del和8个CAPS分子标记,将目的基因定位在s6027和Ba6040两个标记之间物理距离为128 kb的区段。5.基因侯选:利用预测网站和基因序列分析,分析可知在目的区段有21个开放阅读框(ORFs)。其中ORF10、ORF11、ORF12和ORF13与叶绿体发育或叶绿素合成相关,而已有研究表明ORF10的同源基因在拟南芥中导致类囊体形成受到阻碍。在突变体和AC中扩增4个基因的编码序列以及ORF10的启动子序列,均未发现碱基序列上的差异。6.基因时空表达分析:利用q RT-PCR分析除伤诱导相关基因外的12个基因在AC和突变体中的表达量。结果显示ORF9、ORF10和ORF13的表达量显著降低,ORF11的表达量表现显著升高。而ORF9在ORF10的启动子上,ORF10编码类囊体形成蛋白。因此,推测ORF10可能是导致突变表型的目的基因。7.利用VIGS技术验证候选基因功能:构建了ORF10和ORF13的p TRV2病毒表达载体,以AC为浸染受体。结果显示,AC沉默ORF13不会产生突变表型,沉默ORF10使AC表现与突变表型相似的表型。因此,确定ORF10是导致突变表型的目的基因。
[Abstract]:Tomato is an important vegetable crop, has a very important effect on plant photosynthetic efficiency on Tomato Yield and quality, and the leaf photosynthetic organic matter accumulation in place. Leaf color mutation is a relatively common and easy to observe the mutant phenotype, the formation of this type of mutant is due to the chlorophyll content, chloroplast development and the photosynthesis process affected, thus affecting the quality of crops or production. In this study, a strain of tomato genetic transformation process found in variegated leaf mutant VG as the research object, from the physiological, genetic, and molecular phenotype identification of mutants, genetic analysis, gene mapping, research on functional verification. To understand the mechanism of VG mutant variegated leaves, provide a theoretical basis for the study of tomato photosynthetic pathway and efficiency. The main results are as follows: 1. the phenotype of tomato leaf color mutant VG: VG mutation The body is normal seedling after sowing, in a month or so began to leaf color changes, the first is just part of mature leaves appear white, with white leaf seedling growth have increased, and including the growing point of leaf etiolation, plant growth was inhibited. The autumn Ji Yumiao high temperature high light conditions, the phenotype is more obvious analysis of physiological characteristics, the growth potential of weaker.2. tomato leaf color mutant VG: the ultrastructure of chloroplasts found that compared with AC, the abnormal chloroplast morphology of VG mutant leaves, no thylakoid lamella formation; chloroplast morphology of VG mutant white leaf and green leaf basically normal, but the chloroplast thylakoids of white leaf the scattered arrangement, the grana lamella is less; the normal arrangement of green leaf chloroplast was not close. And in the leaves of VG mutants, not visible The starch grains formed. Chlorophyll content of three leaf color were measured, yellow leaf chlorophyll a, chlorophyll b decreased significantly, white leaf chlorophyll b decreased, chlorophyll content of normal green is basically the same. And the content of soluble sugar in leaves of normal green and starch content were significantly reduced in total isolation and genetic analysis.3. tomato leaf color mutant VG showed that Never mind VG mutant phenotype and gene, and not the T-DNA insertion mutations resulting in mutation, but genetic transformation process. By taking LA1589 as female parent and VG as male parent mutant F_2 segregation population construction, analysis suggested that the mutant phenotype of VG mutant gene mapping using isolated populations.4. is a single recessive nuclear gene control: isolated from F_2 group with single mutant phenotypes of 45 strains, and in each chromosome and screened with stable development 2 In Del marked state of the target gene, located in chromosome seventh. Further screening 15 pairs of In Del molecular marker, gene localization results will be in the physical distance between 7-59980 and 7-60636 of two markers was 756 KB. The section expanded separation of F_2 groups, 182 strains were isolated from the plants with mutant phenotype for fine mapping, using 1 In Del and 8 CAPS markers, the target gene was mapped between s6027 and Ba6040 two marks the physical distance of 128 KB candidate region of the.5. gene using predictive analysis and site selection: gene sequence analysis shows that there are 21 open reading frames in the head section (ORFs) the ORF10, ORF11, ORF12 and ORF13 and chloroplast development or chlorophyll synthesis related, it has been reported that ORF10 homologous gene in Arabidopsis led to the formation of thylakoids is hindered. Amplification of 4 genes encoding sequence in the mutant and AC ORF10 and the promoter sequences were not found differential expression analysis of.6. gene sequence on time: the use of Q RT-PCR analysis in the expression of 12 genes induced by injury related genes outside the AC and in mutants. The results showed that the ORF9 expression of ORF10 and ORF13 were significantly decreased and the expression of ORF11 increased significantly while ORF9 in the ORF10 promoter, ORF10 encoding thylakoid formation protein. Therefore, we speculated that ORF10 may lead to.7. gene mutation phenotype using validation of candidate gene VIGS Technology: Construction of ORF10 and ORF13 P TRV2 virus expression vector with AC disseminated receptor. The results showed that AC silencing ORF13 does not produce mutations the silencing of ORF10 phenotype, AC and mutant phenotype similar phenotypes. Therefore, ORF10 is the result of gene mutation phenotype.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S641.2;Q943.2

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