番茄植株螺旋生长突变体hel遗传分析与基因克隆

发布时间：2018-03-07 17:55

本文选题：番茄　切入点：螺旋生长　出处：《华中农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：番茄是一种重要的经济作物。植株的螺旋生长往往对植株的生长发育产生重要的影响。在拟南芥中很多螺旋生长相关的分子机制已经研究清楚,但是对于番茄螺旋生长的调控途径还没有相关的报道。对番茄植株螺旋生长的研究不仅具有重要的理论意义,而且了解突变体螺旋生长的突变机制可以为进一步了解茄科物种的生长习性奠定了良好的基础。螺旋突变体的hel还可以为进一步深入研究番茄微管功能多样性以及激素控制植株株型的研究提供珍贵的实验材料。本研究以hel突变体为研究对象,通过对突变体进行表型鉴定,遗传分析,同时和野生型材料LA1589杂交构建F2代分离群体,开发分子标记克隆目的基因,探究突变体的突变机制。为进一步深入研究番茄微管功能多样以及激素控制植株株型的研究提供一定的理论依据。主要研究结果如下:1、hel突变体表型稳定,其幼苗期子叶和叶柄较正常植株均出现了右手螺旋生长。对突变体整个生育期进行观察发现其茎也出现了右手螺旋的弯曲,该突变表型持续整个生育期。2、对hel突变体幼苗子叶的主叶脉与叶柄之间的夹角测量发现突变体hel两片子叶的螺旋角度平均值分别约为39.8°和40.1°,这表明突变体子叶的螺旋生长是左右对称的。3、取螺旋生长的突变体的叶柄做石蜡切片观察发现叶柄螺旋生长部位的表皮细胞排列不均匀,并且出现了弧形排列。4、以hel突变体为父本,野生型材料LA1589为母本进行杂交,构建F2遗传分离群体。遗传分析表明hel螺旋生长的突变表型是由隐性基因控制。5、通过BSR-Seq的方法进行测序分析发现四号染色体上出现了明显的SNP位点,这表明目的基因在四号染色体上面。6、利用番茄基因组数据在四号染色体上开发三种标记用于目的基因定位。包括InDel、SNP、CAPS标记。首先用8对具有稳定多态性的InDel标记对从hel×LA1589的F2遗传群体中分离出的具有子叶以及茎螺旋生长表型的108棵单株进行遗传连锁分析,将hel基因定位于两个标记CH4-25和CH4-35之间,遗传距离约为2.4cM;进一步扩大F2代作图群体,开发新的SNP、CAPS标记,进一步用从F2代群体中分离的具有突变体表型的1136棵单株用于遗传连锁分析,最终把目的基因hel精确定位于标记SNP4-6和SNP4-2之间约389kb的物理区间内。7、利用基因分析与预测网站,在标记SNP4-6和SNP4-2之间预测到20个开放阅读框(ORFs)。通过对部分开放阅读框(ORFs)在CR291和hel突变体中的表达量分析表明ORF2在突变体hel中的表达量显著低于其背景材料CR291中的表达量。进一步开发覆盖候选基因ORF2编码区全长的特异性引物,以hel突变体以及其背景材料CR291为模板测扩增和测序,通过基因序列的比对我们发现ORF2核苷酸序列在hel突变体以及其背景材料CR291有18个氨基酸的变化。我们进一步预测其氨基酸序列,结果显示hel突变体氨基酸序列较CR291发生了大片段的缺失。8、通过液相色谱法测定hel突变体以及其背景材料CR291生长素含量,结果显示hel突变体中生长素的含量大约只有对照CR291的三分之一,即与其背景材料CR291相比,螺旋生长突变体hel生长素的含量显著降低。9、通过BSR-seq数据分析hel突变体以及CR291中生长素生物合成以及信号转导途径中关键基因表达变化。其中生长素生物合成途径中的P450和ISS1在hel突变体中的表达量显著高于对照材料CR291。AMI1的表达量在hel突变体中的表达量显著低于对照材料CR291。在生长素信号转导途径中,ARF11在hel突变体中的表达量显著低于对照材料CR291。而BPS1和AXR3的结果刚好相反,在hel突变体中的表达量显著高于对照材料CR291。
[Abstract]:Tomato is one of the most important economic crops. Spiral growth plants tend to plant growth and development have an important impact. In Arabidopsis many spiral growth related molecular mechanism has been well studied, but there is no relevant reports for tomato growth. The spiral regulation approach not only has important theoretical significance to study the spiral growth of Tomato plants the mutation and understanding the mechanism of mutant spiral growth can lay a good foundation for the further understanding of the growth habit of Solanaceae species. Spiral mutant hel can also provide valuable experimental materials for further research on Tomato microtubule function diversity and hormone control plant was studied. In this study, the hel mutant as the research object. Through phenotypic identification, genetic analysis of the mutant and the wild type at the same time, the material LA1589 hybrid construct F2 segregating population, open With molecular markers of gene cloning, mutation mechanism of mutants. Provide a theoretical basis for the functional diversity and microtubule hormonal control of plant type research to further study of tomato. The main results are as follows: 1. The hel mutant is stable, the Seedling Cotyledon and petiole than normal plants showed a right spiral growth. The mutants were observed during the whole growth period, the stem also appeared in the right-hand bend, the mutant phenotype continued throughout the growth period of.2, measuring the angle between the main veins and petioles of cotyledons of Hel mutant found helix angle mutant hel two cotyledons were averaged about 39.8 degrees and 40.1 degrees, which indicates that the spiral mutant is symmetric about the growth of cotyledon petiole from.3, spiral growth mutant paraffin sections were observed petiole epidermal cells arranged in spiral growth parts Not even, and the emergence of an arc of.4, using hel mutant as the male parent, wild type material LA1589 as the female parent, the construction of F2 genetic segregation population. Genetic analysis indicated that the hel spiral growth of the mutant phenotype was controlled by recessive genes.5, sequencing analysis showed that there was SNP significant loci on chromosome four by BSR-Seq this method indicated that the target gene on chromosome four, above.6, the development of three kinds of markers for the target gene located on chromosome four using tomato genomic data. Including InDel, SNP, CAPS mark. First used on isolated from F2 hel * LA1589 genetic groups in the cotyledons and stems with spiral growth phenotypes of 108 trees plant genetic linkage analysis of 8 InDel markers with stable polymorphism, hel gene was located between two markers CH4-25 and CH4-35, the genetic distance is about 2.4cM; to further expand the F2 mapping The development of the new group, SNP, CAPS mark, further separated from the F2 population with 1136 mutant plants for genetic linkage analysis, finally the accurate positioning of the target gene hel between markers SNP4-6 and SNP4-2 physical interval within approximately 389kb.7, using gene analysis and prediction of the site, between the markers SNP4-6 and SNP4-2 forecast to 20 open reading frames (ORFs). Through the part of the open reading frame (ORFs) expression in CR291 and hel mutants in the analysis of expression of ORF2 in mutant hel showed significantly lower than the expression of CR291 in the background. The further development of specific primers covering full-length candidate gene ORF2 encoding the hel mutant and its background CR291 template for amplification and sequencing test based on the gene sequence, we found that the nucleotide sequence of ORF2 18 hel and ammonia in the mutant background material CR291 The change of amino acid. We further predicted its amino acid sequence, revealed the large fragment deletion.8 mutant hel amino acid sequence is CR291 method for the determination of Hel mutant and its background material CR291 IAA content by HPLC, the results showed that 1/3 growth hormone content is only about CR291 control of the hel mutants, compared to that of its background material CR291, hel mutant auxin content growth spiral.9 decreased by BSR-seq data analysis, key based hel and CR291 mutant auxin biosynthesis and signal transduction pathways for the expression changes. The expression of the auxin biosynthetic pathway in the expression of P450 and ISS1 in Hel were significantly higher than the expression of CR291.AMI1 hel in the control of materials the mutant was significantly lower than the control material CR291. in the auxin signal transduction pathway, ARF11 mutation in hel The amount of expression in the body was significantly lower than that of the control material CR291., while the results of BPS1 and AXR3 were just opposite, and the expression in the hel mutant was significantly higher than that of the control material CR291..

【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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