發(fā)布時間：2018-03-07 17:55

  本文選題：番茄　切入點：螺旋生長　出處：《華中農(nóng)業(yè)大學》2017年碩士論文　論文類型：學位論文

【摘要】：番茄是一種重要的經(jīng)濟作物。植株的螺旋生長往往對植株的生長發(fā)育產(chǎn)生重要的影響。在擬南芥中很多螺旋生長相關(guān)的分子機制已經(jīng)研究清楚,但是對于番茄螺旋生長的調(diào)控途徑還沒有相關(guān)的報道。對番茄植株螺旋生長的研究不僅具有重要的理論意義,而且了解突變體螺旋生長的突變機制可以為進一步了解茄科物種的生長習性奠定了良好的基礎。螺旋突變體的hel還可以為進一步深入研究番茄微管功能多樣性以及激素控制植株株型的研究提供珍貴的實驗材料。本研究以hel突變體為研究對象,通過對突變體進行表型鑒定,遺傳分析,同時和野生型材料LA1589雜交構(gòu)建F2代分離群體,開發(fā)分子標記克隆目的基因,探究突變體的突變機制。為進一步深入研究番茄微管功能多樣以及激素控制植株株型的研究提供一定的理論依據(jù)。主要研究結(jié)果如下:1、hel突變體表型穩(wěn)定,其幼苗期子葉和葉柄較正常植株均出現(xiàn)了右手螺旋生長。對突變體整個生育期進行觀察發(fā)現(xiàn)其莖也出現(xiàn)了右手螺旋的彎曲,該突變表型持續(xù)整個生育期。2、對hel突變體幼苗子葉的主葉脈與葉柄之間的夾角測量發(fā)現(xiàn)突變體hel兩片子葉的螺旋角度平均值分別約為39.8°和40.1°,這表明突變體子葉的螺旋生長是左右對稱的。3、取螺旋生長的突變體的葉柄做石蠟切片觀察發(fā)現(xiàn)葉柄螺旋生長部位的表皮細胞排列不均勻,并且出現(xiàn)了弧形排列。4、以hel突變體為父本,野生型材料LA1589為母本進行雜交,構(gòu)建F2遺傳分離群體。遺傳分析表明hel螺旋生長的突變表型是由隱性基因控制。5、通過BSR-Seq的方法進行測序分析發(fā)現(xiàn)四號染色體上出現(xiàn)了明顯的SNP位點,這表明目的基因在四號染色體上面。6、利用番茄基因組數(shù)據(jù)在四號染色體上開發(fā)三種標記用于目的基因定位。包括InDel、SNP、CAPS標記。首先用8對具有穩(wěn)定多態(tài)性的InDel標記對從hel×LA1589的F2遺傳群體中分離出的具有子葉以及莖螺旋生長表型的108棵單株進行遺傳連鎖分析,將hel基因定位于兩個標記CH4-25和CH4-35之間,遺傳距離約為2.4cM;進一步擴大F2代作圖群體,開發(fā)新的SNP、CAPS標記,進一步用從F2代群體中分離的具有突變體表型的1136棵單株用于遺傳連鎖分析,最終把目的基因hel精確定位于標記SNP4-6和SNP4-2之間約389kb的物理區(qū)間內(nèi)。7、利用基因分析與預測網(wǎng)站,在標記SNP4-6和SNP4-2之間預測到20個開放閱讀框(ORFs)。通過對部分開放閱讀框(ORFs)在CR291和hel突變體中的表達量分析表明ORF2在突變體hel中的表達量顯著低于其背景材料CR291中的表達量。進一步開發(fā)覆蓋候選基因ORF2編碼區(qū)全長的特異性引物,以hel突變體以及其背景材料CR291為模板測擴增和測序,通過基因序列的比對我們發(fā)現(xiàn)ORF2核苷酸序列在hel突變體以及其背景材料CR291有18個氨基酸的變化。我們進一步預測其氨基酸序列,結(jié)果顯示hel突變體氨基酸序列較CR291發(fā)生了大片段的缺失。8、通過液相色譜法測定hel突變體以及其背景材料CR291生長素含量,結(jié)果顯示hel突變體中生長素的含量大約只有對照CR291的三分之一,即與其背景材料CR291相比,螺旋生長突變體hel生長素的含量顯著降低。9、通過BSR-seq數(shù)據(jù)分析hel突變體以及CR291中生長素生物合成以及信號轉(zhuǎn)導途徑中關(guān)鍵基因表達變化。其中生長素生物合成途徑中的P450和ISS1在hel突變體中的表達量顯著高于對照材料CR291。AMI1的表達量在hel突變體中的表達量顯著低于對照材料CR291。在生長素信號轉(zhuǎn)導途徑中,ARF11在hel突變體中的表達量顯著低于對照材料CR291。而BPS1和AXR3的結(jié)果剛好相反,在hel突變體中的表達量顯著高于對照材料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..

【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S641.2;Q943.2

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