發(fā)布時間：2018-04-27 11:39

  本文選題：黃瓜 + 刺瘤密度�。� 參考：《山東農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：果實刺瘤密度是黃瓜(Cucumis sativus L.)重要的外觀商品性狀。然而到目前為止,還未見調(diào)控黃瓜刺瘤密度的相關(guān)基因被克隆的報道,黃瓜刺瘤密度形成的分子機制仍然未知。因此,對黃瓜稀刺基因定位、克隆,有助于研究黃瓜刺瘤密度形成的分子機制,同時為刺瘤密度分子育種提供理論依據(jù)和技術(shù)支持。本研究從華北型密刺黃瓜CNS2(wild type,WT)中分離出一個稀刺突變體,few spines 1(fs1)。fs1果實刺瘤數(shù)目顯著少于WT,而莖、葉、卷須、花萼等其它部位的表皮毛形態(tài)、密度均無顯著變化,是研究黃瓜果實刺瘤密度形成機理的理想材料。用華北型密刺黃瓜CNS2、9930分別與fs1雜交構(gòu)建了2個F2群體,通過BSA(Bulk Segregant Analysis)法結(jié)合基因組重測序?qū)�fs1進行了初步定位,然后利用圖位克隆技術(shù)分離出稀刺基因。同時,對密刺表型和稀刺表型果實混池進行轉(zhuǎn)錄組測序,找到了部分受稀刺基因影響的基因。為了研究稀刺基因的功能,還進行了黃瓜的遺傳轉(zhuǎn)化進行互補驗證。具體研究結(jié)果如下:1.構(gòu)建了稀刺突變體和其親本的F2群體,取稀刺和密刺表型個體葉片混池,提取DNA后重測序,將獲得的SNV進行關(guān)聯(lián)分析,fs1被定位到黃瓜6號染色體上。2.由于fs1與WT的基因組相似度較高,在開發(fā)標記時未找到合適可用的標記來進行下一步的精細定位工作。因此,將fs1和9930進行雜交,構(gòu)建了新的精細定位群體。利用黃瓜基因組重測序得到的SNP數(shù)據(jù),找到了4個CAPS標記和1個SNP標記。用CAPS1和MM2對5400棵F2單株進行篩選,找到130棵交換個體。用CAPS3、CAPS4和MM2對32個稀刺表型交換單株進行分析,將稀刺基因fs1定位到了MM1和MM2標記之間,這兩個標記之間的物理距離為110.4 kb,有25個注釋基因。3.通過對候選區(qū)間內(nèi)25個基因的編碼區(qū)和啟動子區(qū)域測序,將WT和fs1的序列進行比對,結(jié)果發(fā)現(xiàn),Csa6M514870(CsHDZIV11/CsGL3/Tril)的啟動子區(qū)域內(nèi)發(fā)生了片段的替換,WT中10 bp的片段在fs1中被替換成了812 bp。Csa6M514870屬于HD-ZIP IV亞家族,編碼了一個PDF2相關(guān)蛋白。在開花當(dāng)天的果實中,fs1中的Csa6M514870的表達顯著高于WT。這表明,Csa6M514870是最佳候選基因。4.為了進一步分析CsHDZIV11在黃瓜中的功能,進行了黃瓜的遺傳轉(zhuǎn)化。目前已得到20棵再生植株,有待于進一步驗證。5.對稀刺突變體的轉(zhuǎn)錄組數(shù)據(jù)進行分析并和已發(fā)表的黃瓜表皮毛相關(guān)轉(zhuǎn)錄組或表達譜數(shù)據(jù)比較,找到了四個可能參與黃瓜刺瘤密度形成調(diào)控網(wǎng)絡(luò)的基因:Csa5M606310、Csa6M501990、Csa3M101810和Csa3M824990。6.根據(jù)CsHDZIV11的啟動子區(qū)域內(nèi)的片段替換,開發(fā)了一個分子標記MM3。經(jīng)檢測,這個標記和來自不同地區(qū)不同品種的果實刺瘤密度共分離。另外,這一片段也存在于野生黃瓜(C.sativus var.hardwickii)中,表明CsHDZIV11的啟動子區(qū)域內(nèi)的片段替換是黃瓜馴化過程中決定果刺密度的關(guān)鍵因素。本研究不僅定位、克隆了控制果刺密度的最佳候選基因,為研究黃瓜刺瘤密度形成的分子機制奠定了基礎(chǔ);而且找到了決定刺瘤稀密的關(guān)鍵因素,為黃瓜刺瘤密度分子標記輔助育種提供了一個有效的分子標記。
[Abstract]:The fruit prickle density is an important appearance of Cucumis sativus L.. However, up to now, there has not been a report on the cloning of the related genes regulating the density of cucumbers. The molecular mechanism of the formation of cucumber prickle density is still unknown. Therefore, the location and cloning of the cucumbers' dilute prickly gene can help to study the formation of cucumber tumor density. In this study, a rare spiny mutant was isolated from CNS2 (wild type, WT) in North China type, and the number of few spines 1 (FS1).Fs1 fruits was significantly less than WT, while the density of other parts of the stem, leaf, tendril, calyx and other parts were not significant. The change is an ideal material to study the formation mechanism of the density of the cucumber fruit. 2 F2 populations were constructed with the North China dense prickly cucumber CNS29930 and FS1, respectively. The preliminary location of FS1 was carried out by BSA (Bulk Segregant Analysis) method combined with genome sequencing, and then the thin prickly gene was separated by the technique of graphic cloning. The transcriptional group was sequenced by the phenotypic and dilute phenotypic fruit mixed pool, and some genes affected by the dilute prickly gene were found. In order to study the function of the dilute spiny gene, the genetic transformation of the cucumber was also carried out. The specific results were as follows: 1. the sparse thorn mutants and their parent F2 populations were constructed, and the individual leaves of dilute spines and prickles were obtained. In the mixed pool, the DNA was sequenced and the obtained SNV was associated. The FS1 was located on the cucumber chromosome 6 and.2., because of the high similarity between the FS1 and the WT genome, did not find the appropriate marker for the next step in the development of the marker. Therefore, the FS1 and 9930 were hybridized to a new fine positioning group. Using SNP data obtained from cucumber genome re sequencing, 4 CAPS markers and 1 SNP markers were found. 5400 F2 single strains were screened with CAPS1 and MM2, and 130 exchange individuals were found. CAPS3, CAPS4 and MM2 were used to analyze 32 barbaric phenotypes, and the dilute prickly gene FS1 was determined between MM1 and MM2 markers, and the two markers were between these markers. The physical distance was 110.4 KB, and 25 annotated genes,.3., were sequenced by the sequence of the encoding and promoter regions of the 25 genes in the candidate region. The sequence of WT and FS1 was compared. The results showed that the substitution of fragments occurred in the promoter region of Csa6M514870 (CsHDZIV11/CsGL3/Tril), and the fragment of 10 BP in WT was replaced with 812 bp.Cs in FS1. A6M514870 belongs to the HD-ZIP IV subfamily and encodes a PDF2 related protein. In the fruit of the day of flowering, the expression of Csa6M514870 in FS1 is significantly higher than that of WT., indicating that Csa6M514870 is the best candidate gene,.4. in order to further analyze the function of CsHDZIV11 in cucumbers, and to carry out genetic transformation of yellow gourd. At present, 20 plants have been regenerated, To further verify.5.'s analysis of the transcriptional data of the dilute mutants and compare with the published data of the transcriptional or expression profiles of the cucumber surface fur, four genes may be found that may participate in the regulation network for the formation of cucumber tumor density: Csa5M606310, Csa6M501990, Csa3M101810 and Csa3M824990.6. based on the start of CsHDZIV11 Fragment replacement in the subregion, a molecular marker MM3. was developed to detect the total separation of the fruit prickle density from different varieties of different regions. In addition, this fragment also existed in the wild cucumber (C.sativus var.hardwickii), indicating that the fragment replacement in the promoter region of the CsHDZIV11 was determined during the domestication of the cucumber. This study not only localizes, but also clones the best candidate genes to control the density of fruit thorn, which lays the foundation for the study of the molecular mechanism of the formation of cucumber prickle density, and has found the key factor determining the dilute density of the stab tumor, which provides an effective molecular marker for the molecular marker assisted breeding of cucumber tumor density.

【學(xué)位授予單位】：山東農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S642.2;Q943.2

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