本文選題：油菜　切入點：突變體sfz　出處：《江蘇大學》2017年碩士論文　論文類型：學位論文

【摘要】：油菜作為油料作物之一,在全球各地被廣泛種植。我國是植物油消費大國,大量植物性食用油需要進口,對外進口率超過65%,因此提高油菜產(chǎn)量以減少植物油進口量刻不容緩。油菜分枝與機械化收獲,提高生產(chǎn)效率相關,同時也影響油菜的產(chǎn)量,探究油菜分枝發(fā)育機理意義重大。本研究以少分枝突變體sfz作為實驗對象,克隆出BnSFZ基因,分析該基因的功能,從而豐富對油菜分枝發(fā)育的分子機制的了解。以下是本研究的主要部分:1.油菜突變體sfz的表型分析本實驗室已經(jīng)成功獲得含有激活標簽載體PXT246的少分枝突變體sfz。本研究對油菜sfz植株進行性狀統(tǒng)計得出油菜sfz的一次分枝數(shù)較少,一次分枝高度明顯高于野生型NY12植株。近紅外光譜檢測油菜sfz種子中脂肪酸各組成成分的含量與野生型植株無差別。突變體sfz植株表皮蠟質含量較少在菌核病接菌實驗中,突變體sfz葉片被較快侵染且菌斑面積較大。2.油菜sfz的蛋白質組差異表達分析蛋白二維電泳技術,分析突變體sfz蛋白表達差異,鑒定得到16個差異蛋白點,將差異蛋白點通過二級質譜分析,最終得到8個差異蛋白。對其進行理化性質、功能等分析發(fā)現(xiàn)差異蛋白與能量代謝、抗逆、抗病蟲、能量生成、生長發(fā)育等相關,在油菜生長發(fā)育和蠟質合成過程中可能發(fā)揮重要作用。3.油菜突變體sfz突變基因的克隆hiTAIL-PCR、FPNI-PCR技術擴增突變體sfz的T-DNA插入位點的旁側序列,經(jīng)過大量改進嘗試,最終通過FPNI-PCR方法克隆出側翼序列。軟件分析序列得到插入位點附近基因SFZ1、SFZ2、SFZ3、SFZ4。4.候選基因表達量及生物信息學分析qRT-PCR分析發(fā)現(xiàn)在油菜sfz植株中SFZ4基因表達量升高,其余無變化。將SFZ4作為最終候選基因并命名為BnSFZ。生物信息學分析發(fā)現(xiàn)BnSFZ蛋白屬于AP2大家族,在植物生長發(fā)育和蠟質合成中具有重要作用。5.候選基因互補實驗驗證候選基因BnSFZ的互補實驗驗證,構建BnSFZ的過表達和抑制表達載體,農(nóng)桿菌浸花法轉化到野生型油菜中。6.目標區(qū)域GWAS分析在油菜分枝數(shù)全基因組關聯(lián)分析結果的基礎上,選取插入位點前后20Kb序列中尋找SNPs和Indel位點,進而分析這些位點與植物分枝性狀的相關性。結果得出SFZ-SNP-18409641位點對分枝性狀影響顯著(P0.01),該位點位于BnSFZ基因的啟動子區(qū)域,說明在自然群體關聯(lián)分析中,BnSFZ基因與分枝數(shù)具有相關關系。本研究結果表明,突變體sfz中BnSFZ基因表達量升高,導致植株分枝減少,蠟質含量降低,推測該基因參與植物生長發(fā)育和蠟質合成途徑。本研究的開展有助于了解植物分枝發(fā)育及蠟質合成的分子機理。
[Abstract]:Rape, as one of the oil crops, is widely cultivated around the world. China is a big consumer of vegetable oil, and a large number of vegetable edible oils need to be imported. The foreign import rate is more than 65%, so it is urgent to increase rape yield so as to reduce the import of vegetable oil. Rape branching is related to mechanized harvesting, improving production efficiency, and also affecting rape yield. It is of great significance to explore the mechanism of branching development in rape. In this study, the BnSFZ gene was cloned and the function of the BnSFZ gene was analyzed by using the small branching mutant sfz as the experimental object. The following is the main part of this study: 1. Phenotypic analysis of rapeseed mutant sfz has been successfully obtained in our laboratory with the activation tag vector PXT246, the oligobranched mutant sfz. In this study, the characters of rapeseed sfz plants were analyzed and the results showed that the number of primary branches of sfz in rape was less. The primary branching height was significantly higher than that of wild type NY12 plants. The content of fatty acid components in sfz seeds of rape was not different from that of wild type plants by near infrared spectroscopy. The epidermal wax content of mutant sfz plants was less than that of wild type plants. The mutant sfz leaves were quickly infected and the plaque area was larger. 2.The proteome differential expression analysis of rapeseed sfz by two-dimensional electrophoresis was used to analyze the difference of sfz protein expression in the mutant, and 16 differentially expressed protein spots were identified. Eight differentially expressed proteins were obtained by secondary mass spectrometry. The physical and chemical properties and functions of differential proteins were analyzed. It was found that differential proteins were related to energy metabolism, stress resistance, disease and insect resistance, energy generation, growth and development, etc. It may play an important role in the growth, development and waxy synthesis of rapeseed. 3. Cloning of sfz mutant gene from rapeseed mutant (hiTAIL-PCRN-FPNI-PCR) was used to amplify the flanking sequence of T-DNA insertion site of mutant sfz, and a large number of attempts were made to improve it. Finally, the flanking sequence was cloned by FPNI-PCR method. The gene SFZ1, SFZ2, SFZ3, SFZ4.4near the insertion site was obtained by software analysis. The expression of candidate genes and bioinformatics qRT-PCR analysis showed that the expression of SFZ4 gene was increased in the sfz plants of Brassica napus. SFZ4 was the final candidate gene and named BnSFZ.The bioinformatics analysis found that the BnSFZ protein belonged to the AP2 family. It plays an important role in plant growth, development and waxy synthesis. 5. Candidate gene complementation test verifies candidate gene BnSFZ, constructs BnSFZ overexpression and inhibits expression vector. Transformation of Agrobacterium tumefaciens to wild rapeseed. Based on the results of genome association analysis of branch number of rape, SNPs and Indel loci were selected from 20kb sequence before and after insertion. The results showed that the SFZ-SNP-18409641 locus had a significant effect on the branching traits, which was located in the promoter region of the BnSFZ gene. The results of this study showed that the expression of BnSFZ gene in the mutant sfz increased, resulting in the decrease of plant branching and the decrease of waxy content, and the correlation between the BnSFZ gene and the number of branches was found in the natural population association analysis. It is speculated that this gene is involved in plant growth and waxy synthesis pathway. The development of this study is helpful to understand the molecular mechanism of plant branching and waxy synthesis.
【學位授予單位】：江蘇大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S565.4

【參考文獻】

相關期刊論文 前10條

1 王東陽;邵淑君;季娜娜;張新華;李富軍;;植物表皮蠟質合成與分泌基因研究進展[J];植物生理學報;2016年06期

2 奧娜;趙泓;裴雁曦;王永勤;;大蔥AfCER1基因的克隆及表達分析[J];山西農(nóng)業(yè)科學;2016年05期

3 劉小鳳;安靜波;張立新;王文嬌;徐沖;任華中;;黃瓜調控蠟質合成相關基因CsCER7的克隆與表達分析[J];園藝學報;2014年04期

4 曾秀存;劉自剛;史鵬輝;許耀照;孫佳;方彥;楊剛;武軍艷;孔德晶;孫萬倉;;白菜型冬油菜銅鋅超氧化物歧化酶(Cu/Zn-SOD)基因的克隆及其在低溫條件下的表達[J];作物學報;2014年04期

5 王漢中;;我國油菜產(chǎn)業(yè)發(fā)展的歷史回顧與展望[J];中國油料作物學報;2010年02期

6 朱再清;;我國油菜籽及菜籽油的比較優(yōu)勢及國際競爭力分析[J];華中農(nóng)業(yè)大學學報(社會科學版);2009年06期

7 徐鴻林;翟紅利;王鋒;樸建華;楊曉光;朱禎;;豇豆胰蛋白酶抑制劑基因(cpti)及其在抗蟲轉基因作物中的應用[J];中國農(nóng)業(yè)科技導報;2008年01期

8 宋福南;楊傳平;劉雪梅;李公斌;;鹽脅迫對白榆超氧化物歧化酶(SOD)活性的影響(英文)[J];Journal of Forestry Research;2006年01期

9 王競,康莊,廖海,張年輝,杜林方;白菜型油菜種子胰蛋白酶抑制劑純化及部分性質研究[J];天然產(chǎn)物研究與開發(fā);2005年03期

10 鞏鵬濤,李迪;植物分枝發(fā)育的遺傳控制[J];分子植物育種;2005年02期

相關碩士學位論文 前1條

1 馬全兵;家蠶脂肪體變態(tài)發(fā)育的蛋白質組研究及Hemolin與Yippee蛋白相互作用[D];江蘇大學;2016年

，

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