本文選題：甘藍(lán)型油菜 + 干旱　； 參考：《華中農(nóng)業(yè)大學(xué)》2016年博士論文

【摘要】：油菜(Brassica napus L.)是重要的油料作物之一,地位僅次于大豆,中國(guó)是最大的油菜生產(chǎn)國(guó)。我國(guó)油菜主產(chǎn)區(qū)長(zhǎng)江中下游地區(qū)由于降水不均、季節(jié)性干旱造成油菜矮化、不實(shí),是油菜減產(chǎn)和含油量下降的最主要因素。挖掘油菜抗旱基因是研究油菜抗逆育種的基礎(chǔ)�；蜣D(zhuǎn)錄表達(dá)水平調(diào)節(jié)是植物從分子水平上響應(yīng)干旱脅迫的重要機(jī)制,因此通過(guò)轉(zhuǎn)錄水平上的表達(dá)差異來(lái)篩選和鑒定抗旱相關(guān)的基因,是挖掘抗旱基因、研究抗旱機(jī)理、提高抗旱育種的重要手段。甘藍(lán)型油菜Q2是具有較強(qiáng)抗性的自交品系,在干旱條件下表現(xiàn)較高的相對(duì)含水量(RWC)、抗旱指數(shù)(DRI)以及相對(duì)較低的電解質(zhì)滲漏率。利用Q2應(yīng)對(duì)干旱脅迫,研究干旱脅迫的分子機(jī)理,挖掘重要的抗旱相關(guān)基因以及轉(zhuǎn)錄調(diào)控元件,為抗旱育種提供理論依據(jù)和技術(shù)支持。本研究以抗旱型甘藍(lán)型油菜Q2為實(shí)驗(yàn)材料,對(duì)空氣干旱24h和對(duì)照條件下的根和葉片分別提取RNA,進(jìn)行Illumina第二代高通量測(cè)序,經(jīng)過(guò)序列比對(duì)、分析,篩選干旱脅迫條件下的差異表達(dá)基因,并通過(guò)突變體抗旱性篩選抗旱基因,挑選了5個(gè)候選基因,并對(duì)部分基因進(jìn)行轉(zhuǎn)基因驗(yàn)證,主要結(jié)果如下:1.甘藍(lán)型油菜根和葉片干旱脅迫條件下的轉(zhuǎn)錄組分析以抗旱型甘藍(lán)型油菜Q2為材料,基于Illumina第二代高通量測(cè)序RNA-seq技術(shù),獲得Q2響應(yīng)干旱脅迫轉(zhuǎn)錄本序列信息,根和葉片空氣干旱24h和對(duì)照處理總共4個(gè)樣本分別得到12,308,861、11,763,435、11,905,805、12,168,994條clean reads。測(cè)序質(zhì)量評(píng)估結(jié)果表明,測(cè)序深度達(dá)到10M,而且70%序列比對(duì)到參考基因組,測(cè)序質(zhì)量良好。以白菜基因組數(shù)據(jù)庫(kù)為參考,對(duì)以上兩組轉(zhuǎn)錄組數(shù)據(jù)庫(kù)進(jìn)行比對(duì),根和葉片中分別獲得31,240和31,118個(gè)基因。以FDR≤0.001和|log2Ratio|≥1為標(biāo)準(zhǔn),根和葉片中分別篩選得到6018和5377個(gè)差異表達(dá)基因。其中,根中上調(diào)基因2448個(gè),下調(diào)基因3570個(gè);葉片中上調(diào)基因3770個(gè),下調(diào)基因1607個(gè)。根中下調(diào)基因數(shù)目是葉片中的兩倍,初步顯示了根和葉片干旱脅迫下的調(diào)控模式存在差異。GO功能富集分析發(fā)現(xiàn),大部分差異表達(dá)基因跟脅迫響應(yīng)、刺激響應(yīng)相關(guān);Blast2GO分析表明,根中12條顯著富集的基因功能中,上調(diào)基因富集主要跟脅迫、刺激相關(guān),而下調(diào)基因主要跟細(xì)胞、功能相關(guān);相反地,葉片中上調(diào)基因主要富集在細(xì)胞結(jié)構(gòu)功能,下調(diào)基因主要富集脅迫、刺激功能,顯示了干旱脅迫條件下根和葉片分工應(yīng)答脅迫的模式。KEGG富集結(jié)果表明,差異表達(dá)基因主要富集于植物激素信號(hào)轉(zhuǎn)導(dǎo)路徑和苯丙素合成路徑。根和葉片中大量轉(zhuǎn)錄因子應(yīng)激表達(dá),包括MYB、b HLH、C2H2、AP2/EREBP、NAC等。其中,AP2/EREBP和WRKY在根和葉片相反調(diào)控模式中發(fā)揮作用。轉(zhuǎn)錄因子結(jié)合下游干旱誘導(dǎo)基因啟動(dòng)子區(qū)順式作用元件,誘導(dǎo)下游一些基因表達(dá),包括ERD1、RAB18、RD29B、RD20A、JA-related基因,最終調(diào)控細(xì)胞中Cl-、NO3-、K+、Ca2+等離子水平,改變細(xì)胞膨壓,增加根中細(xì)胞吸水,關(guān)閉氣孔減少葉片蒸騰失水。2.候選基因篩選根據(jù)轉(zhuǎn)錄組結(jié)果log2Ratio和RPKM值,從差異表達(dá)基因中挑選出與干旱相關(guān)的5個(gè)候選基因TMA7(Bra026774)、BAM3(Bra012676)、SERPIN1(Bra015607)、Peroxidase(Bra009221)、Unknown(Bra009013),功能分別為翻譯相關(guān)、B-淀粉酶、絲氨酸蛋白酶抑制劑、過(guò)氧化物酶以及功能未知蛋白。將候選基因CDS序列比對(duì)到甘藍(lán)型油菜數(shù)據(jù)庫(kù),通過(guò)序列比對(duì)、測(cè)序、克隆,最終確定在甘藍(lán)型油菜Q2中的拷貝數(shù):TMA7有6個(gè)拷貝,BAM3有2個(gè)拷貝,SERPIN1有2個(gè)拷貝,Peroxidase有6個(gè)拷貝,Unknown基因1個(gè)拷貝。3.突變體干旱脅迫條件下的表型觀察根據(jù)5個(gè)候選基因與擬南芥同源基因的信息購(gòu)買(mǎi)突變體tma7(SALK_044092C)、bam3(SALK_041214C)、serpin1(SALK_019150C)、peroxidase(SALK_124744)、un-2(SALK_121527C),觀察干旱條件下突變體表型發(fā)現(xiàn),相比較野生型擬南芥(Col),突變體離體葉片失水率更高、花期提前,表現(xiàn)較強(qiáng)的干旱敏感性,干旱復(fù)水后提早結(jié)實(shí);un-2突變體尤其突出,苗子離體失水后快速皺縮萎蔫。4.候選基因的表達(dá)和功能研究將與白菜候選基因同源性最相近的甘藍(lán)型油菜基因TMA7其中一個(gè)拷貝Bna TMA7,BAM3的2個(gè)拷貝Bna BAM3-C1和Bna BAM3-C2,SERPIN1的2個(gè)拷貝Bna SERPIN1-C1和Bna SERPIN1-C2,Peroxidase其中1個(gè)拷貝Bna Peroxidase,unknown基因1個(gè)拷貝Bnaunknown的特異序列位點(diǎn)設(shè)計(jì)引物,q RT-PCR結(jié)果分析表明,隨著干旱時(shí)間延長(zhǎng),根中7個(gè)基因都應(yīng)激上調(diào)表達(dá);而在葉片中表達(dá)趨勢(shì)不盡相同,部分基因下調(diào)表達(dá)。甘藍(lán)型油菜SERPIN1基因啟動(dòng)子GUS染色結(jié)果表明,正常條件下GUS信號(hào)較微弱,主要在子葉葉尖和表皮毛基部、下胚軸、真葉葉尖、以及根中表達(dá),逆境處理后GUS信號(hào)增強(qiáng),開(kāi)始在葉片維管組織中表達(dá)以及根毛生長(zhǎng)點(diǎn)處的表達(dá)量增強(qiáng)。5.Bam3基因的功能研究Bam3是一種編碼β淀粉酶的基因。本研究分別克隆了油菜和擬南芥的Bam3-Bna Bam3-C1、Bna Bam3-C2和Ara Bam3基因,并對(duì)3個(gè)基因在野生型擬南芥中超表達(dá),進(jìn)行初步的功能驗(yàn)證。Bna Bam3-C1-OE和Ara Bam3-OE超表達(dá)植株表現(xiàn)開(kāi)花期提前抽苔期蓮座葉減少,滲透脅迫條件下敏感性增強(qiáng),干旱條件下生育期縮短、生物量較大,表現(xiàn)一定的避旱性。
[Abstract]:Rape (Brassica napus L.) is one of the most important oil crops, second only to soybean, China is the largest producer of rapeseed. In the middle and lower reaches of the Yangtze River, the main factor of rapeseed reduction and decline in oil content is the main factor of rapeseed reduction and decline in oil content due to the uneven precipitation in the middle and lower reaches of the Yangtze River and the seasonal drought. The regulation of gene transcriptional expression level is an important mechanism for plants to respond to drought stress at the molecular level. Therefore, screening and identifying drought related genes through the differential expression on the transcriptional level is an important means to excavate drought resistant genes, study the mechanism of drought resistance and raise the drought resistance breeding. Brassica napus Q2 is an important method. The highly resistant self bred lines showed high relative water content (RWC), drought resistance index (DRI) and relatively low electrolyte leakage rate under drought conditions. Using Q2 to study the molecular mechanism of drought stress, study the molecular mechanism of drought stress, excavate important drought related genes and transcriptional regulation elements, and provide theoretical basis for drought resistance breeding. Technical support. In this study, the drought resistant Brassica napus Q2 was used as the experimental material to extract RNA from the roots and leaves under the air drought 24h and the control conditions respectively. The high throughput sequencing of the second generation of Illumina was carried out. After the sequence alignment, the differential expression of the basis was screened under drought stress, and the drought resistance genes were selected and selected to select the drought resistance genes by the mutant drought resistance. 5 candidate genes were selected and some genes were genetically modified. The main results were as follows: 1. the transcriptional analysis of Brassica napus root and leaf under drought stress was based on the drought resistant Brassica napus Q2 as material, and based on the high throughput sequencing of the Illumina second generation RNA-seq technology, the transcriptional sequence information of drought stress, root and leaf were obtained. The results of 12308861,11763435,11905805,12168994 clean reads. sequencing of 4 samples in total 4 samples were obtained. The results showed that the sequencing depth was 10M, and the 70% sequence was compared to the reference genome, and the sequencing quality was good. The above two sets of transcriptional group data were taken as reference. 31240 and 31118 genes were obtained in the roots and leaves. 6018 and 5377 differentially expressed genes were screened in the roots and leaves of the roots and leaves. Among them, 2448 and 3570 down regulated genes were up to up in the root and the down regulated genes in the leaves were 3770, and the genes were down regulated in the root. The number of down regulated genes in the roots was 31240. The target was two times of the leaf, and it was preliminarily shown that there was a difference of.GO function enrichment analysis under drought stress of root and leaf. Most of the differentially expressed genes were related to the response of stress and stimulation response. Blast2GO analysis showed that the up-regulated gene enrichment was mainly related to stress and stimulation in the 12 significant gene functions in the root. On the contrary, the up-regulated genes in the leaves were mainly enriched in the cell structure function, and the main enrichment and stimulation function of the genes were downregulated. The results of.KEGG enrichment results showed that the differentially expressed genes were mainly enriched in plant hormone signal conversion under drought stress. The stress expression of a large number of transcription factors in roots and leaves, including MYB, B HLH, C2H2, AP2/EREBP, NAC, etc., AP2/EREBP and WRKY play a role in the reverse regulation mode of roots and leaves. Transcription factors combine downstream drought induced gene promoter region cis acting elements to induce some downstream genes to be expressed. Including ERD1, RAB18, RD29B, RD20A, JA-related gene, ultimately regulate the level of Cl-, NO3-, K+, Ca2+ plasma, change the cell bulge, increase the water absorption of the cells in the root, and close the stomata to reduce the leaf transpiration loss of.2. candidate genes, and select the 5 candidate genes related to drought from the differentially expressed genes. Gene TMA7 (Bra026774), BAM3 (Bra012676), SERPIN1 (Bra015607), Peroxidase (Bra009221), Unknown (Bra009013), function respectively for translation related, B- amylase, serine protease inhibitor, peroxidase and functional unknown protein. The candidate base was compared to the Brassica napus database by sequence alignment, sequencing, cloning, Finally determine the copy number in Brassica napus Q2: TMA7 has 6 copies, BAM3 has 2 copies, SERPIN1 has 2 copies, Peroxidase has 6 copies, Unknown gene 1 copies.3. mutants under drought stress condition observation based on the 5 candidate genes and Arabidopsis homologous information to purchase mutant tma7 (SALK_044092C), bam3 (SALK_) 041214C), serpin1 (SALK_019150C), peroxidase (SALK_124744) and un-2 (SALK_121527C), observe the phenotype of mutant phenotype under drought conditions and compare wild type Arabidopsis (Col). The mutant leaves have higher water loss rate, early flowering time, strong drought sensitivity and early fruit setting after drought recovery; un-2 mutant is especially prominent and seedling in vitro. The expression and function of the.4. candidate gene for fast shrinking wilt after water loss, one of the most similar homology of the cabbage candidate gene TMA7, is a copy of Bna TMA7, 2 copies of BAM3 Bna BAM3-C1 and Bna BAM3-C2, and the 2 copies Bna SERPIN1-C1 and 1 copies of SERPIN1. The specific sequence loci of the unknown gene were designed for 1 copies of Bnaunknown. The results of Q RT-PCR analysis showed that the expression of 7 genes in the root was up-regulated with prolonged drought, while the expression trend in the leaves was different and some genes were down regulated. The result of GUS staining of SERPIN1 gene promoter in Brassica napus showed that GUS under normal condition was GUS. The signal is weak, mainly in the cotyledon leaf tip and the epidermis, the hypocotyl, the tip of the true leaf, and the expression in the root. After adversity, the GUS signal is enhanced, the expression in the vascular tissue of the leaves and the expression of the root hair growing point increase the function of the.5.Bam3 gene. Bam3 is a gene encoding beta amylase. This study was cloned respectively. Bam3-Bna Bam3-C1, Bna Bam3-C2 and Ara Bam3 genes of rapeseed and Arabidopsis, and 3 genes overexpressed in wild Arabidopsis thaliana, the preliminary functional verification of the overexpression of.Bna Bam3-C1-OE and Ara Bam3-OE showed that the lotus leaves decreased in the early flowering stage of the flowering stage, and the sensitivity was enhanced under osmotic stress, and the growth period under drought conditions was reduced. Short, large biomass, showing a certain drought resistance.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S565.4

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