甘蓝型油菜响应干旱胁迫的表达谱分析

发布时间：2018-07-12 15:25

本文选题：甘蓝型油菜 + 干旱　；参考：《华中农业大学》2016年博士论文

【摘要】：油菜(Brassica napus L.)是重要的油料作物之一,地位仅次于大豆,中国是最大的油菜生产国。我国油菜主产区长江中下游地区由于降水不均、季节性干旱造成油菜矮化、不实,是油菜减产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种的基础。基因转录表达水平调节是植物从分子水平上响应干旱胁迫的重要机制,因此通过转录水平上的表达差异来筛选和鉴定抗旱相关的基因,是挖掘抗旱基因、研究抗旱机理、提高抗旱育种的重要手段。甘蓝型油菜Q2是具有较强抗性的自交品系,在干旱条件下表现较高的相对含水量(RWC)、抗旱指数(DRI)以及相对较低的电解质渗漏率。利用Q2应对干旱胁迫,研究干旱胁迫的分子机理,挖掘重要的抗旱相关基因以及转录调控元件,为抗旱育种提供理论依据和技术支持。本研究以抗旱型甘蓝型油菜Q2为实验材料,对空气干旱24h和对照条件下的根和叶片分别提取RNA,进行Illumina第二代高通量测序,经过序列比对、分析,筛选干旱胁迫条件下的差异表达基因,并通过突变体抗旱性筛选抗旱基因,挑选了5个候选基因,并对部分基因进行转基因验证,主要结果如下:1.甘蓝型油菜根和叶片干旱胁迫条件下的转录组分析以抗旱型甘蓝型油菜Q2为材料,基于Illumina第二代高通量测序RNA-seq技术,获得Q2响应干旱胁迫转录本序列信息,根和叶片空气干旱24h和对照处理总共4个样本分别得到12,308,861、11,763,435、11,905,805、12,168,994条clean reads。测序质量评估结果表明,测序深度达到10M,而且70%序列比对到参考基因组,测序质量良好。以白菜基因组数据库为参考,对以上两组转录组数据库进行比对,根和叶片中分别获得31,240和31,118个基因。以FDR≤0.001和|log2Ratio|≥1为标准,根和叶片中分别筛选得到6018和5377个差异表达基因。其中,根中上调基因2448个,下调基因3570个;叶片中上调基因3770个,下调基因1607个。根中下调基因数目是叶片中的两倍,初步显示了根和叶片干旱胁迫下的调控模式存在差异。GO功能富集分析发现,大部分差异表达基因跟胁迫响应、刺激响应相关;Blast2GO分析表明,根中12条显著富集的基因功能中,上调基因富集主要跟胁迫、刺激相关,而下调基因主要跟细胞、功能相关;相反地,叶片中上调基因主要富集在细胞结构功能,下调基因主要富集胁迫、刺激功能,显示了干旱胁迫条件下根和叶片分工应答胁迫的模式。KEGG富集结果表明,差异表达基因主要富集于植物激素信号转导路径和苯丙素合成路径。根和叶片中大量转录因子应激表达,包括MYB、b HLH、C2H2、AP2/EREBP、NAC等。其中,AP2/EREBP和WRKY在根和叶片相反调控模式中发挥作用。转录因子结合下游干旱诱导基因启动子区顺式作用元件,诱导下游一些基因表达,包括ERD1、RAB18、RD29B、RD20A、JA-related基因,最终调控细胞中Cl-、NO3-、K+、Ca2+等离子水平,改变细胞膨压,增加根中细胞吸水,关闭气孔减少叶片蒸腾失水。2.候选基因筛选根据转录组结果log2Ratio和RPKM值,从差异表达基因中挑选出与干旱相关的5个候选基因TMA7(Bra026774)、BAM3(Bra012676)、SERPIN1(Bra015607)、Peroxidase(Bra009221)、Unknown(Bra009013),功能分别为翻译相关、B-淀粉酶、丝氨酸蛋白酶抑制剂、过氧化物酶以及功能未知蛋白。将候选基因CDS序列比对到甘蓝型油菜数据库,通过序列比对、测序、克隆,最终确定在甘蓝型油菜Q2中的拷贝数:TMA7有6个拷贝,BAM3有2个拷贝,SERPIN1有2个拷贝,Peroxidase有6个拷贝,Unknown基因1个拷贝。3.突变体干旱胁迫条件下的表型观察根据5个候选基因与拟南芥同源基因的信息购买突变体tma7(SALK_044092C)、bam3(SALK_041214C)、serpin1(SALK_019150C)、peroxidase(SALK_124744)、un-2(SALK_121527C),观察干旱条件下突变体表型发现,相比较野生型拟南芥(Col),突变体离体叶片失水率更高、花期提前,表现较强的干旱敏感性,干旱复水后提早结实;un-2突变体尤其突出,苗子离体失水后快速皱缩萎蔫。4.候选基因的表达和功能研究将与白菜候选基因同源性最相近的甘蓝型油菜基因TMA7其中一个拷贝Bna TMA7,BAM3的2个拷贝Bna BAM3-C1和Bna BAM3-C2,SERPIN1的2个拷贝Bna SERPIN1-C1和Bna SERPIN1-C2,Peroxidase其中1个拷贝Bna Peroxidase,unknown基因1个拷贝Bnaunknown的特异序列位点设计引物,q RT-PCR结果分析表明,随着干旱时间延长,根中7个基因都应激上调表达;而在叶片中表达趋势不尽相同,部分基因下调表达。甘蓝型油菜SERPIN1基因启动子GUS染色结果表明,正常条件下GUS信号较微弱,主要在子叶叶尖和表皮毛基部、下胚轴、真叶叶尖、以及根中表达,逆境处理后GUS信号增强,开始在叶片维管组织中表达以及根毛生长点处的表达量增强。5.Bam3基因的功能研究Bam3是一种编码β淀粉酶的基因。本研究分别克隆了油菜和拟南芥的Bam3-Bna Bam3-C1、Bna Bam3-C2和Ara Bam3基因,并对3个基因在野生型拟南芥中超表达,进行初步的功能验证。Bna Bam3-C1-OE和Ara Bam3-OE超表达植株表现开花期提前抽苔期莲座叶减少,渗透胁迫条件下敏感性增强,干旱条件下生育期缩短、生物量较大,表现一定的避旱性。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S565.4

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