基于RNA-seq进行小桐子在干旱胁迫响应与适应过程中的转录组分析及其关键基因的克隆和功能验证

发布时间：2018-08-12 16:17

【摘要】：小桐子是具有巨大开发潜力的能源植物树种,干旱是限制小桐子产业发展的重要环境因素。因此,弄清小桐子抗旱性形成的分子机制,获取跟抗旱性密切相关的关键基因,并通过基因工程手段进行初步改良已成为急需解决的问题。本研究主要是通过借助Illumina Hiseq 2000高通量测序系统对小桐子模拟干旱锻炼和空气胁迫过程中的转录组进行RNA-seq解析,通过鉴定干旱响应的差异表达基因及其功能富集分析,阐明小桐子在干旱响应和适应过程中的转录组响应全景,进而克隆并获得了两个与抗旱性相关的差异表达关键基因,为揭示小桐子干旱锻炼下抗旱性形成的分子机制和为后续通过基因工程手段改良小桐子提供了坚实的理论和实验基础。研究结果如下:1.以生长三周的小桐子幼苗为实验材料,经10%PEG 6000模拟干旱锻炼48h(设置5个取样点:0 h、6 h、12 h、24 h、48 h),之后转入空气干旱胁迫72h(设置3个取样点:24 h、48 h、72 h),未经干旱锻炼的幼苗同样进行空气干旱胁迫72 h,并于胁迫结束后取样。每份叶片样本均取自5株独立植株,均匀混样,记为三次生物学重复,用于cDNA文库构建和RNA-seq测序。经过滤去除低质量reads后,每个cDNA文库产出clean reads平均数目为11,933,126条,通过与参考基因组和多重数据库比对,共计26,979条获得功能注释。2.使用NOISeq方法进行差异表达基因分析发现,在干旱锻炼阶段,与对照0 h相比,锻炼6 h、12 h、24 h、48 h分别有27、67、74、186个基因表达下调同时分别有36、76、122、166个基因表达上调;在随后进行的空气干旱胁迫24h、48 h、72 h,下调表达基因数量为671、742、696,同时有411、409、375个基因表达上调。共鉴定出1,573个干旱响应基因,其中上调表达基因主要涉及大量渗透调节蛋白、激素生物合成、抗氧化系统、转录因子等。3.对干旱响应的差异表达基因进行KEGG和基因本体富集分析,发现干旱锻炼促进了转录调控和激素信号调节,表现出大量转录因子(如WRKY、bHLH、NF、C3H、HD-ZIP等)以及激素生物合成与信号转导相关元件(如SAMS、ACO、ARPs、GA20 oxidase、SnRK、ZEP等)的上调表达。此外,锻炼诱导基因还广泛参与了渗透调节物质(如半乳糖、海藻糖和甜菜碱等)及抗逆蛋白(如nsLTPs、HSPs等)的生物合成,抗氧化系统(如GST、GPX、SOD、氧还蛋白和金属硫蛋白等)的活化、脂肪酸的生物合成(CER、WSD、GPAT、FAD、细胞色素P450等)以及糖类的代谢和转化(如RuBisco、PGK等)。4.为了验证RNA-seq数据的可信性,我们挑选了22个基因进行qRT-PCR分析,所选基因的表达水平、模式和功能尽量随机。结果显示qPCR数据与RNA-seq数据的平均相关系数为0.8以上,表明RNA-seq数据是可信的。5.LEA蛋白是一类跟抗逆性紧密相关的蛋白,在不同物种中都有大量发现。我们的RNA-seq分析结果显示,共计6个LEA编码基因的表达均受到干旱处理的诱导,其中XM_012232372.1在叶片中的本底表达量相对最高并且变化倍数显著。在此基础上,我们选择该LEA蛋白进行基因克隆及功能验证。序列分析可知该基因CDS序列全长为459 bp,编码152个氨基酸的蛋白,经特定基序和同源性序列比对可知其属于第四组典型亲水性的LEA蛋白,故命名为JcLEA4。实时荧光定量PCR分析发现,该基因在小桐子幼苗的叶中受空气干旱胁迫和ABA诱导显著,并且其异源表达增强了酵母菌对PEG模拟干旱胁迫的耐受能力。6.GDSL酯酶/脂肪酶(GDSL esterase/lipase)是近几年新发现的具有GDSL保守结构域的脂肪酶的亚家族成员。在RNA-seq分析的基础上,我们从小桐子幼苗中克隆得到一个编码GDSL酯酶/脂肪酶的基因JcGLIP,经过保守结构域分析可知其为SGNH水解酶超家族/亚家族一员,CDS序列全长为1,104bp,编码蛋白有367个氨基酸,荧光定量PCR结果可知其在空气干旱胁迫和ABA处理的前期被大量诱导,属于干旱快速响应基因,转基因酵母在PEG模拟干旱胁迫下同样表现出一定的抵抗能力。本研究初步证实了JcLEA4和JcGLIP基因参与了小桐子的干旱响应,并且此过程可能与ABA信号调节有关。
[Abstract]:Jatropha curcas L. is an energy plant species with great potential for development. Drought is an important environmental factor limiting the development of Jatropha curcas L. industry. The transcriptomes of Jatropha curcas during simulated drought training and air stress were analyzed by using Illumina Hiseq 2000 high-throughput sequencing system. The transcriptome response panorama of Jatropha curcas during drought response and adaptation was clarified by identifying differentially expressed genes and their functional enrichment analysis. Two differentially expressed key genes related to drought resistance were cloned and obtained, which provided a solid theoretical and experimental basis for revealing the molecular mechanism of drought resistance formation under drought stress and for subsequent improvement of Jatropha curcas L. by genetic engineering. The results were as follows: 1. After 48 hours of simulated drought stress (5 sampling points: 0 h, 6 h, 12 h, 24 h, 48 h), 3 sampling points (24 h, 48 h, 72 h) were set up. The seedlings without drought stress were sampled for 72 h and after the stress was over. Each leaf sample was taken from 5 independent plants and mixed evenly. After removing low-quality reads, the average number of clean reads per cDNA library was 11,933,126. By comparing with the reference genome and multiple databases, a total of 26,979 functional annotations were obtained. At the stage of drought training, 27,67,74,186 genes were down-regulated and 36,76,122,166 genes were up-regulated respectively at 6,12,24 and 48 hours of exercise, compared with the control at 0 H. At the following 24,48,72 hours of air drought stress, the down-regulated gene number was 671,742,696, and 411,409,375 genes were up-regulated. 573 drought-responsive genes, of which the up-regulated genes mainly involved a large number of osmotic regulatory proteins, hormone biosynthesis, antioxidant system, transcription factors and so on. In addition, exercise-induced genes are involved in the biosynthesis of osmotic regulators (such as galactose, trehalose, betaine, etc.) and stress-resistant proteins (such as nsLTPs, HSPs, etc.). Activation of oxidative systems (such as GST, GPX, SOD, Oxoredoxin and metallothionein, etc.), biosynthesis of fatty acids (CER, WSD, GPAT, FAD, cytochrome P450, etc.), and metabolism and transformation of carbohydrates (such as RuBisco, PGK, etc.). 4. To verify the reliability of RNA-seq data, we selected 22 genes for qRT-PCR analysis, and the expression level of selected genes. The results showed that the average correlation coefficient between qPCR data and RNA-seq data was above 0.8, indicating that the RNA-seq data was reliable. 5. LEA proteins were a class of proteins closely related to stress resistance and were found in a large number of different species. The background expression of XM_0122372.1 in leaves was the highest and varied significantly after drought induction. On this basis, we selected the LEA protein for gene cloning and functional verification. Sequence analysis showed that the CDS sequence of the gene was 459 bp, encoding a protein of 152 amino acids, and was sequenced by specific sequence and homology. The sequence comparison showed that it belonged to the fourth group of typical hydrophilic LEA proteins, so it was named JcLEA4. Real-time fluorescence quantitative PCR analysis showed that the gene was significantly induced by air drought stress and ABA in the leaves of Jatropha curcas seedlings, and its heterologous expression enhanced the tolerance of yeast to PEG simulated drought stress. 6. GDSL esterase / lipase (GDSL es) Terase / lipase is a member of the lipase subfamily with a conserved domain of GDSL. Based on RNA-seq analysis, we cloned a gene JcGLIP encoding GDSL esterase / lipase from Jatropha curcas seedlings. The gene JcGLIP was identified as a member of the SGNH hydrolase superfamily / subfamily and CDS sequence by conserved domain analysis. The total length was 1,104 BP and the coding protein contained 367 amino acids. The results of fluorescence quantitative PCR showed that JcLEA4 and JcGLIP genes were induced in large quantities at the early stage of air drought stress and ABA treatment, and they belonged to drought fast response genes. Transgenic yeasts also showed resistance to PEG simulated drought stress. The drought response of Jatropha curcas L. is related to this process, and this process may be related to ABA signal regulation.
【学位授予单位】：云南师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2;S794.9

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