花生耐盐突变体M34耐盐相关基因的筛选及功能分析

New peanut cultivars with high-yielding and salt-tolerance have become one of important breeding objectives, with the growing soil salinization. However, it is difficult to achieve the new cultivars with high-yielding and salt-tolerance using the conventional hybrid method, for the narrow genetic base and the lack of resources with salt toletance in peanut. Peanut mutant M34 with significantly improved salt tolerance has been obtained by use of chemical mutagenesis and tissue culture screening. The transcriptome sequencing and expression profiling analysis on mutant M34 and control peanuts will be conducted using RNA sequencing technology after they were treated by salt stress. Concerned genes with salt tolerance will be identified and 2-3 important candidate genes will be screened and cloned by use of the cDNA library which has been constructed and RACE method. Subcellular localization on candidate genes will be carried out by ligating them to the vector with GFP, and the response of transgenic plants to salt stress will be conducted by overexpressing these genes in transgenic peanuts. The molecular regulation mechanism of candidate genes on salt tolerance will be clarified by expresssion analysis on candidate genes and functional analysis in transgenetic plants. This project aims to provide new genes for creating new germplasm by gene engineering, supply guidance to study on molecular mechanism of salt tolerance, and provide reliable basis for developing new pathway of stress tolerance breeding in peanut. So this study has both great theoretical and practical significance.

随着土壤盐渍化不断加剧，培育高产耐盐花生新品种成为育种的重要目标之一。然而花生遗传基础狭窄，缺乏耐盐资源，利用常规杂交方法难以实现育种目标。本课题组前期利用化学诱变结合组织培养筛选出耐盐性显著提高的突变体M34。本研究拟以该突变体为材料，应用RNA-Seq技术，在不同盐胁迫处理条件下进行转录组测序及表达谱分析，筛选出差异表达显著的相关序列进行注释及信息比对分析，确定耐盐相关候选基因，并利用所构建的cDNA文库及RACE技术克隆2-3个重要功能基因。将该基因与GFP融合后进行亚细胞定位，并通过遗传转化在花生中超量表达，分析转化子对盐胁迫的反应。结合差异表达分析及转基因功能验证结果，阐明候选基因在盐胁迫处理下的分子调控机制。本项目的开展，旨在为今后通过遗传工程创造耐盐新种质提供新基因，并为花生耐盐分子机理研究提供指导，也为开拓新的花生抗逆育种途径提供可靠依据，因而具有理论和实践双重意义。

花生耐盐突变体M34和野生型对照花育22的种子播种于沙土中萌发生长，对6周龄幼苗用200mM NaCl进行灌溉处理，处理后 0、6、12、24、48h分别取幼叶，投入液氮冷冻保存。使用Trizol reagent（Invitrogen, US）提取每份样品的总RNA，送北京诺禾致源生物信息科技有限公司，利用Illumina HiSeq 2000测序平台进行RNA-sequencing。测序获得的Unigenes 与 NCBI nonredundant（Nr）蛋白质数据库，Swiss-Prot 蛋白质数据库，Kyoto Encyclopedia of Genes and Genomes（KEGG）通路数据库，euKaryotic Ortholog Groups of proteins（KOG）数据库，以及 Blast2GO（http://www.blast2go.rog/) GO 数据库进行比对和注释。对突变体M34及对照亲本花育22的5个不同时间段处理分别进行了数字基因表达谱（Digital Gene Expression，DGEs）分析，共筛选得到112个差异表达的DEGs。其中86个为已经报道的耐盐相关基因，除此外还包括一些未知功能的基因。利用Real-time PCR技术进一步对筛选获得的差异基因在盐胁迫处理后不同时间的表达量进行了分析验证。选择差异表达明显的基因WRKY家族成员AhWRKY75，AhWRKY45，AhWRKY23，以及Lea家族成员AhLea-3，AhLea-Dehydrin进行PCR克隆测序，构建过表达载体构建并通过遗传转化花生品种花育23来进行功能验证。结果表明与非转基因对照植株相比较，过量表达AhWRKY75，AhWRKY45，AhWRKY23，AhLea-3，AhLea-Dehydrin的转基因花生植株在盐胁迫条件下表现为植株生长势较好，叶片萎焉程度较低，光合作用能力增强，抗逆相关基因的表达量增加，SOD、CAT、POD酶活性升高，氧自由基含量和MDA含量降低等多种表型。以上结果初步验证了这些基因的耐盐功能。

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