基于根系形态解剖及定量蛋白质组学的花生耐渍涝机理研究

Waterlogging, as one of main disasters in peanut production in China, occurred frequently in the earlier growing stage in southern areas. To solove this problem, we have developed many achievements in past fifteen years, such as determining physilogical and agronomic effects, establishing indexes and criteria for disaster identification, creating the first database of tolerance identification including near 900 germplasm, waterlogging precautioning and controling with cultivation performance. Although the previous researches around the world carried out common phenotypic analysis for peanut waterlogging, these studies were insufficient in originality and thoroughly discuss. This projepct will focus on roots where waterlogging stress directly, persistently and seriously effects on peanut plants. Morphological characteristics, tissue structures, cellular and subcellular characters of main root, lateral roots and adventitous roots of typical tolerant and susceptible accessions will be studied by morphological and anatomical methods. Further, indentification of differential proteins, pathway analysis for these proteins, expression and verification of tolerant-related genes will be also explored by quantitative proteomics (iTRAQ) technique, LC-MS/MS technique, bioinformatics technique and real time RT-PCR. Hence the relationships between morphological characteristics and proteome of roots will be analysed, which wll illuminate tolerance mechanisms and provid theoretical basis for waterlogging control of peanut production.

作为我国花生的主要灾害之一，渍涝在南方花生生育前期频繁发生。通过持续15年的不断探索，我们在确定渍涝的生理与农艺影响、建立渍涝鉴定指标与标准、耐渍生态育种、创建近900份种质的世界首个耐渍鉴定数据库、灾害栽培防控等方面取得了系列进展。然而，国内外花生渍涝研究多囿于一般性的表型分析，缺乏原创性的深层次探讨。本项目拟聚焦渍涝危害最为直接、持久、严重的根系部位，采用植物形态与解剖学方法，研究渍涝胁迫下耐性差异明显的两类花生品种的主根、侧根、不定根的形态特征、组织结构、细胞与亚细胞特点；采用iTRAQ定量蛋白质组学技术、LC-MS/MS液质联用质谱技术、生物信息学技术和real time RT-PCR技术相结合的方法，研究三类根的差异蛋白质及代谢途径、耐涝相关基因的表达与验证，进而剖析形态解剖分异与蛋白质组、基因表达变化的对应关系，基本探明耐渍花生的主要机理，为花生渍涝灾害治理提供理论依据。

渍涝是我国南方地区花生生育前期的严重问题。试验以渍涝敏感品种中花4号及耐性品种湘花2008、泉花8号、豫花15号根系为试材，在幼苗期进行渍涝处理3-12天和正常灌溉，采用形态解剖方法和定量蛋白组学技术（DIA）探究耐涝机理。结果如下:. （1）利用DIA技术鉴定出差异表达蛋白。4个品种鉴定出3097个淹水与正常灌溉差异蛋白，包括主根1993个，457个上调，1536个下调；侧根1104个，389个上调，715个下调，豫花15最多，中花4号最少，预示着耐性品种对付渍涝有更多的机制或策略。. （2）分析了差异表达蛋白功能及亚细胞定位。GO分析表明，4个品种主、侧根差异表达蛋白主要富集在新陈代谢和细胞进程等过程，主要定位在细胞膜及其组分、细胞及其组分上。KEGG、KOG分析表明，主、侧根差异表达蛋白主要富集在碳水化合物、氨基酸、脂质的新陈代谢和次生代谢物的合成、转运和分解、能源的生产和转换等途径、细胞信号传导等过程，主要定位在叶绿体、细胞质和质膜。. （3）明确与耐涝相关的关键蛋白及对应关键基因。DIA技术结合韦恩图分析得出湘花2008主根出现156个耐涝相关特异性表达蛋白，其中关键蛋白为：乙醇脱氢酶、磷酸甘油酸激酶、茎特异性蛋白和下调表达蛋白IAA氨基化合成酶、IAA氨基酸水解酶，对应的相关基因为Adh，Pgk，Tsjt1，Gh，Ilr。. （4）明确渍涝胁迫下根系形态特征、显微及超微结构特征。.随着淹水时间延长，敏感品种根系严重受损，颜色加深，气味恶化，无明显通气组织，导管不规则分布，细胞器受胁迫较重，加速衰老死亡；3个耐性品种根系影响较轻，颜色较浅，气味较轻，出现了通气组织，且数量增加、横截面加大，木质部基本保持正常，导管呈射线状分布，细胞器受影响较轻。