基于大片段基因组DNA植物表达文库的复苏植物耐脱水机理研究

Resurrection plants inhabit in extream drought habitat and exhibit desiccation tolerance as it can survive after 95% water lost,thus represent a good model for studies on severe drought resistance.Most studies have focused on the dehydration-induced genes and neglected those constitutively expressed genes, and gene clusters, gene interactions and chromosomal modifications and noncoding sequences on the genome. The main reason is that the lack of genetic information on the genome of resurrection plants and effective genetic manipulation system limits the use of "-omics" approaches.Recently, we have construced a library of high-molecular weight(MW) genomic DNA from Boea hygrometrica, a typical resurrection plant distribute widely in China, using a binary vector of BIBAC series that can be directly used to transform Agrobacteria and integrate the inserted large DNA frament into plant genome. In this project,this library will be used to construct a transgenic population of Arabidopsis haboring high-MW genomic DNA fragments of Boea hygrometrica. The exogenous high-MW DNA fragments that are functional to improve transgenic plant drought resistance will be picked out by comparing the sensitivity to dehydration stress. Furthermore,the genes and noncoding sequences in the candidate large DNA fragments and the possible interactions between them, as well as the putative chromosomal modifications that are responsible for the enhancement of transgenic plant drought tolerance will be identified by BAC- shotgun sequensing and subclone library construction. Taking together, this project aims to find the novel genes and noncoding sequences that play important roles in dehydration tolerance from the resurrection plant B. hygrometrica using a transgenic population of Arabidopsis carring high MW genomic DNA fragments of B. hygrometrica, with a special emphasis on the genes with unaltered expression,the gene clusters,gene interactions,noncoding sequences and chromosomal modifications on whole genome level. This study will be helpful to reveal comprehensive mechanisms of dehydration tolerance of resurrection plants and lay a theoretical foundation for improving plant drought tolerance.

复苏植物生活于极端干旱生境，具有极强的耐脱水能力，失水95%以上仍然干而不死，其特殊的抗旱机制值得关注。以往研究主要针对干旱诱导基因，忽视了那些组成型表达的基因,和基因簇、染色质修饰、基因互作、非编码序列等基因组水平上的调控对抗旱性状的影响。这主要是因为复苏植物的遗传信息不清楚、遗传研究的体系不成熟，常规"组学"研究难以深入。本项目在多学科交叉研究的基础上借鉴遗传学策略提出新思路，利用可以携带大片段DNA的BIBAC植物表达体系将复苏植物牛耳草的大片段基因组DNA文库直接转入拟南芥创建转化群体，通过对转化群体中转基因株系进行系统的抗旱性表型比较来鉴定具有提高植物抗旱性功能的大片段DNA，进一步克隆其所包含的关键基因与调控序列，最终在基因组层面上高通量地鉴定旋蒴苣苔的抗旱基因/基因簇/非编码序列，分析其中可能存在的基因互作、修饰和调控，从而深化植物抗旱分子调控理论，为抗旱遗传育种提供基础。

本项目在以往针对干旱诱导基因的研究基础上，关注复苏植物在基因组水平上调控抗旱的机制。利用可以携带大片段DNA的BIBAC植物表达体系将复苏植物牛耳草的大片段基因组DNA文库直接转入拟南芥创建转化群体，通过对转化群体中转基因株系进行系统的抗旱性表型比较来鉴定具有提高植物抗旱性功能的大片段DNA，进一步克隆其所包含的关键基因与调控序列，最终在基因组层面上高通量地鉴定旋蒴苣苔的抗旱基因/基因簇/非编码序列，分析其中可能存在的基因互作、修饰和调控，从而深化植物抗旱分子调控理论，为抗旱遗传育种提供基础。本项目开展四年来，基本按任务书计划展开，已取得的重要进展如下：1）构建了耐旱复苏植物牛耳草的大片段基因组 DNA 的植物表达文库1个，并完成了基因组（合作）和转录组测序；2）通过对转化群体中转基因株系进行系统的抗旱性表型比较鉴定了可以提高抗旱性的大片段 DNA 4个，鸟枪法测序发现其所含有的抗性基因均为逆转座子；3）对其中一个大片段1-4构建亚克隆文库，并对其转基因植物进行表型鉴定，发现其中2 kb序列是1-4和所有具备抗旱表型的亚克隆共有的序列，因此可能是产生抗逆性状的功能序列（命名为OAR1），未发现其与其他片段互作或有协同作用，但有DNA甲基化修饰现象；4）OAR1编码可形成3个发夹结构富集区的不完整反转录转座子，表明这段序列有可能产生小RNA；进一步分析已将目标序列确定到800 bp范围内。本项目的进展已经整理成文，在Plant & Cell physiol.和PLoS one等期刊发表学术论文7篇，申请专利1项，已获得授权。