利用高通量amiRNA拟南芥突变体库及离子组学研究挖掘重金属响应的新基因及构建调控网络

Ionomics is a powerful technique for discovery the important genes that function in heavy metal uptake, transport and signal transduction and will accelerate the engineering and breeding of plants for bioremediation using non crop plants as well as for excluding toxic metals from edible tissues of crops. However, many important genes and pathways that function in heavy metal over-accumulation in plants remain to be identified. In addition, many genetic redundancies in plant genomes cause major limitations in heavy metal response gene discovery. To address redundant genes function on a high throughput, we have designed a genomic scale artificial microRNA (amiRNA) library for genome-wide knockdown of homologous gene family members (including transcription factors, kinases, transporters, metabolic enzymes et al.). Using the high throughput screening the amiRNA library will lead to discovery of new genes and further characterize key plant genes and network mechanisms that function in heavy metal accumulation, resistance and remediation. This project includes the following: (1) high throughput amiRNA screening for altered heavy metal response phenotypes which including growth phenotype and analyzing diverse heavy metal contents in different plant tissues; (2) Confirming candidate phenotype by crossing or CRISPR/Cas9 gene edition; (3) Further functional analyses for two or three new key genes identified in our screening. (4) Systems level analysis of newly discovered genes and published genes using bioinformatics techniques to generate predictive networks of gene functions and associations.

离子组学作为分离植物体内离子吸收、转运和信号转导关键调控因子的核心技术，极大地推进了重金属植物修复技术的基础理论研究，但是其效率严重受限于基因功能的冗余现象。本项目拟利用已经建成的人工 microRNA (amiRNA) 突变体库克服传统离子组学研究的缺陷。该突变体库覆盖了全基因组的十大类基因（转录因子、蛋白激酶、转运蛋白、代谢酶、水解酶等），通过同时靶标多个基因克服功能冗余，非常有利于发现新的调控因子，构建出一个重金属吸收、转运及耐受的遗传调控网络。具体研究内容包括（1）高通量分析突变体库的生长表型及离子谱；（2）利用T-DNA突变体杂交或CRISPR/Cas9编辑技术获得候选amiRNA靶标基因的突变体材料，进行表型再验证；（3）重点解析2-3个关键的新调控基因；（4）利用我们新发现的各类基因及已报道的与重金属抗逆相关的基因，结合生物信息学技术构建重金属吸收、转运及信号转导的调控网络。

植物体内由于同源基因的存在，单个基因的突变往往不能观察到明显的表型，这样严重影响了功能基因的挖掘。amiRNA可以靶标同源性较高的多个基因，克服基因功能冗余的不利影响。本项目利用amiRNA突变体库，利用正向遗传筛选对重金属Cd和As敏感或者耐受表型的突变体，实验发现1-1559和4-123表型与Cd相关，1-1559比野生型耐受Cd胁迫，4-123为Cd敏感；筛选到As（Ⅲ）胁迫敏感的突变体10-67、As（Ⅲ）胁迫耐受的突变体10-9、4-138、4-85、4-89、4-82、4-184。重点研究了4-138、10-67和10-9。amiRNA家系4-138靶标到两类转录因子ERF34、ERF35和CBF1、2、3、4。通过CRISPR基因编辑得到cbf1/2/3三突变体和erf34/35 T-DNA双突变体，实验发现cbf1/2/3表现为As（Ⅲ）耐受表型，而erf34/35没有表现出As（Ⅲ）耐受表型。实验还发现CBF转录因子能够上调PHTs基因转录表达。erf34 erf35突变体对Cd敏感的表型主要源于NRT1.8低的表达量。amiRNA 10-9突变体对砷不敏感，其靶标3个PHTs。本项目挖掘到了多个对Cd和As有响应的新基因，该研究可以为低重金属毒害的农作物分子育种提供借鉴。