荒漠盐生植物盐穗木转录调控因子HcSCL13的耐盐功能及作用机制研究

Mechanism of plant salt tolerance is very complicated and involved in multi- signaling routes, but in which many components are not very clear. The plant-specific GRAS transcription factors played diverse roles in plant development and signaling pathway. However, far little is known of any GRAS functions in plants in response to salt changing stimuli and few have been functionally characterized in halophyte. Recently, a fragment of EST was isolated from desert halophyte Halostachys Caspica by suppression substractive hybridization and its full-length cDNA with 2090 bp was cloned by SMARTTM RACE. HcSCL13 showed up-regulated expression patterns under salt stress by quantitative real-time PCR method, so we hypothesize that HcSCL13 acts as a positive regulator participating in Halostachys Caspica salt tolerance. In order to evaluate the salt-related function of the HcSCL13 gene in Halostachys Caspica, HcSCL13 gene will be respectively transferred into wild type and salt-sensitive mutant atscl13 of Arabidopsis thaliana by floral dip method. HcSCL13 promoter will be cloned by genomic walking method. In an effort to determine the necessary regions of sequence or motif for response to salt in HcSCL13 promoter, the full length promoter and different deletion fragments will be synthesized by PCR and then are substituted for 35S upstream gus in a binary plasmid to construct recombined plasmids of HcSCL13-gus fusions. Transgenic Arabidopsis plants are produced by Agrobacterium-mediated transformation. Gus activity will be analyzed in transgenic plants. We will exploit Arabidopsis gene-chip technology to analyze the functional categories (GO) and metabolic pathways (KEGG) of differentially expressed genes for salt tolerant transgenic plants under the salt treatment. Physiological and biochemical parameters corresponding to metabolic pathways of HcSCL13 in transgenic plants will be also measured. Above researches help us to understand muti-roles for GRAS families and salt tolerance mechanism of halophyte. HcSCL13 may be a candidate gene with potential application in molecular breeding to enhance stress tolerance in crops.

植物的耐盐机制非常复杂，尽管目前已取得了很大进展，但诸多信号通路中的具体组分还不完全清楚。从新疆荒漠盐生植物盐穗木中克隆获得的GRAS家族转录因子基因HcSCL13对盐胁迫显著应答，推测该基因作为盐响应信号通路中的正调节因子参与盐穗木的耐盐调控。因此本项目拟以HcSCL13为靶基因，野生型拟南芥和盐敏感atscl13拟南芥突变体为材料，通过农杆菌介导的遗传转化进行耐盐功能鉴定；采用基因组步移、免疫组化和酶活活性检测手段分析HcSCL13基因启动中的盐响应特定元件，同时结合拟南芥基因芯片对耐盐性提高的转基因植株在盐胁迫下差异表达基因的功能聚类、代谢通路分析及对应通路的生理生化数据，最终确定HcSCL13基因参与盐胁迫的分子机制。以上研究对解析GRAS家族的多种生物学功能及其耐盐机制有重要作用，同时为揭示盐生植物耐盐机理提供理论依据，也将为培育耐盐作物提供有价值的候选基因。

HcSCL13是从盐穗木中克隆获得的一个受盐胁迫积极诱导的GRAS家族转录因子基因。 采用花序浸染法并结合抗生素筛选获得了转HcSCL13基因拟南芥纯系。正常和不同NaCl浓度胁迫处理下，转基因植株的种子萌发率均低于野生型，但萌发后期生长表型及生长指标（根长和鲜重）均优于野生型；种苗期，转基因拟南芥无论在正常还是125 mM NaCl胁迫下其根长、鲜重、叶绿素、可溶性糖及可溶性蛋白含量均显著高于野生型拟南芥；抗氧化系统中，转HcSCL13基因拟南芥在盐胁迫下的MDA含量显著低于野生型拟南芥，且CAT活性显著性高于野生型拟南芥。以上实验充分表明，盐穗木HcSCL13基因能够显著调节植物的生长和在盐胁迫下的抗性。. 通过RNA-Seq技术对正常和盐胁迫条件下的野生型和转基因拟南芥进行转录组测序分析，结果表明，转基因拟南芥在正常条件下较野生型拟南芥差异表达基因279个，其中上调类基因为植物糖类和蛋白等生长发育相关基因，占差异表达基因的37.4%，而下调基因主要为逆境胁迫响应类和生长发育负相关基因；转基因拟南芥在盐胁迫前后差异表达基因110个，GO及KEGG分析表明，差异表达基因中上调基因主要包括生长发育和非生物胁迫响应相关基因，下调基因大部分为生物胁迫响应基因。以上分析提示，盐穗木HcSCL13作为转录因子不仅能够调节植物的生长发育相关基因，也能够调控逆境胁迫响应类基因。. 利用基因组步移法克隆获得了HcSCL13基因2, 200 bp的启动子序列，PlantCARE在线分析结果表明该启动子序列不仅含有核心序列CAAT-box和TATA-box，还包含多个光响应和逆境应答相关调控基序。构建该启动子与GUS基因融合的植物表达载体并转化拟南芥，通过对转基因拟南芥不同发育阶段的GUS组织化学染色，显示该基因启动子在植株的各个生活史阶段和不同组织部位均有启动活性。盐胁迫下转基因拟南芥的GUS酶活显著增强，提示该启动子中具有盐响应相关顺式作用元件。. 综上结果，盐穗木HcSCL13转录因子是控制植物生长发育的重要调控因子，通过调节生长和逆境胁迫相关基因的表达，从而赋予植物在正常和盐胁迫条件下的生长和抗性。