水稻OsbHLH156互作蛋白筛选、鉴定与表达分析  

Screening,identification and expression analysis of rice OsbHLH156-interacting proteins

作　　者：方静 李杰 朱佳美 寿惠霞[1] FANG Jing;LI Jie;Zhu Jiamei;SHOU Huixia(Institute of Plant Biology,College of Life Sciences,Zhejiang University,Hangzhou 310058,China)

机构地区：[1]浙江大学生命科学学院植物研究所,杭州310058

出　　处：《植物生理学报》2022年第8期1485-1495,共11页Plant Physiology Journal

基　　金：浙江省重点研发计划(2021C02053)。

摘　　要：铁(Fe)是植物生长发育必需的微量营养元素,植物一般通过机制I或机制II两种不同途径从土壤中吸收铁。OsbHLH156是调控水稻(Oryza sativa)铁吸收机制II的核心转录因子。为了进一步探究OsbHLH156的功能和调控网络,本研究通过酵母双杂交和双分子荧光互补实验筛选出并验证了两个与OsbHLH156互作的转录因子——OsbHLH168和OsZFP7,并通过亚细胞定位、生物信息学和荧光定量PCR方法对其展开分析。亚细胞定位结果显示OsZFP7定位于细胞核,OsbHLH168定位于细胞核和细胞质。与OsbHLH156共表达时,OsbHLH168主要定位于细胞核。生物信息学分析结果显示OsbHLH168属于bHLH转录因子家族,与拟南芥(Arabidopsis thaliana)中的AtbHLH167和AtbHLH168亲缘关系较近,编码基因包含两个外显子和1个内含子;OsZFP7属于锌指蛋白家族,具有1个保守的C2H2型锌指结构域,与拟南芥中AtZFP4和At ZFP7亲缘关系较近,编码基因中只有1个外显子,没有内含子。荧光定量PCR结果显示,Osb HLH168主要在叶片中表达,在野生型水稻根中受缺铁诱导表达,在osbhlh156突变体的根中表达不受缺铁影响,表明OsbHLH156突变抑制了OsbHLH168在根中的缺铁诱导表达;OsZFP7主要在根中表达,在野生型水稻根中不受缺铁诱导表达,但在突变体osbhlh156根中受缺铁诱导表达,说明OsbHLH156抑制了OsZFP7的缺铁诱导表达。本研究为进一步完善水稻缺铁响应机制奠定基础。Iron(Fe) is an essential micronutrient for plant growth,and plants generally uptake Fe from soil through strategy I or II.OsbHLH156 is a core transcription factor that regulates Fe uptake strategy II in rice(Oryza sativa).In order to further explore the function and regulatory network of OsbHLH156,in this study,we screened a yeast library of rice transcription factors and verified two transcription factors,OsbHLH168and OsZFP7,that interact with OsbHLH156 by yeast two-hybrid and bimolecular fluorescence complementation experiments.OsbHLH168 and OsZFP7 were further analyzed for their subcellular localization,gene structure,phylogenetic information and transcription abundance.Subcellular localization analysis showed that OsZFP7 is localized in the nucleus,while OsbHLH168 is localized in both nucleus and cytoplasm.Co-expression of OsbHLH156 enhanced the nuclear localization of OsbHLH168.Bioinformatics analysis showed that OsbHLH168 belongs to the bHLH transcription factor family and is similar to AtbHLH167 and AtbHLH168 in Arabidopsis thaliana.OsZFP7 belongs to the family of zinc finger proteins and has a conserved C2H2-type zinc finger domain.It is closely related to AtZFP4 and AtZFP7 in A.thaliana,and the gene has one exon without intron.Real-time quantitative PCR showed that OsbHLH168 was mainly expressed in leaves,and can be induced by Fe deficiency in roots.In osbhlh156 mutant,the expression of Osb HLH168 is no longer induced by Fe deficiency,indicating that the mutation of Osb HLH156 directly or indirectly inhibits the expression of OsbHLH168 in roots.Os ZFP7 was mainly expressed in roots,and was not responsive to Fe deficiency in wild type rice roots.However,the expression of Os ZFP7 was induced by Fe deficiency in osbhlh156 mutant roots,suggesting that OsbHLH156 may inhibit the Fe deficiency–induced expression of Os ZFP7.This study can provide a basis for further analysis of the regulatory mechanism in response to Fe deficiency in rice.

关 键 词：水稻 铁 OsbHLH156 互作蛋白 转录因子 

分 类 号：S511[农业科学—作物学]