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草莓FaFIT在拟南芥中异源表达促进根系铁吸收 被引量:3

Heterologous expression of strawberry FaFIT promotes iron uptakes by roots in Arabidopsis thaliana
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摘要 【目的】植物根系吸收铁受到碱性螺旋-环-螺旋(basic helix-loop-helix, bHLH)转录因子的调控,bHLH转录因子FaFIT在草莓中的功能未知,试验初步分析FaFIT基因在草莓根系铁吸收过程中调控作用。【方法】以红颜草莓为试材,基于根系缺铁胁迫转录组数据生物信息学分析结果,克隆草莓根系铁吸收运转调控基因FaFIT;结合实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)分析该基因在不同组织中的表达差异,通过转基因拟南芥进一步验证该基因的功能。【结果】从红颜草莓根系中克隆获得FaFIT基因序列,该基因mRNA编码区序列全长1020 bp,编码339个氨基酸。蛋白结构预测显示,FaFIT蛋白具有保守的HLH区域,属于bHLH转录因子家族。FaFIT基因启动子序列具有bHLH转录因子DNA结合元件、脱落酸(abscisic acid,ABA)等激素调控元件及干旱胁迫诱导元件,推测FaFIT基因可能受到上游b HLH转录因子、ABA和干旱等因素的调控和诱导。qRT-PCR分析显示,FaFIT基因在根系中特异性表达;FaFIT基因在根组织中受到缺铁、高pH值诱导后表达上调,但在铁过量胁迫时表达显著下调。通过FaFIT基因转化拟南芥,结果发现阳性株根系中与铁吸收相关的3个基因AtAHA2、AtFRO2和AtIRT1的表达水平均上调,且与3个基因相对应蛋白酶H~+-ATPase(P-type)、铁还原酶(ferric-chelate reductase, FCR)、铁转运体(iron-regulated transporter,IRT)的酶活性均提高。同时,在pH=5.8和pH=8.0的1/2 MS培养基中转FaFIT基因拟南芥阳性株系根部Fe2+积累显著增加,叶片叶绿素含量提高。【结论】FaFIT基因通过调控AtAHA、AtFRO2和AtIRT1基因表达以及增强H+-ATPase(ptype)、FCR和IRT酶活性促进根系铁的吸收和积累,推测是草莓根系铁吸收和转运过程中的关键调控因子。 【Objective】Iron is involved in the process of physiological and biochemical reactions in plants. Serious iron deficiency can lead to plant chlorosis and even death. Plants have evolved two ways of iron absorption, called strategy Ⅰ and strategy Ⅱ, to overcome iron deficiency. Among all plants,strategyⅠ exists in such plants other than graminaceous ones. Basic helix-loop-helix(bHLH) transcription factor regulates plant root uptake of iron. FIT, a bHLH gene, is a key gene regulating iron absorption in roots of strategyⅠ plants. FIT gene has been well studied in Arabidopsis, but it has not been reported in strawberry, so that the regulation mode of strawberry FaFIT gene is not clear. The function of strawberry FaFIT gene will be identified in this paper.【Methods】The differentially expressed bHLH gene maker-Fvb3-4-snap-gene-100.50 was obtained from the transcriptome data of Benihoppe root under iron deficiency stress. Further search found that maker-Fvb3-4-snap-gene-100.50 and At FIT(Arabidopsis) were homologous genes, so the maker-Fvb3-4-snap-gene-100.50 was named FaFIT. The coding region sequence and promoter sequence of FaFIT were searched in GDR(https://www.rosaceae.org/),and PCR primers were designed in NCBI(https://www.ncbi.nlm.nih.gov/). Total RNA was extracted from the roots of Benihoppe and reverse transcribed for cDNA. The coding sequence of FaFIT gene was cloned with cDNA as template. The promoter sequence of FaFIT gene was cloned with DNA as template. The secondary structure of FaFIT protein was predicted by PredictProtein, the conserved amino acid sequence of FaFIT was analyzed by MEME, and the FaFIT promoter elements were predicted by PlantCare. The Benihoppe strawberry plantlets were treated with iron deficiency, iron excess and high pH. The expression patterns of FaFIT gene under abiotic stress were analyzed by real-time fluorescence quantitative PCR(qRT-PCR). The 35S::FaFIT-GFP fusion overexpression vector was constructed to study the gene function of FaFIT in Arabidopsis. The constructed vector was transferred into the strain of Agrobacterium GV3101, and then was infected into Arabidopsis by the dipping flower method.Arabidopsis positive lines were screened with 1/2 MS medium containing 50 mg · mLkanamycin, and were screened continuously until T2 seeds were obtained. The positive lines of Arabidopsis were identified by PCR. Strategy I of the iron absorption by plant roots is divided into three steps: 1. P-type H+-ATP(p-type) ase pumps protons(H+) to the rhizosphere to acidify the soil and dissolve Feions;2. Ferric-chelate reductase(FCR) reduces Feto Fe;3. Iron-regulated transporter(IRT) transports Feinto root cells. In Arabidopsis, the genes corresponding to these three enzymes are AtAHA2, AtFRO2 and AtIRT1, respectively. Therefore, we used qRT-PCR to determine the gene expression of AtAHA2, AtFRO2 and AtIRT1 in Arabidopsis positive lines, and enzyme-linked immunosorbent assay kit to determine the enzyme activities of H+-ATPase(p-type), FCR and IRT. Arabidopsis positive lines grew on 1/2MS medium with pH=5.8 or pH=8.0 to observe the phenotype of Arabidopsis positive lines.【Results】The total length of FaFIT coding region was 1020 bp and encoded 339 amino-acids. FaFIT protein had a conserved HLH region and belonged to bHLH transcription factor family. The promoter of FaFIT contained bHLH transcription factor DNA binding element, abscisic acid-response elements and drought-stress response elements, which indicated FaFIT might be induced by upstream bHLH transcription factor, ABA and drought-stress. The expression pattern of FaFIT was analyzed by q RT-PCR.The results showed that FaFIT was mainly expressed in roots and induced by iron-deficiency and high p H value but the expression level of FaFIT was significantly down-regulated under excessive iron stress. Benihoppe showed iron chlorotic symptoms under iron deficiency and high pH stress. Excessive iron stress caused harmful impacts on the Benihoppe seedlings. Agrobacterium-mediated transformation of Arabidopsis thaliana showed that the FaFIT-overexpression lines activated the expression of iron absorption related genes AtAHA2, AtFRO2 and AtIRT1, and increased the enzyme activities of H+-ATPase(p-type), ferric-chelate reductase(FCR) and iron regulated transporter(IRT). FaFIT-overexpression lines of Arabidopsis thaliana accumulated more Feon 1/2 MS medium with pH=5.8 and pH=8.0.The results showed that FaFIT was specifically expressed in roots and was induced by iron-deficiency stress and high p H value.【Conclusion】FaFIT was inhibited under excessive iron stress. FaFIT can promote the iron absorption in roots by increasing the enzyme activities of FCR, H+-ATP(p-type)ase and IRT.
作者 陈亚铎 宋艳红 李刚 赵霞 刘丽锋 周厚成 CHEN Yaduo;SONG Yanhong;LI Gang;ZHAO Xia;LIU Lifeng;ZHOU Houcheng(Zhengzhou Fruit Research Institute,CAAS,Zhengzhou 450009,Henan,China)
出处 《果树学报》 CAS CSCD 北大核心 2022年第9期1562-1572,共11页 Journal of Fruit Science
基金 河南省大宗水果产业技术体系项目(S2014-11-G07) 西藏自治区科技重点研发与转化计划项目(XZ201901NB04) 郑州市重大科技创新专项(2019CXZX0084) 中国农业科学院科技创新工程项目(CAAS-ASTIP-2021-ZFRI)。
关键词 草莓 FaFIT基因 铁吸收 表达分析 Strawberry FaFIT Iron uptake Gene expression
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