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锦绣杜鹃热激转录因子基因RpHSFC1a的克隆及功能分析OA北大核心CSTPCD

Cloning and Functional Analysis of the Heat-shock Transcription Factor Gene RpHSFC1aof Rhododendron×pulchrum

中文摘要英文摘要

杜鹃花(Rhododendron)具有极高的观赏价值、经济价值和药用价值,而目前栽培的大多数杜鹃优良品种的耐热性较差,温度的升高会严重影响其正常生长发育.热激转录因子(heat stress transcription factors,HSFs)在植物热激应答或耐热性调控中起核心作用.本研究克隆了锦绣杜鹃(Rhododendron×pulchrum)RpHSFC1a(GenBank No.OQ628046)和八氢番茄红素脱氢酶(phytoene desaturase)基因RpPDS(GenBank No.OQ628048),以RpPDS为报告基因,烟草脆裂叶病毒(Tobacco rattle virus,TRV)为载体,构建锦绣杜鹃病毒诱导的基因沉默(virus-induced gene silencing,VIGS)体系,并对RpHSFC1a基因的生物信息学及其在响应高温胁迫中的功能进行了分析.通过表型观测、PCR和qPCR方法检测发现,TRV病毒可以侵染并在锦绣杜鹃叶片中复制和转移,当RpPDS基因被抑制表达后,锦绣杜鹃叶片会出现黄化现象,表明TRV病毒介导的基因沉默体系可以在锦绣杜鹃中应用.RpHSFC1a基因的CDS共891 bp,编码296个氨基酸,编码蛋白具有HSF_DNA-bind superfamily保守结构域.RpHSFC1a蛋白预测为亲水蛋白和跨膜蛋白.系统发育分析表明,RpHSFC1a蛋白与君迁子(Diospyros lotus)HSFC1同源性最高,为75.8%.热胁迫下,RpHSFC1a基因显著高表达,RpHSFC1a基因沉默株系叶片的叶绿素参数Fv/Fm、PIABS和ETo/RC显著小于对照组;而DIo/RC显著高于对照组.表明RpHSFC1a基因的低表达可导致PSⅡ光能利用的能力降低,进而降低了锦绣杜鹃抵御高温胁迫的能力,推测RpHSFC1a基因参与锦绣杜鹃响应高温胁迫的调控.本研究可为杜鹃耐热性的研究提供理论参考,也为提高杜鹃高温胁迫耐受能力提供基因资源.

Rhododendron species have ornamental,economic and medicinal value,while most of the excellent varieties of Rhododendron cultivated at present have poor heat tolerance,and the increase in temperature will seriously affect the normal growth and development of Rhododendron.Heat stress transcription factors(HSFs)plays a key role in the regulation of plant heat shock response or heat tolerance.In this study,phytoene desaturase(RpPDS,OQ628048)and RpHSFC1a(OQ628046)genes of Rhododendron×pulchrum were cloned,and the virus induced gene silencing(VIGS)system was constructed with RpPDS as the reporter gene and Tobacco rattle virus(TRV)as the vector.The bioinformatics of the RpHSFC1a gene and its function in response to high temperature stress were analyzed.Using phenotypic observation,PCR and qPCR methods,TRV could infect and replicate and metastasize in the leaves of Rhododendron×pulchrum,and yellowing will occurred in leaves when the expression of RpPDS gene is inhibited,the result showed that TRV virus-mediated gene silencing system could be applied in Rhododendron×pulchrum.The CDS of the RpHSFC1a gene length totally 891 bp and encodes 296 amino acids,and RpHSFC1a contained HSF_DNA-bind superfamily conserved domain.RpHSFC1a was predicted to be a hydrophilic and transmembrane protein.Phylogenetic analysis showed that RpHSFC1a protein had the highest homology rate with HSFC1 of Diospyros lotus,with a homology of 75.8%.Under heat stress,RpHSFC1a gene was significantly up-expression,the chlorophyll parameters Fv/Fm,PIABS and ETo/RC in the leaves of the strain of RpHSFC1a gene silenced were significantly lower than those of the control group.The DIo/RC was significantly higher than that of the control group.It was shown that the low expression of RpHSFC1a gene could reduce the ability of Rhododendron×pulchrum to resist high temperature stress,and it is speculated that the RpHSFC1a gene participates in the regulatory mechanism of Rhododendron×pulchrum in response to high temperature stress.This study provides a theoretical reference for the study of heat tolerance,and genetic resources for improving the high temperature stress tolerance of Rhododendron.

申建双;胡玥;李雪芹;程和丰;金松恒

浙江农林大学暨阳学院,诸暨 311800||杭州职业技术学院,杭州 310000浙江农林大学暨阳学院,诸暨 311800

园艺学与植物营养学

锦绣杜鹃病毒诱导的基因沉默(VIGS)热激转录因子(HSFs)叶绿素荧光基因功能分析

Rhododendron×pulchrumVirus-induced gene silencing(VIGS)Heat stress transcription factors(HSFs)Chlorophyll fluorescenceGene functional analysis

《农业生物技术学报》 2024 (006)

1284-1296 / 13

国家重点研发计划(2019YFE0118900);国家自然科学基金(31971641;32201608)

10.3969/j.issn.1674-7968.2024.06.005

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