FabHLH37调控草莓抗灰霉病的功能分析

Function analysis of FabHLH37 regulating strawberry resistance to Botrytis cinerea

[目的]本文旨在通过验证草莓bHLH(basic/helix-loop-helix)家族转录因子FabHLH37的功能,解析其调控草莓抗灰霉病的机制,为草莓的抗病育种提供新的基因资源。[方法]以栽培草莓品种‘红颜’为试验材料,提取灰霉菌侵染后的草莓果实总RNA并反转录为cDNA,克隆FabHLH37编码区序列,并使用在线软件对其进行生物信息学分析;利用实时荧光定量PCR(qPCR)分别检测该基因在草莓不同组织及果实接种灰霉菌后的相对表达量;利用烟草瞬时表达系统研究该基因的亚细胞定位;通过草莓果实瞬时表达系统研究FabHLH37抗灰霉病功能。[结果]FabHLH37基因CDS全长789 bp,编码262个氨基酸,理论等电点(pI)为6.61,相对分子质量为2.9×10^(5)。进化分析显示FabHLH37与月季、苹果等蔷薇科植物bHLH37蛋白同源关系较近。亚细胞定位分析显示FabHLH37特异性定位于细胞核。qPCR分析表明FabHLH37在草莓根中表达量最高,叶次之,灰霉病菌侵染可以显著诱导FabHLH37基因的表达。草莓果实瞬时表达结果显示,与对照相比,过表达FabHLH37的草莓果实接种灰霉菌后感病程度轻,而沉默FabHLH37的草莓果实相反。进一步的qPCR检测发现,在草莓果实瞬时表达体系中,FaPR1/4/5-1、FaBG2-1、FaCHI3-1、FaSOD、FaPAL等多个防御基因的表达量与FabHLH37的表达量呈正相关,即过量表达FabHLH37可显著提高这些基因的表达量,而沉默FabHLH37则显著降低这些基因的表达量。[结论]FabHLH37可通过诱导防御基因的表达从而正向调控草莓灰霉病。

[Objectives]This paper aimed to verify the function of strawberry bHLH(basic/helix-loop-helix)family transcription factor FabHLH37,and analyze the mechanism of its regulation of strawberry resistance to gray mold,so as to provide new gene resources for strawberry disease resistance breeding.[Methods]The total RNA of strawberry‘Benihoppe’was extracted and reversely transcribed into cDNA.The FabHLH37 coding region sequence was cloned and bioinformatic analysis was performed using online software.Real-time quantitative PCR(qPCR)was used to detect the relative expression of this gene in different tissues of strawberry and in fruits inoculated with Botrytis cinerea.The subcellular localization of the gene was studied using the transient expression system of tobacco.The function of FabHLH37 against Botrytis cinerea was studied by transient expression system in strawberry fruits.[Results]The total length of FabHLH37 gene CDS was 789 bp,encoding 262 amino acids.The theoretical isoelectric point(pI)was 6.61,and the relative molecular weight was 2.9×10^(5).Analysis of homologous evolution with different species of bHLH37 protein showed that FabHLH37 protein was closely related to the bHLH37 protein in Rosaceae crops such as rose and apple.The subcellular localization results showed that FabHLH37 was specifically expressed in the nucleus.The qPCR analysis showed that the expression of FabHLH37 was the highest in strawberry roots,followed by leaves,and the expression of FabHLH37 gene was significantly induced by Botrytis cinerea infection.The transient expression of FabHLH37 in strawberry fruits showed that compared with the control,strawberry fruits with overexpression of FabHLH37 were less susceptible to Botrytis cinerea inoculation,while strawberry fruits with silence of FabHLH37 were the opposite.Further qPCR detection showed that the expression levels of FaPR1/4/5-1,FaBG2-1,FaCHI3-1,FaSOD,FaPAL and other defense genes were positively correlated with the expression levels of FabHLH37 in the transient expression system of strawberry fruits.Overexpression of FabHLH37 could significantly increase the expression of these genes,while silencing FabHLH37 could significantly decrease the expression of these genes.[Conclusions]FabHLH37 transcription factor could positively regulate resistance to Botrytis cinerea by inducing the expression of defense genes in strawberry fruits.