黄毛草莓F-box蛋白基因FnFBOX1及其启动子的克隆和表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0295

摘要/Abstract

摘要：

探索黄毛草莓FnFBOX1参与草莓胶孢炭疽菌（Colletotrichum gloeosporioides）侵染过程中的抗病反应和pFnFBOX1启动子的转录活性,为研究FnFBOX1抗炭疽病功能奠定基础。以黄毛草莓（Fragaria nilgerrensis Schidl.）为研究对象,通过RT-PCR技术克隆FnFBOX1的cDNA序列。通过氨基酸序列比对、进化树分析、亚细胞定位、启动子克隆并构建瞬时表达载体、组织特异性表达分析,最后用荧光定量分析了抗病的黄毛草莓和感病的‘妙香 3’草莓在水杨酸（SA）和胶孢炭疽菌胁迫下FnFBOX1的表达水平变化。结果表明,FnFBOX1的ORF全长为1 227 bp,编码408个氨基酸。氨基酸序列比对和进化树分析发现FnFBOX1与森林草莓（Fragaria vesca）FvFBOX1同源性最高。亚细胞定位结果显示FnFBOX1定位于细胞核。克隆的FnFBOX1启动子长度为821 bp,构建pFnFBOX1的瞬时表达载体pFnFBOX1∷GUS。瞬时转化烟草后对其进行组织化学染色分析和GUS活性测定,结果表明,pFnFBOX1具有驱动下游基因转录的活性,且胶孢炭疽菌和SA可促进FnFBOX1启动子启动的GUS活性的提高。荧光定量PCR分析表明,FnFBOX1在黄毛草莓的不同组织中均有表达。抗病黄毛草莓和感病‘妙香 3’草莓均进行接种胶孢炭疽菌和喷施SA处理,结果显示,黄毛草莓接种胶孢炭疽菌12 h后FnFBOX1表达量达到最高,是0 h的6.3倍;SA处理24 h时,其表达量达到最高,是0 h的7.9倍。‘妙香 3’草莓也响应胶孢炭疽菌和SA处理,但FBOX1表达量比黄毛草莓低。

关键词: 黄毛草莓, 炭疽病, FnFBOX1, 启动子, 表达分析

Abstract:

This work is to explore the involvement of Fragaria nilgerrensis Schidl. FnFBOX1 gene in plant-pathogen defense response during pathogen infection of Colletotrichum gloeosporioides and transcriptional activity of pFnFBOX1 promoter,thus laying a foundation for further revealing that FBOX1 gene in the regulation of resistance to anthracnose in strawberry. Having F. nilgerrensis as the experimental material,the cDNA sequence of a F-box protein gene FnFBOX1 was cloned by RT-PCR. Then amino acid sequences alignment,phylogenetic tree,subcellular localization,promoter clone and construction of transient expression vector,and tissue specific expression analysis were conducted. And the expression patterns of FnFBOX1 in F. nilgerrensis and infected ‘Miaoxiang 3’ in response to stresses of salicylic acid and C. gloeosporioides were detected by real-time quantitative PCR. The results showed that the ORF of FnFBOX1 was 1 227 bp length,encoding 408 amino acids. Analysis from amino acid sequence alignment and phylogenetic tree showed that FnFBOX1 had the highest homology with the FvFBOX1 of Fragaria vesca. Subcellular localization assays showed that the FnFBOX1 was located in the nucleus. The 821 bp region of promoter pFnFBOX1 was cloned,and the transient expression vector pFnFBOX1∷GUS was generated. Histochemical staining analysis and GUS activity determination showed that pFnFBOX1 had the activity of driving downstream gene transcription. C. gloeosporioides infection and SA treatment promoted the increase of GUS activity derived by pFnFBOX1. RT-qPCR analysis showed that FnFBOX1 gene was expressed in different tissues of F. nilgerrensis. Both the disease-resistant F. nilgerrensis and the susceptible ‘Miaoxiang 3’ strawberry were treated with C. gloeosporioides and SA,and the result showed that the FnFBOX1 gene expression in the F. nilgerrensis reached the highest at 12 h after inoculation with C. gloeosporioides,which was 6.3 times over than that of 0 h. At 24 h after SA treatment,its expression reached the maximum,which was 7.9 times over than that of 0 h. ‘Miaoxiang 3’ strawberry also responded to C. gloeosporioides and SA treatment,but the response degree was not as high as that of F. nilgerrensis.

Key words: Fragaria nilgerrensis Schidl., anthracnose, FnFBOX1 gene, promoter, expression analysis

时雅倩, 申亚茹, 陈漫影, 何淑敏, 刘予涵, 何天楠, 陈清西, 文志丰. 黄毛草莓F-box蛋白基因FnFBOX1及其启动子的克隆和表达分析[J]. 生物技术通报, 2022, 38(2): 44-56.

SHI Ya-qian, SHEN Ya-ru, CHEN Man-ying, HE Shu-min, LIU Yu-han, HE Tian-nan, CHEN Qing-xi, WEN Zhi-feng. Molecular Cloning and Expression Analysis of a F-box Protein Gene FnFBOX1 and Its Promoter from Fragaria nilgerrensis[J]. Biotechnology Bulletin, 2022, 38(2): 44-56.

图/表 12

表1 PCR引物信息

Table 1 Information for PCR primers

引物名称Primer name	引物序列Primer sequence（5'-3'）	引物用途Primer function
FnFBOX1 -F	ATGCTGTTAACTACTCCTGACCAT	ORF的扩增 ORF amplification
FnFBOX1-R	TCAAACACAAGACAGAGTCTCTAT	ORF的扩增 ORF amplification
FnFBOX1-real-F	CGCCTTCCCGTCAAATCTCT	实时荧光定量PCR Quantitative real-time PCR
FnFBOX1-real-R	TTCCGGCTCTTTCCCAACTC	实时荧光定量PCR Quantitative real-time PCR
FnActin-real-F	GCCAGAAAGATGCTTATGTCGGT	黄毛草莓内参基因 Actin gene in F. nilgerrensis
FnActin -real-R	TGGGGCAACACGAAGCTCAT	黄毛草莓内参基因 Actin gene in F. nilgerrensis
FaActin-real-F	CGAGGCTCAATCCAAAAGAG	栽培草莓内参基因 Actin gene in F.xananassa
FaActin-real-R	TGGCCACATACATAGCAGGA	栽培草莓内参基因 Actin gene in F.xananassa
P-FnFBOX1-F	CGATCGAGTATCCACATTTAAACA	启动子克隆 Promoter cloning
P-FnFBOX1-R	TCATTGGCTTGGGATATAATGAAG	启动子克隆 Promoter cloning
P-FnFBOX1-GUS-F	CGGGATCCCGCGATCGAGTATCCACATTTAAACA	融合GUS载体构建 Infusion GUS vector
P-FnFBOX1-GUS-R	CCCAAGCTTGGGTCATTGGCTTGGGATATAATGAAG	融合GUS载体构建 Infusion GUS vector
FnFBOX1-YFP-F	ATGCTGTTAACTACTCCTGACCA	融合YFP载体构建 Infusion YFP vector
FnFBOX1-YFP-R	AACACAAGACAGAGTCTCTATGAA	融合YFP载体构建 Infusion YFP vector

图1 黄毛草莓FnFBOX1的PCR扩增图 M：Marker 5 000;1-2：FnFBOX1

Fig. 1 Electropherogram of PCR product for FnFBOX1 gene in F. nilgerrensis

图2 黄毛草莓FnFBOX1的二级结构预测（A）与信号肽预测（B） A：红色表示β-折叠,蓝色表示α-螺旋,紫色表示随机卷曲,绿色表示β-转角

Fig. 2 Prediction of secondary structure of FnFBOX1 protein in F. nilgerrensis（A）and signal peptide prediction（B） A：The color red,blue,purple and green represent beta-sheet,alpha helix,random coil and beta-turn,respectively

图3 黄毛草莓FnFBOX1与其他植物氨基酸序列的比对和保守结构域分析 FnFBOX1：黄毛草莓,MN709780;FvFBOX1：森林草莓,XP_024192373;PaFBOX1：樱桃,XP_008223808;RcaFBOX1：月季,XP_021825170。划线部分为F-box保守结构域

Fig. 3 Alignment of FnFBOX1 in F. nilgerrensis with other plant amino acid sequences and analysis of conserved domains FnFBOX1：Fragaria nilgerrensis,MN709780;FvFBOX1：Fragaria vesca,XP_024192373;PaFBOX1：Prunus avium,XP_008223808;RcaFBOX1：Prunus avium,XP_021825170. The underlined part is the F-box conserved domain

图4 黄毛草莓FnFBOX1和其他植物FBOX1序列构建的系统进化树甜橙：Camellia sinensis,KDO52565;可可：Theobroma cacao,EOY22796;木薯：Manihotesculenta,XP_021634763;黄毛草莓：Fragarianilgerrensis,MN709780;森林草莓：Fragariavesca,XP_004301401;月季：Rosa chinensis,XP_024192373;苹果：Malus domestica,XP_008351366;樱桃：Prunusavium,XP_021825170;巨桉：Eucalyptus grandis,XP_010050919;核桃：Juglans regia,XP_018807608

Fig. 4 Phylogenetic tree of FnFBOX1 in F. nilgerrensis with FBOX1 of other plant species

图5 FnFBOX1在洋葱表皮中亚细胞定位

Fig. 5 Subcellular localization of FnFBOX1 in onion epidermis

图6 黄毛草莓FnFBOX1启动子的PCR扩增 M：Marker 5 000;1：FnFBOX1启动子

Fig. 6 PCR product of pFnFBOX1 in F. nilgerrensis M：Marker 5 000;1：pFnFBOX1

图7 黄毛草莓FnFBOX1上游启动子pFnFBOX1序列及预测的顺式作用元件

Fig. 7 Sequence and cis-elements in the promoter of FnFBOX1 gene in F. nilgerrensis

图8 黄毛草莓FnFBOX1启动子融合GUS载体构建 A：BamH I和Hind III双酶切的pMDTM-20T-pFnBOX1重组质粒（M：DL5 000 marker,1：重组质粒）;B：BamH I和Hind III双酶切0380∷GUS（M：DL5 000 marker,1和2：0380∷GUS双酶切）;C：BamH I和Hind III双酶切pFnFBOX1∷GUS（M：DL5 000 marker,1和2：pFnFBOX1∷GUS双酶切

Fig. 8 Construction of pFnFBOX1∷GUS fusion vector in F. nilgerrensis A：BamH I and Hind III double digest the pMDTM-20T-pFnBOX1（M：DL5 000 marker;1：recombined plasmid）;B：BamH I and Hind III double digest the 0380∷GUS（M：DL5 000 marker;1-2：0380∷GUS double digest）;C：BamH I and Hind III double digest the pFnFBOX1∷GUS（M：DL5 000 marker;1-2：pFnFBOX1∷GUS double digest）

图9 黄毛草莓启动子pFnFBOX1∷GUS载体转化烟草后染色图及GUS酶活测定 A：pCaMV35S∷GUS载体转化烟草后染色图;B：pC0380∷GUS载体转化烟草后染色图;C：pFnFBOX1∷GUS载体转化烟草后染色图;D：烟草瞬时表达GUS酶活性

Fig. 9 Staining and GUS activity after pFnFBOX1∷GUS vector transformed into tobacco A：Staining after pCaMV35S∷GUS vector was transformed into tobacco. B：Staining after pC0380∷GUS vector was transformed into tobacco. C：Staining after pFnFBOX1∷GUS vector was transformed into tobacco. D：GUS activity in the leaves of tobacco transiently expressing the pCaMV35S∷GUS or pFnFBOX1∷GUS

图10 不同处理及黄毛草莓不同组织中FnFBOX1的表达 A：黄毛草莓叶片接种胶孢炭疽菌FnFBOX1的表达;B：SA处理后FnFBOX1的表达;C：黄毛草莓不同组织中FnFBOX1的表达。3次生物学重复和3次技术重复,标准误显示在柱形图上,星号表示处理系和对照之间的差异（*P<0.05,**P<0.01）

Fig. 10 Expressions of FnFBOX1 after different treatments and in different tissues of F. nilgerrensis A：Expression of FnFBOX1after the leaves of F. nilgerrensis inoculated with C. gloeosporioides. B：Expression of FnFBOX1 after SA treatment. C：Expression of FnFBOX1 in different tissues of F. nilgerrensis. There are 3 biological and technical replicates,the standard error is displayed on the bar graph,and the asterisk indicates the difference between the treatment line and the control（*P<0.05,**P<0.01）

图11 ‘妙香3’叶片接种胶孢炭疽菌和SA处理后黄毛草莓FBOX1的表达 A：‘妙香3’草莓叶片接种胶孢炭疽菌后FnFBOX1的相对表达;B：SA处理后FnFBOX1的相对表达。3次生物学重复和3次技术重复,标准误显示在柱形图上,小写字母表示处理系和对照之间的差异（P<0.05）

Fig. 11 Expressions of FBOX1 in F. nilgerrensis after inoculation with C. gloeosporioides and SA treatment in ‘Miaoxiang 3’ leaves A：Expression of FnFBOX1 after the leaves of ‘Miaoxiang 3’ inoculated with C. gloeosporioides. B：Expression of FnFBOX1 after SA treatment. There are 3 biological and technical replicates,the standard error is displayed on the bar graph,and lowercase letters indicate the difference between the treatment line and the control（P<0.05）

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