Molecular Cloning and Expression Analysis of a F-box Protein Gene FnFBOX1 and Its Promoter from Fragaria nilgerrensis

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

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

Fig. 5 Subcellular localization of FnFBOX1 in onion epidermis

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

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

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）

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

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）

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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