Expression of S-RNase in vitro and Identification of Its Interacting Protein FviFPA from Fragaria viridis

doi:10.16420/j.issn.0513-353x.2022-1017

Acta Horticulturae Sinica ›› 2023, Vol. 50 ›› Issue (12): 2577-2590.doi: 10.16420/j.issn.0513-353x.2022-1017

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Expression of S-RNase in vitro and Identification of Its Interacting Protein FviFPA from Fragaria viridis

NI Zhiyou¹, XU Linlin², Du Jianke¹, WANG Jing¹, WANG Tao¹, PAN Jiaoyan¹, ZHAO Mizhen², QIAO Yushan¹^,²^,^*()

¹ College of Horticulture，Nanjing Agricultural University，Nanjing 210095，China
² Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement，Institute of Pomology，Jiangsu Academy of Agricultural Sciences，Nanjing 210014，China

Received:2023-03-21 Revised:2023-09-06 Online:2023-12-25 Published:2023-12-29
Contact: QIAO Yushan

Abstract

Abstract:

The self-incompatible style determinants S_a-RNase，S_b-RNase and their interacting protein FviFPA were studied based on the selfing progenies of Fragaria viridis 10-42 line. The results showed that the recombinant S-RNase protein obtained by prokaryotic expression technology could specifically inhibit self-pollen growth，both the pollen germination rate and pollen tube length decreased with the increase of the recombinant protein concentration. FviFPA protein that interacts with S_a-RNase was screened from Fragaria viridis style cDNA library in the early stage. In this study，the full-length of FviFPA was cloned and the bioinformatics was analyzed，the authenticity of the interaction between FviFPA and S-RNase was verified by yeast two-hybrid and bimolecular fluorescence complementation assay，and there was no haplotype specificity in the interaction.

Key words: Fragaria viridis, self-incompatibility, S-RNase, FviFPA, protein interaction

NI Zhiyou, XU Linlin, Du Jianke, WANG Jing, WANG Tao, PAN Jiaoyan, ZHAO Mizhen, QIAO Yushan. Expression of S-RNase in vitro and Identification of Its Interacting Protein FviFPA from Fragaria viridis[J]. Acta Horticulturae Sinica, 2023, 50(12): 2577-2590.

Figures/Tables 8

Table 1 Primer sequences used in this study

目的 Purpose	引物名称 Primer name	引物序列（5′-3′） Primer sequence
原核表达 Prokaryotic expression	pCold-S_a	F：GGATCCTCCTATGAATATTTTAAATTTGTGGTACA
	pCold-S_a	R：TCTAGATAGAATATTGATATCATTATTTCCGCAA
	pCold-S_b	F：GGATCCATGTATCAATATTTCAAGTTTGTTGTGCA
	pCold-S_b	R：TCTAGATAGAATATCAATTGGACTCTTCCTTG
基因克隆 Gene cloning	FviFPA	F：AACACTATGGCGGGTCG;R：AACACTATGGCGGGTCG
实时荧光定量PCR qRT-PCR	q-FviFPA	F：CTCCACCAGCTTCGTTTCCT;R：TGACTGAGGGATGCTCCTGA
实时荧光定量PCR qRT-PCR	EF-1α	F：CATGCGCCAGACTGTTGCTGT;R：GACCGACTCAGAATACTAGTAGC
酵母双杂交 Yeast two-hybrid	pGADT7-FviFPA	F：GTCGACATGGCGGGTCGTGGCGGAG R：GGATCCTCAACTTCCTTTCCCTTGTTG
双分子荧光互补 BiFC	FviFPA-YCE	F：GGCGCGCCATGGCGGGTCGTGGCGG;R：GGATCCACTTCCTTTCCCTTGTTG

Fig. 1 The PCR amplification of S-RNase（A）and its recombinant vector of pCold-TF（B）

Fig. 2 In vitro expression and purification of S-RNase（A，B）and detection of their RNase activity（C） M：Two-color pre-stained protein marker;1-8 indicate recombinant proteins eluted with 20，60，100，200，300，400，500 and 600 mmol · L-1 imidazole eluent. The different small letters mean significant difference at 0.05 level.

Fig. 3 Phenotypes（A），pollen germination rate（B）and mean pollen tube length（C）of self and non-self pollen treated with Fragaria viridis recombinant S-RNase in vitro The different small letters mean significant difference at 0.05 level.

Fig. 4 Cloning of FviFPA（A）and double digestion detection of pGADT7-FviFPA vector（B）and FviFPA-YCE vector（C）

Fig. 5 Interaction between FviFPA and S-RNase in yeast Three biological replicates.

Fig. 6 Interactions between FviFPA and S-RNase in Nicotiana benthamiana were verified by BiFC assay

Fig. 7 Expression of FviFPA in different tissues（A）and at different time of styles after self-pollination（B） The different small letters mean significant difference at 0.05 level.

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Expression of S-RNase in vitro and Identification of Its Interacting Protein FviFPA from Fragaria viridis

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