绿色草莓S-RNase的体外表达及其互作蛋白FviFPA鉴定

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

摘要/Abstract

摘要：

以绿色草莓（Fragaria viridis）10-42的自交二代为试材，对自交不亲和花柱决定因子S_a-RNase和S_b-RNase及其互作蛋白FviFPA进行了研究。结果表明，通过原核表达技术获得的S-RNase重组蛋白可特异性抑制自我花粉生长，花粉萌发率和花粉管长度均随重组蛋白浓度的升高而下降。前期筛选绿色草莓花柱cDNA文库获得了一个与S_a-RNase存在相互作用的FviFPA蛋白。通过FviFPA全长克隆和生物信息分析，利用酵母双杂交和双分子荧光互补试验验证了FviFPA与S-RNase存在相互作用的真实性，且其互作不存在单倍型特异性。

关键词: 绿色草莓, 自交不亲和, S-RNase, FviFPA, 蛋白互作

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

倪知游, 许林林, 杜建科, 王静, 汪涛, 潘娇艳, 赵密珍, 乔玉山. 绿色草莓S-RNase的体外表达及其互作蛋白FviFPA鉴定[J]. 园艺学报, 2023, 50(12): 2577-2590.

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.

图/表 8

表1 本研究中所用引物序列

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

图1 S-RNase（A）及其与pCold-TF的重组载体（B）PCR扩增

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

图2 S-RNase体外表达纯化（A、B）及其活性检测（C） M：双色预染蛋白marker;1 ~ 8分别是用20、60、100、200、300、400、500和600 mmol · L-1咪唑洗脱的重组蛋白。不同小写字母表示差异显著（P < 0.05）。

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.

图3 绿色草莓S-RNase重组蛋白处理的自我与非自我花粉形态（A）、花粉萌发率（B）及花粉管长度（C）不同小写字母表示差异显著（P < 0.05）。

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.

图4 FviFPA克隆（A）及pGADT7-FviFPA载体（B）、FviFPA-YCE载体（C）双酶切检测

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

图5 酵母中FviFPA与S-RNase互作 3个生物学重复。

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

图6 BiFC验证烟草中FviFPA与S-RNase互作

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

图7 绿色草莓FviFPA在不同组织（A）和自花授粉不同时间花柱中（B）的表达量不同小写字母表示差异显著（P < 0.05）。

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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