甘蔗ShPR10基因的克隆及其编码蛋白与甘蔗线条花叶病毒P1蛋白的互作研究

doi:10.13560/j.cnki.biotech.bull.1985.2023-0403

摘要/Abstract

摘要：

病程相关蛋白10（pathogenesis related protein 10, PR10）在植物抵抗病毒侵染中发挥重要作用。前期以甘蔗线条花叶病毒（Sugarcane streak mosaic virus, SCSMV）编码的RNA沉默抑制子P1为诱饵，筛选获得一个甘蔗ShPR10蛋白。为探究ShPR10在甘蔗应答SCSMV侵染过程中的功能，利用同源克隆技术克隆甘蔗ShPR10基因并对其编码蛋白进行生物信息学分析，利用绿色荧光蛋白融合表达法分析ShPR10蛋白的亚细胞定位，采用酵母双杂交和双分子荧光互补技术验证ShPR10与SCSMV P1的互作关系，采用农杆菌共浸润瞬时表达系统和Western blot技术分析ShPR10对P1沉默抑制子活性的影响。结果显示，甘蔗ShPR10基因开放阅读框全长570 bp，编码一个不稳定亲水蛋白，蛋白分子量为21.17 kD，等电点为4.77，含有一个P-loop基序，不含跨膜结构域和信号肽。ShPR10二级结构包含51.85%的无规则卷曲、35.98%的α-螺旋、7.41%的延伸链和4.76%的β-转角。ShPR10蛋白与玉米ZmPR10蛋白的氨基酸序列相似性高达91.53%，两者在进化树上聚为一个分支。ShPR10定位在细胞质和细胞核，与SCSMV P1在酵母细胞和烟草细胞中存在互作关系。ShPR10本身不具有沉默抑制子活性，其表达削弱了P1的沉默抑制子活性，但对P1蛋白的含量无明显影响。综上，ShPR10可能通过结合P1来削弱P1的沉默抑制子活性，从而提高甘蔗对SCSMV的抗性。

Abstract:

Pathogenesis related protein 10（PR10）plays an important role in plant resistance to viral infection. In the early stage, the RNA silencing suppressor P1 encoded by sugarcane streak mosaic virus（SCSMV）was used as bait to screen and obtain a sugarcane ShPR10 protein. In order to explore the function of ShPR10 in sugarcane response to SCSMV infection, the sugarcane ShPR10 gene was cloned by homologous cloning technology, and its coding protein was analyzed via bioinformatics. The subcellular localization of ShPR10 protein was analyzed by fusion expression with green fluorescent protein. The interaction between ShPR10 and SCSMV P1 was validated by yeast two hybrid and bimolecular fluorescence complementation techniques. The effect of ShPR10 on P1 silencing suppressor activity was analyzed using the Agrobacterium tumefaciens co-infiltration transient expression system and Western blot technology. The results showed that the open reading frame of sugarcane ShPR10 gene was 570 bp, which encoded an unstable hydrophilic protein with a molecular weight of 21.17 kD, an isoelectric point of 4.77, one P-loop motif, and no transmembrane domains and signal peptides. The secondary structure of ShPR10 contained 51.85% random coil, 35.98% α-helix, 7.41% extended-strand, and 4.76% β-turn. The amino acid sequence similarity between ShPR10 protein and ZmPR10 protein of Zea mays was as high as 91.53%, and the two proteins were clustered into one branch on the evolutionary tree. ShPR10 was located in the cytoplasm and nucleus, and interacted with SCSMV P1 in yeast and tobacco cells. ShPR10 itself did not have silencing suppressor activity, and its expression weakened the silencing suppressor activity of P1, but had no significant effect on the content of P1 protein. In summary, ShPR10 may weaken the silencing suppressor activity of P1 by binding to P1, thereby improving the resistance of sugarcane to SCSMV.

Key words: pathogenesis related protein, PR10, sugarcane streak mosaic virus, RNA silencing suppressor, P1 protein, sugarcane

黄佳艳, 冯小艳, 沈林波, 王文治, 胡海燕, 张树珍. 甘蔗ShPR10基因的克隆及其编码蛋白与甘蔗线条花叶病毒P1蛋白的互作研究[J]. 生物技术通报, 2023, 39(10): 163-174.

HUANG Jia-yan, FENG Xiao-yan, SHEN Lin-bo, WANG Wen-zhi, HU Hai-yan, ZHANG Shu-zhen. Cloning of Sugarcane ShPR10 Gene and Study on the Interaction Between ShPR10 Protein and P1 Protein Encoded by Sugarcane Streak Mosaic Virus[J]. Biotechnology Bulletin, 2023, 39(10): 163-174.

图/表 11

表1 本研究使用的引物

Table 1 Primers used in this study

引物名称 Primer name	引物序列 Primer sequence（5'-3'）	引物用途 Purpose of primer
ShPR10-F	ATGGGTTGGCGTTGGCACGA	基因克隆Gene cloning
ShPR10-R	TTACACATCTGAAATCTTGC	基因克隆Gene cloning
GFP-p-F	AACACGGGGGACTTTGCAACATGGTGAGCAAGGGCGAGGAG	亚细胞定位载体构建 Vector construction for subcellular localization
GFP-p-R	GCCAACCCATGTACAGCTCGTCCATGCCGTG
ShPR10-p-F	CGAGCTGTACATGGGTTGGCGTTGGCACGAC
ShPR10-p-R	TGAAGACAGAGCTAGTTACATTACACATCTGAAATCTTGCTCCTGAATTCTGAG
ShPR10-JT-F	AGTGGTCTCTGTCCAGTCCTATGGGTTGGCGTTGGCACGA	Y2H、BiFC和植物表达载体构建 Vector construction for Y2H, BiFC and plant expression
ShPR10-JT-R	GGTCTCAGCAGACCACAAGTTTACACATCTGAAATCTTGC	Y2H和植物表达载体构建 Vector construction for Y2H and plant expression
ShPR10-JT-R1	GGTCTCAGCAGACCACAAGTCACATCTGAAATCTTGCTCCTG	BiFC载体构建Vector construction for BiFC
P1-JT-F	AGTGGTCTCTGTCCAGTCCTATGGCTACTATCACTAAGAAGC	BiFC和植物表达载体构建 Vector construction for BiFC and plant expression
P1-JT-R	GGTCTCAGCAGACCACAAGTGTAAAATACTAAATCTTCACAGC	BiFC载体构建Vector construction for BiFC
P1-JT-R1	GGTCTCAGCAGACCACAAGTTCAGTAAAATACTAAATCTTCAC	植物表达载体构建Vector construction for plant expression

图1 ShPR10基因克隆结果 A：ShPR10基因扩增产物，M表示DL2000 DNA marker；B：ShPR10基因的核苷酸序列及其推导的氨基酸序列，P-loop基序用黑色下划线标记

Fig. 1 Cloning results of ShPR10 gene A: Amplification product of ShPR10 gene, M represents DL2000 DNA marker. B: Nucleotide acid sequence and deduced amino acid sequence of ShPR10 gene, P-loop motif is marked with black underline

图2 ShPR10蛋白二级结构预测蓝色为α-螺旋；红色为延伸链；绿色为β-转角；紫色为无规则卷曲

Fig. 2 Secondary structure prediction of ShPR10 protein The blue is α-helix. Red is extended-strand. Green is β-turn. Purple is random coil

图3 ShPR10蛋白跨膜结构预测

Fig. 3 Transmembrane structure prediction of ShPR10 protein

图4 ShPR10蛋白信号肽预测

Fig. 4 Signal peptide prediction of ShPR10 protein

图5 甘蔗ShPR10与其他物种PR10的氨基酸序列比对

Fig. 5 Amino acid sequence alignment between ShPR10 protein from sugarcane and PR10 from other species LpPR10: XP_051226795.1; LrPR10: XP_039849257.1; ObPR10: XP_040382970.1; OgPR10: XP_052140876.1; PvPR10: XP_039810901.1; TaPR10: XP_044357998.1; TdPR10: XP_037414265.1; TuPR10: XP_048564659.1; ZmPR10: NP_001152669.1

图6 甘蔗ShPR10与其他物种PR10蛋白的系统进化树分析

Fig. 6 Phylogenetic tree analysis of ShPR10 protein from sugarcane and PR10 proteins from other species

图7 ShPR10在本氏烟草表皮细胞中的定位

Fig. 7 Localization of ShPR10 in N. benthamiana epidermal cells

图8 Y2H验证ShPR10与SCSMV P1的互作

Fig. 8 Verification of the interaction between ShPR10 and SCSMV P1 by Y2H assay

图9 BiFC验证ShPR10与SCSMV P1的互作

Fig. 9 Verification of the interaction between ShPR10 and SCSMV P1 by BiFC assay

图10 ShPR10对SCSMV P1沉默抑制子活性的影响 A：携带不同质粒的3种农杆菌混合注射后烟草叶片中的荧光表达情况，GFP表示pCam3304-35S-GFP质粒，HA-ShPR10表示pNC-Cam33HN-ShPR10质粒，Flag-P1表示pNC-Cam33FN-P1质粒，HA表示pNC-Cam33HN空质粒，Flag表示pNC-Cam33FN空质粒；B：Western blot检测烟草叶片注射区域的蛋白水平，图片上方表示的质粒组合同图A，HA抗体、Flag抗体和GFP抗体分别用来检测ShPR10、P1和GFP的蛋白水平，考马斯亮蓝（CBB）染色的Rubisco大亚基作为上样量对照，误差代表3次重复实验的标准差

Fig. 10 Effect of ShPR10 on the activity of SCSMV P1 silencing suppressor A: Fluorescence expression in N. benthamiana leaves after co-injection of three A. tumefaciens cultures carrying different plasmids. GFP represents the pCam3304-35S-GFP plasmid, HA-ShPR10 represents the pNC-Cam33HN-ShPR10 plasmid, Flag-P1 represents the pNC-Cam33FN-P1 plasmid, HA represents the pNC-Cam33HN empty plasmid, and Flag represents the pNC-Cam33FN empty plasmid. B: The protein levels in the injection areas of N. benthamiana leaves were detected by Western blot. The plasmid combinations shown at the top of the figure were the same as that in Figure A. Levels of ShPR10, P1, and GFP proteins were confirmed with HA, Flag, and GFP antibodies, respectively. The large subunit of Rubisco stained with Coomassie brilliant blue（CBB）was used as a loading control. The error indicates the standard deviation of three repeated experiments

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