水稻抗潜根线虫基因OsRAI1的克隆及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0362

摘要/Abstract

摘要：

水稻潜根线虫病是江西省水稻生产上危害严重的病害之一。前期通过对水稻细尖潜根线虫侵染抗/感病水稻品种的根部组织进行比较转录组分析,筛选出差异表达上调基因OsRAI1。本研究克隆获得OsRAI1全长,通过蛋白结构预测、亚细胞定位对其功能进行分析,通过IPTG诱导,对OsRAI1蛋白表达条件进行了优化。结果表明,OsRAI1基因ORF全长1 065 bp,编码354个氨基酸,属于bHLH转录因子家族。该蛋白为亲水性蛋白,分子量37.90 kD,pI值4.86,脂肪系数68.33,总平均亲水性（GRAVY）-0.415,为非跨膜蛋白。蛋白互作预测显示该蛋白可能是通过两个蛋白间HLH区域相互结合作用。亚细胞定位结果表明该蛋白在细胞核中表达。可溶性蛋白在0.6 mmol/L IPTG 16℃诱导18 h后表达量最高。本研究结果为进一步阐明水稻抗潜根线虫分子机制奠定基础。

关键词: 水稻潜根线虫, 抗病基因, bHLH转录因子, 原核表达, 亚细胞定位

Abstract:

The plant-parasitic nematode Hirschmanniella mucronata is one of the most serious rice nematodes in Jiangxi province. In the early work of the laboratory,the up-regulated OsRAI1 gene was screened by transcriptome analysis of resistant/susceptible rice root tissues infected by H. mucronata. The full length of the gene was cloned,its protein structure was predicted,and subcellular localization were analyzed. The expression conditions of OsRAI1 protein were optimized by IPTG induction. The results showed that the ORF of OsRAI1 gene was 1 065 bp in length,encoding 354 amino acids,and it belonged to the bHLH transcription factor family. The protein was hydrophilic with molecular weight of 37.90 kD,pI value of 4.86,fat coefficient of 68.33,total average hydrophilicity（GRAVY）of -0.415,and non-transmembrane protein. The results of protein interaction prediction demonstrated that the protein may interact with each other through the HLH region between the two proteins. The results of subcellular localization showed that the protein was expressed in nucleus. The protein expression was the highest when IPTG concentration was 0.6 mmol/L and induced at 16℃ for 18 h. The results lay a foundation for understanding the molecular mechanism of H. mucronate resistance.

Key words: Hirschmanniella mucronata, disease-resistant gene, bHLH transcription factor, prokaryotic expression, subcellular localization

山草梅, 叶蕾, 张连虎, 况卫刚, 孙晓棠, 马建, 崔汝强. 水稻抗潜根线虫基因OsRAI1的克隆及功能分析[J]. 生物技术通报, 2021, 37(7): 146-155.

SHAN Cao-mei, YE Lei, ZHANG Lian-hu, KUANG Wei-gang, SUN Xiao-tang, MA Jian, CUI Ru-qiang. Cloning,and Functional Analysis of Gene OsRAI1 Resistant to Hirschmanniella mucronate in Rice[J]. Biotechnology Bulletin, 2021, 37(7): 146-155.

图/表 12

表1 PCR引物信息

Table 1 Information of PCR primers

名称Name	序列 Sequences（5'-3'）	作用 Function
OsRAI1-F	ATGGAGCTTGACGAG	基因克隆 Gene cloning
OsRAI1-R	CAGACACCTTCCGCC	基因克隆 Gene cloning
T7	CCTATAGTGAGTCGTATTA	原核表达载体鉴定 Identification of prokaryotic expression vectors
T7ter	CTAGCATAACCCCTTGGGGC	原核表达载体鉴定 Identification of prokaryotic expression vectors
pET28a-OsRAI1-F	CAGCAAATGGGTCGCGGATCCATGGAGCTTGACGAG	添加酶切位点 Adding enzyme cutting site
pET28a-OsRAI1-R	ACTCGAGCACCACCAGCGGCCGCCAGACACCTTCCGCC	添加酶切位点 Adding enzyme cutting site
1302-OsRAI1-F	ACCATGGTAGATCTGACTAGTATGGAGCTTGACGAGGAGTCC	添加酶切位点 Adding enzyme cutting site
1302-OsRAI1-R	AAGTTCTTCTCCTTTACTAGTCAGACACCTTCCGCCATAGC	添加酶切位点 Adding enzyme cutting site
pCA-F	TGAGACTTTTCAACAAAGGGTAATATCCGG	过表达载体鉴定 Identification of overexpression vectors
pCA-R	GATCTAGTAACATAGATGACACCGC	过表达载体鉴定 Identification of overexpression vectors

图1 原核表达重组表达载体构建流程图

Fig. 1 Flow chart of construction of recombinant expression vector for prokaryotic expression

图2 过表达重组表达载体构建流程图

Fig. 2 Flow chart of construction of recombinant express-ion vector for overexpression

图3 OsRAI1基因序列及其编码氨基酸序列

Fig. 3 Sequence of gene OsRAI1 and its encoded amino acid sequence

图4 OsRAI1蛋白的三级结构预测

Fig. 4 3D structure prediction of OsRAI1 protein

图5 OsRAI1蛋白系统的发育与进化 A：OsRAI1氨基酸序列的进化树分析;B：OsRAI1蛋白序列保守Motif元件预测

Fig. 5 Development and evolution of OsRAI1 protein system A: Phylogenetic tree of OsRAI1. B: Conserved motif element prediction of OsRAI1 protein sequences

图6 OsRAI1基因PCR扩增图 A：基因OsRAI1原核表达扩增产物;B：基因OsRAI1过表达扩增产物。M为2 000 marker,1-3为扩增产物

Fig. 6 Electropherogram of PCR product for OsRAI1 gene A：Amplified product of gene OsRAI1 via prokaryotic expression; B：Amplified product of gene OsRAI1 via overexpression. M：2 000 marker; 1-3：Amplified product

图7 基因OsRAI1表达载体阳性克隆鉴定 A：基因OsRAI1原核表达载体阳性克隆鉴定,M为2000 marker,1-10为单菌落PCR产物;B：基因OsRAI1过表达载体阳性克隆鉴定,M为5000 marker,1-10为单菌落PCR产物

Fig. 7 Identification of positive clones of OsRAI1 gene via vectors A：Identification of positive clones of gene OsRAI1 via prokaryotic expression vector; M：2000 marker. 1-10：PCR product of single colony. B：Identification of positive clones of gene OsRAI1 via overexpression vector; M：5000 marker. 1-10：PCR product of single colony

图8 基因OsRAI1在烟草叶片中亚细胞定位 A：目标蛋白荧光通道;B：叶绿体荧光通道;C：明场;D：叠加图

Fig. 8 Subcellular localization of gene OsRAI1 in the leaves of Nicotiana benthamiana A：Target protein fluorescence channel. B：Chloroplast fluorescence channel. C：Brightfield. D：Overlay chart

图9 不同浓度IPTG诱导重组蛋白表达产物的WB分析 M：Marker;1：1 mmol/L诱导全菌;2：0.8 mmol/L诱导上清;3：0.8 mmol/L诱导全菌;4：0.6 mmol/L诱导上清;5：0.6 mmol/L诱导全菌;6：0.2 mmol/L诱导上清;7：0.2 mmol/L诱导全菌

Fig. 9 WB analysis of recombinant protein expression products induced by different concentrations of IPTG M：Protein marker. 1：Whole bacteria induced by 1 mmol/L. 2：Supernatant induced by 0.8 mmol/L. 3：Whole bacteria induced by 0.8 mmol/L. 4：Supernatant induced by 0.6 mmol/L. 5：Whole bacteria induced by 0.6 mmol/L. 6：Supernatant induced by 0.2 mmol/L. 7：Whole bacteria induced by 0.2 mmol/L

图10 不同诱导时间和温度诱导重组蛋白表达产物的SDS-PAGE分析 M：Protein marker;1：37℃ 3 h诱导全菌;2：37℃ 6 h诱导全菌;3：37℃ 18 h诱导全菌;4：28℃ 3 h诱导全菌;5：28℃ 6 h诱导全菌;6：28℃ 18 h诱导全菌;7：16℃ 3 h诱导全菌;8：16℃ 6 h诱导全菌;9：16℃ 18 h诱导全菌;10：37℃ 3 h诱导上清;11：37℃ 6 h诱导上清;12：37℃ 18 h诱导上清;13：28℃ 3 h诱导上清;14：28℃ 6 h诱导上清;15：28℃ 18 h诱导上清;16：16℃ 3 h诱导上清;17：16℃ 6 h诱导上清;18：16℃ 18 h诱导上清

Fig. 10 SDS-PAGE analysis of recombinant protein expression products induced by different inducing time and temperature M：Protein Marker. 1：Whole bacteria induced by 37℃ for 3 h. 2：Whole bacteria induced by 37℃ for 6 h. 3：Whole bacteria induced by 37℃ for 18 h. 4：Whole bacteria induced by 28℃ for 3 h. 5：Whole bacteria induced by 28℃ for 6 h. 6：Whole bacteria induced by 28℃ for 18 h. 7：Whole bacteria induced by 16℃ for 3 h. 8：Whole bacteria induced by 16℃ for 6 h. 9：Whole bacteria induced by 16℃ for 18 h. 10：Supernatant induced by 37℃ for 3 h. 11：Supernatant induced by 37℃ for 6 h. 12：Supernatant induced by 37℃ for 18 h. 13：Supernatant induced by 28℃ for 3 h. 14：Supernatant induced by 28℃ for 6 h. 15：Supernatant induced by 28℃ for 18 h. 16：Supernatant induced by 16℃ for 3 h. 17：Supernatant induced by 16℃ for 6 h. 18：Supernatant induced by 16℃ for 18 h

表2 OsRAI1功能互作蛋白预测

Table 2 Functional interaction protein prediction of OsRAI1

基因名称 Gene name	蛋白描述 Protein description	功能结构域 Functional domain	氨基酸数/个 Number of amino acids	互作系数 Interaction coefficient
Os05T0586300-00	Expressed protein;Os03g0137400 protein	HLH	387	0.644
Os04T0486400-01	OJ000223_09.13 protein;Os04g0486400 protein	SANT	385	0.612
Os03T0137400-01	Expressed protein;Os03g0137400 protein	transmembrane helix region	792	0.578
Os01T0752500-00	Putative ethylene responsive protein Os01g0752500 protein	AP2	212	0.570
Os06T0164400-01	annotation not available;Os06g0164400 protein	coiled coil region;HLH	197	0.503

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