Cloning,and Functional Analysis of Gene OsRAI1 Resistant to Hirschmanniella mucronate in Rice

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

Abstract

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

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.

Figures/Tables 12

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

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

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

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

Fig. 4 3D structure prediction of OsRAI1 protein

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

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

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

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

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

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

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

References 21

[1]	de Silva WSI, Perera MMN, Perera KLNS, et al. In silico analysis of osr40c1 promoter sequence isolated from indica variety pokkali[J]. Rice Sci, 2017, 24(4):228-234. doi: 10.1016/j.rsci.2016.11.002 URL
[2]	Wani SH, Kumar Sah S. Biotechnology and abiotic stress tolerance in rice[J]. J Rice Res, 2014, 2(2):e105. DOI: 10.4172/jrr.1000e105. doi: 10.4172/jrr.1000e105
[3]	曾红根. 江西省高产优质直播水稻栽培技术及病虫草害防治探析[J]. 种子科技, 2020, 38(20):98-99.
	Zeng HG. Analysis on cultivation technology of high yield and high quality direct seeding rice and prevention and control of pests and weeds in jiangxi province[J]. Seed Science & Technology, 2020, 38(20):98-99.
[4]	汪家旭, 潘沧桑. 潜根线虫的种类[J]. 厦门大学学报:自然科学版, 1999, 38(2):145-152.
	Wang JX, Pan CS. Studies of rice root Nematodes(Hirschmanniella species)[J]. J Xiamen Univ:Nat Sci, 1999, 38(2):145-152.
[5]	Elling AA. Major emerging problems with minor Meloidogyne species[J]. Phytopathology, 2013, 103(11):1092-1102. doi: 10.1094/PHYTO-01-13-0019-RVW pmid: 23777404
[6]	张绍升, 李茂胜, 严叔平. 水稻潜根线虫的致病性和综合防治技术[J]. 中国水稻科学, 1998, 12(1):31-34.
	Zhang SS, Li MS, Yan SP. The pathogenicity and integrated control measures of rice root Nematodes[J]. Chin J Rice Sci, 1998, 12(1):31-34.
[7]	Sun XT, Zhang L, Tang ZQ, et al. Transcriptome analysis of roots from resistant and susceptible rice varieties infected with Hirschmanniella mucronata[J]. FEBS Open Bio, 2019, 9(11):1968-1982. doi: 10.1002/feb4.v9.11 URL
[8]	于冰, 田烨, 李海英, 等. 植物bHLH转录因子的研究进展[J]. 中国农学通报, 2019, 35(9):75-80.
	Yu B, Tian Y, Li HY, et al. Research progress of plant bHLH transc-ription factor[J]. Chin Agric Sci Bull, 2019, 35(9):75-80.
[9]	Li X, Duan X, Jiang H, et al. Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis[J]. Plant Physiol, 2006, 141(4):1167-1184. doi: 10.1104/pp.106.080580 URL
[10]	刘晓月, 王文生, 傅彬英. 植物bHLH转录因子家族的功能研究进展[J]. 生物技术进展, 2011, 1(6):391-397.
	Liu XY, Wang WS, Fu BY. Research progress of plant bHLH transcription factor family[J]. Curr Biotechnol, 2011, 1(6):391-397.
[11]	Thorstensen T, Grini PE, Mercy IS, et al. The Arabidopsis SET-domain protein ASHR3 is involved in stamen development and interacts with the bHLH transcription factor ABORTED MICROSPORES(AMS)[J]. Plant Mol Biol, 2008, 66(1/2):47-59. doi: 10.1007/s11103-007-9251-y URL
[12]	王艳敏, 白卉, 曹焱. bHLH转录因子研究进展及其在植物抗逆中的应用[J]. 安徽农业科学, 2015, 43(21):34-35, 50.
	Wang YM, Bai H, Cao Y. Research progress of bHLH transcription factor and application in plant abiotic stress tolerance[J]. J Anhui Agric Sci, 2015, 43(21):34-35, 50.
[13]	Wang YJ, Zhang ZG, He XJ, et al. A rice transcription factor OsbHLH1 is involved in cold stress response[J]. Theor Appl Genet, 2003, 107(8):1402-1409. doi: 10.1007/s00122-003-1378-x URL
[14]	Ortolan F, Fonini LS, Pastori T, et al. Tightly controlled expression of OsbHLH35 is critical for anther development in rice[J]. Plant Sci, 2021, 302:110716. doi: 10.1016/j.plantsci.2020.110716 URL
[15]	Wang L, Ying Y, Narsai R, et al. Identification of OsbHLH133 as a regulator of iron distribution between roots and shoots in Oryza sativa[J]. Plant Cell Environ, 2013, 36(1):224-236. doi: 10.1111/pce.2013.36.issue-1 URL
[16]	Lee J, Moon S, Jang S, et al. OsbHLH073 negatively regulates internode elongation and plant height by modulating GA homeostasis in rice[J]. Plants, 2020, 9(4):547. doi: 10.3390/plants9040547 URL
[17]	邵雅芳, 徐非非, 唐富福, 等. 水稻花青素合成相关基因的时空表达研究[J]. 核农学报, 2013, 27(1):9-14.
	Shao YF, Xu FF, Tang FF, et al. The temporal and spatial expression pattern of anthocyanin related genes in RICE(Oryza sativa L.)[J]. J Nucl Agric Sci, 2013, 27(1):9-14.
[18]	Carretero-Paulet L, Galstyan A, Roig-Villanova I, et al. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae[J]. Plant Physiol, 2010, 153(3):1398-1412. doi: 10.1104/pp.110.153593 pmid: 20472752
[19]	Chen ZH, Nimmo GA, et al. BHLH32 modulates several biochemical and morphological processes that respond to Pi starvation in Arabidopsis[J]. Biochem J, 2007, 405(1):191-198. doi: 10.1042/BJ20070102 URL
[20]	Sun XH, Copeland NG, Jenkins NA, et al. Id proteins Id1 and Id2 selectively inhibit DNA binding by one class of helix-loop-helix proteins[J]. Mol Cell Biol, 1991, 11(11):5603-5611. pmid: 1922066
[21]	Qin Y, L i X, Guo M, et al. Regulation of salt and ABA responses by CIPK14, a calcium sensor interacting protein kinase in Arabidopsis[J]. Sci China C Life Sci, 2008, 51(5):391-401.