Soybean Cyst Nematode Resistant Zinc Finger Protein Gene GmC2H2-2like Cloning and Preliminary Functional Analysis

doi:10.7668/hbnxb.20194623

Abstract

Abstract:

In order to study the important role of zinc finger protein C₂H₂ in the stress response to soybean cyst nematode disease(SCN),lay an important foundation for further research on anti-soybean cyst nematode.The transcriptome data of SCN3 roots inoculated with Dongnong L-10(SCN-resistant)and Heinong 37(SCN-susceptible)were utilized in the present study,and the differentially expressed gene,GmC₂H₂-2like,was screened out.The localization of this gene was observed under the inverted fluorescence microscope.The gene was bioinformatically analyzed,and its overexpression vector was cloned and constructed to transform soybean hairy roots to study its resistance function against cyst nematode disease.The results showed that GmC₂H₂-2like encoded 410 amino acid residues,with a molecular weight of 45.77 ku,an isoelectric point of 8.73,and 47 phosphorylation sites,and the protein tertiary structure contained four structures,namely,α-helix(25.61%),random coil(57.32%),extended strand(13.66%),and β-turn(3.14%);the results of the subcellular localization revealed that the encoded protein was located in the nucleus;analysis of the overexpressed trans-GmC₂H₂-2like soybean hairy roots revealed that the number of cyst nematodes per unit area of the root tissue was significantly lower than that of the empty vector control,suggesting that GmC₂H₂-2like has the function of inhibiting cyst nematodes.

Key words: Soybean, Cyst nematode, GmC₂H₂-2like, Bioinformatics, Subcellular localisation

QU Shuo, LIU Fang, SONG Gengchen, HU Shihao, FANG Yaoyao, ZHAO Xue, HAN Yingpeng. Soybean Cyst Nematode Resistant Zinc Finger Protein Gene GmC₂H₂-2like Cloning and Preliminary Functional Analysis[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(3): 195-207. doi: 10.7668/hbnxb.20194623.

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Figures/Tables 17

Tab.1 Primers sequence information

引物名称 Primer name	序列(5'—3') Sequence(5'—3')	酶切位点 Restriction sites	目的片段大小/bp Target fragment size	功能 Functions
GmC₂H₂-2like-F	ATGATGCCAAAGGCCACAAT		1 233	克隆引物
GmC₂H₂-2like-R	TCTTAAACCACCAGCAATAT		1 233
TGmC₂H₂-2like-F	TCGAGCTCCGTCGACAAGCTTATGATGCCAAAGGCCACAAT	Hind Ⅲ	1 233	克隆引物
TGmC₂H₂-2like-R	GCCCTTGCTCACCATAAGCTTTCTTAAACCACCAGCAATAT	Hind Ⅲ	1 233
subGmC₂H₂-2like-F	ACGGGGGACTCTTGACCATGGATGATGCCAAAGGCCACAAT	Nco Ⅰ	1 233	亚细胞引物
subGmC₂H₂-2like-F	TACTAGTCAGATCTACCATGGTCTTAAACCACCAGCAATAT	Nco Ⅰ	1 233
Actin 4-F	GTTTCAAGCTCTTGCTCGTAATCA		104	内参引物
Actin 4-R	GTGTCAGCCATACTGTCCCCATTT
QRT-F	CCTCATCACCTTCACACTCATC		104	定量引物
QRT-R	GGAGACAAGAACACCAACTAGG

Fig.1 Results of GmC₂H₂-2like gene PCR electrophoresis M.DS2000 Marker;1—10.Test samples;A.Gradient PCR;B.Specific PCR.

Fig.2 GmC₂H₂-2like gene bacterial fluid PCR identification results M.DS2000 Marker;A:Trans-DH5α bacterial fluid PCR;B:1—5.Trans-K599 bacterial fluid PCR,6—10.Trans-GV3101 bacterial fluid PCR.

Fig.3 Results of DNAman sequence alignment

Fig.4 Results of bioinformatics research on GmC₂H₂-2like gene A.Signal peptide prediction;B.Phosphorylation analysis;C.Protein transmembrane region analysis;D.Protein secondary structure;E.Protein hydrophilicity;F.Subcellular localization analysis;G.Protein tertiary structure.

Fig.5 Results of phylogenetic tree construction of GmC₂H₂-2like gene

Fig.6 Interaction network of GmC₂H₂-2like gene-encoded proteins

Fig.7 Prediction of AtC₂H₂-2like gene expression pattern in Arabidopsis thaliana A.Subcellular analysis of AtC₂H₂-2like-encoded proteins;B.Position of AtC₂H₂-2like in the chromosome;C.AtC₂H₂-2like gene expression pattern map.

Fig.8 Prediction of GmC₂H₂-2like gene expression in soybean A.Gene horizontal transcript abundance spectrum;B.Gene expression matrix;C.Median and mean bar chart.

Fig.9 Analysis of GmC₂H₂-2like gene promoter and gene structure A.GmC₂H₂-2like gene promoter region analysis;B.C₂H₂-2like domain protein conserved domain.

Tab.2 Analysis of cis-acting elements of GmC₂H₂-2like gene promoter

顺式元件 Cis-element	序列 Sequence	功能 Function	数量 Number
Box 4	TAATAA	参与光响应保守DNA模块	8
ABRE	GTGCA	参与脱落酸反应的顺式作用元件	2
MBS	GTCAAC	干旱诱导相关元件	1
ARE	AAACCA	对厌氧诱导必不可少的顺式调节元件	1
CAAT-box	CCAAT	启动子和增强子区域中常见的顺式作用元件	36
STRE	AGGGG	胁迫相应响应元件	1
I-BOX	CCATATCAAAT	部分光响应元件	1
MYC	CATTTG	参与茉莉酸反应的顺势作用元件	3
MYb	CAACTG	防御响应元件	2
TATA-box	ATATAA	核心启动子元件在转录开始的-30左右	109
TATC-box	TATCCCA	参与赤霉素反应性的顺式作用元件	1
chs-CMA2a	TCACTTGA	光响应元件的一部分	1
chs-CMA1a	TTACTTAA	光响应元件的一部分	1

Fig.10 Expression map of the regulatory network of C₂H₂ transcription factors and target genes

Fig.11 Identification of transgenic plants and results of soybean root BAR test strip A.Not converted Dongnong 50;B.GmC₂H₂-2like overexpression plant;C.Root test strip results.

Fig.12 Distribution of average number of cyst nematodes per unit area GmC₂H₂-2like-OX.Transgenic soybean overexpressing the GmC₂H₂-2like gene;pCAMBIA3300.Control soybean transfected with empty vector.Different capital letters indicate the extremely significant difference(P<0.001).

Tab.3 t-test analysis of variance

处理 Treatments	均值 Average value	标准差 Standard deviation	t值 t-value	均值P值(双尾) Mean P-value (Two-tailed)
GmC₂H₂-2like-OX	2.630	0.500	10.921	0.000
pCAMBIA3300	5.060	0.667

Fig.13 Subcellular localization analysis of GmC₂H₂-2like protein

Fig.14 GmC₂H₂-2like Real-time Quantitative PCR results A.SCN stress treatment of DN50 soybean with GmC₂H₂-2like gene;B.SCN stress treatment of DN50 soybean;C.Color code of Fig.A;D.Color code of Fig.B.

References 23

[1]	刘金洋. 大豆油份相关性状遗传网络构建与基因GmPDAT功能分析[D]. 南京: 南京农业大学, 2020.doi:10.27244/d.cnki.gnjnu.2020.000662.
	Liu J Y. Construction of genetic network for related traits of soybean oil and functional analysis of gene GmPDAT[D]. Nanjing: Nanjing Agricultural University, 2020.
[2]	Peng D L, Jiang R, Peng H, Liu S M. Soybean cyst nematodes:a destructive threat to soybean production in China[J]. Phytopathology Research, 2021, 3(1):19.doi:10.1186/s42483-021-00095-w.
[3]	Yan G P, Baidoo R. Current research status of Heterodera glycines resistance and its implication on soybean breeding[J]. Engineering, 2018, 4(4):534-541.doi:10.1016/j.eng.2018.07.009.
[4]	Dong J, Hudson M E. WI12_Rhg1 interacts with DELLAs and mediates soybean cyst nematode resistance through hormone pathways[J]. Plant Biotechnology Journal, 2022, 20(2):283-296.doi:10.1111/pbi.13709.
[5]	Riggs R D, Schmitt D P. Complete characterization of the race scheme for Heterodera glycines[J]. Journal of Nematology, 1988, 20(3):392-395. pmid: 19290228
[6]	Niblack T L, Arelli P R, Noel G R, Opperman C H, Orf J H, Schmitt D P, Shannon J G, Tylka G L. A revised classification scheme for genetically diverse populations of Heterodera glycines[J]. Journal of Nematology, 2002, 34(4):279-288. pmid: 19265945
[7]	Caldwell B E, Brim C A, Ross J P. Inheritance of resistance of soybeans to the cyst nematode,Heterodera Glycines[J]. Agronomy Journal, 1960, 52(11):635-636.doi:10.2134/agronj1960.00021962005200110007x.
[8]	Matson A L, Williams L F. Evidence of a fourt gene for resistance to the soybean cyst nematodde[J]. Crop Science, 1965, 5(5):477.doi:10.2135/cropsci1965.0011183x000500050032xa.
[9]	Rao-Arelli A P. Inheritance of resistance to Heterodera glycines Race 3 in soybean accessions[J]. Plant Disease, 1994, 78(9):898.doi:10.1094/pd-78-0898.
[10]	Concibido V C, Diers B W, Arelli P R. A decade of QTL mapping for cyst nematode resistance in soybean[J]. Crop Science, 2004, 44(4):1121-1131.doi:10.2135/cropsci2004.1121.
[11]	Niblack T L, Colgrove A L, Colgrove K, Bond J P. Shift in virulence of soybean cyst nematode is associated with use of resistance from PI 88788[J]. Plant Health Progress, 2008, 9(1): doi:10.1094/php-2008-0118-01-rs.
[12]	Miller J, McLachlan A D, Klug A. Repetitive zinc-binding domains in the protein transcription factor IIIA from Xenopus oocytes[J]. The EMBO Journal, 1985, 4(6):1609-1614.doi:10.1002/j.1460-2075.1985.tb03825.x.
[13]	Berg J M, Shi Y G. The galvanization of biology:a growing appreciation for the roles of zinc[J]. Science, 1996, 271(5252):1081-1085.doi:10.1126/science.271.5252.1081. pmid: 8599083
[14]	韩丹, 王丕武, 曲静, 高嵩, 吴楠, 宋阳, 何欢, 刘双, 陈沼汀. 大豆C₂H₂型锌指蛋白基因SCTF-1转化及功能分析[J]. 中国油料作物学报, 2016, 38(3):307-312.doi:10.7505/j.issn.1007-9084.2016.03.006.
	Han D, Wang P W, Qu J, Gao S, Wu N, Song Y, He H, Liu S, Chen Z T. Transformation and functional analysis of soybean SCTF-1 gene encoding a C₂H₂-typezinc finger protein[J]. Chinese Journal of Oil Crop Sciences, 2016, 38(3):307-312.
[15]	汪德州, 莫晓婷, 张霞, 徐妙云, 赵军, 王磊. 玉米逆境响应相关转录因子ZmC2H2-1基因克隆及功能验证[J]. 遗传, 2018, 40(9):767-778.doi:10.16288/j.yczz.18-119.
	Wang D Z, Mo X T, Zhang X, Xu M Y, Zhao J, Wang L. Cloning and functional verification of transcription factor ZmC2H2-1 gene related to maize stress response[J]. The Genetic, 2018, 40(9):767-778.doi:10.16288/j.yCZZ.18-119.
[16]	Weng L, Zhao F F, Li R, Xu C J, Chen K S, Xiao H. The zinc-finger transcription factor SlZFP2 negatively regulates abscisic acid biosynthesis and fruit ripening in tomato[J]. Plant Physiology, 2015, 167(3):931-949. doi: 10.1104/pp.114.255174 pmid: 25637453
[17]	Feng Y, Zhang S, Li J, Pei R, Tian L, Qi J, Azam M, Agyenim-Boateng K G, Shaibu A S, Liu Y, Zhu Z, Li B, Sun J. Dual-function C₂H₂-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean[J]. New Phytologist, 2023, 237(5):1794-1809.doi:10.1111/nph.18610.
[18]	井建玲, 张鹏, 王振宇, 马秋香. 木薯 C₂H₂ 型锌指蛋白转录因子家族全基因组鉴定及表达分析[J]. 植物生理学, 2020, 56(12):2664-2676.doi:10.13592/j.cnki.ppj.2020.0404.
	Jing J L, Zhang P, Wang Z Y, Ma Q X. Genome-wide identification and expression analysis of the C₂H₂-type zinc finger protein transcription factor family in cassava[J]. Plant Physiology Journal, 2020, 56(12):2664-2676.
[19]	Li S, Li Y, Cai Q, Li X, Sun Y, Yu T, Yang J, Zhang J. Analysis of the C₂H₂ gene family in maize(Zea mays L.)under cold stress:identification and expression[J]. Life(Basel), 2022, 31(13):1-122.doi:10.3390/life13010122.
[20]	冯振宇. OsC₂H₂-96过表达转基因水稻的干旱响应和ABA应答分析[D]. 长春: 吉林大学, 2022.
	Feng Z Y. Drought response and ABA response analysis of transgenic rice overexpressed by OSC₂H₂-96[D]. Changchun: Jilin University, 2022.
[21]	马姗姗, 杨静静, 曲德辉, 李梦婕, 张进, 吴凡林, 宿红艳, 王磊. 过表达C₂H₂型锌指蛋白基因PdbZFP26提高山新杨耐盐性[J]. 林业科学, 2023, 59(1):110-118.doi:10.11707/j.1001-7488.LYKX20210971.
	Ma S S, Yang J J, Qu D H, Li M J, Zhang J, Wu F L, Su H Y, Wang L. Overexpression of PdbZFP26,a gene encoding C₂H₂ zinc finger protein,improves salt to lerance of transgenic Populus davidiana × P.bolleana[J]. Scientia Silvae Sinicae, 2023, 59(1):110-118.
[22]	张新宇, 林书岱, 张涛, 裴柳玲, 唐清, 刘峰, 刘永昌. 棉花C₂H₂类型锌指蛋白基因GhSIZ1的克隆及表达分析[J]. 棉花学报, 2015, 27(3):189-197.doi:10.11963/issn.1002-7807.201503001.
	Zhang X Y, Lin S D, Zhang T, Pei L L, Tang Q, Liu F, Liu Y C. Cloning and expression analysis of GhSIZ1,encoding a C₂H₂ zinc finger protein in cotton(Gossypium hirsutum)[J]. Cotton Science, 2015, 27(3):189-197.
[23]	黄凯. 水稻C₂H₂型转录因子OsZFP151在低温胁迫中的功能研究[D]. 长春: 吉林大学, 2018.
	Huang K. Functional study of rice C₂H₂-type transcription factor OsZFP151 in low temperature stress[D]. Changchun: Jilin University, 2018.

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