Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants

doi:10.3969/j.issn.1004-1524.2022.09.16

Acta Agriculturae Zhejiangensis ›› 2022, Vol. 34 ›› Issue (9): 1976-984.DOI: 10.3969/j.issn.1004-1524.2022.09.16

• Plant protection • Previous Articles Next Articles

Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants

XU Li^a(), WANG Qi^b, DING Ting^b, JIANG Teng^c^,^*()

a. College of Life Sciences, Anhui Agricultural University,Hefei 230061, China
b. School of Plant Protection, Anhui Agricultural University,Hefei 230061, China
c. Institute of New Rural Development Research, Anhui Agricultural University,Hefei 230061, China

Received:2021-06-07 Online:2022-09-25 Published:2022-09-30
Contact: JIANG Teng

Abstract

Abstract:

The GRMZM2G455909 gene of maize was discovered in our previous study. In order to determine the molecular characteristics of this gene, bioinformatics analysis and a range of molecular experiments were carried out. The over-expression vector pCAMBIA1301A-GRMZM2G455909 was constructed and transformed into the Arabidopsis thaliana plants by an Agrobacterium-mediated method, then the disease-resistance and its signal pathway of transgenic Arabidopsis thaliana plants with GRMZM2G455909 gene were analyzed after inoculation with Pst DC3000. The results showed that the protein encoded by GRMZM2G455909 gene belonged to the NBS-LRR family, and the number of bacterium in leaves from transgenic Arabidopsis thaliana plants with GRMZM2G455909 gene was significantly decreased after inoculation with Pst DC3000 compared with wild-type plants. These suggested that transformation of Arabidopsis thaliana plants with GRMZM2G455909 gene might increase resistance to Pst DC3000 via a SA-mediated signaling pathway. This study could provide a new genetic and breeding approach for improving the disease resistance of crop.

Key words: GRMZM2G455909 gene, vector construction, transgenic Arabidopsis thaliana, disease-resistance

CLC Number:

S513

XU Li, WANG Qi, DING Ting, JIANG Teng. Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants[J]. Acta Agriculturae Zhejiangensis, 2022, 34(9): 1976-984.

Figures/Tables 10

Table 1 Primers sequences used in qRT-PCR

基因 Gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
AtActin2	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCT TGC
AtTUB4	CGAAAACGCTGACGAGTGTA	CCTTGGGAATGGGATAAGGT
GRMZM2G455909	CGACGAATGAGGCTCCTAGT	CTTGTCCAGCTCATTGTCGG

Table 2 The primers sequences of disease resistance related genes

基因 Gene	上游引物序列 Forward primer sequences(5'→3')	下游引物序列 Reverse primer sequences(5'→3')
PR1	ACACGTGCAATGGAGTTTGT	TGCAACTGATTATGGTTCCA
LOX	AGGAGTTTGGACGGGAGATT	CCGTACTTGCTCGGGTCA
EFR1	CCTTCCGAT CAA ATC CGT AAG	TCCCGAGCC AAA CCC TAA TAC
NPR1	AACGATTCTTCCCGCGCTGTTC	TTCTCCGCAAGCCAGTTGAGTC
AtActin2	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCT TGC
AtTUB4	CGAAAACGCTGACGAGTGTA	CCTTGGGAATGGGATAAGGT

Fig.1 Alignment of homologues amino acid sequences for amino acid sequence coded by GRMZM2G455909 gene

Fig.2 GRMZM2G455909 gene structure(A) and its coding protein domain(B)

Fig.3 Electrophoresis result of GRMZM2G455909 gene by PCR amplification(A) and gene-Blunt Simple by double enzyme digestion(B) M1 was DL5000 DNA marker; 1, 2 were both genes;CK was pEASY-Blunt Simple Cloning vector; 3 was the digestion result of transgenic plants.

Fig.4 Electrophoresis result of the detection for transgenic positive plants M2 was DL2000 DNA marker; L2,L9,Transgenic Arabidopsis plants.

Fig.5 Verification of transgenic Arabidopsis thaliana positive plants with GRMZM2G455909 gene by qRT-PCR WT, Wild Arabidopsis thaliana plant ; L2,L9,Transgenic Arabidopsis plants; Different lowercase letters indicated that the difference of relative expression at 5% level was obvious.

Fig.6 Incidence of transgenic Arabidopsis thaliana plants after inoculated with Pst DC3000 A was the phenotype at the 6 d; B was the disease index at different times. WT, Wild Arabidopsis thaliana plant ; L2,L9,Transgenic Arabidopsis plants.In B, different lowercase letters indicated that the difference of disease index at 5% level was obvious in different treatments on the same day.

Table 3 Number of bacterium in leaves from transgenic Arabidopsis thaliana plants after inoculation with Pst DC3000

处理 Treatments	时间 t/d	细菌数 Number of bacterium/(10⁵ CFU·g^-1)
WT	2	1.13 c
	4	11.23 a
	6	7.27 b
L2	2	0.57 c
	4	6.82 a
	6	1.58 b
L9	2	0.24 c
	4	3.22 a
	6	1.28 b

Fig.7 Expression analysis of disease resistance related genes for transgenic Arabidopsis thaliana plants WT, Wild Arabidopsis thaliana plant ; L2 and L9,Transgenic Arabidopsis plants. Different lowercase letters indicated that the difference of relative expression at 5% level was obvious on the same gene from different treatments.

References 30

[1]	高花雨, 何琪, 贾劲松, 等. 小麦抗条锈基因Yr10的抗病通路研究[J]. 麦类作物学报, 2021, 41(3): 287-294.
	GAO H Y, HE Q, JIA J S, et al. Resistance pathway of wheat stripe rust resistance gene Yr10[J]. Journal of Triticeae Crops, 2021, 41(3): 287-294. (in Chinese with English abstract)
[2]	WANG X D, BI W S, GAO J, et al. Systemic acquired resistance, NPR1, and pathogenesis-related genes in wheat and barley[J]. Journal of Integrative Agriculture, 2018, 17(11): 2468-2477. DOI URL
[3]	GAO J, BI W S, LI H P, et al. WRKY transcription factors associated with NPR1-mediated acquired resistance in barley are potential resources to improve wheat resistance to Puccinia triticina[J]. Frontiers in Plant Science, 2018(9): 1486.
[4]	孙黄兵, 钟年孝, 孔令茹, 等. 杜仲内生细菌DZSY21诱导玉米抗小斑病的系统抗性研究[J]. 生物学杂志, 2018, 35(5): 49-53.
	SUN H B, ZHONG N X, KONG L R, et al. Research on maize system resistance triggered by the endophytic bacterium DZSY21[J]. Journal of Biology, 2018, 35(5): 49-53. (in Chinese with English abstract)
[5]	DING T, SU B, CHEN X J, et al. An endophytic bacterial strain isolated from Eucommia ulmoides inhibits southern corn leaf blight[J]. Frontiers in Microbiology, 2017(8): 903.
[6]	王其, 陈小洁, 顾双月, 等. 杜仲内生拮抗细菌DZSY21诱导玉米抗病基因表达变化的转录组学研究[J]. 浙江农业学报, 2019, 31(3): 345-354. DOI
	WANG Q, CHEN X J, GU S Y, et al. Transcriptome profiling of maize resistance gene in response to DZSY21 induction[J]. Acta Agriculturae Zhejiangensis, 2019, 31(3): 345-354. (in Chinese with English abstract) DOI
[7]	SAITOU N, NEI M. The neighbor-joining method: a new method for reconstructing phylogenetic trees[J]. Molecular Biology and Evolution, 1987, 4(4): 406-425. DOI PMID
[8]	HU B, JIN J P, GUO A Y, et al. GSDS 2.0: an upgraded gene feature visualization server[J]. Bioinformatics, 2014, 31(8): 1296-1297. DOI URL
[9]	KATOH K, MISAWA K, KUMA K I, et al. MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform[J]. Nucleic Acids Research, 2002, 30(14): 3059-3066. PMID
[10]	NIU D D, LIU H X, JIANG C H, et al. The plant growth-promoting rhizobacterium Bacillus cereus AR156 induces systemic resistance in Arabidopsis thaliana by simultaneously activating salicylate- and jasmonate/ethylene-dependent signaling pathways[J]. Molecular Plant-Microbe Interactions: MPMI, 2011, 24(5): 533-542. DOI URL
[11]	曾海红. 壳寡糖诱导拟南芥对Pseudomonas syringae pv tomato DC3000的抗性及其抗性机制的初步研究[D]. 大连: 大连海洋大学, 2016.
	ZENG H H. The primary study of the mechanism of the oligosaccharidesinduced resistance to Pseudomonas syringae pv.tomato DC3000 in Arabidopsis thaliana[D]. Dalian: Dalian Ocean University, 2016. (in Chinese with English abstract)
[12]	刘俊杰, 贾晓晨, 赵小明, 等. SPINDLY在壳寡糖诱导拟南芥抗丁香假单胞菌中的功能[J]. 西北植物学报, 2020, 40(5): 766-772.
	LIU J J, JIA X C, ZHAO X M, et al. Function of SPINDLY in chitosan oligosaccharide induced resistance to pst DC3000 in Arabidopsis[J]. Acta Botanica Boreali-Occidentalia Sinica, 2020, 40(5): 766-772. (in Chinese with English abstract)
[13]	LIU J L, LIU X L, DAI L Y, et al. Recent progress in elucidating the structure, function and evolution of disease resistance genes in plants[J]. Journal of Genetics and Genomics, 2007, 34(9): 765-776. PMID
[14]	王友红, 张鹏飞, 陈建群. 植物抗病基因及其作用机理[J]. 植物学通报, 2005, 40(1): 92-99.
	WANG Y H, ZHANG P F, CHEN J Q. Disease resistance genes and mechanisms in plants[J]. Chinese Bulletin of Botany, 2005, 40(1): 92-99. (in Chinese with English abstract)
[15]	LEAH M, XIAOPING T, PATRICE K, et al. Plant NBS-LRR protein: adaptable guards[J]. Genome Biology, 2006, 7: 212-223.
[16]	姜峰. 拮抗酵母诱导番茄果实抗性基因的分离与功能鉴定[D]. 杭州: 浙江大学, 2008.
	JIANG F. Identication and characterization of differentially expressed genes from cherry tomato fruit after application of the biological control yeast Cryptococcus laurentii[D]. Hangzhou: Zhejiang University, 2008. (in Chinese with English abstract)
[17]	张颖, 王长春, 胡海涛, 等. 水稻白叶枯病候选抗性基因SHNLR的RGAs克隆及分析[J]. 生物技术通报, 2012(4): 51-57.
	ZHANG Y, WANG C C, HU H T, et al. RGAs cloning and analysis of a candidate resistance gene SHNLR to rice bacterial blight[J]. Biotechnology Bulletin, 2012(4): 51-57. (in Chinese with English abstract)
[18]	丁玉梅. 黑籽南瓜对枯萎病菌侵染的应答机制及NBS类抗病基因筛选[D]. 重庆: 西南大学, 2019.
	DING Y M. The response mechanism of Cucurbita ficifolia infected by Fusarium oxysporum f.sp. cucumerinum and selecting of NBS type disease-resistance genes[D]. Chongqing: Southwest University, 2019. (in Chinese with English abstract)
[19]	PARKER J E, COLEMAN M J, SZABÒ V, et al. The Arabidopsis downy mildew resistance gene RPP5 shares similarity to the toll and interleukin-1 receptors with N and L6[J]. The Plant Cell, 1997, 9(6): 879-894. DOI URL
[20]	BENT A F, KUNKEL B N, DAHLBECK D, et al. RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes[J]. Science, 1994, 265(5180): 1856-1860. DOI URL
[21]	SONG W Y, WANG G L, CHEN L L, et al. A receptor kinase-like protein encoded by the rice disease resistance gene, Xa21[J]. Science, 1995, 270(5243): 1804-1806. DOI PMID
[22]	DINESH-KUMAR S P, WHITHAM S, CHOI D, et al. Transposon tagging of tobacco mosaic virus resistance gene N: its possible role in the TMV-N-mediated signal transduction pathway[J]. Proceedings of the National Academy of Sciences of the United States of America, 1995, 92(10): 4175-4180.
[23]	JONES D A, THOMAS C M, HAMMOND-KOSACK K E, et al. Isolation of the tomato Cf-9 gene for resistance to Cladosporium fulvum by transposon tagging[J]. Science, 1994, 266(5186): 789-793. DOI URL
[24]	李宁. 抗小麦黄矮病候选基因的克隆、特性分析及病原诱导启动子的分离[D]. 济南: 山东师范大学, 2008.
	LI N. Cloning and characterization of candidate genes for resistance to wheat yellow dwarf disease and isolation of promoter induced by pathogen[D]. Jinan: Shandong Normal University, 2008. (in Chinese with English abstract)
[25]	王惠梅. 菰遗传多样性与ZlBBR1基因的功能分析[D]. 北京: 中国农业科学院, 2017.
	WANG H M. Genetic diversity of Zizania latifolia griseb(Turcz) and functional analysis of ZlBBR1[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. (in Chinese with English abstract)
[26]	马世伟. 水稻抗稻瘟病新基因的功能分析和Gα参与抗稻瘟病机理研究[D]. 福州: 福建农林大学, 2019.
	MA S W. Functional analysis on two novel blast-resistant genes in rice(Oryza sativa L.) and Gα participating in blast fungus infection[D]. Fuzhou: Fujian Agriculture and Forestry University, 2019. (in Chinese with English abstract)
[27]	KRUSE T, TALLMAN G, ZEIGER E. Isolation of guard cell protoplasts from mechanically prepared epidermis of Vicia faba leaves[J]. Plant Physiology, 1989, 90(4): 1382-1386. DOI URL
[28]	张晓英. 水杨酸信号途径在B型烟粉虱诱导烟草对烟蚜防御反应中的作用[D]. 泰安: 山东农业大学, 2012.
	ZHANG X Y. The role of salicylic acid pathway induced by Bemisia tabaci B-biotype in tobacco defense Myzus persicae[D]. Tai’an: Shandong Agricultural University, 2012. (in Chinese with English abstract)
[29]	周明琦, 吴丽华, 沈忱, 等. ABA、MeJA和SA诱导下的荠菜CBF途径冷响应相关基因表达调控研究[J]. 中国农业科技导报, 2010, 12(6): 75-80.
	ZHOU M Q, WU L H, SHEN C, et al. Regulation of cold-responsive genes in CBF signaling pathway from Capsella Bursa induced by ABA, MeJA and SA[J]. Journal of Agricultural Science and Technology, 2010, 12(6): 75-80. (in Chinese with English abstract)
[30]	景岚, 李凌欣, 裴旭, 等. 寡糖诱导向日葵抗锈病的信号转导途径[J]. 中国油料作物学报, 2012, 34(5): 523-527.
	JING L, LI L X, PEI X, et al. Oligosaccharide-induced signal transduction pathway of rust disease resistance in sunflower[J]. Chinese Journal of Oil Crop Sciences, 2012, 34(5): 523-527. (in Chinese with English abstract)

Cloning of GRMZM2G455909 gene from maize and its functional analysis in transgenic plants

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