Cloning and Expression Analysis of Disease Resistance Gene SbRPM1 in Sorghum

doi:10.7668/hbnxb.20194651

Abstract

Abstract:

In order to explore the role of RPM1 in sorghum disease resistance,a sorghum SbRPM1 gene was obtained from sorghum smut resistant variety SX44B by homologous cloning method.The bioinformatics analysis results showed that the total length of the cDNA of SbRPM1 gene was 2 802 bp,encoding 933 amino acids,and its protein had a theoretical molecular weight of 106.1 ku and an isoelectric point of 7.11,which was a hydrophilic protein.The SbRPM1 protein had no transmembrane structure,and its subcellular localization was in the cytoplasm.Conservative domain analysis showed that SbRPM1 protein contained RX-CC-like,NB-ARC and LRR domains,and belonged to CNL proteins in the NLRs family.Phylogenetic analysis showed that SbRPM1 protein was most closely related to the RPM1 protein of Miscanthus lutarioriparius.The expression pattern of SbRPM1 gene was detected by Real-time quantitative PCR,and the results showed that the expression of SbRPM1 gene was higher in leaves and inflorescence,followed by roots,and the lowest in stem.The expression of SbRPM1 gene was significantly up-regulated at 24—72 h in disease-resistant varieties after inoculation with Sporisorium reilianum pathogen,suggesting that this gene could be induced by S.reilianum and played an important role in sorghum disease resistance.In this study, the CDS sequence of the SbRPM1 gene was cloned for the first time in sorghum, and the structure, nature and expression of the gene were characterized.

Key words: Sorghum, SbRPM1 gene, Gene clone, Bioinformatics analysis, Expression analysis

WANG Huizhen, ZHANG Chaozheng, HUANG Yiming, LI Yaoxin, CHENG Ziyang, YUE Chaoyin. Cloning and Expression Analysis of Disease Resistance Gene SbRPM1 in Sorghum[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(2): 153-160. doi: 10.7668/hbnxb.20194651.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20194651

http://www.hbnxb.net/EN/Y2024/V39/I2/153

Figures/Tables 8

Tab.1 Primer information

引物名称 Primer name	引物序列(5'—3') Primer sequences (5'—3')	功能 Function
SbRPM1-F	AGTGAATTCGAGCTCGGTAC	基因克隆
SbRPM1-R	GCCCGGGATCCGATATCTAG
qRT-SbRPM1-F	CGGTTATGCTGGAGTTAAGA	qRT-PCR
qRT-SbRPM1-R	AACAGCATTACGCAAGTCTA
qRT-SbACTIN-F	CGAAGACATACTGGACGCAACA
qRT-SbACTIN-R	GGGCGGAAAGAATTAGAAGC

Fig.1 PCR products of SbRPM1 gene M.Magic Ruler DNA Marker;1.SbRPM1 DNA segment.

Fig.2 The multiple alignment of SbRPM1 and other plant RPM1 amino acid sequences The red under line is NB-ARC domain.

Fig.3 Phylogenetic tree analysis of SbRPM1 protein and its homologues

Fig.4 Physicochemical property analysis and structure prediction of SbRPM1 protein A.Prediction of affinity/hydrophobicity of SbRPM1 protein;B.Secondary structure of SbRPM1 protein; C.Tertiary structure of SbRPM1 protein;D.Conserved domain of SbRPM1 protein.

Fig.5 SbRPM1 protein signal peptide,transmembrane region and phosphorylation site prediction A.SbRPM1 protein signal peptide prediction;B.SbRPM1 protein transmembrane region prediction; C.SbRPM1 protein phosphorylation site prediction.

Fig.6 Expression of SbRPM1 gene in different tissues of SX44B Different lowercase letters indicate significant difference in different tissue (P<0.05).The same as Fig.7.

Fig.7 Expression of SbRPM1 gene induced by S.reilianumt in resistant and susceptible sorghum varieties

References 31

[1]	王佳, 钟鹏, 姚爽, 李旭业, 吴宪, 金永顺, 朱中华, 徐婷婷, 王宇. 饲用高粱利用关键技术及效果评价[J]. 现代畜牧科技, 2024(2): 73-76. doi:10.19369/j.cnki.2095-9737.2024.02.019.
	Wang J, Zhong P, Yao S, Li X Y, Wu X, Jin Y S, Zhu Z H, Xu T T, Wang Y. Key technologies and effect evaluation of forage sorghum utilization[J]. Modern Animal Science and Technology, 2024(2):73-76.
[2]	张静. 高粱丝黑穗病病害[J]. 现代农业, 2019(8): 55.doi:10.14070/j.cnki.15-1098.2019.08.035.
	Zhang J. Sorghum head smut disease[J]. Modern Agriculture, 2019(8):55.
[3]	杨德卫, 李生平, 崔海涛, 邹声浩, 王伟. 寄主植物与病原菌免疫反应的分子遗传基础[J]. 遗传, 2020, 42(3): 278-294. doi:10.16288/j.yczz.20-015.
	Yang D W, Li S P, Cui H T, Zou S H, Wang W. Molecular and genetic basis of immune response between host plants and pathogenic bacteria[J]. Heredity, 2020, 42(3): 278-294.
[4]	Armijo G, Salinas P, Monteoliva M I, Seguel A, García C, Villarroel-Candia E, Song W, van der Krol A R, Álvarez M E, Holuigue L. A salicylic acid-induced lectin-like protein plays a positive role in the effector-triggered immunity response of Arabidopsis thaliana to Pseudomonas syringae Avr-Rpm1[J]. Molecular Plant-Microbe Interactions, 2013, 26(12):1395-1406.doi:10.1094/MPMI-02-13-0044-R.
[5]	Russell A R, Ashfield T, Innes R W. Pseudomonas syringae effector AvrPphB suppresses AvrB-induced activation of RPM1 but not AvrRpm1-induced activation[J]. Molecular Plant-Microbe Interactions, 2015, 28(6):727-735.doi:10.1094/MPMI-08-14-0248-R.
[6]	Grant M R, Godiard L, Straube E, Ashfield T, Lewald J, Sattler A, Innes R W, Dang J L. Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance[J]. Science, 1995, 269(5225):843-846.doi:10.1126/science.7638602. pmid: 7638602
[7]	Tornero P, Chao R A, Luthin W N, Goff S A, Dangl J L. Large-scale structure function analysis of the Arabidopsis RPM1 disease resistance protein[J]. The Plant Cell, 2002, 14(2):435-450.doi:10.1105/tpc.010393. URL
[8]	Kim M G, Geng X Q, Lee S Y, MacKey D. The Pseudomonas syringae type Ⅲ effector AvrRpm1 induces significant defenses by activating the Arabidopsis nucleotide-binding leucine-rich repeat protein RPS2[J]. The Plant Journal, 2009, 57(4):645-653.doi:10.1111/j.1365-313X.2008.03716.x.
[9]	Geng X Q, Shen M Z, Kim J H, MacKey D. The Pseudomonas syringae type Ⅲ effectors AvrRpm1 and AvrRpt2 promote virulence dependent on the F-box protein COI1[J]. Plant Cell Reports, 2016, 35(4):921-932.doi:10.1007/s00299-016-1932-z. URL
[10]	Nie Y B, Ji W Q. Cloning and characterization of disease resistance protein RPM1 genes against powdery mildew in wheat line N9134[J]. Cereal Research Communications, 2019, 47(3):473-483.doi:10.1556/0806.47.2019.27.
[11]	Nounurai P, Afifah A, Kittisenachai S, Roytrakul S. Phosphorylation of CAD1,PLDdelta,NDT1,RPM1 proteins induce resistance in tomatoes infected by Ralstonia solanacearum[J]. Plants, 2022, 11(6):726.doi:10.3390/plants11060726. URL
[12]	关宇涵, 王雨沙, 李晓明, 李贺. 草莓FveRPM1基因的鉴定及功能分析[J]. 沈阳农业大学学报, 2022, 53(6):641-649.doi:10.3969/j.issn.1000-1700.2022.06.001.
	Guan Y H, Wang Y S, Li X M, Li H. Identification and function analysis of FveRPM1 gene in strawberry[J]. Journal of Shenyang Agricultural University, 2022, 53(6):641-649.
[13]	孟祥鹏. 拟南芥抗病基因RIN4和RPM1的克隆及在大豆中的功能验证[D]. 长春: 吉林农业大学, 2018.doi:10.7666/d.D01653843.
	Meng X P. Cloning of Arabidopsis resistance gene RIN4 and RPM1 and functional verification in soybean[D]. Changchun: Jilin Agricultural University, 2018.
[14]	晏牧熙, 崔海峰, 李帅, 赵金兰, 虞悦, 张雅芬, 俞晓平, 叶子弘. 茭白ZlRPM1.1和ZlPRM1.2基因克隆及其对菰黑粉菌菌丝侵染的响应[J]. 农业生物技术学报, 2021, 29(11):2061-2073.doi:10.3969/j.issn.1674-7968.2021.11.001.
	Yan M X, Cui H F, Li S, Zhao J L, Yu Y, Zhang Y F, Yu X P, Ye Z H. Cloning of ZlRPM1.1 and ZlRPM1.2 genes and responses to the hyphal infection of Ustilago esculenta in Jiaobai[J]. Journal of Agricultural Biotechnology, 2021, 29(11):2061-2073.
[15]	张福耀, 平俊爱, 杜志宏, 程庆军, 侯爱斌, 吕鑫. 山西高平高粱丝黑穗病菌致病力研究[J]. 植物病理学报, 2005, 35(5):475-477.doi:10.13926/j.cnki.apps.2005.05.018.
	Zhang F Y, Ping J A, Du Z H, Cheng Q J, Hou A B, Lü X. Identification for physiological race of Sporisorium reilianumin sorghum[J]. Acta Phytopathologica Sinica, 2005, 35(5):475-477.
[16]	张瑶, 陈磊, 马乐, 张朝政, 乐超银. 高粱SbRLK2基因的克隆及表达模式分析[J]. 分子植物育种, 2020, 18(22):7276-7282.doi:10.13271/j.mpb.018.007276.
	Zhang Y, Chen L, Ma L, Zhang C Z, Yue C Y. Cloning and expression pattern analysis of SbRLK2 gene in sorghum[J]. Molecular Plant Breeding, 2020, 18(22):7276-7282.
[17]	魏鑫, 李玥莹, 关尔鑫. 甜高粱总RNA提取方法的比较研究[J]. 江苏农业科学, 2011, 39(3):45-47.doi:10.15889/j.issn.1002-1302.2011.03.118.
	Wei X, Li Y Y, Guan E X. Comparative study on extraction methods of total RNA from sweet sorghum[J]. Jiangsu Agricultural sciences, 2011, 39(3):45-47.
[18]	Cui H T, Tsuda K, Parker J E. Effector-triggered immunity:from pathogen perception to robust defense[J]. Annual Review of Plant Biology, 2015,66:487-511.doi:10.1146/annurev-arplant-050213-040012.
[19]	闫佳, 刘雅琼, 侯岁稳. 植物抗病蛋白研究进展[J]. 植物学报, 2018, 53(2):250-263.doi:10.11983/CBB17148.
	Yan J, Liu Y Q, Hou S W. Recent advances in disease resistance proteins in plant immunity[J]. Chinese Bulletin of Botany, 2018, 53(2):250-263.
[20]	Jupe F, Witek K, Verweij W, Sliwka J, Pritchard L, Etherington G J, MacLean D, Cock P J, Leggett R M, Bryan G J, Cardle L, Hein I, Jones J D G. Resistance gene enrichment sequencing(RenSeq)enables reannotation of the NB-LRR gene family from sequenced plant genomes and rapid mapping of resistance loci in segregating populations[J]. The Plant Journal, 2013, 76(3):530-544.doi:10.1111/tpj.12307. URL
[21]	Steele J F C, Hughes R K, Banfield M J. Structural and biochemical studies of an NB-ARC domain from a plant NLR immune receptor[J]. PLoS One, 2019, 14(8): e0221226. doi:10.1371/journal.pone.0221226.
[22]	Tameling W I L, Vossen J H, Albrecht M, Lengauer T, Berden J A, Haring M A, Cornelissen B J C, Takken F L W. Mutations in the NB-ARC domain of I-2 that impair ATP hydrolysis cause autoactivation[J]. Plant Physiology, 2006, 140(4):1233-1245.doi:10.1104/pp.105.073510. pmid: 16489136
[23]	Sun Y, Zhu Y X, Balint-Kurti P J, Wang G F. Fine-tuning immunity: players and regulators for plant NLRs[J]. Trends in Plant Science, 2020 (7): 695-713.doi:10.1016/j.tplants.2020.02.008. pmid: 32526174
[24]	胡玉慈, 姚立萍, 张童, 何淑敏, 唐鑫彪, 陈清西, 文志丰. 黄毛草莓编码NB-ARC结构域的FnCN基因和启动子克隆及FnCN基因表达分析[J]. 植物资源与环境学报, 2020, 29(4):1-10.doi:10.3969/j.issn.1674-7895.2020.04.01.
	Hu Y C, Yao L P, Zhang T, He S M, Tang X B, Chen Q X, Wen Z F. Cloning of FnCN gene encoding NB-ARC domain and its promoter from Fragaria nilgerrensis and analysis on FnCN gene expression[J]. Journal of Plant Resources and Environment, 2020, 29(4):1-10.
[25]	MacKey D, Yun D J, Nam J. Proteasome-dependent degradation of RPM1 desensitizes the RPM1-mediated hypersensitive response[J]. Journal of Plant Biology, 2021, 64(3):217-225.doi:10.1007/s12374-021-09296-4.
[26]	Joshi R K, Nayak S. Functional characterization and signal transduction ability of nucleotide-binding site-leucine-rich repeat resistance genes in plants[J]. Genetics and Molecular Research, 2011, 10(4):2637-2652.doi:10.4238/2011.October.25.10. pmid: 22057959
[27]	Tailor A, Bhatla S C. R gene-mediated resistance in the management of plant diseases[J]. Journal of Plant Biochemistry and Biotechnology, 2023, 33(1) :5-23. doi:10.1007/S13562-023-00858-W.
[28]	Abebe D A, van Bentum S, Suzuki M, Ando S, Takahashi H, Miyashita S. Plant death caused by inefficient induction of antiviral R-gene-mediated resistance may function as a suicidal population resistance mechanism[J]. Communications Biology, 2021, 4(1): 947.doi:10.1038/s42003-021-02482-7.
[29]	柯宇航, 刘明洋, 王燕, 梁晓宇, 王萌, 张宇. 橡胶树HbRPM1-3基因克隆及其应答白粉菌侵染的功能分析[J]. 植物生理学报, 2021, 57(11):2167-2178.doi:10.13592/j.cnki.ppj.2021.0141.
	Ke Y H, Liu M Y, Wang Y, Liang X Y, Wang M, Zhang Y. Cloning of HbRPM1-3 gene from Hevea brasiliensis and functional analysis of its response to powdery mildew infection[J]. Plant Physiology Journal, 2021, 57(11):2167-2178.
[30]	白春明, 陆晓春. 高粱抗丝黑穗病分子遗传机制研究进展[J]. 辽宁农业科学, 2017(6):39-43.doi:10.3969/j.issn.1002-1728.2017.06.008.
	Bai C M, Lu X C. Research progress on molecular genetic mechanism of head smut resistance of sorghum[J]. Liaoning Agricultural Sciences, 2017(6):39-43.
[31]	杨慧勇, 赵文博, 王花云, 张福耀. 高粱丝黑穗病菌4号生理小种抗性基因的定位[J]. 中国农业科学, 2015, 48(8):1484-1491.doi:10.3864/j.issn.0578-1752.2015.08.03.
	Yang H Y, Zhao W B, Wang H Y, Zhang F Y. Genetic mapping of the resistance gene for the race 4 of Sphacelotheca reiliana in sorghum[J]. Scientia Agricultura Sinica, 2015, 48(8):1484-1491.

Please choose a citation manager

Content to export