Cloning and Functional Analysis of the Soybean Gene GmRPK2

doi:10.7668/hbnxb.20193317

Abstract

Abstract:

Abiotic stresses such as drought and soil salinization seriously affect the growth,development and yield of crops.GmRPK2 gene was cloned and constructed into the overexpression vector,then the vector was transformed into Arabidopsis thaliana and the homozygotes was screened.Under PEG,NaCl and ABA stress,the resistance of GmRPK2 overexpression Arabidopsis was tested.The results showed that the germination rate of the overexpressed Arabidopsis seeds was significantly lower than wild type.The elongation of the overexpressed Arabidopsis seedlings roots was also shorter than that of wild-type.In the stress resistance test of Arabidopsis plants,it was found that after salt stress and drought stress treatment,the inhibition of transgenic Arabidopsis plants was more obvious than that of wild-type Arabidopsis plants.The stress resistance test at adult stage showed that the growth inhibition of overexpressed Arabidopsis was more obvious under salt and drought stress.In order to explore the internal mechanism of the reduction of its stress resistance,the physiological indexes of GmRPK2 overexpressed Arabidopsis were detected.The results showed that,the chlorophyll content of overexpressed Arabidopsis was significantly lower,the malondialdehyde and conductivity were obviously higher,and the DAB staining was deeper than that of the wild type.After drought stress treatment,the contents of proline and soluble sugar of overexpressed Arabidopsis were obviously lower than those of wild type,and the water loss rate was obviously higher.The results of RT-PCR showed that GmRPK2 overexpressed in Arabidopsis obviously inhibited SAD1,P5CS1 and ADH1 expression.It was preliminarily speculated that it might inhibit the transmission of stress signals by inhibiting the expression of these genes.Therefore,GmRPK2 gene may affect the stress resistance of Arabidopsis by inhibiting CDPK stress resistance signal transduction pathway,reducing proline accumulation and weakening ABA signal transduction pathway.

Key words: Soybean, GmRPK2, Receptor like kinases, Stress resistance function, Physiological index, Signal transduction pathway

GU Yahui, ZHANG Changqing, ZHANG Xuebing, ZHANG Xinyue, ZHANG Xinjie, GE Rongchao. Cloning and Functional Analysis of the Soybean Gene GmRPK2[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(6): 42-49. doi: 10.7668/hbnxb.20193317.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20193317

http://www.hbnxb.net/EN/Y2022/V37/I6/42

Figures/Tables 10

Tab.1 qRT-PCR primer sequences

基因 Gene	引物序列(5'—3') Primer sequence(5'—3')
ESK1	GTAGCGTATAGGAGAGTGATGAGGACTT
	CCTACGCTGAATGGCATTGACATGTTTAG
SOS3	CGCTTCTTCACGAATCCGAACTAGTTC
	GGCAAAGTCATGTTCTTGATGAGCGATG
FRY1	GTGTCAAAGCTCCACCAGTCCGTATT
	CGCCTCTGTAACAACTATAGCACCAG
SAD1	CCGAATATGGAGATAAGGAGCTCGTTG
	GGCGATGTTGTTGCCATTGAGTAGAATC
RD29B	GGTCACTTCTTGGCTCGGTGGT
	TCCCAACACTTCCTCCGGAATCC
ADH1	GACGATGCCTTCAAGACTCATCCGAT
	CCTTGTTGATTTCCGAGAATGGCACTGT
P5CS1	GGTTTCCGATTTGGACTTGGTGCAG
	CCATTGTCTCCGTCGACAACTTGTC
β-Actin	TACAGTGTCTGGATCGGTGGTTCC
	TGTGAACGATTCCTGGACCTGCC

Fig.1 Relative expression levels of GmRPK2 under stress in soybean Different lowercase letters indicate significant differences (P<0.05).The same as Fig.4—5,7—9.

Fig.2 Subcellular localization of GmRPK2 fusion protein

Fig.3 Detection of GmRPK2 overexpressed Arabidopsis A.PCR detection of genome;B.RT-PCR detection; M.DL2000 DNA Marker.

Fig.4 Germination rate of GmRPK2 overexpressed Arabidopsis under stress

Fig.5 Root growth and development of GmRPK2 overexpressed Arabidopsis under stress

Fig.6 Resistance of GmRPK2 overexpressed Arabidopsis adult plants to high-salt and drought

Fig.7 Physiological index of GmRPK2 transgenic Arabidopsis under salt stress

Fig.8 Physiological indicators of GmRPK2 transgenic Arabidopsis under drought stress

Fig.9 qRT-PCR of the expression of stress-related genes in GmRPK2 overexpressed Arabidopsis

References 34

[1]	Assmann S M. Natural variation in abiotic stress and climate change responses in Arabidopsis: Implications for Twenty-First-Century agriculture[J]. International Journal of Plant Sciences, 2013, 174(1): 3-26.doi: 10.1086/667798. doi: 10.1086/667798 URL
[2]	Shiu S H, Bleecker A B. Receptor-like kinases from Arabidopsis form a monophyletic gene family related to animal receptor kinases[J]. Proceedings of the National Academy of Sciences of the United States of America, 2001, 98(19): 10763-10768.doi: 10.1073/pnas.181141598. doi: 10.1073/pnas.181141598 pmid: 11526204
[3]	Morillo S A, Tax F E. Functional analysis of receptor-like kinases in monocots and dicots[J]. Current Opinion in Plant Biology, 2006, 9(5): 460-469.doi:10.1016/j.pbi.2006.07.009. doi: 10.1016/j.pbi.2006.07.009 pmid: 16877029
[4]	Stone J M, Walker J C. Plant protein kinase families and signal transduction[J]. Plant Physiology, 1995, 108(2): 451-457.doi:10.1104/pp.108.2.451. doi: 10.1104/pp.108.2.451 pmid: 7610156
[5]	石翠翠, 高雷更, 惠颖, 胡京蕊, 路佳, 葛荣朝. 植物类受体蛋白激酶的研究进展[J]. 河北师范大学学报(自然科学版), 2010, 34(2): 216-220.doi:10.13763/j.cnki.jhebnu.nse.2010.02.012. doi: 10.13763/j.cnki.jhebnu.nse.2010.02.012
	Shi C C, Gao L G, Hui Y, Hu J R, Lu J, Ge R C. Progress of study on receptor-like protein kinases in plant[J]. Journal of Hebei Normal University (Natural Science Edition), 2010, 34(2): 216-220.
[6]	diGaspero G, Cipriani G. Nucleotide binding site/leucine-rich repeats,Pto-like and receptor-like kinases related to disease resistance in grapevine[J]. Molecular Genetics and Genomics, 2003, 269(5): 612-623.doi:10.1007/s00438-003-0884-5. doi: 10.1007/s00438-003-0884-5 pmid: 12884009
[7]	Becraft P W, Stinard P S, McCarty D R. CRINKLY4: A TNFR-like receptor kinase involved in maize epidermal differentiation[J]. Science, 1996, 273(5280): 1406-1409.doi:10.1126/science.273.5280.1406. doi: 10.1126/science.273.5280.1406 pmid: 8703079
[8]	Shiu S H, Bleecker A B. Plant receptor-like kinase gene family: Diversity,function,and signaling[J]. Science's STKE, 2001, 2001(113): re22.doi:10.1126/stke.2001.113.re22. doi: 10.1126/stke.2001.113.re22
[9]	Kobe B, Deisenhofer J. A structural basis of the interactions between leucine-rich repeats and protein ligands[J]. Nature, 1995, 374(6518): 183-186.doi:10.1038/374183a0. doi: 10.1038/374183a0 URL
[10]	宋婷, 熊炜, 李莹莹, 曹振华, 刘晨曦, 刘薇茵, 栾维江. 一个水稻富亮氨酸重复类受体蛋白激酶的RNAi及表达分析[J]. 天津师范大学学报(自然科学版), 2016, 36(1): 48-51.doi: 10.3969/j.issn.1671-1114.2016.01.011. doi: 10.3969/j.issn.1671-1114.2016.01.011
	Song T, Xiong W, Li Y Y, Cao Z H, Liu C X, Liu W Y, Luan W J. RNAi and expression analysis of a leucine-rich repeat receptor-like protein kinase in rice[J]. Journal of Tianjin Normal University (Natural Science Edition), 2016, 36(1): 48-51.
[11]	Tsuwamoto R, Fukuoka H, Takahata Y. GASSHO1 and GASSHO2 encoding a putative leucine-rich repeat transmembrane-type receptor kinase are essential for the normal development of the epidermal surface in Arabidopsis embryos[J]. The Plant Journal, 2008, 54(1): 30-42.doi: 10.1111/j.1365-313X.2007.03395.x. doi: 10.1111/j.1365-313X.2007.03395.x pmid: 18088309
[12]	Sijacic P, Liu Z C. Novel insights from live-imaging in shoot meristem development[J]. Journal of Integrative Plant Biology, 2010, 52(4): 393-399.doi:10.1111/j.1744-7909.2010.00941.x. doi: 10.1111/j.1744-7909.2010.00941.x
[13]	Zha X J, Luo X J, Qian X Y, He G M, Yang M F, Li Y, Yang J S. Over-expression of the rice LRK1 gene improves quantitative yield components[J]. Plant Biotechnology Journal, 2009, 7(7): 611-620.doi:10.1111/j.1467-7652.2009.00428.x. doi: 10.1111/j.1467-7652.2009.00428.x URL
[14]	吴允哲. 拟南芥富亮氨酸重复类受体激酶表达图谱分析及ELE调节叶间期发育研究[D]. 兰州: 兰州大学, 2016.doi: 10.7666/d.D01032868. doi: 10.7666/d.D01032868
	Wu Y Z. Genome-wide expression pattern analyses of LRR-RLKs and study of ELE regulating plastochron length of leaf in Arabidopsis[D]. Lanzhou: Lanzhou University, 2016.
[15]	Torii K U, Mitsukawa N, Oosumi T, Matsuura Y, Yokoyama R, Whittier R F, Komeda Y. The Arabidopsis ERECTA gene encodes a putative receptor protein kinase with extracellular leucine-rich repeats[J]. The Plant Cell, 1996, 8(4): 735-746.doi:10.1105/tpc.8.4.735. doi: 10.1105/tpc.8.4.735
[16]	Dievart A, Perin C, Hirsch J, Bettembourg M, Lanau N, Artus F, Bureau C, Noel N, Droc G, Peyramard M, Pereira S, Courtois B, Morel J B, Guiderdoni E. The phenome analysis of mutant alleles in Leucine-Rich Repeat Receptor-Like Kinase genes in rice reveals new potential targets for stress tolerant cereals[J]. Plant Science, 2016, 242: 240-249.doi: 10.1016/j.plantsci.2015.06.019. doi: S0168-9452(15)30002-9 pmid: 26566841
[17]	Shimizu N, Ishida T, Yamada M, Shigenobu S, Tabata R, Kinoshita A, Yamaguchi K, Hasebe M, Mitsumasu K, Sawa S. Bam 1 and receptor-like protein kinase 2 constitute a signaling pathway and modulate cle peptide-triggered growth inhibition in Arabidopsis root[J]. The New Phytologist, 2015, 208(4): 1104-1113.doi:10.1111/nph.13520. doi: 10.1111/nph.13520 URL
[18]	Tanaka H, Osakabe Y, Katsura S, Mizuno S, Maruyama K, Kusakabe K, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K. Abiotic stress-inducible receptor-like kinases negatively control ABA signaling in Arabidopsis[J]. The Plant Journal, 2012, 70(4): 599-613.doi:10.1111/j.1365-313X.2012.04901.x. doi: 10.1111/j.1365-313X.2012.04901.x URL
[19]	Hua D P, Wang C, He J N, Liao H, Duan Y, Zhu Z Q, Guo Y, Chen Z Z, Gong Z Z. A plasma membrane receptor kinase,GHR1,mediates abscisic acid-and hydrogen peroxide-regulated stomatal movement in Arabidopsis[J]. The Plant Cell, 2012, 24(6): 2546-2561.doi:10.1105/tpc.112.100107. doi: 10.1105/tpc.112.100107 URL
[20]	Yang T B, Chaudhuri S, Yang L H, Du L Q, Poovaiah B W. A calcium/calmodulin-regulated member of the receptor-like kinase family confers cold tolerance in plants[J]. The Journal of Biological Chemistry, 2010, 285(10): 7119-7126.doi:10.1074/jbc.M109.035659. doi: 10.1074/jbc.M109.035659 URL
[21]	Sicilia A, Testa G, Santoro D F, Cosentino S L, Lo Piero A R. RNASeq analysis of giant cane reveals the leaf transcriptome dynamics under long-term salt stress[J]. BMC Plant Biology, 2019, 19(1): 355.doi:10.1186/s12870-019-1964-y. doi: 10.1186/s12870-019-1964-y pmid: 31416418
[22]	Osakabe Y, Maruyama K, Seki M, Satou M, Shinozaki K, Yamaguchi-Shinozaki K. Leucine-rich repeat receptor-like Kinase1 is a key membrane-bound regulator of abscisic acid early signaling in Arabidopsis[J]. The Plant Cell, 2005, 17(4): 1105-1119.doi:10.1105/tpc.104.027474. doi: 10.1105/tpc.104.027474 URL
[23]	Osakabe Y, Mizuno S, Tanaka H, Maruyama K, Osakabe K, Todaka D, Fujita Y, Kobayashi M, Shinozaki K, Yamaguchi-Shinozaki K. Overproduction of the membrane-bound receptor-like protein kinase 1,RPK1,enhances abiotic stress tolerance in Arabidopsis[J]. The Journal of Biological Chemistry, 2010, 285(12): 9190-9201.doi:10.1074/jbc.M109.051938. doi: 10.1074/jbc.M109.051938 URL
[24]	李晶岚, 陈鑫欣, 石翠翠, 刘方惠, 孙静, 葛荣朝. OsRPK1基因过表达和RNA干涉对水稻苗期耐盐性的影响[J]. 作物学报, 2020, 46(8): 1217-1224.doi:10.3724/SP.J.1006.2020.92060. doi: 10.3724/SP.J.1006.2020.92060
	Li J L, Chen X X, Shi C C, Liu F H, Sun J, Ge R C. Effects of OsRPK1 gene overexpression and RNAi on the salt-tolerance at seed-Ling stage in rice[J]. Acta Agronomica Sinica, 2020, 46(8): 1217-1224. doi: 10.3724/SP.J.1006.2020.92060 URL
[25]	Shi C C, Feng C C, Yang M M, Li J L, Li X X, Zhao B C, Huang Z J, Ge R C. Overexpression of the receptor-like protein kinase genes AtRPK1 and OsRPK1 reduces the salt tolerance of Arabidopsis thaliana[J]. Plant Science, 2014, 217/218: 63-70.doi:10.1016/j.plantsci.2013.12.002. doi: 10.1016/j.plantsci.2013.12.002 URL
[26]	Saijo Y, Hata S, Kyozuka J, Shimamoto K, Izui K. Over-expression of a single Ca²⁺-dependent protein kinase confers both cold and salt/drought tolerance on rice plants[J]. The Plant Journal, 2000, 23(3): 319-327.doi:10.1046/j.1365-313x.2000.00787.x. doi: 10.1046/j.1365-313x.2000.00787.x URL
[27]	Ramegowda V, Basu S, Gupta C, Pereira A. Regulation of grain yield in rice under well-watered and drought stress conditions by GUDK[J]. Plant Signaling & Behavior, 2015, 10(11): e1034421.doi:10.1080/15592324.2015.1034421. doi: 10.1080/15592324.2015.1034421
[28]	Wani K I, Naeem M, Castroverde C D M, Kalaji H M, Albaqami M, Aftab T. Molecular mechanisms of nitric oxide(NO)signaling and Reactive oxygen species(ROS)homeostasis during abiotic stresses in plants[J]. International Journal of Molecular Sciences, 2021, 22(17): 9656.doi:10.3390/ijms22179656. doi: 10.3390/ijms22179656 URL
[29]	Wang Y Y, Liu Z Y, Wang P L, Jiang B, Lei X J, Wu J, Dong W F, Gao C Q. Correction to: A 2-Cys peroxiredoxin gene from Tamarix hispida improved salt stress tolerance in plants[J]. BMC Plant Biology, 2020, 20(1): 382.doi:10.1186/s12870-020-02600-3. doi: 10.1186/s12870-020-02600-3 URL
[30]	Ashraf M, Foolad M R. Roles of glycine betaine and proline in improving plant abiotic stress resistance[J]. Environmental and Experimental Botany, 2007, 59(2): 206-216.doi: 10.1016/j.envexpbot.2007.12.006. doi: 10.1016/j.envexpbot.2007.12.006 URL
[31]	Zhu J K. Plant salt tolerance[J]. Trends in Plant Science, 2001, 6(2): 66-71.doi: 10.1016/S1360-1385(00)01838-0. doi: 10.1016/s1360-1385(00)01838-0 pmid: 11173290
[32]	Delauney A J, Verma D P. A soybean gene encoding Δ'-pyrroline-5-carboxylate reductase was isolated by functional complementation in Escherichia coli and is found to be osmoregulated[J]. Molecular & General Genetics, 1990, 221(3): 299-305.doi:10.1007/BF00259392. doi: 10.1007/BF00259392
[33]	张斌. 大豆GmPP2C89基因在盐胁迫中的功能分析[J]. 华北农学报, 2022, 37(4): 20-27. doi: 10.7668/hbnxb.20193021. doi: 10.7668/hbnxb.20193021
	Zhang B. Functional analysis of soybean GmPP2C89 gene under salt stress[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(4):20-27.
[34]	Xiong L, Bh L, Ishitani M, Lee H, Zhang C, Zhu J K. FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis[J]. Genes & Development, 2001, 15(15): 1971-1984.doi:10.1101/gad.891901. doi: 10.1101/gad.891901 URL

Please choose a citation manager

Content to export