Analysis of Sequence Characteristics and Expression Patterns of PbKRP2 in Pyrus betulaefolia

doi:10.7668/hbnxb.20196258

Abstract

Abstract:

The Kip-related protein(KRP)gene family acts as a key regulator of the cell cycle,modulating cell division and endoreduplication by inhibiting cyclin-dependent kinase(CDK)activity,and participates in plant abiotic stress responses.To investigate the structural characteristics and environmental stress response profile of PbKRP2 in Pyrus betulifolia,the gene was cloned using reverse transcription PCR(RT-PCR).Bioinformatic analysis was conducted to elucidate its sequence features,predicted protein interactions,and phylogenetic relationships.Quantitative Real-time PCR(qRT-PCR)was employed to assess the tissue-specific expression characteristics of PbKRP2 and its expression patterns under various stress conditions.Results showed that PbKRP2 was 585 bp in length,contained 4 exons and 3 introns,and encoded a 194-amino acid protein.The predicted protein had 25 potential phosphorylation sites,a O-glycosylation site,and a conserved C-terminal cyclin-dependent kinase inhibitor(CDI)domain.Phylogenetic analysis indicated that PbKRP2 was closely related to PyKRP2 in Prunus yedoensis var.nudiflora,PpKRP2 in Prunus persica,and MdKRP2 in Malus domestica.Protein interaction network screening identified potential interactions between PbKRP2 and 10 proteins.Analysis of the static structure of potential interacting proteins suggested that PbKRP2 formed spatial interaction conformations with its target proteins PbCDKB1;1 and PbCYCD7;1,respectively.The promoter region of PbKRP2 contained multiple cis-acting regulatory elements responsive to environmental factors and hormonal signals.qRT-PCR analysis demonstrated that PbKRP2 transcript levels were significantly higher in stems and leaves compared to roots,petals,and fruits.Furthermore,PbKRP2 transcription levels were consistently upregulated under low-temperature stress and abscisic acid(ABA)treatment,but significantly downregulated under dehydration and salt stress.In summary,these findings provide a candidate gene for stress-resistant breeding of pear and genetic improvement of fruit trees by elucidating the sequence characteristics of PbKRP2 and its differential response patterns to abiotic stresses.

Key words: Pyrus betulaefolia, PbKRP2 gene, Gene cloning, Abiotic stress, Expression analysis

CLC Number:

JIN Cong, XU Lianli, LIU Qi, ZHANG Xiaoxiao, ZHANG Yushuang, XU Yuan, CHEN Yuqi, REN Yanan, WANG Jizhong. Analysis of Sequence Characteristics and Expression Patterns of PbKRP2 in Pyrus betulaefolia[J]. Acta Agriculturae Boreali-Sinica, 2026, 41(2): 36-46. doi: 10.7668/hbnxb.20196258.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.20196258

http://www.hbnxb.net/EN/Y2026/V41/I2/36

Figures/Tables 12

Fig.1 Electrophoretogram of amplification product of the PbKRP2 gene M.DL2000 DNA Marker;1—2.PbKRP2 amplification products.

Fig.2 Nucleotide sequence of PbKRP2 and its encoded amino acid sequence

Fig.3 Prediction of the PbKRP2 gene structure(A)and conserved domains(B)

Fig.4 Tertiary structure prediction of PbKRP2 protein The purple highlighted regions represent conserved CDI structural domains.

Fig.5 Prediction of phosphorylation and glycosylation site of PbKRP2 protein

Fig.6 Predicted protein-protein interaction network of PbKRP2

Fig.7 Structural prediction of the static complex formed by PbKRP2 interaction with its target proteins A.The PbKRP2-PbCDKB1;1 complex;B.The PbKRP2-PbCYCD7;1 complex.

Fig.8 Multiple sequence alignment of PbKRP2 with other homologous proteins Areas marked with solid lines indicate the nuclear localization signal;positions marked with asterisks(*)denote the acidic-hydrophobic cluster.

Fig.9 Phylogenetic analysis of PbKRP2 and other homologous proteins(A)and conserved motif analysis(B)

Fig.10 Cis-acting element analysis of the PbKRP2 gene

Fig.11 Expression patterns of the PbKRP2 gene in different tissues Different lowercase letters above bars indicate significant differences among treatments(P<0.05).The same as Fig.12.

Fig.12 Expression profiling of PbKRP2 in response to various abiotic stresses A.4 ℃ treatment;B.Dehydration treatment;C.200 mmol/L NaCl treatment;D.100 μmol/L ABA treatment.

References 39

[1]	Harashima H, Dissmeyer N, Schnittger A. Cell cycle control across the eukaryotic Kingdom[J]. Trends in Cell Biology, 2013, 23(7):345-356.doi:10.1016/j.tcb.2013.03.002. pmid: 23566594
[2]	Inzé D, De Veylder L. Cell cycle regulation in plant development[J]. Annual Review of Genetics, 2006, 40:77-105.doi:10.1146/annurev.genet.40.110405.090431. pmid: 17094738
[3]	Sherr C J, Roberts J M. CDK inhibitors:positive and negative regulators of G1-phase progression[J]. Genes & Development, 1999, 13(12):1501-1512.doi:10.1101/gad.13.12.1501. URL
[4]	Wang H, Fowke L C, Crosby W L. A plant cyclin-dependent kinase inhibitor gene[J]. Nature, 1997, 386(6624):451-452.doi:10.1038/386451a0.
[5]	De Veylder L, Beeckman T, Beemster G T S, Krols L, Terras F, Landrieu I, Van Der Schueren E, Maes S, Naudts M, Inzé D. Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis[J]. The Plant Cell, 2001, 13(7):1653-1668.doi:10.1105/tpc.010087. URL
[6]	Wang H, Qi Q G, Schorr P, Cutler A J, Crosby W L, Fowke L C. ICK1,a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts with both Cdc2a and CycD3,and its expression is induced by abscisic acid[J]. The Plant Journal, 1998, 15(4):501-510.doi:10.1046/j.1365-313x.1998.00231.x. URL
[7]	Wang H, Zhou Y M, Fowke L C. The emerging importance of cyclin-dependent kinase inhibitors in the regulation of the plant cell cycle and related processes[J]. Canadian Journal of Botany, 2006, 84(4):640-650.doi:10.1139/b06-043. URL
[8]	Coelho C M, Dante R A, Sabelli P A, Sun Y J, Dilkes B P, Gordon-Kamm W J, Larkins B A. Cyclin-dependent kinase inhibitors in maize endosperm and their potential role in endoreduplication[J]. Plant Physiology, 2005, 138(4):2323-2336.doi:10.1104/pp.105.063917. pmid: 16055680
[9]	Barrôco R M, Peres A, Droual A M, De Veylder L, Nguyen L S L, De Wolf J, Mironov V, Peerbolte R, Beemster G T S, Inzé D, Broekaert W F, Frankard V. The cyclin-dependent kinase inhibitor Orysa;KRP1 plays an important role in seed development of rice[J]. Plant Physiology, 2006, 142(3):1053-1064.doi:10.1104/pp.106.087056. pmid: 17012406
[10]	Jasinski S, Leite C S, Domenichini S, Stevens R, Raynaud C, Perennes C, Bergounioux C, Glab N. NtKIS2,a novel tobacco cyclin-dependent kinase inhibitor is differentially expressed during the cell cycle and plant development[J]. Plant Physiology and Biochemistry, 2003, 41(6/7):667-676.doi:10.1016/S0981-9428(03)00082-2. URL
[11]	Nafati M, Frangne N, Hernould M, Chevalier C, G vaudant F. Functional characterization of the tomato cyclin-dependent kinase inhibitor SlKRP1 domains involved in protein-protein interactions[J]. New Phytologist, 2010, 188(1):136-149.doi:10.1111/j.1469-8137.2010.03364.x.
[12]	Pettkó-Szandtner A, Mészáros T, Horváth G V, Bakó L, Csordás-Tóth E, Blastyák A, Zhiponova M, Miskolczi P, Dudits D. Activation of an alfalfa cyclin-dependent kinase inhibitor by calmodulin-like domain protein kinase[J]. The Plant Journal, 2006, 46(1):111-123.doi:10.1111/j.1365-313x.2006.02677.x. URL
[13]	Torres Acosta J A, Fowke L C, Wang H. Analyses of phylogeny,evolution,conserved sequences and genome-wide expression of the ICK/KRP family of plant CDK inhibitors[J]. Annals of Botany, 2011, 107(7):1141-1157.doi:10.1093/aob/mcr034. URL
[14]	Ormenese S, de Almeida Engler J, De Groodt R, De Veylder L, Inzé D, Jacqmard A. Analysis of the spatial expression pattern of seven Kip related proteins(KRPs)in the shoot apex of Arabidopsis thaliana[J]. Annals of Botany, 2004, 93(5):575-580.doi:10.1093/aob/mch077. pmid: 15037450
[15]	Jun S E, Okushima Y, Nam J, Umeda M, Kim G T. Kip-related protein 3 is required for control of endoreduplication in the shoot apical meristem and leaves of Arabidopsis[J]. Molecules and Cells, 2013, 35(1):47-53.doi:10.1007/s10059-013-2270-4. URL
[16]	Jégu T, Latrasse D, Delarue M, Mazubert C, Bourge M, Hudik E, Blanchet S, Soler M N, Charon C, De Veylder L, Raynaud C, Bergounioux C, Benhamed M. Multiple functions of Kip-related protein5 connect endoreduplication and cell elongation[J]. Plant Physiology, 2013, 161(4):1694-1705.doi:10.1104/pp.112.212357. pmid: 23426196
[17]	Guan C F, Ji J, Guan W Z, Li X Z, Jin C, Li J, Wang Y R, Wang G. LcKRP,a cyclin-dependent kinase inhibitor from Lycium chinense,is involved in an ABA-dependent drought stress-signaling pathway[J]. Plant and Soil, 2014, 382(1):43-59.doi:10.1007/s11104-014-2134-5. URL
[18]	Liu G Z, Guan Z F, Ma M X, Wang H Y, Liu X F, Song S Y, Dai N Y, Ma F F, Bao Z L. Genome-wide identification and molecular characterization of SlKRP family members in tomato and their expression profiles in response to abiotic stress[J]. Vegetable Research, 2023, 3(1):27.doi:10.48130/vr-2023-0027.
[19]	Shen L, Yang S X, Xia X, Nie W F, Yang X. Genome-wide identification of Kip-related protein(KRP)gene family members in eggplant and the function of SmKRP3 under salt stress[J]. Vegetable Research, 2024, 4(1):e013.doi:10.48130/vegres-0024-0012.
[20]	Guo B H, Chen L, Dong L, Yang C H, Zhang J H, Geng X Y, Zhou L J, Song L. Characterization of the soybean KRP gene family reveals a key role for GmKRP2a in root development[J]. Frontiers in Plant Science, 2023, 14:1096467.doi:10.3389/fpls.2023.1096467. URL
[21]	张绍铃, 谢智华. 我国梨产业发展现状、趋势、存在问题与对策建议[J]. 果树学报, 2019, 36(8):1067-1072.doi:10.13925/j.cnki.gsxb.20190033.
	Zhang S L, Xie Z H. Current status,trends,main problems and the suggestions on development of pear industry in China[J]. Journal of Fruit Science, 2019, 36(8):1067-1072.
[22]	董星光, 曹玉芬, 王昆, 田路明, 张莹, 齐丹. 中国3个主要梨砧木资源木质部导管分子结构及分布比较[J]. 植物学报, 2015, 50(2):227-233.doi:10.3724/SP.J.1259.2015.00227.
	Dong X G, Cao Y F, Wang K, Tian L M, Zhang Y, Qi D. Comparison of the characters and distribution of vessel elements in xylem among three main pear rootstocks in China[J]. Chinese Bulletin of Botany, 2015, 50(2):227-233. doi: 10.3724/SP.J.1259.2015.00227 URL
[23]	Chen C J, Wu Y, Li J W, Wang X, Zeng Z H, Xu J, Liu Y L, Feng J T, Chen H, He Y H, Xia R. TBtools-Ⅱ:a “one for all,all for one” bioinformatics platform for biological big-data mining[J]. Molecular Plant, 2023, 16(11):1733-1742.doi:10.1016/j.molp.2023.09.010. URL
[24]	Szklarczyk D, Kirsch R, Koutrouli M, Nastou K, Mehryary F, Hachilif R, Gable A L, Fang T, Doncheva N T, Pyysalo S, Bork P, Jensen L J, von Mering C. The STRING database in 2023:protein protein association networks and functional enrichment analyses for any sequenced genome of interest[J]. Nucleic Acids Research, 2023, 51(D1):D638-D646.doi:10.1093/nar/gkac1000.
[25]	Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A, Ronneberger O, Willmore L, Ballard A J, Bambrick J, Bodenstein S W, Evans D A, Hung C C, O'Neill M, Reiman D, Tunyasuvunakool K, Wu Z, Žemgulytè A, Arvaniti E, Beattie C, Bertolli O, Bridgland A, Cherepanov A, Congreve M, Cowen-Rivers A I, Cowie A, Figurnov M, Fuchs F B, Gladman H, Jain R, Khan Y A, Low C M R, Perlin K, Potapenko A, Savy P, Singh S, Stecula A, Thillaisundaram A, Tong C, Yakneen S, Zhong E D, Zielinski M, Žídek A, Bapst V, Kohli P, Jaderberg M, Hassabis D, Jumper J M. Accurate structure prediction of biomolecular interactions with AlphaFold 3[J]. Nature, 2024, 630(8016):493-500.doi:10.1038/s41586-024-07487-w.
[26]	Seeliger D, de Groot B L. Ligand docking and binding site analysis with PyMOL and Autodock/Vina[J]. Journal of Computer-Aided Molecular Design, 2010, 24(5):417-422.doi:10.1007/s10822-010-9352-6. pmid: 20401516
[27]	Tamura K, Stecher G, Kumar S. MEGA11:molecular evolutionary genetics analysis version 11[J]. Molecular Biology and Evolution, 2021, 38(7):3022-3027.doi:10.1093/molbev/msab120. URL
[28]	冯清香, 朱哈修, 杜洋, 徐艳茹, 王晨晨, 刘雪, 李大勇, 周军, 张彬. 菊苣ANS基因的克隆及表达分析[J]. 华北农学报, 2025, 40(3):51-59.doi:10.7668/hbnxb.20195328.
	Feng Q X, Zhu H X, Du Y, Xu Y R, Wang C C, Liu X, Li D Y, Zhou J, Zhang B. Cloning and expression analysis of ANS gene in Cichorium intybus[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(3):51-59.
[29]	桑莹莹, 李珊珊, 鲍薇, 徐东, 张雪, 赵艳. 大豆P34蛋白基因启动子克隆及其调控活性分析[J]. 华北农学报, 2025, 40(1):22-28.doi:10.7668/hbnxb.20195348.
	Sang Y Y, Li S S, Bao W, Xu D, Zhang X, Zhao Y. Cloning and regulatory activity analysis of soybean P34 protein gene promoter[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(1):22-28. doi: 10.7668/hbnxb.20195348
[30]	Yanai I, Benjamin H, Shmoish M, Chalifa-Caspi V, Shklar M, Ophir R, Bar-Even A, Horn-Saban S, Safran M, Domany E, Lancet D, Shmueli O. Genome-wide midrange transcription profiles reveal expression level relationships in human tissue specification[J]. Bioinformatics, 2005, 21(5):650-659.doi:10.1093/bioinformatics/bti042. pmid: 15388519
[31]	Lim S, Kaldis P. Cdks,cyclins and CKIs:roles beyond cell cycle regulation[J]. Development, 2013, 140(15):3079-3093.doi:10.1242/dev.091744. URL
[32]	Nakai T, Kato K, Shinmyo A, Sekine M. Arabidopsis KRPs have distinct inhibitory activity toward cyclin D2-associated kinases,including plant-specific B-type cyclin-dependent kinase[J]. FEBS Letters, 2006, 580(1):336-340.doi:10.1016/j.febslet.2005.12.018. URL
[33]	Zhou Y M, Wang H, Gilmer S, Whitwill S, Fowke L C. Effects of co-expressing the plant CDK inhibitor ICK1 and D-type cyclin genes on plant growth,cell size and ploidy in Arabidopsis thaliana[J]. Planta, 2003, 216(4):604-613.doi:10.1007/s00425-002-0935-x.
[34]	Zhou Y M, Li G Y, Brandizzi F, Fowke L C, Wang H. The plant cyclin-dependent kinase inhibitor ICK1 has distinct functional domains for in vivo kinase inhibition,protein instability and nuclear localization[J]. The Plant Journal, 2003, 35(4):476-489.doi:10.1046/j.1365-313x.2003.01821.x. URL
[35]	Peres A, Churchman M L, Hariharan S, Himanen K, Verkest A, Vandepoele K, Magyar Z, Hatzfeld Y, Van Der Schueren E, Beemster G T S, Frankard V, Larkin J C, Inzé D, De Veylder L. Novel plant-specific cyclin-dependent kinase inhibitors induced by biotic and abiotic stresses[J]. Journal of Biological Chemistry, 2007, 282(35):25588-25596.doi:10.1074/jbc.M703326200. pmid: 17599908
[36]	Zhou Y M, Niu H S, Brandizzi F, Fowke L C, Wang H. Molecular control of nuclear and subnuclear targeting of the plant CDK inhibitor ICK1 and ICK1-mediated nuclear transport of CDKA[J]. Plant Molecular Biology, 2006, 62(1):261-278.doi:10.1007/s11103-006-9019-9. URL
[37]	Wen B, Nieuwland J, Murray J A H. The Arabidopsis CDK inhibitor ICK3/KRP5 is rate limiting for primary root growth and promotes growth through cell elongation and endoreduplication[J]. Journal of Experimental Botany, 2013, 64(4):1135-1144.doi:10.1093/jxb/ert009. pmid: 23440171
[38]	Cheng Y, Liu H, Cao L, Wang S, Li Y P, Zhang Y Y, Jiang W, Zhou Y M, Wang H. Down-regulation of multiple CDK inhibitor ICK/KRP genes promotes cell proliferation,callus induction and plant regeneration in Arabidopsis[J]. Frontiers in Plant Science, 2015, 6:825.doi:10.3389/fpls.2015.00825. pmid: 26528298
[39]	Lui H, Wang H, Delong C, Fowke L C, Crosby W L, Fobert P R. The Arabidopsis Cdc2a-interacting protein ICK2 is structurally related to ICK1 and is a potent inhibitor of cyclin-dependent kinase activity in vitro[J]. The Plant Journal, 2000, 21(4):379-385.doi:10.1046/j.1365-313x.2000.00688.x. URL

Please choose a citation manager

Content to export