Cloning,Expression Analysis and Expression Vector Construction of CDPK,a Key Gene in Response to Abiotic Stress in Watermelon

doi:10.7668/hbnxb.20195255

Abstract

Abstract:

In order to investigate the function of watermelon calcium-dependent protein kinase (CDPK) in grafted seedlings and abiotic stress environments, this study used RT-PCR technology to clone the ClCDPK(Cla97C01G019720) gene from watermelon grafted seedlings and performed bioinformatics analysis on it. Further designed specific primers with Kpn Ⅰ and Sal Ⅰ enzyme cleavage sites based on the ClCDPK sequence,conducted amplification and double enzyme cleavage, and connected with pCAMBIA1300 to successfully construct the expression vector pCAMBIA1300-35S-ClCDPK for the target gene.Using RT-qPCR technology, the gene expression levels of ClCDPK were measured in self rooted seedlings (ZG) and grafted seedlings (JJ) after being subjected to salt and drought stress, respectively.The results showed that the ORF of ClCDPK gene was 1 647 bp, encoding 548 amino acids. Its protein contained STKc_CAMK and FRQ1 functional domains, and was a hydrophilic protein. Subcellular localization prediction showed that the protein was located in the nucleus. Evolutionary tree analysis of ClCDPK with CDPK from six other plants revealed that it was closely related to CDPK from Cucurbitaceae melons and pumpkins, with protein sequence homology alignment exceeding 92.64%, indicating high homology.The RT-qPCR expression results showed that the expression level of ClCDPK in grafted seedlings was significantly higher than that in self rooted seedlings. With the duration of stress, the expression levels of ClCDPK in grafted and self rooted seedlings first increased and then decreased, and under the same stress treatment, the expression level of ClCDPK in grafted seedlings was higher than that in self rooted seedlings.This study indicated that ClCDPK responded positively to salt and drought stress, and the ability of grafted seedlings to resist stress was higher than that of self rooted seedlings. It is speculated that ClCDPK is one of the key factors in watermelon's response to grafting, thereby improving the salt and drought resistance of watermelon grafted seedlings.

Key words: Watermelon, ClCDPK, Abiotic stress, Vector construction, Expression analysis

SONG Jiaxin, LI Mingxuan, LI Ai, SU Chaijing, ZHANG Weihua, CAI Zeyu, WU Ying. Cloning,Expression Analysis and Expression Vector Construction of CDPK,a Key Gene in Response to Abiotic Stress in Watermelon[J]. Acta Agriculturae Boreali-Sinica, 2024, 39(6): 40-46. doi: 10.7668/hbnxb.20195255.

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Figures/Tables 10

Tab.1 Reference and gene-specific primers for RT-qPCR

基因名称 Gene name	引物序列(5'—3') Primer sequence (5'—3')
ClCDPK	F: TTCAAGTCAGCCAAAGAG
	R: GACATAGTATGCACTACCG
ACTIN	F: CCATGTATGTTGCCATCCAG
	R: GGATAGCATGGGGTAGAGCA

Fig.1 ORF amplification of ClCDPK M.DL2000 DNA Marker;1.Fragment of ClCDPK;2.Negative control(H₂O).

Fig.2 Conserved domain analysis of ClCDPK protein

Fig.3 The secondary(A)and tertiary(B)structure model of ClCDPK protein

Fig.4 The sinteraction network prediction of ClCDPK protein

Fig.5 Comparison of multiple sequences between ClCDPK and other plant CDPK homologous proteins

Fig.6 The phylogenetic tree analysis between other plants CDPK and ClCDPK

Fig.7 Recombinant plasmid double digestion verification of ClCDPK M.DL10000 plus DNA Marker;1—2.Double digestion validation of recombinant plasmids using Kpn Ⅰ and Sal Ⅰ.

Fig.8 Expression analysis of ClCDPK gene under salt(A)and drought(B)stress at different times Different lowercase letters indicate significant differences under different treatments at the same time (P<0.05).

Fig.9 Expression analysis of ClCDPK gene in different treatments of salt(A)and drought(B)stress Different lowercase letters indicate significant differences in the same treatment at different times(P<0.05).

References 24

[1]	张磊, 武竞春, 张海英, 宫国义, 柳唐镜, 张洁, 许勇. 西瓜重要性状功能基因研究进展[J]. 园艺学报, 2023, 50(12):2748-2764.doi:10.16420/j.issn.0513-353x.2022-1218.
	Zhang L, Wu J C, Zhang H Y, Gong G Y, Liu T J, Zhang J, Xu Y. Advances in functional genes for important traits in watermelon[J]. Acta Horticulturae Sinica, 2023, 50(12):2748-2764. doi: 10.16420/j.issn.0513-353x.2022-1218
[2]	陈甦, 段海明, 李帅, 杨胜雨, 周慧, 高青海. 3株芽孢杆菌及其混合发酵液对西瓜幼苗生长的影响[J]. 中国瓜菜, 2024, 37(2):46-51.doi:10.16861/j.cnki.zggc.202423.0324.
	Chen S, Duan H M, Li S, Yang S Y, Zhou H, Gao Q H. Effects of three strains of Bacillus and their mixed fermentation broth on the growth of watermelon seedlings[J]. China Cucurbits and Vegetables, 2024, 37(2):46-51.
[3]	Ghani M I, Yi B L, Rehmani M S, Wei X, Ali Siddiqui J, Fan R D, Liu Y J, El-Sheikh M A, Chen X, Ahmad P. Potential of melatonin and Trichoderma harzianum inoculation in ameliorating salt toxicity in watermelon:insights into antioxidant system,leaf ultrastructure,and gene regulation[J]. Plant Physiology and Biochemistry, 2024,211:108639.doi:10.1016/j.plaphy.2024.108639.
[4]	高博文, 孙德玺, 袁高鹏, 安国林, 李卫华, 刘君璞, 朱迎春. 121份西瓜材料幼苗期耐盐性鉴定[J]. 果树学报, 2022, 39(9):1597-1606.doi:10.13925/j.cnki.gsxb.20210701.
	Gao B W, Sun D X, Yuan G P, An G L, Li W H, Liu J P, Zhu Y C. Identification of salt tolerance of 121 watermelon(Citrullus lanatus L.) germplasm resources[J]. Journal of Fruit Science, 2022, 39(9):1597-1606.
[5]	何文, 潘鹤立, 潘腾飞, 汤浩茹, 王小蓉, 潘东明. 果树砧穗互作研究进展[J]. 园艺学报, 2017, 44(9):1645-1657.doi:10.16420/j.issn.0513-353x.2017-0147.
	He W, Pan H L, Pan T F, Tang H R, Wang X R, Pan D M. Research progress on the interaction between scion and rootstock in fruit trees[J]. Acta Horticulturae Sinica, 2017, 44(9):1645-1657. doi: 10.16420/j.issn.0513-353x.2017-0147
[6]	Wang Y, Zhou J Q, Wen W X, Sun J, Shu S, Guo S R. Transcriptome and proteome analysis identifies salt stress response genes in bottle gourd rootstock-grafted watermelon seedlings[J]. Agronomy, 2023, 13(3):618.doi:10.3390/agronomy13030618.
[7]	Hu Z, Wang F S, Yu H, Zhang M M, Jiang D, Huang T J, Xiang J S, Zhu S P, Zhao X C. Effects of scion-rootstock interaction on Citrus fruit quality related to differentially expressed small RNAs[J]. Scientia Horticulturae, 2022,298:110974.doi:10.1016/j.scienta.2022.110974.
[8]	Zhou Z, Yuan Y Q, Wang K T, Wang H J, Huang J Q, Yu H, Cui X. Rootstock-scion interactions affect fruit flavor in grafted tomato[J]. Horticultural Plant Journal, 2022, 8(4):499-510.doi:10.1016/j.hpj.2022.01.001.
[9]	陈东升, 王洪旭, 王振雨, 黄健, 陈宗光. 北京地区5种砧木对小果型西瓜生长、果实品质及产量的影响[J]. 中国瓜菜, 2023, 36(5):66-71.doi:10.16861/j.cnki.zggc.2023.0085.
	Chen D S, Wang H X, Wang Z Y, Huang J, Chen Z G. Effects of five rootstocks on growth,fruit quality and yield of mini-water-melon in Beijing[J]. China Cucurbits and Vegetables, 2023, 36(5):66-71.
[10]	曹绍玉, 王艳芳, 苏婉玉, 张琳, 张应华, 许俊强. 类钙调蛋白在植物生长发育及逆境胁迫中的功能研究进展[J]. 植物生理学报, 2018, 54(10):1517-1526.doi:10.13592/j.cnki.ppj.2018.0053.
	Cao S Y, Wang Y F, Su W Y, Zhang L, Zhang Y H, Xu J Q. Research progress on functions of calmodulin-like proteins in processes of plant growth and developments and stresses[J]. Plant Physiology Journal, 2018, 54(10):1517-1526.
[11]	Jing X, Cai C J, Fan S H, Luo H Y. Physiological response characteristics of moso bamboo under drought stress based on calcium signal[J]. Forests, 2021, 12(12):1699.doi:10.3390/f12121699.
[12]	Liu Y, Xu C J, Zhu Y F, Zhang L N, Chen T Y, Zhou F, Chen H, Lin Y J. The calcium-dependent kinase OsCPK24 functions in cold stress responses in rice[J]. Journal of Integrative Plant Biology, 2018, 60(2):173-188.doi:10.1111/jipb.12614.
[13]	Tao X C, Lu Y T. Loss of AtCRK1 gene function in Arabidopsis thaliana decreases tolerance to salt[J]. Journal of Plant Biology, 2013, 56(5):306-314.doi:10.1007/s12374-012-0352-z.
[14]	Fu X, Lü C Y, Zhang Y Y, Ai X Z, Bi H G. Comparative transcriptome analysis of grafting to improve chilling tolerance of cucumber[J]. Protoplasma, 2023, 260(5):1349-1364.doi:10.1007/s00709-023-01854-6. pmid: 36949344
[15]	张雪莹. 黄蒿嫁接影响菊花抗蚜性的机制研究[D]. 泰安: 山东农业大学, 2020.doi:10.27277/d.cnki.gsdnu.2020.000546.
	Zhang X Y. Mechanism in effects of grafting onto Artemisia annua on aphid resistance in chrysanthemum[D]. Taian: Shandong Agricultural University,2020.
[16]	黄金艳. 薄皮甜瓜嫁接优势的生理机制与蛋白质组学研究[D]. 南宁: 广西大学, 2020.
	Huang J Y. Physiological mechanism of grafting superiority of thin-skinned melon and protein genomics[D]. Nanning: Guangxi University, 2020.
[17]	Li H, Guo Y L, Lan Z X, Zhang Z X, Ahammed G J, Chang J J, Zhang Y, Wei C H, Zhang X. Melatonin antagonizes ABA action to promote seed germination by regulating Ca²⁺efflux and H₂O₂ accumulation[J]. Plant Science, 2021,303:110761.doi:10.1016/j.plantsci.2020.110761.
[18]	张桐. 补光通过钙信号抑制番茄花柄脱落的作用研究[D]. 沈阳: 沈阳农业大学, 2023.doi:10.27327/d.cnki.gshnu.2023.000936.
	Zhang T. Study on the effect of supplementary light on inhibiting tomato flower stalk shedding through calcium signal[D]. Shenyang: Shenyang Agricultural University,2023.
[19]	Hossain M S, Li J, Sikdar A, Hasanuzzaman M, Uzizerimana F, Muhammad I, Yuan Y H, Zhang C J, Wang C Y, Feng B L. Exogenous melatonin modulates the physiological and biochemical mechanisms of drought tolerance in Tartary buckwheat(Fagopyrum tataricum(L.)gaertn)[J]. Molecules, 2020, 25(12):2828.doi:10.3390/molecules25122828.
[20]	Durian G, Sedaghatmehr M, Matallana-Ramirez L P, Schilling S M, Schaepe S, Guerra T, Herde M, Witte C P, Mueller-Roeber B, Schulze W X, Balazadeh S, Romeis T. Calcium-dependent protein kinase CPK1 controls cell death by in vivo phosphorylation of senescence master regulator ORE1[J]. The Plant Cell, 2020, 32(5):1610-1625.doi:10.1105/tpc.19.00810. pmid: 32111670
[21]	张悦婧, 桑鹤天, 王涵琦, 石珍珍, 李丽, 王馨, 孙坤, 张继, 冯汉青. 植物对非生物胁迫系统性反应中信号传递的研究进展[J]. 植物学报, 2024, 59(1):122-133.doi:10.11983/CBB23063.
	Zhang Y J, Sang H T, Wang H Q, Shi Z Z, Li L, Wang X, Sun K, Zhang J, Feng H Q. Research progress of plant signaling in systemic responses to abiotic stresses[J]. Bulletin of Botany, 2024, 59(1):122-133. doi: 10.11983/CBB23063
[22]	Cheng H K, Pan G Y, Zhou N, Zhai Z K, Yang L Q, Zhu H F, Cui X, Zhao P Y, Zhang H F, Li S J, Yang B, Jiang Y Q. Calcium-dependent Protein Kinase 5(CPK5)positively modulates drought tolerance through phosphorylating ABA-responsive element binding factors in oilseed rape(Brassica napus L.)[J]. Plant Science, 2022,315:111125.doi:10.1016/j.plantsci.2021.111125.
[23]	马成. Ca²⁺/CPKs参与褪黑素调控黄瓜幼苗耐寒性的研究[D]. 兰州: 西北师范大学, 2023.doi:10.27410/d.cnki.gxbfu.2023.002313.
	Ma C. Studies on the involvement of the Ca²⁺/CPKs in melatonin-regulated low temperature tolerance of cucumber seedlings[D]. Lanzhou: Northwest Normal University, 2023.
[24]	刘剑君. 烤烟嫁接传导抗性的生理响应与产质效应研究[D]. 郑州: 河南农业大学, 2013.doi:10.7666/d.Y2692380.
	Liu J J. The research of the conduction resistance physiological mechanisms and effect on yield quality in grafting flue-cured tobacco[D]. Zhengzhou: Henan Agricultural University,2013.

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