| 348 | 1 | 144 |
| 下载次数 | 被引频次 | 阅读次数 |
【目的】在芍药中克隆钙离子(Ca2+)响应基因PlCDPK24并进行表达分析，为探究CDPK基因调控芍药远缘杂交花粉管生长的机制奠定基础。【方法】利用已有芍药转录组RNA-seq数据库对钙依赖性蛋白基因家族进行鉴定，以芍药‘粉玉奴’柱头为试验材料克隆PlCDPK24基因，对其基因结构及蛋白的理化性质进行生信分析；通过亚细胞定位确定其在细胞内作用的位置；通过实时荧光定量PCR(RT-qPCR)技术分析PlCDPK24在芍药柱头内花粉管生长过程中的表达模式，及其对Ca2+以及Ca2+抑制剂乙二醇二乙醚二胺四乙酸(EGTA)的响应。【结果】鉴定筛选出15个在杂交不亲和组合中高表达的PlCDPK基因，基因编码区均含有STKc-CAMK结构域、EF-hand手性结构域以及motif 1～10，其中芍药PlCDPK24与在拟南芥中已报道花粉管生长相关AtCDPK24同源，其基因序列完整，长1 599 bp，编码532个氨基酸，理论等电点为5.69，是一种带负电荷的亲水性蛋白。PlCDPK24定位在细胞核，且与牡丹PsCDPK24蛋白同源性最高。实时荧光定量结果表明，相比于芍药其他部位，PlCDPK24在芍药柱头高表达；相比于自交组合，PlCDPK24在杂交后期(24和36 h)表达量显著上调，显著高于自交后期的表达；Ca2+以及Ca2+抑制剂EGTA处理后的杂交柱头，PlCDPK24的表达量相对于自交柱头显著上调，Ca2+及EGTA处理均会不同程度降低PlCDPK24的表达量。【结论】PlCDPK24可能参与调控了花粉在柱头的萌发和花粉管的生长过程。
Abstract：【Objective】The calcium-responsive gene PlCDPK24 was cloned and analysed for expression, laying the foundation for exploring the mechanism by which the CDPK gene regulates the growth of pollen tubes in Paeonia L distant hybridization. 【Method】Using the existing RNA-seq database, the calcium-dependent protein family genes in the unaffiliated transcriptome of Paeonia lactiflora were identified. The PlCDPK24 gene was cloned from its stigma and bioinformatics analysis was performed on the gene structure and physicochemical properties of the encoded proteins. The intracellular localization of PlCDPK24 was determined, and its expression pattern during pollen tube growth in the stigma was assessed using RT-qPCR. Additionally, its response to Ca2+ and the Ca2+ inhibitor ethylene glycol tetraacetic acid(EGTA) was analyzed. 【Result】Fifteen PlCDPK genes that were highly expressed in hybrid unaffinity combinations were identified and screened, and the coding regions of the genes all contained STKc-CAMK structural domains, EF-hand chiral structural domains, and motifs 1 to 10. Among them, the sequence of the Paeonia lactiflora PlCDPK24 gene, which was homologous to the pollen-tube-growth-associated AtCDPK24 that had been reported in Arabidopsis thaliana, was complete, with an open reading frame of 1 599 bp and encoding a 5. 69 theoretical isoelectric point, and 532 amino acids, characterized by a negatively charged hydrophilic protein. The PlCDPK24 protein is localised in the nucleus and has the highest homology with the peony PsCDPK24 protein. The results of real-time fluorescence quantification showed that PlCDPK24 was highly expressed in peony stigmas compared with other parts of peony; The expression of PlCDPK24 was significantly up-regulated in the late hybridisation stage(24 and 36 h) compared with the self-cross combination, which was significantly higher than that in the late self-crossing stage; The expression of PlCDPK24 was significantly up-regulated in the hybrid stigmas treated with Ca2+ as well as with the Ca2+ inhibitor, EGTA, relative to the The expression of PlCDPK24 was significantly up-regulated in self-crossed stigmas, and both Ca2+ and EGTA treatments reduced the expression of PlCDPK24 to different degrees. 【Conclusion】PlCDPK24 was highly expressed in the stigma of Paeonia lactis, and increased with time after cross-pollination. The expression level of PlCDPK24 was significantly lower than that of the self-pollinated stigma, suggesting that PlCDPK24 was involved in regulating the growth and elongation of pollen tubes in the stigma to a large extent.
[1] YANG Y, LI B, FENG C Y, et al. Chemical mechanism of flower color microvariation in Paeonia with yellow flowers[J]. Horticultural Plant Journal, 2020, 6(3):179-190.
[2]万映伶，朱梦婷，刘爱青，等.中国观赏芍药表型多样性解析与资源评价[J].中国农业科学，2022, 55(18):3629-3639.WAN Y L, ZHU M T, LIU A Q, et al. Phenotypic diversity analysis of Chinese ornamental herbaceous peonies and its germplasm resource evaluation[J]. Scientia Agricultura Sinica, 2022, 55(18):3629-3639.
[3]王慧娟，符真珠，李艳敏，等.观赏芍药杂交育种研究进展[J].北方园艺，2021(16):144-149.WANG H J, FU Z Z, LI Y M, et al. Research progress on the crossbreeding of ornamental herbaceous peony[J]. Northern Horticulture, 2021(16):144-149.
[4]赵银鸽，袁欣，高杰，等.牡丹ARF基因家族鉴定及表达分析[J].河南农业科学，2024, 53(11):117-126.ZHAO Y G, YUAN X, GAO J, et al. Identification and expression analysis of auxin response factor(ARF)gene family in tree peony[J]. Journal of Henan Agricultural Sciences, 2024, 53(11):117-126.
[5]梁长安，王二强，韩鲲，等.简述伊藤杂种的来源及与牡丹的异同点和应用[J].现代园艺，2022(7):65-67.LIANG C A, WANG E Q, HAN K, et al. The origin,similarities and differences between Itoh hybrid and peony and its application were briefly described[J].Contemporary Horticulture, 2022(7):65-67.
[6]刘建鑫，于晓南.芍药与牡丹远缘杂交花粉萌发与花粉管生长行为观察[J].北京林业大学学报，2016,38(9):80-86.LIU J X, YU X N. Ultrastructural investigations of Paeonia pollen activation and pollen tube growth after intersectional hybridization[J]. Journal of Beijing Forestry University, 2016, 38(9):80-86.
[7]贺丹，张佼蕊，何松林，等.芍药属远缘杂交不亲和PlABCF3基因克隆及表达分析[J].华北农学报，2020, 35(6):81-89.HE D, ZHANG J R, HE S L, et al. Cloning and expression analysis of distant hybridization incompatibility PlABCF3 gene from Paeonia[J]. Acta Agriculturae Boreali-Sinica, 2020, 35(6):81-89.
[8]贺丹，尤啸龙，何松林，等.芍药胼胝质合成酶基因家族鉴定及PlCalS5功能分析[J].中国农业科学，2023, 56(16):3183-3198.HE D, YOU X L, HE S L, et al. Identification of callose synthetase gene family and functional analysis of PlCalS5 in Paeonia lactiflora[J]. Scientia Agricultura Sinica, 2023, 56(16):3183-3198.
[9] BREWBAKER J L, KWACK B H. The essential role of calcium ion in pollen germination and pollen tube growth[J]. American Journal of Botany, 1963, 50(9):859.
[10]卢鑫蕊，沈绍琴，常雅薇，等.钙传感器调控花粉管生长的研究进展[J].分子植物育种，2022, 20(7):2320-2325.LU X R, SHEN S Q, CHANG Y W, et al. Research progress of calcium sensor regulating pollen tube growth[J]. Molecular Plant Breeding, 2022, 20(7):2320-2325.
[11] YANG H, YOU C, YANG S Y, et al. The role of calcium/calcium-dependent protein kinases signal pathway in pollen tube growth[J]. Frontiers in Plant Science,2021, 12:633293.
[12]于晓俊，曹绍玉，董玉梅，等.钙结合蛋白对花粉生长发育调控研究进展[J].西北植物学报，2016, 36(10):2121-2127.YU X J, CAO S Y, DONG Y M, et al. Research progress of calcium binding proteins in pollen growth and development[J]. Acta Botanica Boreali-Occidentalia Sinica, 2016, 36(10):2121-2127.
[13] LI J, LI Y H, DENG Y L, et al. A calcium-dependent protein kinase, ZmCPK32, specifically expressed in maize pollen to regulate pollen tube growth[J]. PLoS One, 2018, 13(5):e0195787.
[14] BOUDSOCQ M, SHEEN J. CDPKs in immune and stress signaling[J]. Trends in Plant Science, 2013, 18(1):30-40.
[15]周子馨，兰海燕.植物CDPK信号转导途径及其互作组分的功能研究进展[J].新疆大学学报（自然科学版）（中英文），2021(6):705-714.ZHOU Z X, LAN H Y. CDPK signaling pathway and its interaction components in plants[J]. Journal of Xinjiang University(Natural Science Edition in Chinese and English), 2021(6):705-714.
[16] ESTRUCH J J, KADWELL S, MERLIN E, et al. Cloning and characterization of a maize pollen-specific calcium-dependent calmodulin-independent protein kinase[J]. Proceedings of the National Academy of Sciences of the United States of America, 1994, 91(19):8837-8841.
[17] WANG Q N, XU Y N, ZHAO S S, et al. Activation of actin-depolymerizing factor by CDPK16-mediated phosphorylation promotes actin turnover in Arabidopsis pollen tubes[J]. PLoS Biology, 2023, 21(4):e3002073.
[18] ZHAO L N, SHEN L K, ZHANG W Z, et al. Ca2+-dependent protein kinase11 and 24 modulate the activity of the inward rectifying K+channels in Arabidopsis pollen tubes[J]. The Plant Cell, 2013, 25(2):649-661.
[19] ZHOU L M, LAN W Z, JIANG Y Q, et al. Acalciumdependent protein kinase interacts with and activates a calcium channel to regulate pollen tube growth[J].Molecular Plant, 7(2):369-376.
[20] YOON G M, DOWD P E, GILROY S, et al. Calciumdependent protein kinase isoforms in Petunia have distinct functions in pollen tube growth, including regulating polarity[J]. The Plant Cell, 2006, 18(4):867-878.
[21]白晓璟，廉小平，王玉奎，等.甘蓝SI相关基因BoCDPK14的克隆与分析[J].作物学报，2019, 45(12):1773-1783.BAI X J, LIAN X P, WANG Y K, et al. Cloning and analysis of BoCDPK14 in self-incompatibility Brasscia olerace[J]. Acta Agronomica Sinica, 2019, 45(12):1773-1783.
[22]李东阳.渗透胁迫下钙调控玉米幼苗水分平衡机制[D].咸阳：西北农林科技大学，2023.LI D Y. Mechanism of calcium regulating water balance under osmotic stress in maize seedli[D]. Xianyang:Northwest A&F University, 2023.
[23] ZHANG L L, HUANG J B, SU S Q, et al. FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa[J]. Current Biology:CB, 2021, 31(14):3004-3016.
[24]杨琳.受体激酶SRK与FERONIA调控大白菜远缘杂交生殖隔离的分子机制[D].泰安：山东农业大学，2023.YANG L. SRK and FERONIA receptor kinases control distant hybridization reproductive barriers in the brassicaceae[D]. Taian:Shandong Agricultural University,2023.
[25] HE D, LOU X Y, HE S L, et al. Isobaric tags for relative and absolute quantitation-based quantitative proteomics analysis provides novel insights into the mechanism of cross-incompatibility between tree peony and herbaceous peony[J]. Functional Plant Biology:FPB,2019, 46(5):417-427.
[26] XU W W, HUANG W C. Calcium-dependent protein kinases in phytohormone signaling pathways[J]. International Journal of Molecular Sciences, 2017, 18(11):2436.
[27] ZHAO P C, LIU Y J, KONG W Y, et al. Genome-wide identification and characterization of Calcium-dependent protein kinase(CDPK)and CDPK-related kinase(CRK)gene families in Medicago truncatula[J]. International Journal of Molecular Sciences, 2021, 22(3):1044.
[28] SHI S J, LI S G, ASIM M, et al. The Arabidopsis calcium-dependent protein kinases(CDPKs)and their roles in plant growth regulation and abiotic stress responses[J]. International Journal of Molecular Sciences, 2018, 19(7):1900.
[29] YIP DELORMEL T, BOUDSOCQ M. Properties and functions of calcium-dependent protein kinases and their relatives in Arabidopsis thaliana[J]. The New Phytologist, 2019, 224(2):585-604.
[30] SCHEIBLE N, HENNING P M, MCCUBBIN A G.Calmodulin-domain protein kinase PiCDPK1 interacts with the 14-3-3-like protein NtGF14 to modulate pollen tube growth[J]. Plants, 2024, 13(3):451.
[31] NIE S S, ZHENG S J, LüC S, et al. Calcium/calmodulin modulates pollen germination and pollen tube growth and self-incompatibility response in Chinese cabbage(Brassica rapa L.)[J]. Scientia Horticulturae, 2023,308:111607.
[32] KONRAD K R, MAIERHOFER T, HEDRICH R.Spatio-temporal aspects of Ca2+signalling:lessons from guard cells and pollen tubes[J]. Journal of Experimental Botany, 2018:26-30.
[33]杨瑞，高赛，王金金，等.外源硼和钙对“索邦”百合花粉萌发和花粉管生长的影响[J].电子显微学报，2014, 33(4):368-372.YANG R, GAO S, WANG J J, et al. Effects of exogenous boron and calcium on pollen germination and tube growth of Lilium oriental‘Sorbonne’[J]. Journal of Chinese Electron Microscopy Society, 2014, 33(4):368-372.
[34]雷雨，丁春元. 4种液肥对提高大樱桃坐果率及果实品质试验[J].甘肃林业科技，2008, 33(1):75-76.LEI Y, DING C Y. Experiment of 4 kinds of liquid fertilizer on improving fruit setting rate and fruit quality of big cherry[J]. Journal of Gansu Forestry Science and Technology, 2008, 33(1):75-76.
[35]汪加魏，李毓琦，施侃侃，等.花期叶面喷施B和Ca对油橄榄完全花比率和坐果率的影响[J].果树学报，2017, 34(8):1016-1024.WANG J W, LI Y Q, SHI K K, et al. Effects of foliar application of boron and calcium on perfect flower rate and fruit setting rate in olive[J]. Journal of Fruit Science, 2017, 34(8):1016-1024.
[36]贺丹，曹健康，何松林，等.芍药远缘杂交不亲和PlABCG15基因的克隆及表达分析[J].河南农业大学学报，2021, 55(6):1074-1080.HE D, CAO J K, HE S L, et al. Cloning and expression analysis of distant hybridization incompatibility PlABCG15 gene from Paeonia lactiflora[J]. Journal of Henan Agricultural University, 2021, 55(6):1074-1080.
[37]张佼蕊，贺丹，何松林，等.芍药PlSPL3基因的克隆与表达分析[J].江苏农业学报，2020, 36(6):1537-1542.ZHANG J R, HE D, HE S L, et al. Cloning and expression analysis of PlSPL3 gene from Paeonia lactiflora[J]. Jiangsu Journal of Agricultural Sciences, 2020, 36(6):1537-1542.
基本信息:
DOI：10.16445/j.cnki.1000-2340.20240912.002
中图分类号:S682.12
引用信息:
[1]贺丹,尤啸龙,周平西等.芍药钙依赖性蛋白激酶基因PlCDPK24的鉴定及表达分析[J].河南农业大学学报,2025,59(04):602-613.DOI:10.16445/j.cnki.1000-2340.20240912.002.
基金信息:
国家自然科学基金项目(32371952); 河南省自然科学基金项目(232300420006); 河南省科技攻关项目(242102110256); 河南省高等学校重点科研项目(23A220001)