鹅掌楸LcPIN1a基因的克隆及其对植株生长发育的影响

郝兆东; 马筱筱; 王丹丹; 陆叶; 施季森; 陈金慧

doi:10.12302/j.issn.1000-2006.202404005

鹅掌楸LcPIN1a基因的克隆及其对植株生长发育的影响

郝兆东, 马筱筱, 王丹丹, 陆叶, 施季森, 陈金慧

南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (6) : 51-61.

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南京林业大学学报（自然科学版） ›› 2024, Vol. 48 ›› Issue (6) : 51-61. DOI: 10.12302/j.issn.1000-2006.202404005

研究论文

鹅掌楸LcPIN1a基因的克隆及其对植株生长发育的影响

作者信息 +

Cloning of the Liriodendron chinense LcPIN1a genes and its effect on plant growth and development

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文章历史 +

摘要

【目的】PIN-FORMED(PIN)属于生长素转运蛋白,能够介导生长素的极性运输,在植物的生长和发育过程中扮演着关键角色。本研究旨在解析鹅掌楸PIN1基因对植物生长和发育的影响。【方法】通过蛋白序列同源比对、系统发育树构建以及蛋白结构域预测的方法,鉴定鹅掌楸中PIN1的同源蛋白LcPIN1s。然后,通过转录组数据解析LcPIN1s基因的组织特异性表达情况,并通过实时荧光定量逆转录PCR(RT-qPCR)方法研究LcPIN1s在不同苗龄再生植株的根、茎和叶组织中的表达动态。此外,通过转录组数据解析LcPIN1s基因在低温、高温以及干旱胁迫条件下的时序动态表达,并进一步利用RT-qPCR方法研究LcPIN1s响应干旱胁迫与内源ABA合成之间的关联。最后,通过克隆鹅掌楸叶片中高表达的PIN1同源基因LcPIN1a,构建由CaMV35S启动子驱动的过表达载体(35S:LcPIN1a),通过异源转化拟南芥(Arabidopsis thaliana)和同源转化杂种鹅掌楸(Liriodendron × sinoamericanum),筛选并获得的异源过表达(LcPIN1a-HO)和同源过表达(LcPIN1a-OE)阳性再生植株,分别进行生长和发育性状的测定和分析。【结果】通过生物信息学方法在鹅掌楸基因组中成功鉴定到了3个PIN1同源蛋白,分别命名为LcPIN1a、LcPIN1b和LcPIN1c。组织特异性表达分析显示,LcPIN1a主要在叶片中高表达,而LcPIN1b和LcPIN1c主要在茎和根以及成年后的雌蕊中高表达。另外,鹅掌楸3个PIN1同源基因均受到低温(4 ℃)和干旱(质量分数15% PEG6000)处理的诱导而表现出先上调后下调的表达模式,但在高温(40 ℃)胁迫下则会急剧下调表达。利用聚乙二醇(PEG)、脱落酸(ABA)以及ABA合成抑制剂氟啶酮(Flu)处理,发现LcPIN1s在响应干旱诱导时表现出不同的模式,即LcPIN1a不依赖于内源ABA的合成,而LcPIN1b和LcPIN1c则依赖于内源ABA的合成。最后,通过对拟南芥异源过表达株系(LcPIN1a-HO)再生植株的生长性状统计分析发现,其在根长和株高方面均显著低于野生型,同时雄蕊数发生显著变异,从野生型的6枚雄蕊变为以5枚为主。在杂交鹅掌楸过表达株系(LcPIN1a-OE)中,其体胚成苗率显著降低,并且成苗后的再生植株在根长和株高方面均显著低于野生型,同时根系结构发生明显变化,主根不明显。【结论】鹅掌楸PIN1蛋白在植株的营养和生殖生长方面发挥重要作用,过量表达不利于植株的正常生长和发育。

Abstract

【Objective】This study aimed to explore the role of the auxin transporter PIN1 in plant growth and development in Liriodendron chinense.【Method】Three PIN1 homologous proteins were identified in the Liriodendron genome using bioinformatic methods, and expression pattern analyses of the three LcPIN1s genes were performed in different tissues and response to various abiotic stresses. An overexpression vector driven by the CaMV35S promoter was then constructed and transformed into Arabidopsis and Liriodendron×sinoamericanum, followed by phenotypic determination of growth and developmental traits in the transgenic positive lines.【Result】Three PIN1 homologous proteins were identified in the Liriodendron genome, named LcPIN1a, LcPIN1b and LcPIN1c. Expression pattern analyses showed that LcPIN1a was mainly expressed in leaves, while LcPIN1b and LcPIN1c were primarily expressed in roots and stems and stigmas when the plantlets transitioned into reproductive growth. In addition, all three LcPIN1 genes transcriptionally responded to drought stress, with LcPIN1b and LcPIN1c showing dependence on the biosynthesis of endogenous ABA, while LcPIN1a does not. Root length and plant height were significantly reduced in LcPIN1a-heterologous overexpression (LcPIN1a-HO) lines compared to wild-type Arabidopsis. The number of stamens was predominantly five in LcPIN1a-HO lines, whereas wild-type Arabidopsis typically contained six stamens. The regeneration of plantlets in LcPIN1a-overexpressing (LcPIN1a-OE) Liriodendron×sinoamericanum was significantly reduced compared to wild-type plants. In addition, the root length and plant height of LcPIN1a-OE regenerated seedlings were significantly lower than those of the wild type. The root structure of LcPIN1a-OE plants was significantly changed, with the taproot being less distinct.【Conclusion】The PIN1 proteins of L. chinense play a crucial role in vegetative and reproductive growth. Overexpression of LcPIN1 genes can be detrimental to normal plant growth and development.

导出引用

郝兆东, 马筱筱, 王丹丹, 等. 鹅掌楸LcPIN1a基因的克隆及其对植株生长发育的影响[J]. 南京林业大学学报（自然科学版）. 2024, 48(6): 51-61 https://doi.org/10.12302/j.issn.1000-2006.202404005

HAO Zhaodong, MA Xiaoxiao, WANG Dandan, et al. Cloning of the Liriodendron chinense LcPIN1a genes and its effect on plant growth and development[J]. JOURNAL OF NANJING FORESTRY UNIVERSITY. 2024, 48(6): 51-61 https://doi.org/10.12302/j.issn.1000-2006.202404005

中图分类号： S718;S68

参考文献

列表( 原文顺序 | 文献年度倒序 | 文中引用次数倒序 ) 可视化分析

[1]	ZHAO Y D. Auxin biosynthesis and its role in plant development[J]. Annu Rev Plant Biol, 2010,61:49-64.DOI: 10.1146/annurev-arplant-042809-112308. 本文引用 [1]

[2]	FUKUI K, HAYASHI K I. Manipulation and sensing of auxin metabolism,transport and signaling[J]. Plant Cell Physiol, 2018, 59(8):1500-1510.DOI: 10.1093/pcp/pcy076. 本文引用 [1]

[3]	NARAMOTO S. Polar transport in plants mediated by membrane transporters:focus on mechanisms of polar auxin transport[J]. Curr Opin Plant Biol, 2017, 40:8-14.DOI: 10.1016/j.pbi.2017.06.012. 本文引用 [1]

[4]	TAN S T, LUSCHNIG C, FRIML J. Pho-view of auxin:reversible protein phosphorylation in auxin biosynthesis,transport and signaling[J]. Mol Plant, 2021, 14(1):151-165.DOI: 10.1016/j.molp.2020.11.004. 本文引用 [2]

[5]	ADAMOWSKI M, FRIML J. PIN-dependent auxin transport:action,regulation,and evolution[J]. Plant Cell, 2015, 27(1):20-32.DOI: 10.1105/tpc.114.134874. 本文引用 [1]

[6]	BLILOU I, XU J, WILDWATER M, et al. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots[J]. Nature, 2005, 433(7021):39-44.DOI: 10.1038/nature03184. 本文引用 [1]

[7]	FRIML J, BENKOVÁ E, BLILOU I, et al. AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis[J]. Cell, 2002, 108(5):661-673.DOI: 10.1016/s0092-8674(02)00656-6. 本文引用 [2]

[8]	GÄLWEILER L, GUAN C, MÜLLER A, et al. Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue[J]. Science, 1998, 282(5397):2226-2230.DOI: 10.1126/science.282.5397.2226. 本文引用 [2]

[9]	FRIML J, VIETEN A, SAUER M, et al. Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis[J]. Nature, 2003, 426(6963):147-153.DOI: 10.1038/nature02085. 本文引用 [2]

[10]	MÜLLER A, GUAN C, GÄLWEILER L, et al. AtPIN2 defines a locus of Arabidopsis for root gravitropism control[J]. EMBO J, 1998, 17(23):6903-6911.DOI: 10.1093/emboj/17.23.6903. 本文引用 [1]

[11]	FRIML J, WISNIEWSKA J, BENKOVÁ E, et al. Lateral relocation of auxin efflux regulator PIN3 mediates tropism in Arabidopsis[J]. Nature, 2002, 415(6873):806-809.DOI: 10.1038/415806a. 本文引用 [1]

[12]	DING Z J, WANG B J, MORENO I, et al. ER-localized auxin transporter PIN8 regulates auxin homeostasis and male gametophyte development in Arabidopsis[J]. Nat Commun, 2012,3:941.DOI: 10.1038/ncomms1941. 本文引用 [1]

[13]	SIMON S, SKŮPA P, VIAENE T, et al. PIN6 auxin transporter at endoplasmic reticulum and plasma membrane mediates auxin homeostasis and organogenesis in Arabidopsis[J]. New Phytol, 2016, 211(1):65-74.DOI: 10.1111/nph.14019. 本文引用 [1]

[14]	OKADA K, UEDA J, KOMAKI M K, et al. Requirement of the auxin polar transport system in early stages of Arabidopsis floral bud formation[J]. Plant Cell, 1991, 3(7):677-684.DOI: 10.1105/tpc.3.7.677. 本文引用 [1]

[15]	GOVINDARAJU P, VERNA C, ZHU T B, et al. Vein patterning by tissue-specific auxin transport[J]. Development, 2020, 147(13):dev187666.DOI: 10.1242/dev.187666. 本文引用 [1]

[16]	NOH B, BANDYOPADHYAY A, PEER W A, et al. Enhanced gravi-and phototropism in plant mdr mutants mislocalizing the auxin efflux protein PIN1[J]. Nature, 2003, 423(6943):999-1002.DOI: 10.1038/nature01716. 本文引用 [1]

[17]	LI Y, ZHU J S, WU L L, et al. Functional divergence of PIN1 paralogous genes in rice[J]. Plant Cell Physiol, 2019, 60(12):2720-2732.DOI: 10.1093/pcp/pcz159. 本文引用 [2]

[18]	郝日明, 贺善安, 汤诗杰, 等. 鹅掌楸在中国的自然分布及其特点[J]. 植物资源与环境, 1995, 4(1):1-6. HAO R M, HE S A, TANG S J, et al. Geographical distribution of Liridendron chinense in China and its significance[J]. J Plant Resour Environ, 1995, 4(1):1-6. 本文引用 [1]

[19]	PARKS C R, MILLER N G, WENDEL J F, et al. Genetic divergence within the genus Liriodendron (Magnoliaceae)[J]. Ann Mo Bot Gard, 1983, 70(4):658.DOI: 10.2307/2398983. 本文引用 [1]

[20]	PARKS C R, WENDEL J F. Molecular divergence between Asian and north American species of Liriodendron (Magnoliaceae) with implications for interpretation of fossil floras[J]. Am J Bot, 1990, 77(10):1243.DOI: 10.2307/2444585. 本文引用 [1]

[21]	李火根, 施季森. 杂交鹅掌楸良种选育与种苗繁育[J]. 林业科技开发, 2009, 23(3):1-5. LI H G, SHI J S. Breeding and propagation of Liriodendron hybrids[J]. J For Eng, 2009, 23(3):1-5.DOI: 10.3969/j.issn.1000-8101.2009.03.001. 本文引用 [1]

[22]	向其柏, 王章荣. 杂交马褂木的新名称:亚美马褂木[J]. 南京林业大学学报(自然科学版), 2012, 36(2):1-2. SHANG C B, WANG Z R. A new scientific name of hybrid Liriodendron: L. sino americanum[J]. J Nanjing For Univ (Nat Sci Ed), 2012, 36(2):1-2.DOI: 10.3969/j.issn.1000-2006.2012.02.001. 本文引用 [1]

[23]	CHEN J H, HAO Z D, GUANG X M, et al. Liriodendron genome sheds light on angiosperm phylogeny and species-pair differentiation[J]. Nat Plants, 2019, 5(1):18-25.DOI: 10.1038/s41477-018-0323-6. 本文引用 [2]

[24]	陈金慧, 施季森, 诸葛强, 等. 杂交鹅掌楸体细胞胚胎发生研究[J]. 林业科学, 2003, 39(4):49-53,177. CHEN J H, SHI J S, ZHUGE Q, et al. Studies on the somatic embryogenesis of Liriodendron hybrids (L. chinense × L. tulipifera)[J]. Sci Silvae Sin, 2003, 39(4):49-53,177.DOI: 10.3321/j.issn:1001-7488.2003.04.008. 本文引用 [2]

[25]	LI M P, WANG D, LONG X F, et al. Agrobacterium-mediated genetic transformation of embryogenic callus in a Liriodendron hybrid (L.chinense × L.tulipifera)[J]. Front Plant Sci, 2022,13:802128.DOI: 10.3389/fpls.2022.802128. 本文引用 [2]

[26]	HU L F, WANG P K, LONG X F, et al. The PIN gene family in relic plant L.chinense:genome-wide identification and gene expression profiling in different organizations and abiotic stress responses[J]. Plant Physiol Biochem, 2021, 162:634-646.DOI: 10.1016/j.plaphy.2021.03.030. 本文引用 [1]

[27]	LI R, PAN Y, HU L F, et al. PIN3 from Liriodendron may function in inflorescence development and root elongation[J]. Forests, 2022, 13(4):568.DOI: 10.3390/f13040568. 本文引用 [1]

[28]	BERARDINI T Z, REISER L, LI D H, et al. The Arabidopsis information resource:making and mining the “gold standard” annotated reference plant genome[J]. Genesis, 2015, 53(8):474-485.DOI: 10.1002/dvg.22877. 本文引用 [2]

[29]	JOHNSON M, ZARETSKAYA I, RAYTSELIS Y, et al. NCBI BLAST:a better web interface[J]. Nucleic Acids Res, 2008, 36(Web Server issue):5-9.DOI: 10.1093/nar/gkn201. 本文引用 [1]

[30]	GOODSTEIN D M, SHU S Q, HOWSON R, et al. Phytozome:a comparative platform for green plant genomics[J]. Nucleic Acids Res, 2012, 40(Database issue):D1178-D1186.DOI: 10.1093/nar/gkr944. 本文引用 [1]

[31]	SIEVERS F, HIGGINS D G. Clustal Omega for making accurate alignments of many protein sequences[J]. Protein Sci, 2018, 27(1):135-145.DOI: 10.1002/pro.3290. 本文引用 [1]

[32]	TOGKOUSIDIS A, KOZLOV O M, HAAG J, et al. Adaptive RAxML-NG:accelerating phylogenetic inference under maximum likelihood using dataset difficulty[J]. Mol Biol Evol, 2023, 40(10):msad227.DOI: 10.1093/molbev/msad227. 本文引用 [1]

[33]	CHEN C J, WU Y, LI J W, et al. TBtools-Ⅱ:a“one for all,all for one” bioinformatics platform for biological big-data mining[J]. Mol Plant, 2023, 16(11):1733-1742.DOI: 10.1016/j.molp.2023.09.010. 本文引用 [1]

[34]	LETUNIC I, BORK P. Interactive tree of life (iTOL) v5:an online tool for phylogenetic tree display and annotation[J]. Nucleic Acids Res, 2021, 49(W1):293-296.DOI: 10.1093/nar/gkab301. 本文引用 [1]

[35]	OMASITS U, AHRENS C H, MÜLLER S, et al. Protter:interactive protein feature visualization and integration with experimental proteomic data[J]. Bioinformatics, 2014, 30(6):884-886.DOI: 10.1093/bioinformatics/btt607. 本文引用 [1]

[36]	LI T T, YUAN W G, QIU S, et al. Selection of reference genes for gene expression analysis in Liriodendron hybrids’ somatic embryogenesis and germinative tissues[J]. Sci Rep, 2021, 11(1):4957.DOI: 10.1038/s41598-021-84518-w. 本文引用 [1]

[37]	LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C(T)) Method[J]. Methods, 2001, 25(4):402-408.DOI: 10.1006/meth.2001.1262. 本文引用 [1]

[38]	MAVRODIEV E V, DERVINIS C, WHITTEN W M, et al. A new,simple,highly scalable,and efficient protocol for genomic DNA extraction from diverse plant taxa[J]. Appl Plant Sci, 2021, 9(3):e11413.DOI: 10.1002/aps3.11413. 本文引用 [1]

[39]	CLOUGH S J, BENT A F. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. Plant J, 1998, 16(6):735-743.DOI: 10.1046/j.1365-313x.1998.00343.x. 本文引用 [1]

[40]	KRECEK P, SKUPA P, LIBUS J, et al. The PIN-FORMED (PIN) protein family of auxin transporters[J]. Genome Biol, 2009, 10(12):249.DOI: 10.1186/gb-2009-10-12-249. 本文引用 [1]

[41]	SANCHO-ANDRÉS G, SORIANO-ORTEGA E, GAO C J, et al. Sorting motifs involved in the trafficking and localization of the PIN1 auxin efflux carrier[J]. Plant Physiol, 2016, 171(3):1965-1982.DOI: 10.1104/pp.16.00373. 本文引用 [1]

[42]	MARCOTE M J, SANCHO-ANDRÉS G, SORIANO-ORTEGA E, et al. Sorting signals for PIN1 trafficking and localization[J]. Plant Signal Behav, 2016, 11(8):e1212801.DOI: 10.1080/15592324.2016.1212801. 本文引用 [1]

[43]	HUANG F, ZAGO M K, ABAS L, et al. Phosphorylation of conserved PIN motifs directs Arabidopsis PIN1 polarity and auxin transport[J]. Plant Cell, 2010, 22(4):1129-1142.DOI: 10.1105/tpc.109.072678. 本文引用 [1]

[44]	WU W H, ZHU S, XU L, et al. Genome-wide identification of the Liriodendron chinense WRKY gene family and its diverse roles in response to multiple abiotic stress[J]. BMC Plant Biol, 2022, 22(1):25.DOI: 10.1186/s12870-021-03371-1. 本文引用 [1]

[45]	WU W H, ZHU S, ZHU L M, et al. Characterization of the Liriodendron chinense MYB gene family and its role in abiotic stress response[J]. Front Plant Sci, 2021,12:641280.DOI: 10.3389/fpls.2021.641280. 本文引用 [1]

[46]	MROUE S, SIMEUNOVIC A, ROBERT H S. Auxin production as an integrator of environmental cues for developmental growth regulation[J]. J Exp Bot, 2018, 69(2):201-212.DOI: 10.1093/jxb/erx259. 本文引用 [1]

[47]	GU Z L, STEINMETZ L M, GU X, et al. Role of duplicate genes in genetic robustness against null mutations[J]. Nature, 2003, 421(6918):63-66.DOI: 10.1038/nature01198. 本文引用 [1]

[48]	TESSI T M, MAURINO V G, SHAHRIARI M, et al. AZG1 is a cytokinin transporter that interacts with auxin transporter PIN1 and regulates the root stress response[J]. New Phytol, 2023, 238(5):1924-1941.DOI: 10.1111/nph.18879. 本文引用 [1]

[49]	BAWA G, LIU Z X, WU R, et al. PIN1 regulates epidermal cells development under drought and salt stress using single-cell analysis[J]. Front Plant Sci, 2022,13:1043204.DOI: 10.3389/fpls.2022.1043204. 本文引用 [1]

[50]	ZHANG J, VANNESTE S, BREWER P B, et al. Inositol trisphosphate-induced Ca²⁺ signaling modulates auxin transport and PIN polarity[J]. Dev Cell, 2011, 20(6):855-866.DOI: 10.1016/j.devcel.2011.05.013. 本文引用 [2]

[51]	XU M, ZHU L, SHOU H X, et al. A PIN1 family gene,OsPIN1,involved in auxin-dependent adventitious root emergence and tillering in rice[J]. Plant Cell Physiol, 2005, 46(10):1674-1681.DOI: 10.1093/pcp/pci183. 本文引用 [1]

[52]	MRAVEC J, KUBES M, BIELACH A, et al. Interaction of PIN and PGP transport mechanisms in auxin distribution-dependent development[J]. Development, 2008, 135(20):3345-3354.DOI: 10.1242/dev.021071. 本文引用 [1]

[53]	LI K, KAMIYA T, FUJIWARA T. Differential roles of PIN1 and PIN2 in root meristem maintenance under low-B conditions in Arabidopsis thaliana[J]. Plant Cell Physiol, 2015, 56(6):1205-1214.DOI: 10.1093/pcp/pcv047. 本文引用 [1]

[54]	XU K J, SUN F L, WANG Y F, et al. The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass[J]. Genet Mol Biol, 2016, 39(1):62-72.DOI: 10.1590/1678-4685-GMB-2014-0300. 本文引用 [1]

[55]	GUAN L, LI Y J, HUANG K H, et al. Auxin regulation and MdPIN expression during adventitious root initiation in apple cuttings[J]. Hortic Res, 2020, 7(1):143.DOI: 10.1038/s41438-020-00364-3. 本文引用 [1]

[56]	ROTH O, YECHEZKEL S, SERERO O, et al. Slow release of a synthetic auxin induces formation of adventitious roots in recalcitrant woody plants[J]. Nat Biotechnol, 2024.DOI: 10.1038/s41587-023-02065-3. 本文引用 [1]