马尾松PmPIN1基因的克隆及功能分析

doi:10.11707/j.1001-7488.20200319

摘要/Abstract

摘要：

目的: PIN1基因通过调控生长素极性运输的方式在植物根系生长发育中发挥作用。对马尾松PmPIN1基因进行全长克隆和功能分析，有助于在分子水平揭示马尾松的生根机制。方法: 运用PCR和RACE技术成功克隆马尾松PmPIN1基因cDNA序列；利用生物信息学软件分析PmPIN1基因的核苷酸序列及其编码蛋白的氨基酸序列，并构建系统进化树；通过实时荧光定量分析PmPIN1基因在10年生马尾松幼根、1年生枝条、新叶、花中的表达量差异；利用农杆菌侵染法将PmPIN1基因转入烟草获得转基因植株，比较转基因烟草与转pCAMBIA-1302空白载体烟草之间的表型差异；用激光共聚焦显微镜观察PmPIN1-GFP荧光蛋白在转基因烟草根中的表达模式；酶联免疫法测定野生型和转基因烟草的根、根茎结合处及叶中的生长素含量；用不同浓度的生长素极性运输抑制剂1-N-萘基邻氨甲酰苯甲酸（NPA）处理野生型与转基因烟草，分析烟草根、根茎结合处、叶中的生长素含量变化；利用PEG介导法将16318-hGFP-PmPIN1重组载体转化至拟南芥原生质体中进行亚细胞定位分析。结果: PmPIN1基因全长2 914 bp，包含2 085 bp的ORF，编码的蛋白由695个氨基酸组成，N端与C端均有膜运输蛋白。系统进化树显示马尾松与油松在发育树同一支上。通过实时荧光定量发现PmPIN1在不同组织中的表达量差异达到极显著，1年生枝条与幼根中表达量较高，花中最低。农杆菌介导法转化烟草获得的转基因烟草，较转空载烟草株高增长，生根量增加且根系变长。激光共聚焦显微镜观察得出转基因烟草根部中间有明显绿色荧光富集现象。酶联免疫法测定结果表明转基因烟草根、根茎结合处生长素含量高于野生型，且叶较根和根茎结合处减少。不同浓度NPA处理后，野生型烟草根中生长素含量变化达极显著，且生长素含量随NPA处理浓度的增加而减少；处理浓度为10 nmol·L^-1时，叶中生长素含量最高；处理浓度为2 nmol·L^-1时，根茎结合处生长素含量增加较多。不同浓度NPA处理后，转基因烟草不同部位生长素含量变化均未达到极显著。亚细胞定位分析表明PmPIN1蛋白主要分布于细胞膜上。结论: 马尾松PmPIN1与油松PIN1亲缘关系最近。PmPIN1属于膜蛋白。PmPIN1转基因烟草可能提高生长素由地上部位向地下部位的运输能力，减少NPA对生长素含量变化的影响，表明PmPIN1可能调控生长素的极性运输；PmPIN1-GFP绿色荧光富集在根部中间部位，PmPIN1在幼根中表达量较高，转基因烟草较转空载植株发根量多，根长长。研究结果表明PmPIN1基因可能在根系发育中发挥重要作用。

关键词: 马尾松, PmPIN1基因, 转基因, 功能分析

Abstract:

Objective: The PIN1 gene plays a role in the growth and development of plant roots by regulating the polar transport of auxin. In this study, the full-length cloning and functional analysis of the PmPIN1 gene of Pinus massoniana was carried out in order to reveal the molecular mechanism of rooting for cutting propagation of P. massoniana. Method: The cDNA sequence of PmPIN1 gene of P. massoniana was successfully cloned by PCR and RACE. Bioinformatics software was used to analyze the nucleic acid sequence, amino acid sequence and construct a phylogenetic tree of PmPIN1 gene. The expression of PmPIN1 gene in young roots, annual branch, young leaf and flowers of 10-year-old P. massoniana was analyzed by real-time polymerase chain reaction. The PmPIN1 gene was overexpressed in tobacco(Nicotiana tabacum)by Agrobacterium infection method to obtain transgenic plants, and the phenotypic differences between transgenic plants and pCAMBIA-1302 blank vector were compared, and PmPIN1-GFP fluorescent protein was observed in transgenic tobacco roots by laser scanning confocal microscope. The expression of auxin in roots, rhizomes and leaves of transgenic tobacco and wild tobacco was determined by enzyme-linked immunosorbent assay (ELISA). Treatment of wild-type and transgenic tobacco with different concentrations of auxin polar transport inhibitor (1-N-naphthylphthalamic acid, NPA), the changes of auxin content in tobacco roots, rhizome junctions and leaves were analyzed. 16318-hGFP-PmPIN1 recombinant vector was transferred to Arabidopsis thaliana protoplasts by PEG-mediated method for subcellular localization analysis. Result: The PmPIN1 gene is 2 914 bp in length and contains a 2 085 bp ORF, which encodes a protein consisting of 695 amino acids, with a membrane transport protein at the N-terminus and C-terminus. The phylogenetic tree showed that P. massoniana and P. tabulaeformis are on the same branch of the development tree. The difference in expression of PmPIN1 in different tissues was found to be extremely significant by real-time polymerase chain reaction, high expression in annual branch and young roots, lowest in flowers; Transgenic tobacco obtained by Agrobacterium rhizogenes-mediated transformation had higher growth, increased rooting and longer root length compared with a blank vector. Laser confocal microscopy showed that there was obvious green fluorescence enrichment around the root of transgenic tobacco. ELISA was used to determine the content of auxin in the roots and rhizomes of transgenic tobacco, and the content was found higher than that in wild type, and less in leaves than in roots and rhizomes. After treatment with different concentrations of NPA, the auxin content of wild type tobacco roots changed extremely significantly, and the auxin content decreased with the increase of NPA treatment concentration. When the treatment concentration was 10 nmol·L^-1, the auxin content in leaves was the largest. When the concentration was 2 nmol·L^-1, the content of auxin in the rhizome junction increased more. After treatment with different concentrations of NPA, the changes of auxin content in different parts of transgenic tobacco did not reach extremely significant. Subcellular localization analysis indicated that PmPIN1 protein was mainly distributed on the cell membrane. Conclusion: PmPIN1 of P. massoniana and PIN1 of P. tabulaeformis had a higher affinity. PmPIN1 belongs to membrane protein. PmPIN1 transgenic tobacco increases the transport capacity of auxin from aboveground to underground. Transgenic tobacco reduces the effect of NPA on auxin content, indicating that PmPIN1 likely regulates the polar transport of auxin. PmPIN1-GFP green fluorescence was enriched in the middle of the root, PmPIN1 was highly expressed in young roots. Transgenic tobacco had more roots and longer roots than transgenic plasmid. The above findings suggest that the PmPIN1 gene plays an important role in root development.

Key words: Pinus massoniana, PmPIN1 gene, transgenic, functional analysis

中图分类号:

S718.46

武星,胡兴峰,陈佩珍,孙晓波,吴帆,季孔庶. 马尾松PmPIN1基因的克隆及功能分析[J]. 林业科学, 2020, 56(3): 184-192.

Xing Wu,Xingfeng Hu,Peizhen Chen,Xiaobo Sun,Fan Wu,Kongshu Ji. Cloning and Functional Analysis of PmPIN1 Gene from Pinus massoniana[J]. Scientia Silvae Sinicae, 2020, 56(3): 184-192.

图/表 12

表1

图1

图2

图3

图4

图5

图6

图7

图8

图9

表2

图10

参考文献 0

	陈赢男. 2012.生长素转运蛋白OsPIN2对水稻株型、根系生长和磷素营养的调控作用.南京:南京农业大学博士学位论文.
	Chen Y N. 2012. Regulatory effect of OsPIN2, a putative auxin efflux transporter, on rice architecture, root morphology and phosphorus nutrition. Nanjing: PhD thesis of Nanjing Agricultural University.[in Chinese]
	季孔庶, 王章荣, 陈天华, 等. 几种生长调节剂对马尾松插穗促根的效应. 福建林学院学报, 2001. 21 (2): 120- 123. doi: 10.3969/j.issn.1001-389X.2001.02.007
	Ji K S , Wang Z R , Chen T H , et al. Effects of root inducing regulators in rooting of cuttings from Pinus massoniana Lamb. Journal of Fujian College of Forestry, 2001. 21 (2): 120- 123. doi: 10.3969/j.issn.1001-389X.2001.02.007
	李俊华, 种康. 植物生长素极性运输调控机理的研究进展. 植物学通报, 2006. 23 (5): 466- 477. doi: 10.3969/j.issn.1674-3466.2006.05.004
	Li J H , Chong K . Research progress on the regulation mechanism of auxin polar transport. Botanical Bulletin, 2006. 23 (5): 466- 477. doi: 10.3969/j.issn.1674-3466.2006.05.004
	吕素慧, 郗琳, 聂静, 等. 菊花CmPIN1同源基因的克隆与表达分析. 中国农业大学学报, 2016. 21 (3): 58- 66.
	Lü S H , Xi L , Nie J , et al. Isolation and expression analysis of auxin efflux transporter CmPIN1 in Chrysanthemum(Chrysanthemum morifolium 'Jinba'). Journal of China Agricultural University, 2016. 21 (3): 58- 66.
	倪为民, 陈晓亚, 许智宏, 等. 生长素极性运输研究进展. 植物学报, 2000. 42 (3): 221- 228. doi: 10.3321/j.issn:1672-9072.2000.03.001
	Ni W M , Chen X Y , Xu Z H , et al. Advances in study of polar auxin transport. Acta Botanica Sinica, 2000. 42 (3): 221- 228. doi: 10.3321/j.issn:1672-9072.2000.03.001
	苏江, 郭天玮, 季孔庶. 马尾松PmTIR1基因的克隆及编码蛋白质的结构预测. 分子植物育种, 2015. 13 (6): 1355- 1362.
	Su J , Guo T W , Ji K S . Cloning and preliminary analysis of TIR1 and AUX/IAA family genes in Pinus massoniana. Molecular Plant Breeding, 2015. 13 (6): 1355- 1362.
	伍家荣, 谭著明, 蒋丽娟, 等. 马尾松播种苗截根提高造林成活率研究. 林业科技通讯, 1994. (6): 16- 17.
	Wu J R , Tan Z M , Jiang L J , et al. Study on the improvement of afforestation survival rate by cutting roots of Pinus massoniana seedlings. Forest Science and Technology, 1994. (6): 16- 17.
	武星, 季子钧, 吴晓刚, 等. 马尾松PmPIN2基因的克隆及实时定量分析. 分子植物育种, 2018. 16 (15): 4880- 4889.
	Wu X , Ji Z J , Wu X G , et al. Cloning and real-time quantitative analysis of PmPIN2 gene from Pinus massoniana. Molecular Plant Breeding, 2018. 16 (15): 4880- 4889.
	姚瑞玲, 王胤, 吴幼媚. 马尾松组培生根关键因子分析. 广西植物, 2016. 36 (11): 1288- 1294.
	Yao R L , Wang Y , Wu Y M . Key factors affecting rooting of Pinus massoniana by tissue culture. Guihaia, 2016. 36 (11): 1288- 1294.
	朱沛煌. 2016.马尾松GRAS家族SHR和SCR同源基因的克隆与功能初步分析.南京:南京林业大学硕士学位论文.
	Zhu P H. 2016. Cloning and analysis on SHR, SCR and homologous genes form Pinus massoniana GRAS family. Nanjing: MS thesis of Nanjing Forestry University.[in Chinese]
	Benková E , Michniewicz M , Sauer M , et al. Local, efflux-dependent auxin gradients as a common module for plant organ formation. Cell, 2003. 115 (5): 591- 602. doi: 10.1016/S0092-8674(03)00924-3
	Blilou I , Xu J , Wildwater M , et al. The PIN auxin efflux facilitator network controls growth and patterning in Arabidopsis roots. Nature, 2005. 433 (7021): 39- 44. doi: 10.1038/nature03184
	Chen R , Hilson P , Sedbrook J , et al. The Arabidopsis thaliana AGRAVITROPIC 1 gene encodes a component of the polar-auxin-transport efflux carrier. Proceedings of the National Academy of Sciences of the United States of America, 1998. 95 (25): 15112- 15117. doi: 10.1073/pnas.95.25.15112
	Friml J , Benková E , Blilou I , et al. AtPIN4 mediates sink-driven auxin gradients and root patterning in Arabidopsis. Cell, 2002a. 108 (5): 661- 673. doi: 10.1016/S0092-8674(02)00656-6
	Friml J , Palme K . Polar auxin transport-old questions and new concepts?. Plant Mol Biol, 2002b. 49 (3/4): 273- 284. doi: 10.1023/A:1015248926412
	Friml J , Vieten A , Sauer M , et al. Efflux-dependent auxin gradients establish the apical-basal axis of Arabidopsis. Nature, 2003. 426 (6963): 147- 153. doi: 10.1038/nature02085
	Gälweiler L , Guan C , Müller A . Regulation of polar auxin transport by AtPIN1 in Arabidopsis vascular tissue. Science, 1998. 282 (5397): 2226- 2230. doi: 10.1126/science.282.5397.2226
	Kepinski S , Leyser O . Plant development:auxin in loops. Curr Biol, 2005. 15 (6): 208- 210. doi: 10.1016/j.cub.2005.03.012
	Krecek P , Sku°pa P , Libus J , et al. The PIN-FORMED (PIN) protein family of auxin transporters. Genome Biology, 2009. 10 (12): 249. doi: 10.1186/gb-2009-10-12-249
	Maher E P , Martindale S J . Mutants of Arabidopsis thaliana with altered responses to auxins and gravity. Biochem Genet, 1980. 18 (11/12): 1041- 1053.
	Muday G K , Murphy A S . An emerging model of auxin transport regulation. Plant Cell, 2002. 14 (2): 293- 299. doi: 10.1105/tpc.140230
	Paponov I A , Teale W D , Trebar M , et al. The PIN auxin efflux facilitators:evolutionary and functional perspectives. Trends Plant Sci, 2005. 10 (4): 170- 177. doi: 10.1016/j.tplants.2005.02.009
	Peer W A , Bandyopadhyay A , Blakeslee J J , et al. Variation in expression and protein localization of the PIN family of auxin efflux facilitator proteins in flavonoid mutants with altered auxin transport in Arabidopsis thaliana. Plant Cell, 2004. 16 (7): 1898- 1911. doi: 10.1105/tpc.021501
	Pérez-Flores P , Valencia-Cantero E , Altamirano-Hernández J , et al. Bacillus methylotrophicus M4-96 isolated from maize (Zea mays) rhizoplane increases growth and auxin content in Arabidopsis thaliana via emission of volatiles. Protoplasma, 2017. 254 (6): 2201- 2213. doi: 10.1007/s00709-017-1109-9
	Sabatini S , Beis D , Wolkenfelt H , et al. An auxin-dependent distal organizer of pattern and polarity in the Arabidopsis root. Cell, 1999. 99 (5): 463- 472. doi: 10.1016/S0092-8674(00)81535-4
	Vieten A , Vanneste S , Wisniewska J , et al. Functional redundancy of PIN proteins is accompanied by auxin-dependent cross-regulation of PIN expression. Development, 2005. 132 (20): 4521- 4531. doi: 10.1242/dev.02027
	Woodward A W , Bartel B . Auxin:regulation, action, and interaction. Annals of Botany, 2005. 95 (5): 707- 735. doi: 10.1093/aob/mci083
	Xu M . A PIN1 family gene, OsPIN1, involved in auxin-dependent adventitious root emergence and tillering in rice. Plant and Cell Physiology, 2005. 46 (10): 1674- 1681. doi: 10.1093/pcp/pci183
	Zazímalová E , Krecek P , Sku°pa P , et al. Polar transport of the plant hormone auxin-the role of PIN-FORMED(PIN) proteins. Cellular & Molecular Life Sciences, 2007. 64 (13): 1621- 1637.

引物名称 Primer name	引物序列 Sequence(5′—3′)
F1	TGCATAATTTGGTATACTCTCATGCT
R1	AATTACTGTTATGGTTGTTGTCAGGA
F2	GATGTCTATTCATTCCATTCCTCCCG
R2	AAAAACTCCTCGAAGGCCAACAGCA
3′RACE primer	TTCCTCCTCCCCCTCTCTGTC
5′RACE primer	AGCTTCCCGTCTTCTCCGACTTGCG
F3	GCATTGTTCCCTTTGTGTTTG
R3	TCTTGTAGTTCCCGTCGTCCT
Actin2-F	GGTAGCTCCACCTGAGAGGAAGT
Actin2-R	GCCTTTGCAATCCACATCTGT
pCAMBIA-1302-PmPIN1F	ACGGGGGACTCTTGACCATGGATGATTAATGGAGCAGACATATACAACG
pCAMBIA-1302-PmPIN1R	ACTAGTCAGATCATACCATGGAACTCCAAGAAGAAAGTAATACACAAGAG
16318-hGFP-PmPIN1F	TGGAGAGGACAGCCCAAGCTTATGATTAATGGACCAGACATATACAACG
16318-hGFP-PmPIN1R	GCTCACCATGGATCCTCTAGAAACTCCAAGAAGAATGTAATACACAAGAG

烟草类型 Type of tobacco	NPA浓度 NPA concentration/(nmol·L^-1)	根 Root	根茎结合处 Rhizome junction	叶 Leaf
转基因 Transgenic	0	56.05±14.94	49.71±15.71	39.55±4.71
	0.4	50.86±7.95	46.62±9.96	46.21±12.65
	2	53.27±9.92	42.38±2.17	41.74±1.56
	10	49.12±13.04	39.92±2.58	51.95±11.84
野生型 Wild	0	55.91±4.34 a	45.30±6.14 b	46.89±15.40 b
	0.4	48.65±1.55 b	45.10±4.95 b	56.46±3.29 ab
	2	45.05±2.60 b	54.98±5.35 a	53.72±3.56 ab
	10	36.48±2.21 c	49.46±1.52 ab	66.01±1.91 a

[1]	闫小莉,胡文佳,马远帆,霍昱帆,王拓,马祥庆. 异质性供氮环境下杉木、马尾松、木荷氮素吸收偏好及其根系觅氮策略[J]. 林业科学, 2020, 56(2): 1-11.
[2]	袁秀锦, 肖文发, 雷静品, 潘磊, 王晓荣, 崔鸿侠, 胡文杰. 三峡库区马尾松林穿透雨和树干茎流空间变异特征[J]. 林业科学, 2020, 56(1): 10-19.
[3]	张超, 王进茂, 赵洁, 庞丁玮, 张德健, 杨敏生. 转多基因欧美杨Bt基因表达特征[J]. 林业科学, 2019, 55(9): 61-70.
[4]	叶钰倩, 赵家豪, 刘畅, 关庆伟. 间伐强度对马尾松人工林根际与非根际土壤中性糖特征的影响[J]. 林业科学, 2019, 55(8): 28-35.
[5]	邓秀秀, 肖文发, 曾立雄, 雷蕾, 施征. 马尾松幼苗光合产物的运输与分配特征[J]. 林业科学, 2019, 55(7): 27-34.
[6]	金国庆, 张振, 余启新, 冯随起, 丰忠平, 赵世荣, 周志春. 马尾松2个世代种子园6年生家系生长的遗传变异与增益比较[J]. 林业科学, 2019, 55(7): 57-67.
[7]	李志能, 江英杰, 陈竟, 李婷, 眭顺照, 李名扬. 蜡梅CpWOX13基因的表达及功能[J]. 林业科学, 2019, 55(7): 77-85.
[8]	李亚藏, 冯仲科. 气候敏感的马尾松生物量相容性方程系统研建[J]. 林业科学, 2019, 55(5): 65-73.
[9]	王好运, 吴峰, 朱小坤, 谢维斌. 叶型对马尾松幼苗生长及叶绿素荧光特征的影响[J]. 林业科学, 2019, 55(3): 183-192.
[10]	陈金磊, 方晰, 辜翔, 李雷达, 刘兆丹, 王留芳, 张仕吉. 中亚热带2种森林群落组成、结构及区系特征[J]. 林业科学, 2019, 55(2): 159-172.
[11]	杜会聪,王瑶,方加兴,张珍荫,张苏芳,刘福,张真,孔祥波. 马尾松毛虫线粒体全基因组的测定与分析[J]. 林业科学, 2019, 55(12): 162-172.
[12]	王金池,黄清麟,马志波,黄如楚,郑群瑞. 永安市半天然马尾松阔叶混交林的树种组成与多样性[J]. 林业科学, 2019, 55(11): 19-26.
[13]	陈坦, 张振, 楚秀丽, 金国庆, 周志春, 丰忠平. 马尾松二代无性系种子园的花期同步性[J]. 林业科学, 2019, 55(1): 146-156.
[14]	罗晓蔓, 丁贵杰, 王艺. 菌根化马尾松苗木根际土壤浸提物有效成份及其对种子萌发的影响[J]. 林业科学, 2018, 54(8): 32-38.
[15]	姚虹宇, 刘亚敏, 张盛楠, 刘玉民, 周文颖, 王针针. 外源柠檬酸对铝胁迫下马尾松生理特性的影响[J]. 林业科学, 2018, 54(7): 155-164.