木霉诱导下山新杨PodaPIN9基因的组织表达调控

doi:10.7525/j.issn.1673-5102.2019.02.013

摘要/Abstract

摘要： PIN蛋白具有多个跨膜结构域，影响着高等植物生长素的外向运输和众多生长发育过程。木霉菌是能促进植物生长、提高其对多种病害防御作用的生防因子。研究木霉菌对木本植物山新杨生长素的极性分布的影响有重要意义。克隆了山新杨PodaPIN9基因，对其核酸和蛋白序列进行分析；同时，构建的进化树显示PodaPIN9与6个物种的9条PIN基因具有高度一致性（>80%）。qRT-PCR分析表明，PodaPIN9在山新杨茎尖、成熟叶和根中均有表达。在根中表达量极低；在茎尖和叶中的表达量极高，分别是根中的503和346倍。该基因受木霉影响在茎尖和叶中的表达量均显著下调；而根中的表达量显著上调，达到对照的32.01倍。并发现根际接种木霉48 h会使杨树茎尖、叶和根中生长素含量降低。说明木霉菌能影响杨树茎尖、叶和根中IAA水平及PodaPIN9的表达量。并且Pearson相关性分析表明在茎尖、叶和根中，PodaPIN9表达量与IAA水平具有不同的相关性。

关键词: 山新杨, 棘孢木霉菌, PIN基因, qRT-PCR, IAA

Abstract: The PIN proteins contain multiple transmembrane domains, and are involved in the efflux of auxin, affecting many growth and development-related processes in higher plants. Trichoderma is a biocontrol agent that promotes the growth and improves the defense mechanisms of plants against various diseases. Therefore, it is important to study the effects of Trichoderma on the distribution of auxin in the woody species Populus davidiana×P.alba var. pyramidlis(Shanxin poplar). In this study, the gene PodaPIN9 was cloned from Shanxin poplar, and its nucleotide and protein sequences were analyzed. The constructed phylogenetic tree showed that PodaPIN9 has a high identity with 9 PIN genes from 6 species(>80%). By the qRT-PCR analysis, PodaPIN9 was expressed in the stem tips, mature leaves, and roots of the plant. The expression of PodaPIN9 was significantly low in the roots, but was significantly high in the stem tips and leaves(503 and 346 times higher than that in the roots, respectively). Following inoculation with Trichoderma, the expression of PodaPIN9 was significantly downregulated in the stem tips and leaves, whereas its expression was significantly upregulated in the roots. The expression of PodaPIN9 in the roots was 32.01 times higher than the control group. Additionally, we found that the levels of auxin in the stem tips, leaves, and roots of the plant were decreased at 48 h after inoculating the Trichoderma. It demonstrated that Trichoderma can affect auxin levels and the expression of PodaPIN9 in the stem tips, leaves, and roots of poplar. Furthermore, by Pearson correlation analysis, the correlation between the expression of PodaPIN9 and auxin levels differed in the stem tips, leaves, and roots.

Key words: Populus davidiana×P.alba var. pyramidlis, trichoderma asperellum, PIN, qRT-PCR, IAA

中图分类号:

S792.114

李枢航, 苗蕊, 常媛, 李俊男, 燕晓杰, 刘照莹, 张荣沭. 木霉诱导下山新杨PodaPIN9基因的组织表达调控[J]. 植物研究, 2019, 39(2): 267-275.

LI Shu-Hang, MIAO Rui, CHANG Yuan, LI Jun-Nan, YAN Xiao-Jie, LIU Zhao-Ying, ZHANG Rong-Shu. Differential Expression of PodaPIN9 Gene in Tissues of Populus davidiana×P.alba var. pyramidlis Induced by Trichoderma[J]. Bulletin of Botanical Research, 2019, 39(2): 267-275.

参考文献

1. 何克军. 澳大利亚昆士兰州松树杂交育种和造林[J]. 广东林业科技,1996,12(3):34-38. He K J. Hybrid breeding and planting of exotic pine in Queensland[J]. Forestry Science and Technology of Guangdong Province,1996,12(3):34-38.
2. 湛江市林科所. 加勒比松与湿地松杂交育种试验[J]. 广东林业科技,1994(4):23-27. Zhanjiang Forestry Institute. Cross breeding experiment of Pinus caribaea Morelet and Pinus elliottii[J]. Forestry Science and Technology of Guandong Province,1994(4):23-27.
3. 沈熙环,黄永权. 广东省湿加松良种选育和推广进展[J]. 林业科技通讯,2008(1):74-75. Shen X H,Huang Y Q. Progress in breeding and extension of improved Pinus elliottii×P. caribaea varieties in Guangdong Province[J]. Forest Science and Technology,2008(1):74-75.
4. 万述伟,宋凤景,郝俊杰,等. 271份豌豆种质资源农艺性状遗传多样性分析[J]. 植物遗传资源学报,2017,18(1):10-18. Wan S W,Song F J,Hao J J,et al. Genetic Diversity of agronomic traits in 271 Pea germplasm resources[J]. Journal of Plant Genetic Resources,2017,18(1):10-18.
5. Shen Z. A study on the phenotypic genetic diversity of camellia germplasm resources in Macheng city[J]. Asian Agricultural Research,2017,9(4):77-80.
6. 蒋会兵,宋维希,矣兵,等. 云南茶树种质资源的表型遗传多样性[J]. 作物学报,2013,39(11):2000-2008. Jiang H B,Song W X,Yi B,et al. Genetic Diversity of tea germplasm resources in Yunnan province based on phenotypic characteristics[J]. Acta Agronomica Sinica,2013,39(11):2000-2008.
7. 王海岗,贾冠清,智慧,等. 谷子核心种质表型遗传多样性分析及综合评价[J]. 作物学报,2016,42(1):19-30. Wang H G,Jia G Q,Zhi H,et al. Phenotypic diversity evaluations of foxtail millet core collections[J]. Acta Agronomica Sinica,2016,42(1):19-30.
8. 代攀虹,孙君灵,何守朴,等. 陆地棉核心种质表型性状遗传多样性分析及综合评价[J]. 中国农业科学,2016,49(19):3694-3708. Dai P H,Sun J L,He S P,et al. Comprehensive evaluation and genetic diversity analysis of phenotypic traits of core collection in upland cotton[J]. Scientia Agricultura Sinica,2016,49(19):3694-3708.
9. Kisua J,Mwikamba K,Makobe M,et al. Genetic diversity of sweet and grain sorghum populations using phenotypic markers[J]. International Journal of Biosciences,2015,6(9):34-46.
10. 王黎明,焦少杰,姜艳喜,等. 不同来源甜高粱种质资源的表型遗传多样性分析[J]. 植物遗传资源学报,2014,15(2):411-416. Wang L M,Jiao S J,Jiang Y X,et al. Genetic diversity analysis on sweet sorghum germplasm resources of different origins based on agronomical traits[J]. Journal of Plant Genetic Resources,2014,15(2):411-416.
11. 徐斌,彭莉霞,杨会肖,等. 杜鹃红山茶叶片主要性状的遗传多样性分析[J]. 植物研究,2015,35(5):730-734. Xu B,Peng L X,Yang H X,et al. Genetic diversity analysis for leaf main traits of Camellia azalea[J]. Bulletin of Botanical Research,2015,35(5):730-734.
12. 李义良,赵奋成,林昌明,等. 湿地松、加勒比松人工林优树选择研究[J]. 广东林业科技,2015,31(6):29-34. Li Y L,Zhao F C,Lin C M,et al. Selection of superior trees of Pinus elliottii and P. caribaea in Plantation[J]. Guangdong Forestry Science and Technology,2015,31(6):29-34.
13. 林元震. R与ASReml-R统计学[M]. 北京:中国林业出版社,2017. Lin Y Z. R and ASReml-R statistics[M]. Beijing:China Forestry Publishing House,2017.
14. 卢纹岱. SPSS统计分析:4版[M]. 北京:电子工业出版社,2010. Lu W D. SPSS statistical analysis:4th ed[M]. Beijing:Publishing House of Electronics Industry,2010.
15. 张岩,王萍,赵清岩,等. 籽用南瓜种质资源农艺性状遗传多样性及亲缘关系研究[J]. 内蒙古农业大学学报,2010,31(4):34-39. Zhang Y,Wang P,Zhao Q Y,et al. Stuey of agronomic traits diversity and genetic relationship on germplasm resources of seed-used pumpkin[J]. Journal of Inner Mongolia Agricultural University,2010,31(4):34-39.
16. Campbell D R. Using phenotypic manipulations to study multivariate selection of floral trait associations[J]. Annals of Botany,2009,103(9):1557-1566.
17. 李斌,顾万春. 松属植物遗传多样性研究进展[J]. 遗传,2003,25(6):740-748. Li B,Gu W C. Review on genetic diversity in Pinus[J]. Heredity,2003,25(6):740-748.
18.1. 刘宇明,韩国辉,王晓冬,等. 山新杨嫩枝扦插繁殖技术的研究[J]. 吉林林业科技,2005,34(4):4-5,44. Liu Y M,Han G H,Wang X D,et al. Raising young plants by softwood cutting for Populus davidiana×bolleana[J]. Jilin Forestry Science and Technology,2005,34(4):4-5,44.
2. 刘文萍,韩玉琴,南相日,等. 山新杨组织培养快繁技术研究[J]. 中国农学通报,2005,21(2):101-102,121. Liu W P,Han Y Q,Nan X R,et al. Study on rapid multiplication techniques by tissue culture in Shanxinyang(Populus davidiana×P. bollena)[J]. Chinese Agricultural Science Bulletin,2005,21(2):101-102,121.
3. Monfil V O,Casas-Flores S. Molecular mechanisms of biocontrol in Trichoderma spp. and their applications in agriculture[M]. //Gupta V K,Schmoll M,Herrera-Estrella A,et al. Biotechnology and biology of trichoderma. Amsterdam:Elsevier B. V.,2014:429-453.
4. Joeniarti E,Ni'Matuzahroh,Kusriningrum. Tolerance of Trichoderma asperellum isolates to chemical fungicide and their antagonistic activity against Phytophthora infestans[J]. International Journal of Plant & Soil Science,2014,3(1):36-46.
5. Yedidia I,Shoresh M,Kerem Z,et al. Concomitant induction of systemic resistance to Pseudomonas syringae pv. lachrymans in cucumber by Trichoderma asperellum(T-203) and accumulation of phytoalexins[J]. Applied and Environmental Microbiology,2003,69(12):7343-7353.
6. Korolev N,David D R,Elad Y. The role of phytohormones in basal resistance and Trichoderma-induced systemic resistance to Botrytis cinerea in Arabidopsis thaliana[J]. Biocontrol,2008,53(4):667-683.
7. 丁懿,石彩娟,王万军. 水稻PIN家族的生物信息学分析[J]. 安徽农业科学,2012,40(27):13238-13242. Ding Y,Shi C J,Wang W J. Bioinformatics analysis of PIN-formed family in Oryza sativa[J]. Journal of Anhui Agricultural Sciences,2012,40(27):13238-13242.
8. 邹纯雪,门淑珍. 生长素的外输载体PIN蛋白家族研究进展[J]. 中国细胞生物学学报,2013,35(4):574-582. Zou C X,Men S Z. Research advances in auxin efflux carrier PIN proteins[J]. Chinese Journal of Cell Biology,2013,35(4):574-582.
9. Steinmann T,Geldner N,Grebe M,et al. Coordinated polar localization of auxin efflux carrier PIN1 by GNOM ARF GEF[J]. Science,1999,286(5438):316-318.
10. Forestan C,Farinati S,Varotto S. The maize PIN gene family of auxin transporters[J]. Frontiers in Plant Science,2012,3:16.
11. Pattison R J,Catalá C. Evaluating auxin distribution in tomato(Solanum lycopersicum) through an analysis of the PIN and AUX/LAX gene families[J]. The Plant Journal,2012,70(4):585-598.
12. Vanneste S,Friml J. Auxin:a trigger for change in plant development[J]. Cell,2009,136(6):1005-1016.
13. Chen R J,Hilson P,Sedbrook J,et al. The Arabidopsis thaliana AGRAVITROPIC1 gene encodes a component of the polar-auxin-transport efflux carrier[J]. Proceedings of the National Academy of Sciences of the United States of America,1998,95(25):15112-15117.
14. Ni W M,Chen X Y,Xu Z H,et al. Isolation and functional analysis of a Brassica juncea gene encoding a component of auxin efflux carrier[J]. Cell Research,2002,12(3-4):235-245.
15. Zhang R S,Wang Y C,Wang C,et al. Time-course analysis of levels of indole-3-acetic acid and expression of auxin-responsive GH3 genes in Betula platyphylla[J]. Plant Molecular Biology Reporter,2011,29(4):898-905.
16. Yao Z H,Baloch A M,Liu Z H,et al. Cloning and characterization of an AUX/IAA gene in Populus davidiana×P. alba var. Pyramidalis and the correlation between its time-course expression and the levels of indole-3-acetic in saplings inoculated with Trichoderma[J]. Pakistan Journal of Botany,2018,50(1):169-177.
17. Livak K J,Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCt method[J]. Methods,2001,25(4):402-408.
18. Dhonukshe P,Aniento F,Hwang I,et al. Clathrin-mediated constitutive endocytosis of PIN auxin efflux carriers in Arabidopsis[J]. Current Biology,2007,17(6):520-527.
19. Křeček P,Skůpa P,Libus J,et al. The PIN-FORMED(PIN) protein family of auxin transporters[J]. Genome Biology,2009,10(12):249-249.
20. 赵瑞瑞,申琳,生吉萍. 番茄PIN基因家族的生物信息学分析及其表达模式[J]. 食品科学,2017,38(4):1-5. Zhao R R,Shen L,Sheng J P. Bioinformatic analysis of the PIN gene family in tomato and its expression pattern[J]. Food Science,2017,38(4):1-5.
21. 周大喜. 生长素极性运输在水稻根发育中的作用及水稻PIN同源基因的克隆[D]. 上海:上海交通大学,2003. Zhou D X. Affection of auxin polar transport on rice root development and cloning of a PIN gene from rice[D]. Shanghai:Shanghai Jiaotong University,2003.
22. Carraro N,Tisdale-Orr T E,Clouse R M,et al. Diversification and expression of the PIN,AUX/LAX,and ABCB families of putative auxin transporters in Populus[J]. Frontiers in Plant Science,2012,3:17.
23. Nisar N,Cuttriss A J,Pogson B J,et al. The promoter of the Arabidopsis PIN6 auxin transporter enabled strong expression in the vasculature of roots,leaves,floral stems and reproductive organs[J]. Plant Signaling & Behavior,2014,9(1):e27898. 刘雨. 宁夏贺兰山杜松(Juniperus rigida)表型多样性与化感作用研究[D]. 杨凌:西北农林科技大学,2011. Liu Y. Phenotypic Diversity and allelopathic effects of Juniperus rigida in Helan mountain,Ningxia[D]. Yangling:Northwest A&F University,2011.
19. Kowalczyk J. Comparison of phenotypic and genetic selections in Scots pine(Pinus sylvestris L.) single tree plot half-sib progeny tests[J]. Dendrobiology,2005,53:45-56.
20. Hong Y P,Hipkins V D,Strauss S H. Chloroplast DNA diversity among trees,populations and species in the California closed-cone pines(Pinus radiata,Pinus muricata and Pinus attenuata)[J]. Genetics,1993,135(4):1187-1196.
21. KovačevićD,NikolićB,MladenovićDrinićS,et al. Genetic relationships among some Pinus,Picea and Abies species revealed by RAPD markers[J]. Genetika,2013,45(2):493-502.
22. 倪州献,白天道,蔡恒,等. 马尾松基因组SSR标记在松属其他树种中的通用性分析[J]. 分子植物育种,2015,13(12):2811-2817. Ni Z X,Bai T D,Cai H,et al. The transferability of Pinus massoniana SSR in other Pinus species[J]. Molecular Plant Breeding,2015,13(12):2811-2817.
23. 许玉兰,蔡年辉,陈诗,等. 云南松天然群体遗传变异与生态因子的相关性[J]. 生态学杂志,2016,35(7):1767-1775. Xu Y L,Cai N H,Chen S,et al. Relationships between the genetic diversity of Pinus yunnanensis Franch. Natural populations and ecological factors[J]. Chinese Journal of Ecology,2016,35(7):1767-1775.
24. 张悦,易雪梅,姬兰柱. 从松属相关物种筛选红松微卫星标记及其种群遗传多样性分析[J]. 生态学杂志,2013,32(9):2307-2313. Zhang Y,Yi X M,Ji L Z. Screening of Pinus koraiensis microsatellite makers from relative species of Pinus and analysis of population genetic diversity[J]. Chinese Journal of Ecology,2013,32(9):2307-2313.
25. 陈家媛,靖晶,高嵩,等. 草河口林场红松人工林遗传多样性的ISSR分析[J]. 植物研究,2009,29(5):633-636. Chen J Y,Jing J,Gao S,et al. Analysis of genetic diversity of Pinus koraiensis plantation in CaoHekou forest farm by ISSR marker[J]. Bulletin of Botanical Research,2009,29(5):633-636.
26. 钟岁英,赵奋成,王炳新,等. 湿加松无性系生长性状的遗传变异及选择效果研究初报[J]. 广东林业科技,2011,27(3):14-19. Zhong S Y,Zhao F C,Wang B X,et al. A primary study on the genetic variation and selection gain for growth traits of clones of Pinus elliottii var. elliottii×P. caribaea var. hondurensis[J]. Guangdong Forestry Science and Technology,2011,27(3):14-19.
27. 梁德洋,金允哲,赵光浩,等. 50个红松无性系生长与木材性状变异研究[J]. 北京林业大学学报,2016,38(6):51-59. Liang D Y,Jin Y Z,Zhao G H,et al. Variance analyses of growth and wood characteristics of 50 Pinus koraiensis clones[J]. Journal of Beijing Forestry University,2016,38(6):51-59.
28. 李义良,赵奋成,李宪政,等. 湿地松、加勒比松种质资源遗传多样性分析[J]. 广东林业科技,2014,30(6):9-14. Li Y L,Zhao F C,Li X Z,et al. Analysis of genetic diversity of Pinus elliottii and P. caribaea germplasm resources[J]. Guangdong Forestry Science and Technology,2014,30(6):9-14.
29. 王丽侠,程须珍,王素华,等. 中国绿豆核心种质资源在不同环境下的表型变异及生态适应性评价[J]. 作物学报,2014,40(4):739-744. Wang L X,Cheng X Z,Wang S H,et al. Adaptability and Phenotypic Variation of Agronomic Traits in mungbean core collection under different environments in China[J]. Acta Agronomica Sinica,2014,40(4):739-744.
30. 李义良,赵奋成,吴惠姗,等. 湿加松亲本间遗传距离与杂种优势的相关性分析[J]. 林业科学研究,2012,25(2):138-143. Li Y L,Zhao F C,Wu H S,et al. Relationship between growth traits heterosis and genetic distance among parents of Pinus elliottii×P. caribaea based on SSR molecular markers[J]. Forest Research,2012,25(2):138-143.

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