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- 2018
盐芥ThPHT1;8基因的克隆和功能分析
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Abstract:
磷(P)是植物生长发育过程中重要的矿质营养元素之一, PHT1磷酸转运蛋白家族负责调控植物从土壤中吸收磷以及细胞间无机磷(Pi)的转运．本文以盐芥幼苗根cDNA为材料, 克隆到盐芥磷酸转运蛋白ThPHT1;8基因．生物信息学分析结果表明, ThPHT1;8蛋白编码525个氨基酸残基, 蛋白分子质量为58.1 ku, 等电点6.33, 是一个定位在细胞膜上的疏水、无信号肽的非分泌性蛋白, 含有高亲和力磷酸转运蛋白典型的跨膜结构．实时定量PCR结果显示, 低磷胁迫能促进ThPHT1;8基因在盐芥根部的表达, 而且不同磷浓度处理下ThPHT1;8基因表达的模式不同．ThPHT1;8基因在转基因拟南芥中的生物学功能研究表明, 不同浓度低磷胁迫处理下, 与野生型拟南芥相比, 35S：ThPHT1;8转基因拟南芥幼苗的主根根长显著增加、侧根密度显著降低, 叶绿素含量、无机磷和总磷含量提高, 花青素含量降低．上述结果表明, 盐芥ThPHT1;8基因确实参与低磷胁迫时植物的应答, 能够提高转基因拟南芥的耐低磷能力, 为土壤磷高效利用提供了一种有潜力的候选功能基因．
Phosphorus(P) is one of the most limiting mineral nutrients for plant growth and development. In plants，the uptake from soil and intercellular transport of inorganic phosphate(Pi) are mediated by the PHT1 family. In this work，we obtained the ThPHT1;8 gene through cloning from cDNA of Thellungiella salsuginea seedings roots. Bioinformatics analysis indicated that ThPHT1;8 protein had 525 amino acid residues. The molecular weight of the protein was 58.1 ku and the isoelectric point was 6.33. It was a hydrophobic and non-secreted protein with no signal peptide. ThPHT1;8 possessed the major characteristics of high-affinity phosphate transporters，such as transmembrane structure. Real-time quantitative PCR showed that the expression of ThPHT1;8 was strongly enhanced by low phosphorus stress in roots of Thellungiella salsuginea and the patterns of expression were different under different phosphorus stresses. The biological function of ThPHT1;8 gene indicated that 35S: ThPHT1;8 transgenic Arabidopsis had longer primary roots and less lateral root density than wild-type Arabidopsis under deficient phosphorus conditions. The contents of inorganic phosphorus，total phosphorus and chlorophyll increased in the 35S: ThPHT1;8 transgenic Arabidopsis，but the content of anthocyanin decreased. To conclude，ThPHT1;8 could enhance the tolerance of plants to low environmental phosphorus and improve transgenic Arabidopsis resistance to low phosphorus capacity. The study offers a functional candidate gene for efficient utilization of soil phosphorus
| [1] | Schachtman D P, Reid R J, Ayling S M. Phosphorus uptake by plants:From soil to cell[J]. <i>Plant Physiology</i>, 1998, 116(2):447-453. |
| [2] | Shen J, Yuan L, Zhang J, et al. Phosphorus dynamics:From soil to plant[J]. <i>Plant Physiology</i>, 2011, 156(3):997-1005. |
| [3] | M?odzi？？ska E, Zboi？？ska M. Phosphate uptake and allocation―A closer look at <i>Arabidopsis thaliana L</i>. and <i>Oryza sativa L.</i>[J]. <i>Frontiers in Plant Science</i>, 2016, 7:doi:10. 3389/fpls. 2016. 01198. |
| [4] | Kumar S, Nei M, Dudley J, et al. MEGA:A biologist-centric software for evolutionary analysis of DNA and protein sequences[J]. <i>Briefings in Bioinformatics</i>, 2008, 9(4):299-306. |
| [5] | Liu Yan, Meng Zhigang, Sun Guoqing, et al. Cloning and function analysis gene <i>GhPYR1</i> in <i>gossypium hirsutum L.</i>[J]. <i>Biotechnology Bulletin</i>, 2016, 32(2):90-99(in Chinese). |
| [6] | 张宇航, 李永光, 王雪松, 等. 大豆<i>GmGLP</i>10基因的克隆及生物信息学分析[J]. 大豆科学, 2016, 35(3):388-393. |
| [7] | 胡可, 韩科厅, 戴思兰. 环境因子调控花青素苷合成及呈色的机理[J]. 植物学报, 2010, 45(3):307-317. |
| [8] | Mudge S R, Rae A L, Diatloff E, et al. Expression analysis suggests novel roles for members of PHT1 family of phosphate transporters in <i>Arabidopsis</i>[J]. <i>Plant Journal</i>, 2002, 31(3):341-353. |
| [9] | Nussaume L, Kanno S, Javot H, et al. Phosphate import in plants:Focus on the PHT1 transporters[J]. <i>Frontiers in Plant Science</i>, 2011, 2(83):1-12. |
| [10] | Daram P, Brunner S, Persson B L, et al. Functional analysis and cell specific expression of a phosphate transporter from tomato[J]. <i>Planta</i>, 1998, 206(2):225-233. |
| [11] | Sun S, Gu M, Cao Y, et al. A constitutive expressed phosphate transporter, OsPht1；1, modulates phosphate uptake and translocation in Pi-replete rice[J]. <i>Plant Physiology</i>, 2012, 159(4):1571-1581. |
| [12] | Liu F, Chang X J, Ye Y, et al. Comprehensive sequence and whole-life-cycle expression profile analysis of the phosphate transporter gene family in rice[J]. <i>Mol Plant</i>, 2011, 4(6):1105-1122. |
| [13] | Remy E, Cabrito T R, Batista R A, et al. The PHT1；9 and PHT1；8 transporters mediate inorganic phosphate acquisition by the <i>Arabidopsis thaliana</i> root during phosphorus starvation[J]. <i>New Phytol</i>, 2012, 195(2):356-371. |
| [14] | Zhang Yuhang, Li Yongguang, Wang Xuesong, et al. Cloning and bioinformatics analysis of <i>GmGLP10<i> in soybean[J]. <i>Soybean Science</i>, 2016, 35(3):388-393(in Chinese). |
| [15] | 赵乐, 马利刚, 韩杜菀, 等. 独行菜<i>LaSPS</i>基因的克隆与生物信息学分析及原核表达[J]. 北方园艺, 2016(6):92-99. |
| [16] | Misson J, Raghothama K G, Jain A, et al. A genome-wide transcriptional analysis using <i>Arabidopsis thaliana<i> affymetrix gene chips determined plant responses to phosphate deprivation[J]. <i>Proc Natl Acad Sci USA</i>, 2005, 102(33):11934-11939. |
| [17] | Morcuende R, Bari R, Gibon Y, et al. Genome-wide reprogramming of metabolism and regulatory networks of <i>Arabidopsis</i> in response to phosphorus[J]. <i>Plant Cell Environ</i>, 2007, 30(1):85-112. |
| [18] | Nagarajan V K, Jain A, Poling M D, et al. <i>Arabidopsis<i> PHT1；5 mobilizes phosphate between source and sink organs, and influences the interaction between phosphate homeostasis and ethylene signaling[J]. <i>Plant Physiology</i>, 2011, 156(3):1149-1163.</i></i></i></i></i></i></i></i></i></i></i></i> |
| [19] | Cordell D, Drangert J O, White S. The story of phosphorus:Global food security and food for thought[J]. <i>Global Environ Chang</i>, 2009, 19(2):292-305. |
| [20] | Shin H, Shin H S, Dewbre G R, et al. Phosphate transport in <i>Arabidopsis</i>:PHT1；1 and PHT1；4 play a major role in phosphate acquisition from both low and high-phosphate environments[J]. <i>Plant Journal</i>, 2004, 39(4):629-642. |
| [21] | Jia H, Ren H, Gu M, et al. The phosphate transporter gene <i>OsPht1;8</i> is involved in phosphate homeostasis in rice[J]. <i>Plant Physiology</i>, 2011, 156(3):1164-1175. |
| [22] | Lapis-Gaza H R, Jost R, Finnegan P M<i>. Arabidopsis<i> PHOSPHATE TRANSPORTER1 genes <i>PHT1;8</i> and <i>PHT1;9</i> are involved in root-to-shoot translocation of orthophosphate[J]. <i>BMC Plant Biology</i>, 2014, 14:334. |
| [23] | Feng Ren, Zhao Caizhi, Liu Chunsen, et al. A <i>Brassica napus<i> PHT1 phosphate transporter, BnPht1；4, promotes phosphate uptake and affects roots architecture of transgenic <i>Arabidopsis</i>[J]. <i>Plant Mol Biol</i>, 2014, 86(6):595-607. |
| [24] | 高亚平. 盐芥磷高效利用的生理以及分子机制[D]. 济南:山东师范大学生命科学学院, 2008. |
| [25] | Gao Yaping. Physiological and Molecular Mechanism of the High Phosphorous Utility Efficiency in <i>Thellungiella salsuginea</i>[D]. Jinan:College of Life Science, Shandong Normal University, 2008(in Chinese). |
| [26] | 谢全喜. 盐芥在低磷胁迫下的基础生理研究[D]. 济南:山东师范大学生命科学学院, 2009. |
| [27] | Xie Quanxi. Research on Basic Physiology of <i>Thellungiella Ssalsuginea</i> Under Low-Phosphate Stress[D]. Jinan:College of Life Science, Shandong Normal University, 2009(in Chinese). |
| [28] | 王荣春. 盐芥钠磷转运体基因在转基因烟草中的功能鉴定[D]. 济南:山东师范大学生命科学学院, 2008. |
| [29] | Wang Rongchun. Functional Analysis of Na<sup>+</sup>/Pi Transporter Gene from <i>T. halophila</i> in Transgenic Tobacco [D]. Jinan:College of Life Science, Shandong Normal University, 2008(in Chinese). |
| [30] | 高强. 盐芥转运体基因的功能鉴定和FMO基因的克隆及功能分析[D]. 济南:山东师范大学生命科学学院, 2009. |
| [31] | Gao Qiang. Function Identity of a Na<sup>+</sup>/Pi Transporter Gene(ThNPT)and Cloing and Function Analysis of Flavin-Containing Monooxygenase(FMO)Genes from <i>T. halophila</i>[D]. Jinan:College of Life Science, Shandong Normal University, 2009(in Chinese). |
| [32] | Tamura K, Dudley J, Nei M, et al. Molecular evolutionary genetics analysis(MEGA) software version 4.0[J]. <i>Mol Biol Evol</i>, 2007, 24(8):1596-1599. |
| [33] | Finn R D, Tate J, Mistry J, et al. The Pfam protein families database[J]. <i>Nucleic Acids Research</i>, 2010, 38:211-222. |
| [34] | 李磊, 罗杰, 李红, 等. 毛果杨全基因组铵转运蛋白家族成员及其序列分析[J]. 西北农林科技大学学报:自然科学版, 2011, 39(2):133-142. |
| [35] | Li Lei, Luo Jie, Li Hong, et al. Genome-wide analysis of the ammonium transporter gene family in <i>Populus trichocarpa</i>[J]. <i>Journal of Northwest A&F University</i>:<i>Nat Sci Ed</i>, 2011, 39(2):133-142(in Chinese). |
| [36] | 刘妍, 孟志刚, 孙国清, 等. 陆地棉<i>GhPYR</i>1基因的克隆和功能分析[J]. 生物技术通报, 2016, 32(2):90-99. |
| [37] | Zhao Le, Ma Ligang, Han Duwan, et al. Clone and bioinformatic analysis and prokaryotic expression of <i>LaSPS<i> gene from <i>Lepidium apetalum</i>[J]. <i>Northern Horticulture</i>, 2016(6):92-99(in Chinese). |
| [38] | Carter G A, Knapp A K. Leaf optical properties in higher plants:Linking spectral characteristics to stress and chlorophyll concentration[J]. <i>American Journal of Botany</i>, 2001, 88(4):677-684. |
| [39] | Hu Ke, Han Keting, Dai Silan. Regulation of plant anthocyanin synthesis and pigmentation by environmental factors[J]. <i>Bulletin of Botany</i>, 2010, 45(3):307-317(in Chinese). |
| [40] | Raghothama K G, Karthikeyan A S. Phosphate acquisition[J]. <i>Plant Soil</i>, 2005, 274(1/2):37-49. |
