假俭草EoNLA基因克隆与其转基因拟南芥在不同磷水平下的表型鉴定

doi:10.12302/j.issn.1000-2006.2021006013

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南京林业大学学报（自然科学版） ›› 2022, Vol. 46 ›› Issue (3): 134-142.doi: 10.12302/j.issn.1000-2006.2021006013

假俭草EoNLA基因克隆与其转基因拟南芥在不同磷水平下的表型鉴定

何青青¹(), 刘传强¹, 李建建², 王晶晶², 姚祥², 周圣浩³, 陈英¹^,^*(), 王浩然²^,^*()

1.南京林业大学杨树种质创新与品种改良重点实验室,南方现代林业协同创新中心,江苏南京 210037
2.江苏省中国科学院植物研究所,江苏南京 210014
3.江苏省泰兴市农村能源技术开发服务站,江苏泰兴 225400

收稿日期:2021-06-08 接受日期:2021-10-08 出版日期:2022-05-30 发布日期:2022-06-10
通讯作者: 陈英,王浩然
基金资助:
国家自然科学基金青年项目(31902046);国家自然科学基金青年项目(31902060);国家自然科学基金面上项目(32072608);江苏省自然科学基金青年项目(BK20180315)

Cloning of EoNLA gene in Eremochloa ophiuroides and the transgenic Arabidopsis phenotypic characterization under various phosphorus levels

HE Qingqing¹(), LIU Chuanqiang¹, LI Jianjian², WANG Jingjing², YAO Xiang², ZHOU Shenghao³, CHEN Ying¹^,^*(), WANG Haoran²^,^*()

1. Poplar Germplasm Enhancement & Variety Improvement Lab, Co-Innovation Center for Sustainable Forestry in Southern China, Nanjing Forestry University, Nanjing 210037, China
2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China
3. Taixing Rural Energy Technology Development Service Station of Jiangsu Province, Taixing 225400, China

Received:2021-06-08 Accepted:2021-10-08 Online:2022-05-30 Published:2022-06-10
Contact: CHEN Ying,WANG Haoran

摘要/Abstract

摘要：

【目的】磷元素是植物必需的大量营养元素,在植物的生长发育中必不可少。NLA(nitrogen limitation adaptation)蛋白是一种RING型E3泛素连接酶,参与磷转运蛋白的泛素化调控,在植物体的磷素平衡调节方面发挥重要作用。以假俭草这类天然适应低磷土壤条件的植物为研究材料,通过研究假俭草EoNLA的磷高效转运分子机制,为草坪草适应酸性土壤生境的磷高效转运分子育种和栽培调控提供理论依据。【方法】通过RACE方法克隆获得EoNLA基因,应用生物信息学分析确定基因的全长序列及编码氨基酸序列,采用原生质体瞬时表达体系确定EoNLA蛋白膜定位;通过qRT-PCR方法分析EoNLA基因低磷诱导下的表达模式,并通过农杆菌介导转化拟南芥进行基因功能鉴定。【结果】EoNLA基因序列全长1 353 bp,编码一个长度为331个氨基酸的蛋白。该蛋白具有NLA蛋白典型的RING结构域和SPX结构域,EoNLA蛋白定位于细胞膜,EoNLA基因在根组织的表达量显著高于在茎和叶的表达。【结论】EoNLA基因具有根组织表达特异性,且基于拟南芥转基因植株进行的功能鉴定,进一步显示EoNLA基因具有磷素调控相关的功能。

关键词: 假俭草, 磷素调控, EoNLA, 功能分析

Abstract:

【Obiectives】 Phosphorus is a macronutrient of plants which is essential for growth and development. Nitrogen limitation adaptation (NLA), a RING-type E3 ubiquitin ligase involved in the ubiquitin regulation of phosphorus transporters, plays an important role in the regulation of phosphorus balance in plants. 【Method】In this study, we used Eremochloa ophiuroides, which naturally adapts to low-phosphorus soil conditions, to clone the EoNLA gene using RACE. 【Result】The EoNLA gene is 1 353 bp long and encodes a protein of 331 amino acids. Amino acid sequence analysis showed that the NLA protein had a typical RING domain and an SPX domain. Cell membrane localization of the EoNLA protein was observed using a protoplast transient expression system. Agrobacterium-mediated transformation of Arabidopsis thaliana was carried out to verify gene function 【Conclusion】The qRT-PCR analysis showed that expression levels of EoNLA were significantly higher in roots than in stems and leaves, suggesting that EoNLA expression is root-specific. Furthermore, functional identification based on Arabidopsis thaliana transgenic plants suggested that EoNLA is associated with phosphorus regulation in plants.

Key words: Eremochloa ophiuroides, phosphorus regulation, EoNLA, functional analysis

中图分类号:

Q786
S763

何青青,刘传强,李建建,等. 假俭草EoNLA基因克隆与其转基因拟南芥在不同磷水平下的表型鉴定[J]. 南京林业大学学报（自然科学版）, 2022, 46(3): 134-142.

HE Qingqing, LIU Chuanqiang, LI Jianjian, WANG Jingjing, YAO Xiang, ZHOU Shenghao, CHEN Ying, WANG Haoran. Cloning of EoNLA gene in Eremochloa ophiuroides and the transgenic Arabidopsis phenotypic characterization under various phosphorus levels[J].Journal of Nanjing Forestry University (Natural Science Edition), 2022, 46(3): 134-142.DOI: 10.12302/j.issn.1000-2006.2021006013.

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参考文献 29

[1]	郑玉红, 刘建秀. 假俭草(Eremochl ophiuroides(Munro.) Hack.)种质资源改良研究进展[J]. 植物学通报, 2004, 21(5):587-594.
	ZHENG Y H, LIU J X. Study progress in germplasm resources of Eremochloa ophiuroides (Munro.) Hack[J]. Chin Bull Bot, 2004, 21(5):587-594.DOI: 10.3969/j.issn.1674-3466.2004.05.010. doi: 10.3969/j.issn.1674-3466.2004.05.010
[2]	HANNA W W. Centipedegrass: diversity and vulnerability[J]. Crop Sci, 1995, 35(2):332-334.DOI: 10.2135/cropsci1995.0011183X003500020007x. doi: 10.2135/cropsci1995.0011183X003500020007x
[3]	宗俊勤, 牛佳伟, 刘建秀, 等. 假俭草花序发育的形态学观察及其与物候期和积温的对应关系[J]. 植物资源与环境学报, 2021, 30(5):50-57.
	ZONG J Q, NIU J W, LIU J X, et al. Morphological observation on inflorescence development of Eremochloa ophiuroides and its corresponding relationships with phenophase and accumulated temperature[J]. J Plant Resour Environ, 2021, 30(5):50-57. DOI: 10.3969 /j.issn.1674-7895.2021.05.06. doi: 10.3969 /j.issn.1674-7895.2021.05.06
[4]	宣继萍, 郭海林, 刘建秀, 等. 中国假俭草种质资源抗寒性初步鉴定[J]. 草业学报, 2003, 12(6):110-114.
	XUAN J P, GUO H L, LIU J X, et al. Initial identification of cold tolerance in the Eremochloa ophiuroides germ plasm resource[J]. Acta Prataculturae Sin, 2003, 12(6):110-114.DOI: 10.3321/j.issn:1004-5759.2003.06.019. doi: 10.3321/j.issn:1004-5759.2003.06.019
[5]	SUN L L, TIAN J, ZHANG H Y, et al. Phytohormone regulation of root growth triggered by P deficiency or Al toxicity[J]. J Exp Bot, 2016, 67(12):3655-3664.DOI: 10.1093/jxb/erw188. doi: 10.1093/jxb/erw188
[6]	RAGHOTHAMA K G. Phosphate acquisition[J]. Annu Rev Plant Physiol Plant Mol Biol, 1999, 50:665-693.DOI: 10.1146/annurev.arplant.50.1.665. doi: 10.1146/annurev.arplant.50.1.665
[7]	WU P, SHOU H X, XU G H, et al. Improvement of phosphorus efficiency in rice on the basis of understanding phosphate signaling and homeostasis[J]. Curr Opin Plant Biol, 2013, 16(2):205-212.DOI: 10.1016/j.pbi.2013.03.002. doi: 10.1016/j.pbi.2013.03.002
[8]	陈隆升, 陈永忠, 杨小胡, 等. 低磷胁迫对不同油茶无性系幼苗生长及养分利用效率的影响[J]. 南京林业大学学报(自然科学版), 2014, 38(3):45-49.
	CHEN L S, CHEN Y Z, YANG X H, et al. Effects of low phosphorus stress on the growth and nutrient utilization efficiency of different Camellia oleifera clones[J]. J Nanjing For Univ (Nat Sci Ed), 2014, 38(3):45-49.DOI: 10.3969/j.issn.1000-2006.2014.03.009. doi: 10.3969/j.issn.1000-2006.2014.03.009
[9]	GU M, CHEN A Q, SUN S B, et al. Complex regulation of plant phosphate transporters and the gap between molecular mechanisms and practical application:what is missing?[J]. Mol Plant, 2016, 9(3):396-416.DOI: 10.1016/j.molp.2015.12.012. doi: 10.1016/j.molp.2015.12.012
[10]	LÓPEZ-ARREDONDO D L, LEYVA-GONZÁLEZ M A, GONZÁLEZ-MORALES S I, et al. Phosphate nutrition:improving low-phosphate tolerance in crops[J]. Annu Rev Plant Biol, 2014, 65:95-123.DOI: 10.1146/annurev-arplant-050213-035949. doi: 10.1146/annurev-arplant-050213-035949
[11]	ZHENG N, SCHULMAN B A, SONG L Z, et al. Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex[J]. Nature, 2002, 416(6882):703-709.DOI: 10.1038/416703a. doi: 10.1038/416703a
[12]	刘卫霞, 彭小忠, 袁建刚, 等.SCF(Skp1-Cul1-F-box蛋白)复合物及其在细胞周期中的作用[J]. 中国生物工程杂志, 2002, 22(3):1-3.
	LIU W X, PENG X Z, YUAN J G, et al.SCF (Skp1-Cul1-F-box protein) complex and its function in cell cycle[J]. Prog Biotechnol, 2002, 22(3):1-3.DOI: 10.13523/j.cb.20020301. doi: 10.13523/j.cb.20020301
[13]	HANNAM C, GIDDA S K, HUMBERT S, et al. Distinct domains within the nitrogen limitation adaptation protein mediate its subcellular localization and function in the nitrate-dependent phosphate homeostasis pathway[J]. Botany, 2018, 96(2):79-96.DOI: 10.1139/cjb-2017-0149. doi: 10.1139/cjb-2017-0149
[14]	AUESUKAREE C, HOMMA T, KANEKO Y, et al. Transcriptional regulation of phosphate-responsive genes in low-affinity phosphate-transporter-defective mutants in Saccharomyces cerevisiae[J]. Biochem Biophys Res Commun, 2003, 306(4):843-850.DOI: 10.1016/S0006-291X(03)01068-4. doi: 10.1016/S0006-291X(03)01068-4
[15]	GIOTS F, DONATON M C V, THEVELEIN J M. Inorganic phosphate is sensed by specific phosphate carriers and acts in concert with glucose as a nutrient signal for activation of the protein kinase: a pathway in the yeast Saccharomyces cerevisiae[J]. Mol Microbiol, 2003, 47(4):1163-1181.DOI: 10.1046/j.1365-2958.2003.03365.x. doi: 10.1046/j.1365-2958.2003.03365.x.
[16]	HÜRLIMANN H C, STADLER-WAIBEL M, WERNER T P, et al. Pho91 is a vacuolar phosphate transporter that regulates phosphate and polyphosphate metabolism in Saccharomyces cerevisiae[J]. Mol Biol Cell, 2007, 18(11):4438-4445.DOI: 10.1091/mbc.e07-05-0457. doi: 10.1091/mbc.e07-05-0457
[17]	STONE S L, HAUKSDÓTTIR H, TROY A, et al. Functional analysis of the RING-type ubiquitin ligase family of Arabidopsis[J]. Plant Physiol, 2005, 137(1):13-30.DOI: 10.1104/pp.104.052423. doi: 10.1104/pp.104.052423
[18]	SECCO D, WANG C, ARPAT B A, et al. The emerging importance of the SPX domain-containing proteins in phosphate homeostasis[J]. New Phytol, 2012, 193(4):842-851.DOI: 10.1111/j.1469-8137.2011.04002.x. doi: 10.1111/j.1469-8137.2011.04002.x.
[19]	ZHOU Z P, WANG Z Y, LV Q D, et al. SPX proteins regulate Pi homeostasis and signaling in different subcellular level[J]. Plant Signal Behav, 2015, 10(9):e1061163.DOI: 10.1080/15592324.2015.1061163. doi: 10.1080/15592324.2015.1061163
[20]	QI W J, BALDWIN S A, MUENCH S P, et al. Pi sensing and signalling:from prokaryotic to eukaryotic cells[J]. Biochem Soc Trans, 2016, 44(3):766-773.DOI: 10.1042/BST20160026. doi: 10.1042/BST20160026
[21]	KANT S, PENG M S, ROTHSTEIN S J. Genetic regulation by NLA and microRNA827 for maintaining nitrate-dependent phosphate homeostasis in Arabidopsis[J]. PLoS Genet, 2011, 7(3):e1002021.DOI: 10.1371/journal.pgen.1002021. doi: 10.1371/journal.pgen.1002021
[22]	LIU W W, SUN Q, WANG K, et al. Nitrogen Limitation Adaptation (NLA) is involved in source-to-sink remobilization of nitrate by mediating the degradation of NRT1.7 in Arabidopsis[J]. New Phytol, 2017, 214(2):734-744.DOI: 10.1111/nph.14396. doi: 10.1111/nph.14396
[23]	YAN J, CHEN J B, ZHANG T T, et al. Evaluation of aluminum tolerance and nutrient uptake of 50 centipedegrass accessions and cultivars[J]. Hort Science, 2009, 44(3):857-861.DOI: 10.21273/hortsci.44.3.857. doi: 10.21273/hortsci.44.3.857
[24]	SCHMITTGEN T D, LIVAK K J. Analyzing real-time PCR data by the comparative C(T) method[J]. Nat Protoc, 2008, 3(6):1101-1108.DOI: 10.1038/nprot.2008.73. doi: 10.1038/nprot.2008.73
[25]	沈仁芳. 铝在土壤-植物中的行为及植物的适应机制[M]. 北京: 科学出版社, 2008.
[26]	VIGÂ A C, DEVÂ G. Phosphorus adsorption characteristics of some acid and alkaline soils[J]. J Indian Soc Soil Sci, 1984, 32(2):235-239.
[27]	LIN W Y, HUANG T K, CHIOU T J. Nitrogen limitation adaptation,a target of microRNA827,mediates degradation of plasma membrane-localized phosphate transporters to maintain phosphate homeostasis in Arabidopsis[J]. Plant Cell, 2013, 25(10):4061-4074.DOI: 10.1105/tpc.113.116012. doi: 10.1105/tpc.113.116012
[28]	PARK B S, SEO J S, CHUA N H. Nitrogen limitation adaptation recruits phosphate2 to target the phosphate transporter PT2 for degradation during the regulation of Arabidopsis phosphate homeostasis[J]. Plant Cell, 2014, 26(1):454-464.DOI: 10.1105/tpc.113.120311. doi: 10.1105/tpc.113.120311
[29]	YANG S Y, LU W C, KO S S, et al. Upstream open reading frame and phosphate-regulated expression of rice OsNLA1 controls phosphate transport and reproduction[J]. Plant Physiol, 2020, 182(1):393-407.DOI: 10.1104/pp.19.01101. doi: 10.1104/pp.19.01101

功能 prime function	引物 primer	上游序列(5'-3') forward sequence	下游序列(5'-3') reverse sequence
EoNLA 基因克隆 EoNLA cloning	3'RACE Outer	GACGACCGCCGCTGCACGACTGGC	Takara试剂盒配套引物
	3'RACE Inner	GAGCTTCTTCACGTCTC- TTCTGAACGAGATGTC	Takara试剂盒配套引物
	5'RACE Outer	Takara试剂盒配套引物	TGCCATCAGCTCACAGAGCCA
	5'RACE Inner	Takara试剂盒配套引物	TGTGGAGGCTCTGAG- CTTGAGCTTTGAATTC
	ORF	ATGAAGTTCGGCAA- GAAGTACGAGGC	TATGCCTAAAAATGCT- CTGCACTGTGA
qRT-PCR 检测 analysis	EoNLA	CAAGAAGGGTGACAAGAGCCA	TATGAGGGAACAGTCG- CCAAA
qRT-PCR 检测 analysis	Actin	GCACGGAATCGTC- AGCAA	CCCTCGTAGATGGGGACAGT

假俭草EoNLA基因克隆与其转基因拟南芥在不同磷水平下的表型鉴定

Cloning of EoNLA gene in Eremochloa ophiuroides and the transgenic Arabidopsis phenotypic characterization under various phosphorus levels

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