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

doi:10.12302/j.issn.1000-2006.2021006013

JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2022, Vol. 46 ›› Issue (3): 134-142.doi: 10.12302/j.issn.1000-2006.2021006013

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 E-mail:2954272790@qq.com;ychen@njfu.edu.cn;njlydxwhr@163.com

Abstract

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

CLC Number:

Q786
S763

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, 2022, 46(3): 134-142.

Figures/Tables 8

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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

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

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