矮珍珠硝酸盐转运蛋白基因GeNRT2.1的克隆和功能研究

doi:10.19586/j.2095-2341.2021.0033

生物技术进展 ›› 2022, Vol. 12 ›› Issue (2): 256-264.DOI: 10.19586/j.2095-2341.2021.0033

矮珍珠硝酸盐转运蛋白基因GeNRT2.1的克隆和功能研究

黄赳(), 师双峰(), 张二特, 李梅, 于跃, 刘昱辉()

中国农业科学院生物技术研究所，北京 100081

收稿日期:2021-03-10 接受日期:2021-11-08 出版日期:2022-03-25 发布日期:2022-03-25
通讯作者: 刘昱辉
作者简介:黄赳与师双峰为本文共同第一作者。黄赳 E-mail：1101768783@qq.com
师双峰 E-mail：shishuangfeng321@163.com。
基金资助:
国家转基因生物新品种培育重大专项(2016ZX08009003-005)

Cloning and Functional Studies of Nitrate Transporter Gene GeNRT2.1 in Glossostigma elatinoides

Jiu HUANG(), Shuangfeng SHI(), Erte ZHANG, Mei LI, Yue YU, Yuhui LIU()

Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2021-03-10 Accepted:2021-11-08 Online:2022-03-25 Published:2022-03-25
Contact: Yuhui LIU

摘要/Abstract

摘要：

硝酸盐转运蛋白（nitrate transporter，NRT）是植物识别、吸收和转运硝酸盐的关键蛋白，对促进作物根系发育、提高产量具有重要作用。通过筛选水生植物，利用NRT蛋白的保守区设计简并引物，并通过PCR和RACE技术，首次从矮珍珠（Glossostigma elatinoides）中克隆得到GeNRT2.1基因。进化分析结果表明，GeNRT2.1与烟草NRT2.1在进化关系上距离最近。qRT-PCR结果表明，GeNRT2.1在矮珍珠根中表达量最高，其次是叶和茎，此外，低浓度硝酸盐（0.5 mmol·L^-1）处理后，GeNRT2.1在根、叶、茎中的表达量分别是高浓度硝酸（2 mmol·L^-1）处理后的1.89、1.93和2.07倍。功能互补实验发现，GeNRT2.1能使缺陷型酵母Δynr恢复生长，具有硝酸盐转运蛋白的功能。通过丰富NRT基因资源，以期为培育氮肥高效利用转基因作物，发展绿色农业，保证我国的粮食安全和环境安全提供理论依据。

关键词: 矮珍珠, 硝酸盐转运蛋白, GeNRT2.1基因, 功能互补实验

Abstract:

Nitrate transporter （NRT） is a key protein for plants to recognize， absorb and transport nitrate， which plays an important role in promoting crop root development and crop yield improving. In this study， through the screening of aquatic plants， we used Glossostigma elatinoides as materials， designed degenerate primers based on the conserved regions of NRT proteins in different species， used PCR and RACE technology to clone GeNRT2.1 gene for the first time. Evolutionary analysis results showed that GeNRT2.1 was most closely related to Nicotiana tabacumNRT2.1 in evolutionary relationship. qRT-PCR results showed that the expression of GeNRT2.1 in roots was the highest， followed by leaves and stems. In addition， the expression of GeNRT2.1 in roots， leaves， and stems of low concentration nitrate （0.5 mmol·L^-1） were 1.89， 1.93 and 2.07 times than that of high concentration nitrate （2 mmol·L^-1）. Functional complementation experiments results showed that GeNRT2.1 could restore the growth of defective yeast Δynr， and has the function of nitrate transporter. By enriching NRT genetic resources， this study hopes to provide a theoretical basis for cultivating genetically modified crops with high-efficiency utilization of nitrogen fertilizer， developing green agriculture， and ensuring food security and environmental safety in our country.

Key words: Glossostigma elatinoides, nitrate transporter, GeNRT2.1 gene, functional complementarity experiment

中图分类号:

Q943.2

黄赳, 师双峰, 张二特, 李梅, 于跃, 刘昱辉. 矮珍珠硝酸盐转运蛋白基因GeNRT2.1的克隆和功能研究[J]. 生物技术进展, 2022, 12(2): 256-264.

Jiu HUANG, Shuangfeng SHI, Erte ZHANG, Mei LI, Yue YU, Yuhui LIU. Cloning and Functional Studies of Nitrate Transporter Gene GeNRT2.1 in Glossostigma elatinoides[J]. Current Biotechnology, 2022, 12(2): 256-264.

图/表 9

参考文献 24

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引物名称	引物序列（5'→3'）	引物用途
DeF1	5'⁃CARGAYYTNCCNGAYGGNAA⁃3'	GeNRT基因EST序列的扩增
DeR1	5'⁃CATNSWNCCCCAYTGNGG⁃3'
DeF2	5'⁃GGNGCNGAYCARTTYGAYG⁃3'’
DeR2	5'⁃CATNCCYTTNGGRCAYTC⁃3'
DeF3	5'⁃GGNGCNGAYCARTTYGAYG⁃3'
DeR3	5'⁃AVYTCYTCNACYTGNGTNAC⁃3'
H3outer	5'⁃TTATGGACACTATGGACCCTCCAAACCCTAG⁃3'	3'RACE的基因特异性引物
H3inner	5'⁃CCCTTAGCCATCACATTCATG⁃3'	3'RACE的基因特异性引物
H5 outer	5'⁃GTCTTTGGCGACATCACCCTTCTTCTG⁃3'	5'RACE的基因特异性引物
H5 inner	5'⁃CTTCTGAAGCGCACTCAGATTTCC⁃3'	5'RACE的基因特异性引物
AP1	5'⁃CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT⁃3'	5'RACE和3'RACE的通用引物
AP2	5'⁃CTAATACGACTCACTATAGGGC⁃3'	5'RACE和3'RACE的通用引物
qGeNRT5	5'⁃ACAGGTTCGATCTCCAATCCACAC⁃3'	qRT⁃PCR中GeNRT2.1基因的检测
qGeNRT3	5'⁃CGGTGAAATCAGAAGCGAAGCCTCC⁃3'	qRT⁃PCR中GeNRT2.1基因的检测
Actin5	5'⁃ATCCTGCGTCTTGATCTTGCTGGTC⁃3'	qRT⁃PCR中OsActin基因的检测
Actin3	5'⁃ACGCTCCGCTGAAGTAGTGAAAGAG⁃3'	qRT⁃PCR中OsActin基因的检测
GeNRT5	5'⁃GTCGACATGGCTGATATAGAAGGCT⁃3'	构建酵母穿梭载体时扩增GeNRT2.1基因
GeNRT3	5'⁃ACTAGTTCAAACTCGAGACGGTGTC⁃3'	构建酵母穿梭载体时扩增GeNRT2.1基因
GeNRT⁃F	5'⁃GAATTCATGGCTGATATAGAAGGCT⁃3'	构建亚细胞定位载体时扩增GeNRT2.1基因
GeNRT⁃R	5'⁃TCTAGAAACTCGAGACGGTG TCG⁃3'	构建亚细胞定位载体时扩增GeNRT2.1基因
yz⁃Ge2.1F	5'⁃GATACGGTTGCACATTCTTG⁃3'	转基因水稻的鉴定
yz⁃Ge2.1R	5'⁃AGATCGAACCTGTCATAGAAGTA⁃3'	转基因水稻的鉴定

引物名称	引物序列（5'→3'）	引物用途
DeF1	5'⁃CARGAYYTNCCNGAYGGNAA⁃3'	GeNRT基因EST序列的扩增
DeR1	5'⁃CATNSWNCCCCAYTGNGG⁃3'
DeF2	5'⁃GGNGCNGAYCARTTYGAYG⁃3'’
DeR2	5'⁃CATNCCYTTNGGRCAYTC⁃3'
DeF3	5'⁃GGNGCNGAYCARTTYGAYG⁃3'
DeR3	5'⁃AVYTCYTCNACYTGNGTNAC⁃3'
H3outer	5'⁃TTATGGACACTATGGACCCTCCAAACCCTAG⁃3'	3'RACE的基因特异性引物
H3inner	5'⁃CCCTTAGCCATCACATTCATG⁃3'	3'RACE的基因特异性引物
H5 outer	5'⁃GTCTTTGGCGACATCACCCTTCTTCTG⁃3'	5'RACE的基因特异性引物
H5 inner	5'⁃CTTCTGAAGCGCACTCAGATTTCC⁃3'	5'RACE的基因特异性引物
AP1	5'⁃CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT⁃3'	5'RACE和3'RACE的通用引物
AP2	5'⁃CTAATACGACTCACTATAGGGC⁃3'	5'RACE和3'RACE的通用引物
qGeNRT5	5'⁃ACAGGTTCGATCTCCAATCCACAC⁃3'	qRT⁃PCR中GeNRT2.1基因的检测
qGeNRT3	5'⁃CGGTGAAATCAGAAGCGAAGCCTCC⁃3'	qRT⁃PCR中GeNRT2.1基因的检测
Actin5	5'⁃ATCCTGCGTCTTGATCTTGCTGGTC⁃3'	qRT⁃PCR中OsActin基因的检测
Actin3	5'⁃ACGCTCCGCTGAAGTAGTGAAAGAG⁃3'	qRT⁃PCR中OsActin基因的检测
GeNRT5	5'⁃GTCGACATGGCTGATATAGAAGGCT⁃3'	构建酵母穿梭载体时扩增GeNRT2.1基因
GeNRT3	5'⁃ACTAGTTCAAACTCGAGACGGTGTC⁃3'	构建酵母穿梭载体时扩增GeNRT2.1基因
GeNRT⁃F	5'⁃GAATTCATGGCTGATATAGAAGGCT⁃3'	构建亚细胞定位载体时扩增GeNRT2.1基因
GeNRT⁃R	5'⁃TCTAGAAACTCGAGACGGTG TCG⁃3'	构建亚细胞定位载体时扩增GeNRT2.1基因
yz⁃Ge2.1F	5'⁃GATACGGTTGCACATTCTTG⁃3'	转基因水稻的鉴定
yz⁃Ge2.1R	5'⁃AGATCGAACCTGTCATAGAAGTA⁃3'	转基因水稻的鉴定

物种	第0天/g	第7天/g	第14天/g	第21天/g	第28天/g	28 d内鲜重增加率/%
小水榕	0.5	1.02±0.01	1.53±0.02	1.49±0.02	2.51±0.03	402.00
黑藻	0.5	0.81±0.01	0.73±0.01	1.01±0.01	1.09±0.01	376.00
金鱼藻	0.5	0.68±0.01	0.63±0.01	1.05±0.01	1.51±0.01	372.00
矮珍珠	0.5	1.22±0.01	1.61±0.01	1.76±0.01	2.31±0.01	362.00
香菇草	0.5	0.84±0.01	1.41±0.01	2.47±0.01	2.36±0.01	356.00
虎耳草	0.5	0.66±0.01	0.59±0.01	0.71±0.01	1.02±0.01	292.00
南美青竹	0.5	0.68±0.01	0.71±0.01	0.59±0.01	0.90±0.01	276.00
绿菊	0.5	0.96±0.01	1.70±0.01	2.29±0.01	2.38±0.01	270.00
百叶草	0.5	0.81±0.01	0.68±0.01	1.39±0.01	1.79±0.01	258.00
草茨藻	0.5	0.61±0.01	1.02±0.01	1.43±0.01	1.79±0.01	258.00
宫廷草	0.5	0.85±0.02	1.47±0.02	1.58±0.02	1.96±0.01	228.00
草甸排草	0.5	0.59±0.01	1.26±0.01	1.29±0.01	1.59±0.01	218.00
马来眼子菜	0.5	0.72±0.01	0.85±0.01	1.02±0.01	1.85±0.01	202.00
小对叶草	0.5	0.78±0.01	1.14±0.01	1.02±0.01	1.21±0.01	142.00
青苹果草	0.5	0.68±0.01	0.70±0.01	1.21±0.01	1.96±0.01	118.00
直立石龙尾	0.5	0.42±0.01	0.58±0.01	0.62±0.01	0.87±0.01	104.00
红菊	0.5	0.81±0.02	0.67±0.01	1.64±0.01	2.28±0.03	92.00
水蓑衣	0.5	0.74±0.01	0.99±0.01	1.58±0.01	1.64±0.01	80.00
水芹	0.5	0.52±0.01	0.80±0.01	1.76±0.01	1.88±0.02	74.00
五色苋	0.5	0.48±0.01	0.68±0.01	0.67±0.01	0.71±0.01	42.00

物种	第0天/g	第7天/g	第14天/g	第21天/g	第28天/g	28 d内鲜重增加率/%
小水榕	0.5	1.02±0.01	1.53±0.02	1.49±0.02	2.51±0.03	402.00
黑藻	0.5	0.81±0.01	0.73±0.01	1.01±0.01	1.09±0.01	376.00
金鱼藻	0.5	0.68±0.01	0.63±0.01	1.05±0.01	1.51±0.01	372.00
矮珍珠	0.5	1.22±0.01	1.61±0.01	1.76±0.01	2.31±0.01	362.00
香菇草	0.5	0.84±0.01	1.41±0.01	2.47±0.01	2.36±0.01	356.00
虎耳草	0.5	0.66±0.01	0.59±0.01	0.71±0.01	1.02±0.01	292.00
南美青竹	0.5	0.68±0.01	0.71±0.01	0.59±0.01	0.90±0.01	276.00
绿菊	0.5	0.96±0.01	1.70±0.01	2.29±0.01	2.38±0.01	270.00
百叶草	0.5	0.81±0.01	0.68±0.01	1.39±0.01	1.79±0.01	258.00
草茨藻	0.5	0.61±0.01	1.02±0.01	1.43±0.01	1.79±0.01	258.00
宫廷草	0.5	0.85±0.02	1.47±0.02	1.58±0.02	1.96±0.01	228.00
草甸排草	0.5	0.59±0.01	1.26±0.01	1.29±0.01	1.59±0.01	218.00
马来眼子菜	0.5	0.72±0.01	0.85±0.01	1.02±0.01	1.85±0.01	202.00
小对叶草	0.5	0.78±0.01	1.14±0.01	1.02±0.01	1.21±0.01	142.00
青苹果草	0.5	0.68±0.01	0.70±0.01	1.21±0.01	1.96±0.01	118.00
直立石龙尾	0.5	0.42±0.01	0.58±0.01	0.62±0.01	0.87±0.01	104.00
红菊	0.5	0.81±0.02	0.67±0.01	1.64±0.01	2.28±0.03	92.00
水蓑衣	0.5	0.74±0.01	0.99±0.01	1.58±0.01	1.64±0.01	80.00
水芹	0.5	0.52±0.01	0.80±0.01	1.76±0.01	1.88±0.02	74.00
五色苋	0.5	0.48±0.01	0.68±0.01	0.67±0.01	0.71±0.01	42.00

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矮珍珠硝酸盐转运蛋白基因GeNRT2.1的克隆和功能研究

Cloning and Functional Studies of Nitrate Transporter Gene GeNRT2.1 in Glossostigma elatinoides

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