玉米中ClassⅠ和ClassⅡ NAS蛋白之间的BiFC互作研究

doi:10.19586/j.2095-2341.2022.0047

生物技术进展 ›› 2022, Vol. 12 ›› Issue (5): 728-736.DOI: 10.19586/j.2095-2341.2022.0047

玉米中ClassⅠ和ClassⅡ NAS蛋白之间的BiFC互作研究

肖克¹(), 周晓今², 陈茹梅², 逄森¹()

^1.中国农业大学理学院应用化学系，北京 100193
^2.中国农业科学院生物技术研究所，北京 100081

收稿日期:2022-03-29 接受日期:2022-05-05 出版日期:2022-09-25 发布日期:2022-09-30
通讯作者: 逄森
作者简介:肖克 E-mail：S20193101820@cau.edu.cn；
基金资助:
国家转基因生物新品种培育科技重大专项(2016ZX08003-002)

Interactions Between ClassⅠand ClassⅡ NAS Proteins in Maize Using Bimolecular Fluorescence Complementation (BiFC) Assay

Ke XIAO¹(), Xiaojin ZHOU², Rumei CHEN², Sen PANG¹()

^1.Department of Applied Chemistry，College of Science，China Agricultural University，Beijing 100193，China
^2.Biotechnology Research Institute，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2022-03-29 Accepted:2022-05-05 Online:2022-09-25 Published:2022-09-30
Contact: Sen PANG

摘要/Abstract

摘要：

烟草胺合成酶（nicotianamine synthase，NAS）能够催化合成植物体内铁运输所需的螯合物烟草胺（nicotianamine，NA），在植物维持铁稳态方面发挥重要的作用。玉米、小麦和大麦等禾本科植物的NAS蛋白进化为ClassⅠ和ClassⅡ两个亚家族，可能分别参与调节铁的吸收和运输，其家族成员之间蛋白序列同源性较高，ClassⅡ NAS具有特异的N端可变结构域。通过进化分析分析玉米NAS的两个亚家族，以及建模预测两类亚家族代表基因ZmNAS1（ClassⅠ）和ZmNAS3（ClassⅡ）的蛋白结构，结果表明ZmNAS1和ZmNAS3的三维结构高度相似，推测可能通过同源或异源二聚体化发挥功能；进一步通过双分子荧光互补（bimolecular fluorescence complementation，BiFC）分析ZmNAS1和ZmNAS3的相互作用，结果表明，ZmNAS1和ZmNAS3蛋白可以互作，删除N端可变结构域的ZmNAS3?N只能与ZmNAS3蛋白互作而不与ZmNAS1互作，推测ZmNAS可以形成同源二聚体，而形成异源二聚体需要ClassⅡ家族蛋白的N端可变结构域。研究结果揭示了玉米中ClassⅡ NAS调控铁稳态的机制，其分子机制仍需进一步研究。

关键词: 玉米, 烟草胺合成酶, N端可变结构域, 双分子荧光互作

Abstract:

Nicotianamine synthase （NAS）catalyzes the synthesis of nicotianamine （NA）， a chelate required for iron transport in plants， which plays an important role in remaining iron homeostasis in plants. The NAS proteins of graminaceous plants，such as maize，wheat and barley，evolved into two subfamilies，Class Ⅰ and Class Ⅱ， which may participate in iron absorption and transport respectively. There is high homology between family members， Class Ⅱ NAS have specific N-terminal variable domains. This study analyzed evolution of two subfamilies of NAS， and predicted three-dimensional structures of the representative proteins（ZmNAS1 and ZmNAS3） in Class Ⅰ and Class Ⅱ through modeling. The result suggested that， three-dimensional structures of ZmNAS1 and ZmNAS3 were highly similar， speculating that the function of NAS proteins might through homologous or heterologous dimerization. Furthermore， the interaction between ZmNAS1 and ZmNAS3 was also studied using bimolecular fluorescence complementation （BiFC） through transient expression in mesophyll protoplasts. BiFC results showed that ZmNAS1 interacted with ZmNAS3， while ZmNAS3ΔN （lack of the N-terminal variable domain） only interacted with ZmNAS3 but not with ZmNAS1. In summary，ZmNAS can form homologous dimers， while the formation of heterologous dimers requires the N-terminal variable domain of Class Ⅱ family proteins. Therefore， the results revealed the mechanism of Class Ⅱ NAS regulating homeostasis in maize and the molecular mechanism remains to be further studied.

Key words: maize, NAS, N-terminal variable domain, BiFC

中图分类号:

S513

肖克, 周晓今, 陈茹梅, 逄森. 玉米中ClassⅠ和ClassⅡ NAS蛋白之间的BiFC互作研究[J]. 生物技术进展, 2022, 12(5): 728-736.

Ke XIAO, Xiaojin ZHOU, Rumei CHEN, Sen PANG. Interactions Between ClassⅠand ClassⅡ NAS Proteins in Maize Using Bimolecular Fluorescence Complementation (BiFC) Assay[J]. Current Biotechnology, 2022, 12(5): 728-736.

图/表 7

表1 PCR扩增所用引物

Table 1 Primers used in PCR

基因名称	引物名称	引物序列（5'→3'）
ZmNAS1	ZmNAS1-F	5'-ATGGAGGCCCAGAACGTGGA-3'
	ZmNAS1-R	5'-CCACGACAGAGCTGTCCATC-3'
	ZmNAS1-NF	5'-CATTTGGAGAGGACCTCGAGATGGAGGCCCAGAACGTGGA-3'
	ZmNAS1-NR	5'-AACTTTTGCTCCATTCTAGAGATGGACAGCTCTGTCGTGG-3'
	ZmNAS1-CF	5'-CATTTGGAGAGGACCTCGAGATGGAGGCCCAGAACGTGGA-3'
	ZmNAS1-CR	5'-TCGTATGGGTACATTCTAGAGATGGACAGCTCTGTCGTGG-3'
ZmNAS3	ZmNAS3-F	5'-CAGCAAAGTGATCAGTGTGA-3'
	ZmNAS3-R	5'-CTAGTAGCCAGGTCAATCTG-3'
	ZmNAS3-NF	5'-CATTTGGAGAGGACCTCGAGATGGCCGTCATGGGCAAGGA-3'
	ZmNAS3-NR	5'-AACTTTTGCTCCATTCTAGAGGACGGCAGCTGCTCCATGG-3'
	ZmNAS3-CF	5'-CATTTGGAGAGGACCTCGAGATGGCCGTCATGGGCAAGGA-3'
	ZmNAS3-CR	5'-TCGTATGGGTACATTCTAGAGGACGGCAGCTGCTCCATGG-3'
	ZmNAS3∆N-F	5'-TCTTCACCGACCTCGTCACG-3'
	ZmNAS3∆N-R	5'-CCATGGAGCAGCTGCCGTCC-3'
	ZmNAS3∆N-NF	5'-CATTTGGAGAGGACCTCGAGATGCGCGCGGGCCTCATCCG-3'
	ZmNAS3∆N-NR	5'-AACTTTTGCTCCATTCTAGAGGACGGCAGCTGCTCCATGG-3'
	ZmNAS3∆N-CF	5'-CATTTGGAGAGGACCTCGAGATGCGCGCGGGCCTCATCCG-3'
	ZmNAS3∆N-CR	5'-TCGTATGGGTACATTCTAGAGGACGGCAGCTGCTCCATGG-3'

图1 载体构建示意图注：YFPN—黄色荧光蛋白的N端片段，YFPC—黄色荧光蛋白的C端片段，Ter—terminator终止信号。

Fig. 1 Diagram of vector construction

图2 玉米NAS家族成员的蛋白序列比对

Fig. 2 Alignment of protein sequences of ZmNAS family members

图3 ZmNAS蛋白结构分析A：ZmNAS1蛋白结构；B：ZmNAS3蛋白结构；C：ZmNAS3?N蛋白结构；D：ZmNAS1和ZmNAS3蛋白结构比对；E：ZmNAS1和ZmNAS3?N蛋白结构比对；F：ZmNAS3和ZmNAS3?N蛋白结构比对

Fig. 3 ZmNAS protein structure analysis

图4 ZmNAS1、ZmNAS3和ZmNAS3?N基因的克隆注：M1—DL2000 DNA ladder；1—ZmNAS1；2—ZmNAS3；3—ZmNAS3?N。

Fig. 4 Genes clone of ZmNAS1、ZmNAS3 and ZmNAS3?N

图5 载体酶切验证注：M1—1 kb DNA ladder；M2—100 bp DNA ladder；1—pZmNAS1-YFPN；2—pZmNAS1-YFPC；3—pZmNAS3-YFPN；4—pZmNAS3-YFPC；5—pZmNAS3?N-YFPN；6—pZmNAS3?N-YFPC。

Fig. 5 Enzyme digestion verification of vectors

图6 ZmNAS1、ZmNAS3和ZmNAS3?N的互作分析注：Chl—叶绿体自发荧光通道；Bright—明场通道；Merge—YFP、Chl和明场的叠加结果；标尺为10 μm在应用激光共聚焦显微镜拍照记录整理成图片时，为使细胞展示的大小一致，故选择了不同的放大倍数，图中标尺也会随之改变。

Fig. 6 Analysis to interactions between ZmNAS1, ZmNAS3 and ZmNAS3?N

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玉米中ClassⅠ和ClassⅡ NAS蛋白之间的BiFC互作研究

Interactions Between ClassⅠand ClassⅡ NAS Proteins in Maize Using Bimolecular Fluorescence Complementation (BiFC) Assay

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