Crops ›› 2023, Vol. 39 ›› Issue (5): 71-80.doi: 10.16035/j.issn.1001-7283.2023.05.011

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Cloning and Analysis of the Structure and Function of the Key Enzyme Genes StGOGATs of Potato Nitrogen Assimilation

Han Zhijun1(), Zhao Yanfei1, Lu Yue1, Li Shuang2, Zhang Jiayue1, Han Yuzhu1(), Zhang Jingying1()   

  1. 1College of Horticulture, Jilin Agricultural University, Changchun 130118, Jilin, China
    2Teaching and Research Base Management Office, Jilin Agricultural University, Changchun 130118, Jilin, China
  • Received:2022-04-12 Revised:2023-07-17 Online:2023-10-15 Published:2023-10-16

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Abstract:

Two glutamate synthase genes (StFd-GOGAT, StNADH-GOGAT) were cloned and their structures and functions were analyzed using potato cultivar Chunshu 4. The results showed that the StFd- GOGAT and StNADH-GOGAT consisted of 1622 and 2210 amino acids, respectively, with protein molecular weights of 176.106 and 242.021kDa, respectively. Prediction showed that StGOGATs were stable and hydrophilic proteins. Analysis of their protein domains showed that StGOGATs proteins belonged to the gltB family in the first 1700 amino acid positions, which played a role in nitrogen metabolism and could catalyze the conversion of amide nitrogen from glutamine. It was hydrolyzed into glutamic acid and ammonia and transferred to suitable substrates. Gene mapping showed that StGOGATs were located in the first 850kb of potato chromosome 3. Promoter cis-acting element analysis of StGOGATs showed that they were related to plant photosynthesis and the synthesis of nitrogen compounds. It was found that they had a high homology with Solanum lycopersicum and Solanum pennellii by constructing a phylogenetic tree. The protein interaction prediction showed that there was a direct interaction between StFd-GOGAT and StNADH-GOGAT. The tertiary structure prediction showed that only StNADH-GOGAT had its own homodimer structure. Subcellular localization speculated that StFd-GOGAT and StNADH-GOGAT function in the cytoplasm and chloroplast, respectively. The properties, structure and function of StGOGATs were preliminarily clarified, laying a theoretical foundation for the subsequent exploration of their molecular regulation mechanism in the GS/GOGAT cycle.

Key words: GS/GOGAT cycle, StGOGATs, Protein structure, Protein interaction, Promoter

Table 1

Primer sequence"

引物名称Primer name 引物序列Primer sequence
StFd-GOGAT F ATGGCGGTGAATTCCGTGGC
StFd-GOGAT R TTATTTTAATGGCATCTCTGCAGACTG
StNADH-GOGAT F GTGGTGGTGTTGCCTATGTTCTTGA
StNADH-GOGAT R TTGTGTTACGCTGGTGTTGCTGTAT

Table 2

Online analysis software name and website"

工具Tool 网址/软件URL/Software 功能Function
ProtParam http://web.expasy.org/protparam/ 蛋白理化性质分析
ProtScale https://web.expasy.org/protscale/ 蛋白亲疏水性分析
TMHMM 2.0 http://www.cbs.dtu.dk/services/TMHMM/ 氨基酸跨膜结构预测
SignalP 5.0 http://www.cbs.dtu.dk/services/SignalP/ 信号肽预测
NetPhos 3.1 http://www.cbs.dtu.dk/services/NetPhos/ 磷酸化位点预测
InterProScan http://www.ebi.ac.uk/interpro/search/sequence/ 蛋白结构域预测
CD-Search https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi 保守结构域搜索
SOPMA https://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html 蛋白二级结构预测
SWISS-MODEL https://swissmodel.expasy.org/interactive 蛋白三级结构预测
PSORT Ⅱ https://psort.hgc.jp/form2.html 亚细胞定位预测
Cytoscape https://cytoscape.org/ 蛋白互作预测
MG2C http://mg2c.iask.in/mg2c_v2.1/ 染色体定位查看
Plant CARE http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ 启动子元件预测
MEGA 7.0 https://www.megasoftware.net/ 系统进化树构建

Fig.1

StFd-GOGAT cloning vector bacterial liquid PCR verification M: DL15000; 1: negative control; 2: positive control; 3-8: StFd- GOGAT bacterial solution"

Fig.2

PCR verification for StNADH-GOGAT cloning vector bacterial liquid M: DL15000; 1: positive control; 2: negative control; 3-5: StNADH- GOGAT bacterial solution"

Table 3

Analysis of physicochemical properties of StGOGATs protein"

名称
Name		 氨基酸
数量
Number of
amino acids		 分子量
Molecular
weight
(kDa)		 理论等电点
Theoretical
isoelectric
point		 原子数
Atomic
number		 不稳定
系数
Instability
factor		 脂肪
指数
Fat
index		 亲水性平均值
Average
hydro philicity
(GRAVY)		 负电荷残基
Negatively
charged residues
(Asp+Glu)		 正电荷残基
Positively
charged residues
(Arg+Lys)
StFd-GOGAT 1622 177.106 6.20 24891 39.78 89.86 -0.167 184 171
StNADH-GOGAT 2210 242.021 6.42 34005 37.34 84.05 -0.279 280 265

Fig.3

Analysis of StGOGATs amino acid composition"

Fig.4

Hydrophilic and hydrophobic analysis of StGOGATs protein"

Fig.5

Prediction of the amino acid transmembrane structure of StGOGATs"

Fig.6

StGOGATs protein signal peptide prediction"

Fig.7

Prediction of potential phosphorylation sites of StGOGATs amino acid residues"

Fig.8

StGOGATs protein domain prediction"

Fig.9

Prediction of the secondary structure of StGOGATs protein"

Fig.10

The proportion of the secondary structural elements of StGOGATs protein"

Fig.11

Prediction of the tertiary structure of StGOGATs protein"

Fig.12

Prediction of StNADH-GOGAT protein homodimer structure"

Fig.13

StGOGATs protein mutual production network"

Fig.14

StGOGATs chromosomal location map"

Table 4

Table of cis-acting elements of StGOGATs promoter"

元件
Element		 功能
Function		 数量Number
StFd-GOGAT StNADH-GOGAT
TGACG-motif 参与MeJA反应的顺式调节元件 1 8
CGTCA-motif 参与MeJA反应的顺式调节元件 1 8
TC-rich repeats 参与防御和压力反应的顺式作用元件 2 0
Box 4 参与光反应的保守DNA模块的一部分 1 0
G-Box 参与光反应的顺式调节元件 1 11
GCN4-motif 参与胚乳表达的顺式调节元件 0 3
ABRE 参与脱落酸反应的顺式作用元件 1 8
O2-site 参与玉米醇溶蛋白代谢调节的顺式调节元件 2 0
P-box 赤霉素反应元件 3 2
TGA-element 生长素反应元件 1 1
AuxRR-core 参与生长素反应的顺式调节元件 0 1
AuxRE 生长素反应元件的一部分 0 1
AE-box 光响应模块的一部分 0 3
GT1-motif 光响应元件 2 2
LAMP-element 光响应元件的一部分 1 0
I-box 光响应元件的一部分 2 0
TCT-motif 光响应元件的一部分 1 2
GATA-motif 光响应元件的一部分 0 2
TCCC-motif 光响应元件的一部分 0 1
GTGGC-motif 光响应元件的一部分 1 0
circadian 参与昼夜节律控制的顺式调节元件 1 0
CAT-box 与分生组织表达相关的顺式调节元件 2 4
MSA-like 参与细胞周期调控的顺式作用元件 0 1
ARE 厌氧诱导所必需的顺式调节元件 2 7
MBS 与干旱诱导有关的MYB结合位点 5 5
TCA-element 与水杨酸反应有关的顺式作用元件 0 4
CCAAT-box MYBHv1结合位点 1 1
MRE 参与光反应的MYB结合位点 1 0
AT-rich element 富含AT的DNA结合蛋白（ATBP-1）结合位点 1 1
HD-Zip 1 参与栅栏叶肉细胞分化的元素 1 0

Fig.15

Cis-acting elements of StGOGATs promoter"

Fig.16

StGOGATs phylogenetic tree"

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