马铃薯氮同化关键酶基因StGOGATs的克隆及结构与功能分析

doi:10.16035/j.issn.1001-7283.2023.05.011

摘要/Abstract

摘要：

以马铃薯品种春薯4号组培苗为材料，对谷氨酸合成酶基因StFd-GOGAT、StNADH-GOGAT进行克隆及结构与功能分析。结果表明，StFd-GOGAT、StNADH-GOGAT分别由1622及2210个氨基酸组成，分子量分别为176.106和242.021kDa，预测表明，StGOGATs蛋白均为稳定、亲水性蛋白。蛋白结构域分析发现，StGOGATs蛋白在前1700个氨基酸位置上共同属于gltB家族，在氮代谢中起作用，可以催化酰胺氮从谷氨酰胺水解成谷氨酸和氨并转移到合适的底物上;基因定位显示,StGOGATs均在马铃薯3号染色体前850kb的位置上;对StGOGATs进行启动子顺式作用元件分析,发现其与植物光合作用及含氮化合物的合成等有关;构建了系统进化树，发现其与番茄及番茄野生种具有较高的同源性。蛋白互作预测显示，StFd-GOGAT与StNADH-GOGAT蛋白间存在直接的互作关系;三级结构预测显示,只有StNADH-GOGAT具有自身同源二聚体结构;亚细胞定位推测StFd-GOGAT和StNADH-GOGAT分别在细胞质及叶绿体中行使功能。初步明确StGOGATs蛋白特性、结构与功能，为后续探究其在GS/GOGAT循环中分子调控机制奠定理论基础。

关键词: GS/GOGAT循环, StGOGATs, 蛋白质结构, 蛋白互作, 启动子

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

韩祉君, 赵艳菲, 卢悦, 李爽, 张嘉越, 韩玉珠, 张婧颖. 马铃薯氮同化关键酶基因StGOGATs的克隆及结构与功能分析[J]. 作物杂志, 2023 (5): 71–80

Han Zhijun, Zhao Yanfei, Lu Yue, Li Shuang, Zhang Jiayue, Han Yuzhu, Zhang Jingying. Cloning and Analysis of the Structure and Function of the Key Enzyme Genes StGOGATs of Potato Nitrogen Assimilation[J]. Crops, 2023(5): 71–80

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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

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

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

元件 Element	功能 Function	数量Number
元件 Element	功能 Function	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