蓝莓3-O-葡萄糖基转移酶基因克隆及生物信息学分析

doi:10.7668/hbnxb.20193980

摘要/Abstract

摘要：

为研究蓝莓3-O-葡萄糖基转移酶基因(3GT)的性质和功能,探明3-O-葡萄糖基转移酶在花青素合成过程中发挥的作用,以成熟的蓝莓果实为材料,利用同源克隆获得了3GT基因的单一拷贝,运用生物信息学软件分析预测蓝莓3GT的序列特征。结果表明:3GT基因长度为1 371 bp,编码456个氨基酸,位于蓝莓的4号染色体末端,含有3个外显子和2个内含子。蛋白的分子式为C₂₂₇₉H₃₅₂₁N₅₉₅O₆₅₀S₁₅,理论等电点(pI)为6.14。信息分析预测3GT蛋白有37个磷酸化位点,亲水指数小于0为亲水性蛋白,不稳定系数大于40为不稳定蛋白,无跨膜区和信号肽;蛋白质二级结构由α-螺旋(Hh)、延伸链(Ee)、β-转角(Tt)和无规则卷曲(Cc)4种结构形式组成,其中α-螺旋占比最高,达40%;3GT属植物糖基转移酶家族(GTB-型),PSPG结构域中第44位是组氨酸,糖基供体可能为UDP-半乳糖;与猕猴桃亲缘关系最近,同源性为67%;分子对接显示蓝莓3GT与矢车菊素分子存在4种相互作用力;蓝莓3GT启动子上游可能具有与光响应、抗胁迫响应及特定激素响应相关的顺式作用元件。本研究克隆得到了蓝莓3GT基因,通过在线网站和预测软件对其进行了生物信息学分析和功能预测。

关键词: 蓝莓, 3GT, 基因特征分析, 花青素合成

Abstract:

In order to study the function of 3-O-glucosyltransferase gene(3GT) in blueberry and to explore the roles of 3-O-glucosyltransferase in anthocyanin synthesis, a single copy of 3GT was cloned from the cDNA of blueberry fruit by RT-PCR, which was 1 371 bp and encoded 456 amino acids. 3GT was located at the end of chromosome Ⅳ in blueberry and contains 3 exons and 2 introns. The molecular formula of the protein was C₂₂₇₉H₃₅₂₁N₅₉₅O₆₅₀S₁₅, and the theoretical isoelectric point (pI) was 6.14. Information analysis predicted that 3GT was one hydrophilic protein and unstable protein with 37 phosphorylation sites, without signal peptide,or transmembrane domain. The secondary structure of protein consists of α-helix (Hh), extended chain (Ee), β-angle (Tt) and random curl (Cc), among which α-helix accounted for 40%.As one number of GTB-type glycosyltransferase family, the 44th position in the PSPG domain of 3GT was histidine, and the glycosylase donor might be UDP-galactosewas. 3GT was closely related to Kiwifruit, with the homology of amino acids reached 67%. Auto-Dock showed that 3GT had four interaction forces with zymolyte of cyanidin. There were multiple cis-regulatory elements that might relate to light response,stress resistance response and specific hormone response in the promoter of 3GT. The blueberry 3GT gene was cloned.The bioinformatics analysis and function prediction were carried out through online website and prediction software to study the characteristics and function of 3GT.

Key words: Blueberry, 3GT, Gene characterization analysis, Anthocyanin biosynthesis

刘娜, 田雨菁, 冯哲瀚, 李虎良, 张蕾, 黄金海, 华德平. 蓝莓3-O-葡萄糖基转移酶基因克隆及生物信息学分析[J]. 华北农学报, 2023, 38(2): 120-128. doi: 10.7668/hbnxb.20193980.

LIU Na, TIAN Yujing, FENG Zhehan, LI Huliang, ZHANG Lei, HUANG Jinhai, HUA Deping. Cloning and Bioinformatics Analysis of UDP-glucose: Anthocyanidin 3-O-glucosetransferase Gene in Blueberry[J]. Acta Agriculturae Boreali-Sinica, 2023, 38(2): 120-128. doi: 10.7668/hbnxb.20193980.

http://www.hbnxb.net/CN/Y2023/V38/I2/120

图/表 7

图1 蓝莓Vc3GT的克隆 A.Vc3GT基因PCR扩增产物的琼脂糖凝胶电泳检测示意图;B.Vc3GT连接入T载体质粒图谱; C.Vc3GT位于蓝莓的4号染色体,含有3个外显子与2个内含子。

Fig.1 The cloning of Vc3GT from the cDNA of blueberry A.The diagram of agarose gel electrophoresis for the PCR amplification of Vc3GT gene;B.The map of the Vc3GT-T vector;C.The Vc3GT localizes on chromosome IV and contains 3 exons and 2 introns.

图2 Vc3GT氨基酸序列分析 A.Vc3GT氨基酸保守域分析;B.多个物种中Vc3GT氨基酸序列比对。

Fig.2 The amino acid sequence analysis of Vc3GT A.The diagram of conserved domain for Vc3GT;B.The amino acid sequence of Vc3GT protein in several species.

图3 Vc3GT蛋白亲/疏水性、蛋白信号肽和跨膜结构域预测 A.预测Vc3GT为亲水性蛋白;B.Vc3GT蛋白无信号肽;C.Vc3GT蛋白无跨膜结构域。

Fig.3 Hydrophilic/hydrophobic analysis, signal peptide prediction and transmembrane region prediction of Vc3GT A.Vc3GT is predicted to be a hydrophilic protein;B.Vc3GT has no signal peptide;C.Vc3GT has no transmembrane region.

图4 Vc3GT蛋白质的结构预测 A:蓝色.α-螺旋,红色.延伸链,绿色.β-转角,黄色.无规则卷曲;B.葡萄3GT蛋白质三级结构(PDB ID:2C1X);C.Vc3GT蛋白质的三级结构预测。

Fig.4 Structure prediction of Vc3GT A:Blue.α-helix, Red.Extended chain, Green.β-turn, Yellow.Random coiling;B.Tertiary structure of grape 3GT(PDB ID: 2C1X);C.Prediction of tertiary structure for Vc3GT.

图5 Vc3GT氨基酸序列系统进化树

Fig.5 The phylogenetic tree was constructed using the amino acid sequence of Vc3GT in multiple species

图6 Vc3GT和矢车菊素分子对接模拟 A.Vc3GT酶和矢车菊素分子对接最低能量模型;B.Vc3GT酶和矢车菊素分子对接存在的模拟相互作用力及二维结果。

Fig.6 Simulation of docking between Vc3GT and cyanidin A.The lowest energy model of molecular docking between Vc3GT and cyanidin;B.Simulated interaction and two-dimensional results were shown using molecule docking between Vc3GT and cyanidin.

表1 Vc3GT启动子区顺式作用元件预测

Tab.1 Prediction of cis-acting elements in the promoter region of Vc3GT

顺式元件 Cis-elements	序列 Sequence	元素数量/个 Numbers of element	功能 Function
CAAT-box	CAAT/CCAAT/CAAAT/TGCCAAC	17	转录起始及增强元件
TATA-box	TATA/TATAA/TATAAA/ATATAT/TATACA/TATATTTATATTT	16	转录起始关键调控位点
MYB	TAACTG/CAACCA	3	MYB结合位点
ACE	GACACGTATG	1	光信号响应元件
G-Box	CACGTG/CACGTC	3	光信号响应元件
GT1-motif	GGTTAAT/GGTTAA	2	光信号响应元件
Box 4	ATTAAT	1	光信号响应元件
TCCC-motif	TCTCCCT	1	光信号响应元件
Sp1	GGGCGG	3	光信号响应元件
TCT-motif	TCTTAC	1	光信号响应元件
I-box	GTATAAGGCC	2	光信号响应元件
DRE1	ACCGAGA	1	干旱响应元件
WRE3	CCACCT	1	损伤诱导元件
STRE	AGGGG	2	胁迫响应元件
P-box	CCTTTTG	1	赤霉素响应元件
TCA-element	CCATCTTTTT	1	水杨酸响应元件
ABRE	CACGTG/ACGTG	3	脱落酸响应元件
TGA-element	AACGAC	1	生长素响应元件

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