Cloning and Bioinformatic Analysis of Anthocyanidin Synthase Gene from Hanzhong Black Rice
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摘要: 花青素合成酶（Anthocyanidin Synthase，ANS）是植物花青苷生物合成途径末端的关键酶，催化无色花色素到有色花色素的转变。为研究汉中黑稻的ANS多样性及起源，采用克隆测序的方法对汉中7种品种黑稻的ANS基因序列进行分析。采用生物信息学方法，对该基因序列进行对比，并构建系统进化树和同源树，对其编码蛋白从基本理化性质、亚细胞定位、跨膜结构域、信号肽、导肽、二级结构和三级结构等方面进行预测和分析。结果表明7种汉中黑稻存在黑稻1、黑稻2两种基因序列，开放阅读框均为1 128 bp，编码375个氨基酸。进化树表明黑稻1和黑稻2与籼稻、粳稻、浦竹仔、小麦等禾本科植物较近的亲缘关系，同源性分析表明黑稻1和黑稻2与籼稻等植物的ANS具有高度的同源性。氨基酸序列比对发现黑稻1和黑稻2仅有326位氨基酸不同。黑稻1的ANS蛋白含有2OG-FeⅡ_Oxy加氧酶的保守结构域。ANS蛋白三级结构预测，发现黑稻1和籼稻存在差异，推测ANS可能是花青苷合成中起重要作用的关键酶。这些结果表明ANS基因是一个古老的基因，可以作为种属鉴定的参考基因，可能是影响黑稻花青苷生物合成的主要基因之一。Abstract: Anthocyanidin synthase (ANS) is one of the key enzymesthat catalyzes the biosynthesis that converts leucoanthocyanidin into colored anthocyanidin in plants. To study the diversity and origin of ANS, the ANS genes from 7 varieties of Hanzhong Black Rice were analyzed by PCR cloning and sequencing. Through the bioinformatics, sequences of theANS genes were compared. By constructing the NJ and homology trees, the sequence alignment of ANS protein, basic physical and chemical properties, subcellular localization, transmembrane region, signal peptide, guide peptideas well as the secondary and tertiary structures of ANS protein were studied. The results showed that the ANS genes of theserice varietiescould be divided into two categories, namely, Black Rice 1(BR1) and Black Rice 2(BR2). They had a same length on the open reading frame of 1, 128 bp encoded 375 amino acids. Their phylogenetic treessuggested that BR1 and BR2 were genetically close to other gramineous plants, such as Indica and Japonica, Indosasa hispida, and Triticum aestivum. The sequence of amino acids (AA) revealed that only the 326th AA was different between BR1 and BR2. A conserved domain search showed that the ANS protein hada 2OG-FeⅡ_Oxy domain. Its predicted tertiary structure indicated that BR1 differed from Indica, and suggested that the ANS gene might play a key role in the anthocyanin synthase. It was concluded that the ANS genewas ancient and could be served as a reference gene for species identification, and thatits presence in Hanzhong Black Rice could be one of the main reasons why the anthocyanin synthesis was found in the rice.
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Key words:
- Hanzhong Black Rice /
- anthocyanidin synthase /
- gene cloning /
- bioinformatic analysis
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图 2 基于ANS序列构建的NJ树和同源树
注：A为NJ树；B为同源树。
Figure 2. NJ and homology trees based on ANS sequence
表 1 水稻花青素合成酶的理化性质
Table 1. Physicochemical properties of ANS gene from rice
项目 籼稻 黑稻1 黑稻2 粳稻 分子量 40598.9 40792.1 40734.0 40708.0 正电荷残基 38 39 39 38 负电荷残基 52 53 52 54 等电点 5.52 5.58 5.65 5.38 总平均疏水性 -0.189 -0.236 -0.227 -0.195 分子量 35.68 37.91 38.13 35.15 
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表 2 ANS蛋白的亚细胞定位
Table 2. Subcellular location of ANS protein
定位 概率 细胞质 0.652 线粒体 0.174 细胞核 0.087 高尔基体 0.043 细胞骨架 0.043
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表 3 黑稻1 ANS蛋白信号肽预测
Table 3. Predict signal peptide of BR1 ANS protein
指标 位置 分值 信号肽 max. C 19 0.532 max. Y 19 0.145 max. S 24 0.254 Mean S 1(18 0.140 D 1(18 0.143 无 注：mean S：信号肽分值的平均值；D：mean S和max.Y的加权平均值。
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表 4 黑稻1 ANS蛋白的二级结构预测
Table 4. Predicted secondary structure of BR1 ANS protein
二级结构 氨基酸/个 百分比/% α螺旋 157 41.87 延伸链 58 15.47 β转角 44 11.73 无规则卷曲 116 30.93
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