三角梅黄烷酮3-羟化酶基因的克隆及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2022-0048

摘要/Abstract

摘要：

为探究三角梅产生飞燕草素的可能性，根据前期获得的三角梅转录组数据，克隆花青素合成途径关键酶黄烷酮3-羟化酶基因（flavanone 3-hydroxylase，F3H），并通过荧光定量PCR技术检测其在不同颜色三角梅叶片及苞片中的表达量差异。结果显示成功克隆获得了一条cDNA全长1 089 bp，编码363个氨基酸的基因序列，将其命名为BsF3H，登陆GeneBank（OL957093）。BsF3H相对分子质量为41.14 kD，理论等电点为5.48，含有2OG-FeⅡ_Oxy加氧酶结构域，不具有信号肽及跨膜结构，细胞定位于细胞质中，最多的二级结构为α螺旋，三级结构预测显示为单体蛋白。RT-qPCR结果显示该基因在不同颜色三角梅中表达量均不高，相对而言在紫色安格斯（Elizabeth Angus）苞片中表达量最高。研究结果表明三角梅具有产生各类花青素的潜力，可利用基因工程手段对其颜色进行改造。

关键词: 三角梅, 黄烷酮3-羟化酶, 生物信息学, 表达量分析

Abstract:

In order to explore the feasibility of Bougainvillea spectabilis producing delphinidin，the gene of key enzyme flavanone 3-hydroxylase（F3H）gene in anthocyanin synthesis pathway was cloned according to the high-throughput sequencing data of transcriptome from B. spectabilis leaves. The quantitative real-time PCR assay was used to examine the gene relative transcription levels in different color B. spectabilis leaves and bracts. The results showed that a gene designated BsF3H were cloned. It contained a 1 089 bp open reading frame encoding 363 aa. Its GenBank accession number was OL957093. The BsF3H protein had a calculated molecular mass of 41.14 kD and an isoelectric point of 5.48. It possessed a 2OG-FeⅡ_Oxy oxygenase domain with no signal peptide and transmembrane structure. Subcellular localization analysis indicated that it was cytoplasmic protein. The most abundant secondary structure was alpha helices. In addition，the tertiary structure prediction showed it was a monomeric protein. RT-qPCR analysis showed that the gene was not highly expressed in different color B. spectabiliss，and was relatively expressed higher in the purple Elizabeth Angus bracts. The research results indicated that B. spectabilis has the potential to produce anthocyanins，and its color can be modified by means of genetic engineering.

Key words: Bougainvillea spectabilis Willd., flavanone 3-hydroxylase, bioinformatics, expression analysis

孙蓉, 刘姗, 高静雷. 三角梅黄烷酮3-羟化酶基因的克隆及表达分析[J]. 生物技术通报, 2022, 38(11): 122-128.

SUN Rong, LIU Shan, GAO Jing-lei. Cloning and Expression Analysis of Flavanone 3-hydroxylase Gene from Bougainvillea spectabilis[J]. Biotechnology Bulletin, 2022, 38(11): 122-128.

图/表 7

图1 RNA提取及PCR扩增电泳图 A：M：D2000 DNA marker；1：RNA样品；B：M：D2000 DNA marker；1：目的条带

Fig. 1 Electrophoresis detection results of RNA extraction and PCR amplification products A：M：D2000 DNA marker；1：RNA sample；B：M：D2000 DNA marker；1：target band

图2 BsF3H与其他植物F3H多序列比对下划线：2OG-FeⅡ_Oxy加氧酶结构域；红色方框：Fe2+结合位点（His215，Asp217，His273）；蓝色方框：2-O-酮戊二酸结合位点（Arg283-X-Ser285）

Fig. 2 Alignment analysis of of BsF3H and F3Hs from other plants Underline：2OG-Fe（Ⅱ）oxygenase domain. Red box：Binding site of Fe2+（His215，Asp217，His273）. Blue box：Binding site of 2-O-oxoglutarate（Arg283-X-Ser285）

图3 BsF3H二级结构预测

Fig. 3 Prediction of the secondary structure of BsF3H proteins

图4 BsF3H功能域预测

Fig. 4 Prediction of the domains of BsF3H protein

图5 BsF3H蛋白的三级结构及其与底物结合位点

Fig. 5 Tertiary structure of BsF3H protein and its substr-ate binding site

图6 三角梅F3H与其余植物F3H同源蛋白系统进化树

Fig. 6 Phylogenetic relationships of F3H in B. spectabilis and F3Hs from other plants

图7 BsF3H在不同颜色三角梅中的表达量分析不同的小写字母代表样品间的差异显著（P<0.05）

Fig. 7 Analysis of BsF3H expressions in different color B. spectabilis Different lowercase letters above the bars indicate significant differences（P<0.05）among the samples

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