比利时杜鹃花黄烷酮3-羟化酶基因克隆及功能分析

Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort.

摘要:

黄烷酮3-羟化酶（flavanone 3-hydroxylase，F3H）是植物花青素（anthocyanin）合成过程中的关键酶。本研究以红色比利时杜鹃花（Rhododendron hybridum Hort.）不同发育时期的花瓣为实验材料，利用反转录PCR （reverse transcription PCR，RT-PCR）和cDNA末端快速克隆（rapid-amplification of cDNA ends，RACE）技术对比利时杜鹃花黄烷酮3-羟化酶（Rhododendron hybridum Hort.flavanone 3-hydroxylase，RhF3H）基因进行克隆，并进行生物信息学分析；利用qRT-PCR技术对不同发育时期花瓣RhF3H基因表达量进行分析；构建pET-28a-RhF3H原核表达载体对RhF3H蛋白进行制备和纯化；构建pCAMBIA1302-RhF3H过表达载体，通过农杆菌介导法进行拟南芥遗传转化研究。结果表明，比利时杜鹃花RhF3H基因全长为1 245 bp，开放阅读框（open reading frame，ORF）为1 092 bp，编码363个氨基酸，含有Fe²⁺结合基序和2-酮戊二酸结合基序的双加氧酶超家族。系统进化分析表明，比利时杜鹃花RhF3H蛋白与越橘F3H蛋白亲缘关系最近；实时荧光定量PCR （quantitative real-time polymerase chain reaction，qRT-PCR）分析表明，在不同发育时期花瓣中，红色比利时杜鹃花RhF3H基因的表达水平呈先上升后下降的趋势，在初开期表达量最高；原核表达结果表明，构建原核表达载体pET-28a-RhF3H诱导蛋白大小约为40 kDa，与理论值相近；成功获得转基因RhF3H拟南芥植株，PCR鉴定和β-葡萄糖苷酸酶（β-glucuronidase，GUS）染色证明RhF3H基因整合到拟南芥植株基因组中。qRT-PCR、总黄酮和花青素含量分析显示，相对于野生型，RhF3H在转基因拟南芥植株中显著高表达，其总黄酮和花青素含量也显著增加。本研究为探究杜鹃花RhF3H基因功能提供一定的理论基础，对杜鹃花花色的分子机理有重要的研究价值。

Abstract:

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe²⁺ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.