Cloning and Expression Analysis of Flavanone 3-hydroxylase Gene from Bougainvillea spectabilis

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

Abstract

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

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.

Figures/Tables 7

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

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）

Fig. 3 Prediction of the secondary structure of BsF3H proteins

Fig. 4 Prediction of the domains of BsF3H protein

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

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

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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