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| Cloning and Expression Analysis of a 1, 2Rhamnosyl-transferase Gene (Cm1, 2RhaT) from Citrus maxima cv.Liangping |
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Abstract Naringin is a bittering agent of kinds of hydrotestosterone flavonoids and the rhamnosyltransferase is a key enzyme in the synthesis of naringin. In this study we cloned a 1,2rhamnosyltransferase gene(Cm1,2RhaT) which wa from Citrus maxima(Burm.) Merr. cv. Liangpin Yu. The Cm1, 2RhaT gene expression vector pET28a-CmRhaT was constructed by the gene fragment cloned into the prokaryotic expression vector pET-28a(+), which was then transformed into Escherichia coli BL21(DE3). The objective fusion protein was induced and expressed. Further fusion protein was induced to express and purified. The SYBR Green Real-time qRT-PCR was employed to analyze the expression differences of Cm1,2RhaT in the fruits and leaves's different developmental stages of Citrus maxima (Burm.) Merr. cv. Liangpin Yu and other four citrus varieties.The results showed that Cm1,2RhaT was isolated and characterized from Citrus maxima (Burm.) Merr. cv. Liangpin Yu. The accessed cDNA (GenBank accession No. JQ217381) was 1 436 bp in length encoding 453 deduced amino acids with a predicted molecular mass of 51.2 kD. We obtained the fusion protein after isolation and purification. Its concentration and purity were 0.526 mg/mL and 95%, respectively. Real-time fluorescence qPCR(RT-PCR)showed that the Cm1,2 RhaT expressed in the Liangpingyou's fruits and leaves. The expression level of Cm1,2 RhaT was reduced gradually with the fruit developing from 30~80 d after flowering. Its homologous genes in other species also showed the same as volatility expression patterns as in Liangpingyou. Cm1, 2RhaT which was amplified from Citrus maxima (Burm.) Merr. cv. Liangpin Yu expressed a kind of enzyme with typical glycosyl transfer group. It is a key enzyme in the synthesis of fiavanone which is one of Citrus′s bitter substances. The enzyme involves in secondary metabolism process of Citrus. To sum up, the research and results has certain significance to not-bitter citrus's varieties improvement.
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Received: 09 August 2012
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