RESUMO
Objective To clone the unknown sequence of terpene synthase (TPS) AvTPS1 promoter from Amomum villousm and analyze its activity. Methods In this research, AvTPS1 DNA sequence was amplified and cloned from genomic DNA (gDNA) of A. villousm leaves. Furthermore, the promoter of AvTPS1 was cloned by FPNI-PCR and the sequence was analyzed. The recombinant vector pCAM-AvTPS1p with AvTPS1 promoter for the expression of GUS gene was constructed. The activity of AvTPS1 promoter was verified by transient expression of Agrobacterium-mediated infiltration using the leaves of Nicotiana benthamiana. Results The gene sequence of AvTPS1 was 2 444 bp including seven exons and six introns. The 568 bp AvTPS1 promoter was successfully cloned using FPNI-PCR. Furthermore the sequence had ten kinds of cis-elements including conserved elements TATA-box, CAAT-box, MYC2 related element G-box and other elements. Finally, the GUS staining showed the tobacco leaves infiltrated by the pCAM-AvTPS1p were blue. Conclusion The AvTPS1 promoter can drive the transcription of GUS gene and then it was verified to have the promoter activity. These results give foundation for future research on the function of AvTPS1 involved in the terpenoid biosynthesis and its relationship with the transcription factors.
RESUMO
Objective To identify the reliable reference genes for gene expression analysis of the pericarp and seed of Amomum villosum Lour. by using real-time fluorescence quantitative polymerase chain reaction ( qRT-PCR). Methods Using the fruits ( separated into peels and seeds) of A. villosum at three different developmental periods as the experimental material, 5 candidate reference genes (β-actin, EF-1α, GAPDH, PGK, TUA) with steady expression were screened out by the high throughout sequencing of transcriptome and expression profile data. The qRT-PCR technique was applied to study the expression levels of 5 candidate reference genes in different samples. The stability of the candidate reference genes were evaluated by GeNorm and NormFinder software. Results The 5 reference genes had different stabilities in the pericarp and seed of A. villosum Lour. at different development periods . The order of the steadiness of reference genes showed by GeNorm was EF-1α = TUA>PGK>GAPDH>β-actin. The results of NormFinder revealed that EF-1α was the most stable, followed by TUA, and the order of the other three genes was as same as the results of GeNorm. Conclusion EF-1αand TUA could be used as double reference genes for the normalization of gene expression in A. villosum fruits at different developmental periods by using qRT-PCR.