Molecular Cloning And Expression Analysis Of Chalcone Synthase Gene In Blueberry

Anthocyanin is one of the most important antioxidant and bioactive substance in blueberry (Vaccinium spp.) fruits, it conduced to human health. The biosynthesis of anthocyanin in blueberry is considered to be one of the most important secondary metabolic pathways during the fruit development. Isolating and assaying key enzyme and related genes is the important methods to understand and regulate anthocyanin synthesis in blueberry fruits. Chalcone synthase (chalcone synthase, CHS, EC2.3.1.74) was the first heterozyme in the biosynthetic pathway of anthocyanin and provided basal carbon skeleton, so it is a key enzyme gene in the pathway. So far, no reports in the aspect of CHS gene have been seen until now in blueberry. In this study, a full-length cDNA sequence of chalcone synthase gene (CHS) was cloned from skins of blueberry’Northland’fruits using RT-PCR and RACE based on Unigene23486(440bp)in Illumina/Solexa sequencing library. And the gene expression during the blueberry fruit development was analyzed by real-time PCR technique. In this study, the key enzyme gene in the biosynthesis of anthocyain in blueberry was obtained, which will lay the molecular biology foundation for further research on the molecular mechanism of anthocyanin bisynthesis and new varieties breeding of blueberry. Major results are follows:1Total RNA was isolated firstly from blueberry fruit with the improved CTAB method. Their concentration from pulp and skin of blueberry fruit were80.0-130.0ng/μL and235.0-420.0ng/μL, their yield were8.0-13.0μg/g and23.5-42.0μg/g, the ratios of A260/A280were1.9-2.1and2.0-2.2. The total RNA isolating by this method was enough good to be used in molecular biology experimentation such as RT-PCR and RACE.2A full-length cDNA sequence of chalcone synthase (CHS) gene was cloned firstly from the skin of blueberry fruits using RT-PCR and RACE, named VcCHS and its accession number in Genbank was JN654702.3Sequence analysis indicated that VcCHS was1438bp in full-length cDNA sequence and contained a5’-untranslated region (5’-UTR) of107bp, a3’-untranslated region (3’-UTR) of71bp, and an opening reading reading frame (ORF) of1260bp encoding a protein of419amino acids. Sequence analysis showed that amino acids encoded by VcCHS has up to86.76%of consistency with that encoded by other species. The cDNA sequence possessed the conserved structural domain of cd00831and PLN03173. It possessed the sequence of the family signature: RLMMYQQGCFAGGTVLR and the conserved active sites for the CHS function:Cys(C)164, His(H)303and Asn(N)336. Sequence alignment and phylogenetic tree analyses showed that VcCHS had the closest phylogenetic relationship with the Ericales.4The expression of VcCHS gene in different periods of the blueberry fruit development (from blooming to maturation period) and in different tissues (pulp and skin) were studied firstly by real-time PCR technique. The results showed that VcCHS could be detected on different periods of fruit development and in different tissues of blueberry, exhibiting stronger in the stage of blue fruits, lower in the stage of blooming and pink fruits, the lowest in the stage of green fruit. The expression of VcCHS gene in the blueberry skins was stronger obviously than that in the pulp.5The content of anthocyanins in blueberry was assaied. The results showed that anthocyanins could be detected on different periods of fruit development, exhibiting stronger in the stage of blue fruits, the lowest in the stage of blooming and green fruit. The content of anthocyanins in blueberry skins was the highest and higher obviously than that in the pulp. Meanwhile, the content of anthocyanins had significant consistency with the relative expression of VcCHS, and they both showed the highest transcript abundance in the skins.