Cloning The Full Length CDNA Of Anthocyanins Biosynthasis Key Genes And CHS Gene Promoter From Brunfelsia Acuminate Flowers

Brunfelsia acuminate flowers' color changes from dark purple to white inseveral days and has high ornamental value. Because of each flower blossomingflowering at different time, several different colors can be seen at one plant. In thispresent study, the full length cDNA sequence of chalcone synthase(CHS), chalconeisomerase (CHI), flavonoid3′5′-hydroxylase(F3′5′H) were cloned by RT-PCR and RACEfrom Brunfelsia acuminate flowers. The5′-upstream sequence of CHS gene was clonedby genomic walking. The key genes of anthocyanins biosynthaesis and CHS gene promoterwas cloned and analysied to get further acknowledge of the mechanism of theanthocyanins biosynthasis, with an aim to establish the basis for changing the flower colorby genetic engineering.1Cloning the full length cDNA of CHS from Brunfelsia acuminate flowersThe CHS gene cDNA sequence from Brunfelsia acuminate flowers was cloned byRT-PCR and RACE. Its Genbank accession number was JN966986. The full length cDNAof CHS was1415bp with an open reading frame (ORF) of1170bp,73bp5′UTR and172bp3′UTR, encoding389amino acids. Nucleotide homology analysis showed that thesequence of Brunfelsia acuminata flower CHS has90%,88%,85%,84%,79%respectively with that of Petunia hybrida, Nicotiana tabacum, Solanum lycopersicum,Capsicum annuum, Camellia sinensis.The bioinformatics analysis showed that the relative molecular weight of CHSprotein was42,699D, the isoelectric point was6.57. It was a stable and hydrophilic protein,and the molecular fomula was C1901H3046N516O562S18. The secondary structure prediction ofthe protein showed that it had abundance of α-helices and β-sheet. It didn't contain signalpeptide, but two transmembrane domains at208²25and369³88site. And itssub-cellular localization was most probably in cytoplasm. Conserved Domain Searchreveals that Brunfelsia acuminata flower CHS protein has a cond_enzymes superfamily domain.Using Genomic walking,769bp5′-upstream sequence of CHS gene was cloned.Though Blast program in NCBI, the result showed the had87%identity with Petunia×hybrida CHS promoter from-113to+26, either had75%identity with Nicotiana tabacumfrom-209to-8. SoftBerry software predicted the transcription start site(TSS) was the baseA at653bp. Though PlantCARE software, it was predicted that besides the core-elementslike TATA-box(TATATAAA) and CAAT-box(TGCCAAC), there were several lightcis-action elements such as G-box, Box I, Box IV, I-box, GT1-motif, several hormoneresponsiveness elements such as ABRE,ARE,P-box,TGACG-motif,TGA-element, andsome other elements envolved in circadian, heat stress, endosperm expression. All above isconsistent with the response of the flower pigment accumulation, therefore, it can bepredicted that the5′-upsteam sequence cloned had a characteristic of a common plantpromoter.2Cloning the full length cDNA of CHI from Brunfelsia acuminate flowersThe CHI gene cDNA sequence from Brunfelsia acuminate flowers was cloned byRT-PCR and RACE. Its Genbank accession number was JN887637. The full length cDNAof CHI was1051bp with an open reading frame (ORF) of792bp,22bp5′UTR and237bp3′UTR, encoding263amino acids. Nucleotide homology analysis showed that thesequence of Brunfelsia acuminata flower CHI has84%,82%,76%,81%,72%,73%,75%,73%,72%,71%respectively with that of Petunia hybrida, Nicotiana tabacum, Camellianitidissima, Solanum melongena, Canarium album cultivar Changying, Prunus avium,Rhododendron simsii, Paeonia suffruticosa, Paeonia lactiflora, Chrysanthemum xmorifolium.The bioinformatics analysis showed that the relative molecular weight of CHI proteinwas28.54kD, the isoelectric point was4.91. It was an unstable and hydrophilic protein,and the molecular fomula was C₁₂₈₄H₂0₂₅N₃₁₉O₄0₃S₅. The secondary structure prediction ofthe protein showed that it had abundance of α-helices and β-sheet. It didn't contain signalpeptide, but a transmembrane domain at149¹67site. And its sub-cellular localization wasmost probably in cytoplasm. Conserved Domain Search reveals that Brunfelsia acuminata flower CHI protein has a chalcone superfamily domain.3Cloning the full length cDNA of F3′5′H from Brunfelsia acuminate flowersThe F3′5′H gene cDNA sequence from Brunfelsia acuminate flowers was cloned byRT-PCR and RACE. Its Genbank accession number was JQ678765. The full length cDNAof F3′5′H was1051bp with an open reading frame (ORF) of1702bp,120bp5′UTR and61bp3′UTR, encoding506amino acids. Nucleotide homology analysis showed that thesequence of Brunfelsia acuminata flower F3′5′H has91%,87%,83%,76%,74%,73%respectively with that of Petunia hybrida, Nierembergia sp., Solanum tuberosum,Catharanthus roseus, Camellia sinensis, Eustoma grandiflorum.The bioinformatics analysis showed that the relative molecular weight of F3'5'Hprotein was56.57kD, the isoelectric point was8.78. It was a stable and hydrophilic protein,and the molecular fomula was C₂₅₄₁H₄₀₅₅N₆₈₁O₇₁₄S₂₈. The secondary structure prediction ofthe protein showed that it had abundance of α-helices and β-sheet. It didn't contain signalpeptide, but four transmembrane domains at1²6,39⁵8,170¹90,442⁴64. And itssub-cellular localization was most probably in endoplasmic reticulum membrane.Conserved Domain Search reveals that Brunfelsia acuminata flower CHI protein has aCypX superfamily domain.