Cloning and expression analysis of genes in the active ingredient biosynthesis pathways of cultivated Lycium ruthenicum

[Objective]Present study was conducted to determine the optimum harvesting period of Lycium ruthenicum Murr.berry by analyzing the content of chemical components and the expression of key enzyme genes in different periods. The cloning and expression analysis of key enzyme genes in biosynthetic pathway would establish a foundation for revealing the biosynthetic mechanism of anthocyanins and proanthocyanidins in L. ruthenicum[Methods]The content of anthocyanins and proanthocyandins in L. ruthenicum at different growth period were determined by pH differential method and vanillin-hydrochloric acid method respectively. The full-length cDNA sequences of four key enzyme genes LrDFR,LrANS,LrLAR and LrANR were amplified by RT-PCR,and the expression of those four genes in L. ruthenicum at different developmental stages was analyzed by qRT-PCR. Bioinformatics analysis of gene coding sequences was performed using software DNAMAN,ORF finder and ProtParam,while phylogenetic tree was constructed using MEGA 7. 0 software.[Results]Our results showed that the highest content of anthocyanidins and prothocyanidins in L. ruthenicum at four different developmental stages were approximately 6. 08 mg/g DW at black fruit maturity stage and 22. 3 mg/g DW at half purple and half black fruit stage. The full length sequences of four key genes LrDFR,LrANS,LrLAR and LrANR were 1140 bp,1251 bp,1002 bp and 1017 bp,coding proteins of 379,416,333 and 338 amino acids,respectively. Sequence analysis showed that the amino acids encoded by the four genes had the typical characteristics of the protein family they belonged to. The highest content of anthocyanins and proanthocyanidins were detected in the fruit maturity period and half-purple and half-black fruit period,respectively. With the development of fruits and the increase of anthocyanin content,the expression level of LrDFR and LrANS also gradually increased,while mRNA levels of LrLAR and LrANR were increased first and then decreased during fruit development.[Conclusion]The biosynthesis of proanthocyanidins in L. ruthenicum is controlled by two pathways,which are mainly synthesized by LAR pathway in the early stage of fruit growth and by ANR in the later stage. When the fruit of L. ruthenicum was used for extraction of proanthocyanidins,the harvest period could be appropriately advanced. The acquisition of four key enzyme genes is expected to lay the foundation for the discovery of the biosynthesis mechanism of anthocyanins and proanthocyanidins in L. ruthenicum.