De Novo Transcriptome Sequencing Of Blue Honeysuckle Fruit And Analysis Of Major Genes Involved In Anthocyanin Biosynthesis

Blue honeysuckle(Lonicera caerulea L.), which is known as honeyberry and haskap, is a shrub small berry with a strong cold resistance and distributes in northern hemisphere boreal zone, North America and the Nordic countries. In China, blue honeysuckle main distributes in Changbai Mountain in Jilin Province, eastern of Heilongjiang Province Daxinganling Mountains, Neimenggu, Qinghai, Gansu, Ningxia and Xinjiang Provinces. This berry is a specialties small berry in north of China with an economically important and health value. Its flavonoid and anthocyanin contents are generally higher than these of other fruits and RNA-seq technology provide a method to fill the vacancies of the lacking of genome for analysis experimental materials in specific time and space.In this paper, anthocyanins in different species blue honeysuckle fruit were analyzed qualitativl and quantitative and correlation between total anthocyanin content and antioxidant activity was calculated. Berel(green ripe period and veraison period) was chosen for RNA-sequencing by Illumina Hi SeqTM 2000 program and de novo assemble. Combined with total anthocyanin content and expression levels of related genes including structure genes and transcription factors, the process of anthocyanin biosynthesis was discussed initial and the results was as follows:(1) HPLC-DAD-MS2 program was used for anthocyanin analyzing of different cultivars of blue honeysuckle(berel, HSY-4 and Japan-5) and 8 anthocyanins were detected in a wavelength of 525 nm. Berel fruit has the highest TAC(500.14±62.43mg/100 g FW) while Japan-5 fruit was the lowest(162.79±3.75 mg/100 g FW). Cyanidin 3-glucoside was identified as the mainly anthocyanin in blue honeysuckle fruit and reached 76.61-86.67% of TAC.(2) The result of antioxidant activity(DPPH,FRAP,TPC,SOD) suggested that berel fruit had the highest antioxidant activity while Japan-5 showed the lowest antioxidant activity. The total anthocyanin content of fruits showed a positively correlated with antioxidant activity in blue honeysuckle and the higher TAC, the stronger antioxidant activity.(3) A total of 66,610 All-unigenes were obtained in this study from different periods of blue honeysuckle fruit(green ripe period and veraison period) through transcriptome sequencing by Illumina Hi Seq TM 2000 and de novo assemble program. Aligned to available databases(NR,Swiss-prot,KEGG,COG and GO) on internet and 43,202 All-unigenes(64.86%) were annotated and showed a high similarity with the known protein.(4) Quantitative real-time PCR(q RT-PCR) technology was used to determine genes expression levels among 5 periods(young fruit, green ripe fruit, half-veraison fruit, veraison fruit and soften fruit) of fruit developed in this paper. The result showed that, in general, genes including structure genes and transcription factor in the half-veraison period performed the highest expression level in 5 periods and consistently with anthocyanin accumulation. The genes PAL, ANS and b HLH showed a high abundance expression levels during color changing and might play important roles in anthocyanin biosynthesis.(5) To determine genes expression level among different cultivars(berel, HSY-4 and Japan-5), q RT-PCR technology was used in this paper and 3 cultivars exhibited the same expression model almost. Different cultivars had different and could explained the process of anthocyanin biosynthesis basically.