RESUMO
@#Transcriptome sequencing was performed for the first time on Anoectochilus roxburghii(AR)in different harvesting periods using RNA-seq high-throughput sequencing technique, and the results were verified and analyzed by Q-PCR and HPLC. A total of 51, 370 genes were obtained by transcriptome sequencing and annotated to the database of Nr, GO, Swiss-Prot, KEGG and KOG. The species that were sequenced according to the homology sequence were the same as AR monocotyledon plants. Through comparison of AR transcriptome in different periods, it was found that the differences were mainly in flavonoid biosynthesis-related genes. The expression levels of flavonoid biosynthesis-related genes(trans-cinnamate 4-monooxygenase, caffeoyl-CoA O-methyltransferase, chalcone synthase, flavonol synthase, shikimate O-hydroxycinnamoyltransferase and flavonoid 3′, 5′-hydroxylase)were verified by Q-PCR, and the results were consistent with those of transcriptome sequencing. The contents of 6 flavonoids(rutin, isoquercitrin, narcissin, quercetin, kaempferol and isorhamnrtin)were determined by HPLC. The results showed that the expression of flavonoid synthetic gene in AR increased with the growth time, and the variation trend of flavonoid compound content and gene expression were basically consistent. Combined with transcriptome data, the biosynthetic pathway of flavonoid content in AR was plotted. This study provides important genetic resources for the key genes of flavonoid synthesis in AR and the biosynthesis of flavonoids, as well as the basis for the development of its medicinal value.
RESUMO
Objective To analyse the transcriptome sequencing results of Eucommia ulmoides calli under light and dark culture condition, and verify the expression level of the related genes of flavonoid biosynthesis from transcriptome sequencing results. Methods The transcriptome high-throughput sequencing of E. ulmoides calli was performed by using the Illumina HiSeqTM 2500 sequencing platform, and de novo assembly of the transcriptome sequencing results was finished by Trinity software. The sequencing results of Unigenes were compared with the databases of NR, Swiss-Prot, GO, COG, KOG, KEGG and Pfam by BLAST software. The gene expression abundance was estimated by FPKM value. qRT-PCR was used to verify the expression level of the related genes of flavonoid biosynthesis. Results About 13.00 Giga base pairs (Gbp) clean data (more than 6.02 Gbp, respectively) were obtained, and de novo assembly generated 62 030 Unigenes with an average length of 736.40 bp. A total of 25 167 Unigenes were annotated to Nr. Totally 4 794 Unigenes and their associated enzymes (enzyme commission numbers) were annotated to 82 KEGG pathway. There were 1 986 genes identified as significantly and differentially expressed genes between the two calli under light and dark culture. Among them, 1 139 (57.35%) were up-regulated and 847 (42.65%) were down-regulated in the calli under light culture. Metabolic pathway analysis revealed that seven Unigenes were predicted to be responsible for the flavonoid biosynthesis, six Unigenes of which were up-regulated in the calli under light culture, encoding chalcone isomerase (EC 5.5.1.6), chalcome synthase (EC 2.3.1.74), flavonoid 3’-monooxygenase (EC1.14.13.21), trans-cinnamate 4-monooxygenase (EC 1.14.13.11), Flavonol synthase (EC 1.14.11.23), shikimate-O-hydroxycinnamoyl transferase (EC 2.3.1.133). One Unigene was down-regulated in the calli under light culture, which encoded leucocyanidin oxygenase (EC 1.14.11.19). qRT-PCR analysis showed that the expression of the seven genes related with flavonoid biosynthesis was coincident with the transcriptome high-throughput sequencing results. Conclusion The white light (12 000 lx, 16 h light and 8 h dark) could improve the production capacity of some metabolic intermediate of flavonoid biosynthesis pathway of E. ulmoides calli.