Research On The Discovery Of Functional Genes Related To Veratrum Steroid Alkaloid Synthesis

Veratrum nigrum L.is mainly distributed in northeast China and other northern regions.It is a perennial monocotyledons of Liliaeeae.It is a famous traditional Chinese medicine in China and has a variety of physiological functions such as anti-tumor,blood pressure lowering,analgesia and anti-inflammatory.Its main active substance is the steroid alkaloids,the latest discovery of steroid alkaloids on the medulloblastoma,breast cancer,esophageal cancer and other kinds of cancer has a significant inhibitory effect,so the market demand is huge.At present,the research on the alkaloids of Veratrum mainly focuses on the separation and purification of active substances,pharmacological functions,chemical synthesis,etc.In this study,a transcriptome library of different tissues of the same variety or the same tissue of different varieties was constructed using the hairy panicle Veratrum maackii Regel and Veratrum nigrum as research materials,and transcriptome data were obtained by Illumina high-throughput sequencing.Through the analysis of transcriptome database,some candidate genes involved in the biosynthesis of steroid alkaloids were discovered.The main conclusions are as follows:(1)By sequencing the transcriptomes of two Veratrum samples,a total of 79.12Gb Clean Data was obtained.The Clean Data of each sample reached 5.40Gb and the Q30 base percentage was above 92.23%.After de novo assembly,a total of 437,820 Transcript and 203,912 Unigene were obtained.The N50 of Transcript and Unigene were 871 and 780,of which 33,066 Unigene had a length of 1kb or more.The functional annotation of Unigene has obtained 97 207 annotation results in 9 public databases,accounting for 47.67%of the total Unigene..(2)In the upstream of the steroid alkaloids synthesis pathway to the 2,3-oxidized squalene synthesis stage,this transcriptional database annotated 9 Unigene,DN130520_c3_g5 and DN132006_c4_gl as AACT,DN143017_c4_g1 as HMGs,DN149166_c0_g1 as HMGR,DN146180_c2_g2 as MK,and DN154703_c1_g4 as MPK.DN149586_c1_gl was annotated as MVD,DN126122_c1_g3 as SQS,and DN149201_c0_g2 as SQE,and it was verified by qRT-PCR.The RQ value in the roots of the two Veratrum was generally higher than that in the leaves,which was consistent with the value of FPKM.(3)In the 2,3-squalene oxide to cholesterol synthesis stage in the pathway of alkaloid synthesis of Veratrumin,4 Unigene were annotated to this transcriptome database,namely CAS(DN141351_c3_g4 and DN159671_c4_g1),CPI(DN154936_c1_g3)and CYP51(DN140403_c0_g1).qRT-PCR was used to verify them.Except CAS,the expression of other enzyme genes in roots was higher than that in leaves.(4)From the cholesterol to the alkaloid synthesis stage of Veratrum in the alkaloid synthesis pathway,four Unigene were obtained through analysis and mining,respectively annotated as CYP90B27(DN140151_c4_g1),CYP90G1(DN142431_c1_g7 and DN157938_c1_g1)and CYP90N(DN158282_c4_g2).qRT-PCR was used to analyze the expression patterns of CYP90B27,CYP90G1 and CYP90N Unigene in the roots and leaves of Veratrum,and it was found that the expression levels in the roots of both Veratrum were higher than the leaves,and the number of transcriptome was consistent with the results of qRT-PCR.(5)In the screening of related transcription factors in the alkaloid synthesis pathway of veratro,a total of 866 Unigene annotations were obtained as transcription factors,covering 16 transcription factor families.The maximum number of transcription factors annotated as MYB family was 168 Unigenen,followed by the largest number of Unigene annotated as bZIP and bHLH,which were 152 and 12 respectively.Among 866 Unigene,278 differentially expressed genes were screened.