【作者】 王丹；

【导师】 马瑞； 李虎林；

【作者基本信息】 延边大学 ， 作物学， 2020， 硕士

【摘要】 藜芦(Veratrum nigrum L.)主要分布在我国东北等北方地区,属百合科的多年生的单子叶植物,是我国著名的传统中药,具有抗肿瘤、降血压、镇痛消炎等多种药理作用。最新发现藜芦生物碱对母髓质细胞瘤、乳腺癌、食道癌等多种癌症有着显著的抑制作用,因此市场需求量巨大。目前藜芦生物碱的研究主要集中在活性物质的分离纯化、药理功能、化学合成等方面,对于藜芦生物碱的合成途径以及相关基因的调控至今未被完全解析与挖掘。本研究以毛穗藜芦和藜芦为研究材料,构建了同一品种不同组织和不同品种同一组织的转录组文库,并通过Illumina高通量测序获得转录组数据。经过对转录组数据库的分析,挖掘出了一些参与藜芦生物碱生物合成的候选基因。研究主要结论如下:(1)通过对2种藜芦样品的转录组测序,共获得79.12Gb Clean Data,各样品Clean Data均达到5.40Gb,Q30碱基百分比在92.23%及以上。De novo组装后共获得 437 820 条 Transcript 和 203 912 条 Unigene,Transcript 与 Unigene 的 N50 分别为871和780,其中Unigene长度在1kb以上的Unigene有33 066条。对Unigene进行功能注释,在9个公共数据库中共获得97 207条Unigene的注释结果,占总数的47.67%。(2)在藜芦甾体生物碱合成途径中上游至2,3-氧化角鲨烯合成阶段,本转录组数据库共注释到 9 条 Unigene,DN130520_c3_g5 和 DN132006_c4_g1 注释为 AACT,DN143017_c4_g1 注释为 HMGs,DN149166_c0_g1 注释为 HMGR,DN146180_c2_g2注释为 MK,DN154703_c1_g4 注释为 MPK,DN149586_c1_g1 注释为 MVD,DN126122_c1_g3 注释为 SQS,DN149201_c0_g2 注释为 SQE。qRT-PCR 验证发现,两种藜芦根中的RQ值普遍高于叶中,与FPKM值相一致。(3)在藜芦生物碱合成途径中的2,3-氧化角鲨烯至胆固醇合成阶段,本转录组数据库共注释到4条Unigene,分别注释为CAS(DN141351_c3_g4和DN159671_c4_g1),CPI(DN154936_c1_g3)和 CYP51(DN140403_c0_g1)。利用qRT-PCR对它们进行验证,除CAS外,其他酶基因在根中的表达量均高于叶片中。(4)在藜芦生物碱合成途径中的胆固醇至藜芦生物碱合成阶段,通过对转录组数据分析挖掘得到4条Unigene,分别注释为CYP90B27(DN140151_c4_g1),CYP90G1(DN142431_c1_g7 和 DN157938_c1_g1)以及 CYP90N(DN158282_c4_g2)。并利用qRT-PCR分析了 CYP90B27、CYP90G1、CYP90N Unigene在毛穗藜芦与藜芦的根和叶片中的表达模式,发现在两种藜芦根中的表达量均超过叶片,且FPKM值与qRT-PCR结果相一致。(5)在藜芦生物碱合成途径中对相关转录因子的筛选中,共得到866条Unigene注释为转录因子,覆盖16个转录因子家族,注释为MYB家族的转录因子最多,为168条Unigenen,其次是注释为bZIP和bHLH的Unigene,分别为152和126条。在866条Unigene中,筛选出278个差异表达基因。更多还原

【Abstract】 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.更多还原