【作者】 宋涛；

【导师】 周嘉裕；

【作者基本信息】 西南交通大学 ， 生物化学与分子生物学， 2015， 硕士

【摘要】 川芎(Ligusticum chuanxiong Hort)是伞形科一年生传统药用植物,干燥根状茎为川芎的主要药用部位,已有上千年的药用历史,主要用于治疗头痛,风湿痹痛,心脑血管疾病,月经紊乱,肿胀疼痛等；由于川芎能够改善皮肤的颜色和改变香味,因此也成为化妆品的主要成分。川芎中有多种重要的活性化合物,包括阿魏酸,挥发油,萜类化合物和生物碱等。开展川芎转录组学研究,对于系统认识川芎活性成分代谢途径、发育及抗逆相关机制具有重要的理论意义。本文通过Illumina高通量测序获得了川芎根茎、叶的转录组数据,并对转录组数据进行分析、验证,为发掘川芎活性成分代谢通路和相关的酶基因以及川芎的发育和抗逆相关研究提供遗传学背景。本文的主要结果如下：1)首次对川芎根茎、叶进行转录组测序,分别获得了川芎根茎与叶(共计5.36Gb)的原始数据,分别获得13,717,234、12,823,395clean reads,平均长度为101 nt,通过Trinity软件组装最终获得57,705、50,326个Unigenes,平均长度为713 nt、798 nt。将这些Unigene序列与蛋白质公共数据库进行比对注释,包括Nr、Swiss-Prot、GO、COG和KEGG。分别有35,952、31,421条Unigenes获得功能注释。通过KEGG代谢通路注释分类,分别发现有700,659个相关基因参与苯丙素的生物合成、苯丙氨酸代谢、甘油酯代谢、丙酸代谢、乙醛酸代谢等次生代谢途径。这些相关基因中包含已经被克隆阐明代谢功能的基因,也包含潜在候选基因,有助于通过基因工程和分子生物学手段提高相关通路代谢产物的含量。2)比较川芎根茎与叶的转录组数据,发现有4,680个差异表达基因,其中1,714个基因在根茎中上调,2,966个基因在叶中上调,并对它们进行分析,包括GO分析,COG分析,转录因子及根茎发育相关基因分析。3)对川芎进行qRT-PCR内参基因选择。先从转录组数据库中筛选7个常用的内参基因RPL11,ATC7,RPS18,BTF3,CYB5,GAPDH和UBC,分析它们在根茎与叶中的相对表达水平。然后利用GeNorm与NormFinder软件分析,发现RPL11在川芎根茎与叶的表达水平最为稳定,因此选择RPL11作为后续定量分析的内参基因；通过geNorm计算出最稳定的内参基因组合,即RPL11和BTF3,这为后期的川芎相关基因的表达分析提供一个可靠的均一化标准。4)利用筛选出的定量PCR引物,分析了BDP,Lea1P,ECP40,C3HC4等Unigene在根茎与叶中的表达模式,结果发现PAL在叶中的表达超过根茎,而BDP,Len1P,ECP40, C3HC4,COMT-1和COMT-2在根茎中的表达超过叶,定量PCR结果与转录组数据分析结果一致。构建川芎中阿魏酸生物合成途径,由于差异表达基因OMT-1(c55080.graph_c0)在根茎中的表达水平是叶中3倍且表达量较高,因此推测COMT-1可能为阿魏酸生物合成的关键酶。更多还原

【Abstract】 Ligusticum chuanxiong Hort was a well-known medicinal herb in china, which belonged to Umbelliferae plant. The dry rhizome of L. chuanxiong, also known as chuanxiong, has been widely used as traditional medicine for the treatment of headache, rheumatic arthralgia, menstrual disorder, swelling pain, cardiovascular and cerebrovascular diseases in China, Japan and Korea for thousands of years. Meanwhile, the rhizome of L. chuanxiong has also been used as the components of cosmetics for improving the skin color and fragrance. There were a variety of significant pharmacological compounds, including ferulic acid, essential oil, terpenoids and alkaloids, reported in the rhizome of L. chuanxiong. It was important to cognize metabolic pathways of active components, development and resistance mechanism by transcriptome sequencing of L.chuanxiong. In this study, It was obtained transcriptome data of the rhizome and leaf in L. chuanxiong by Illumina high-throughput sequencing, to complete the analysis and verification, which provided genetic background for exploring key genes of development, stress resistance, active ingredient and metabolic pathways. The main results of this paper were as follows:1) The rhizome and leaf of L. chuanxiong was firstly subjected to transcriptome sequencing and obtained raw data respectively (a total of 5.36 Gb).13,717,234 and 12,823,395 clean reads were generated from the rhizome and leaf (average length was 101 nt), respectively. It eventually won 57,705 unigenes from the rihzome (average length 713 nt) and 50,326 unigenes from leaf (average length 798 nt) by the Trinity software assembly, and then the unigenes were subjected to functional classification and annotation by Nr, Swiss-Prot, GO, COG and KEGG,35,952 and 31,421 unigenes got functional annotation, respectively. In KEGG metabolic pathways,700,659 unigenes was found to involve in secondary metabolic pathways, such as phenylpropanoid biosynthesis, phenylalanine metabolism, glycerophospholipid metabolism, propanoate metabolism, glyoxylate and dicarboxylate metabolism. Some genes had been cloned, which was clear about molecular function in biosynthesis pathway, and some genes were potential candidate genes. It was very helpful to improve the related metabolism by genetic engineering and molecular biology methods.2) To comparative transcriptome of rhizome and leaf in L. chuanxiong, there were totally 4,680 differentially expressed genes between the rhizome and leaf, including 1,714 up-regulated unigenes in rhizome and 2,966 up-regulated unigenes in leaf, meanwhile, to analyze them about GO analysis, COG analysis, transcription factors and so on.3) The suitable reference genes of L. chuanxiong in different tissues were firstly selected and evaluated. Seven conventional were chosen as candidate reference genes to do quantitative analysis from the transcriptome database, like RPLll, ATC7, RPS18, BTF3, CYB5, GAPDH and UBC. By the geNorm, NormFinder software analysing and valuing, RPLll was the most stable genes which would be used as reference genes in L. chuanxiong. And the same time, RPL11 and BTF3 were considered as the the most stable gene combination by geNorm calculating. The results provided a reliable standard for gene expression analysis of L. chuanxiong.4) Expression patterns of some unigenes were analyzed in rhizome and leaf of L.chuanxiong, such as BDP, LealP, ECP40, C3HC4, PAL, COMT-1 COMT-2. The results showed that PAL was up-regulated in leaf, nevertheless, the other genes were up-regulated in rhizome, for instance, BDP, Lea1P, ECP40, C3HC4 and COMT-1, the results of qRT-PCR seemed to be in tune with reaults of transcriptome data analysis. Unigenes related to ferulic acid biosynthesis pathway in L. chuanxiong, because the expression level of COMT-1 (c55080.graph_c0) is higher in rhizome, which was differentially expressed gene, to deduce that COMT-1 could belong to the key enzymes in the biosynthesis of ferulic acid.更多还原