摘要
菘蓝(Isatis indigotica Fort.),十字花科二年生草本。其根入药为板蓝根,其叶入药为大青叶,有清热解毒、凉血消斑、利咽止痛的功效,临床用于流行性感冒、流行性乙型脑炎、流行性腮腺炎、急慢性肝炎、带状疱疹等。前期研究表明,落叶松脂素和落叶松脂素苷正是我国传统中药板蓝根发挥抗病毒功效的重要活性物质基础,然而其含量相对较低。到目前为止,菘蓝中木脂素生源合成途径研究很少,该途径上的一些重要的酶基因尚未得到分离。
本研究采用cDNA末端快速扩增(Rapid amplification of cDNA ends, RACE)技术,首次从菘蓝植物中克隆得到落叶松脂素生源合成途径的五个关键酶基因：肉桂酸-4-羟基化酶(cinnamic acid 4-hydroxylase, C4H)、4-香豆酸：辅酶A连接酶(4-coumarate coenzyme A ligase,4CL)、肉桂酰辅酶A还原酶(cinnamoyl-CoA reductase, CCR)、肉桂醇脱氢酶(cinnamyl alcohol dehydrogenase, CAD)、UDP-葡萄糖基转移酶(UDP-glucosyltransferase, UGT)的全长cDNA序列和基因组DNA序列。对相关基因进行了蛋白结构的预测和初步的生物信息学分析,对Iiccr进行了原核表达的功能分析。
Iic4h基因：肉桂酸-4-羟基化酶(C4H, EC 1.14.13.11)属于细胞色素单加氧酶P450超家族,是公共苯丙烷途径中的第二个关键酶,催化反式肉桂酸转变为p-羟基肉桂酸。其全长cDNA为1674 bp (GenBank登录号：GU014562),包含一个1530 bp的开放阅读框(Open reading frame, ORF),编码509个氨基酸残基。对应的基因组分析表明,lic4h含两个内含子,三个外显子。在根茎叶各器官中均有表达,其中根中最多。考察了各种刺激因素对Iic4h表达的影响,结果显示紫外照射(Ultraviolet-B radiation, UV-B)、茉莉酸甲酯(Methyl jasmonate, MeJA)、脱落酸(abscisic acid,ABA)和赤霉素(Gibberellins, GA3)在一定程度上能够诱导Iiccr的表达上调。
Iiccr基因：肉桂酰辅酶A还原酶(CCR, EC 1.2.1.44)是木脂素特异合成途径的第一个关键酶,可催化3种羟基肉桂酸的辅酶A酯的还原成相应的肉桂醛。其cDNA序列全长1368 bp (GenBank登录号：GQ872418),包含长度为79 bp的5’非编码区(Untranslated Regions, UTR),263 bp的3’UTR,包含一个完整的1026 bp的ORF,编码341个氨基酸残基。对应的基因组分析表明,Iiccr无内含子。该基因在菘蓝中为组成型表达,且根中的表达最高。考察了各种刺激因素对Iiccr表达的影响,结果显示UV-B、MeJA和GA3在一定程度能够诱导Iiccr的表达上调,而ABA却能够抑制Iiccr的表达。构建其原核表达载体pET-32a(+),利用大肠杆菌E.coli BL21(DE3)表达其重组蛋白。
Iiugt基因：UDP-葡萄糖基转移酶(UGT, EC 2.4.1.91)催化相关的木脂素单体成苷。其全长cDNA为1576 bp(GenBank登录号：GU434222),包含一个1431 bp的ORF,编码476个氨基酸残基。对应的基因组分析表明,Iigut无内含子。
Ii4cl基因：4-香豆酸：辅酶A连接酶(4CL, EC 6.2.1.12)是苯丙烷代谢途径的最后一个酶,在苯丙烷代谢途径中处于终端位置,催化4-香豆酸生成4-香豆酰辅酶A。其cDNA全长为1967bp (GenBank登录号：GU937875),包含一个1632 bp的ORF,编码543个氨基酸残基。对应的基因组分析表明,Ii4cl含有五个内含子,六个外显子。
Iicad基因：肉桂醇脱氢酶(CAD, EC 1.1.195)催化肉桂醛生成肉桂醇,其cDNA全长为1402 bp (GenBank登录号：GU937874),包含一个1083 bp的ORF,编码360个氨基酸残基。对应的基因组分析表明,Iicad含有三个内含子,四个内含子。
菘蓝中木脂素生源合成途径五个关键酶基因的克隆与特征分析,为全面阐述菘蓝中木脂素类化合物的代谢流特征,开展植物次生代谢基因工程提高菘蓝中抗病毒活性成分含量打下良好基础。
Isatis indigotica Fort. has been used in traditional Chinese medicine since ancient times. Its leaves and roots, known as Daqingye and Banlangen, respectively, are frequently used as anti-leukemia, antipyretic, anti-inflammatory and anti-influenza agents. By investigation on its chemical constituents and antivirus activity study, we found that the lignans inⅠ. indigotica, including Lariciresinol and Larch Lignan glycosides, showed significant antivirus activity. However, their content inⅠ. indigotica is very low. Until now, there is few research report on the lignan biosynthesis inⅠ. indigotica, and many important enzymes involved in the pathway have not been isolated.
In the present study, five genes involved in the lignan biosynthesis pathway inⅠ. indigotica have been firstly cloned, using the method of rapid amplification of cDNA ends (RACE). Characterization, structure prediction and bioinformatics analysis of these five novel genes, as well as expression of Iiccr in E.coli were performed and reported as follows:
1. Iic4h gene:Cinnamate 4-hydroxylase (C4H, EC 1.14.13.11) is the first P450 upstream in the phenylpropanoid pathway, which catalyses the hydroxylation of cinnamic acid to coumaric acid. The full-length cDNA of Iic4h was 1368 bp (GenBank accession no. GQ872418) long with an open reading frame (ORF) of 1026 bp encoding a polypeptide of 341 amino acid residues. The comparison of Ii4h genomic DNA sequences and Iic4h cDNA sequence revealed that the Iic4h genomic DNA contained two introns. Southern-blot analysis indicated that Iic4h is a multiple copy gene. Iic4h expression could be detected in all tissues at different expression levels, with the strongest expression in roots. Further expression analysis revealed that the signaling components of defense/stress pathways, such as ultraviolet-B radiation (UV-B), methyl jasmonate (MeJA), abscisic acid (ABA)and Gibberellins (GA3) could up-regulate the Iic4h transcript levels compared with the control.
2. Iiccr gene:Cinnamoyl coenzyme A reductase (CCR, EC 1.2.1.44), one of the key enzymes in the biosynthesis of lignins monomers, catalyzes the NADPH-dependent reduction of cinnamoyl-CoA esters to their corresponding cinnamaldehydes. The full-length cDNA of Iiccr was 1674 bp (GenBank accession no. GU014562) long with an ORF of 1233 bp encoding a polypeptide of 411 amino acid residues. The analyses of genomic DNA sequences revealed that Iiccr cDNA sequence and the genomic sequence were identical, which indicated that the Iiccr gene did not contain introns. Southern-blot analysis indicated that Iiccr is a multiple copy gene.IiCCR expression could be detected in all tissues at different expression levels, with the strongest expression in roots. Further expression analysis revealed that UV-B, MeJA and GA3 could up-regulate the Iiccr transcript levels compared with the control, while ABA down-regulate the Iiccr transcript levels. The expression construct of Iiccr with pET-32a(+) was transformed into E.coli BL21(DE3), and the recombinant IiCCR protein was detected by SDS-PAGE analysis.
3.Iiugt gene:UDP-glucosyltransferase (UGT, EC 2.4.1.91) catalyzes the addition of the glycosyl group from a UTP-sugar to a small hydrophobic molecule. The full-length cDNA of liugt was 1576 bp (GenBank accession no. GU434222) long with an ORF of 1431 bp encoding a polypeptide of 476 amino acid residues. The analyses of genomic DNA sequences revealed that Iiugt cDNA sequence and the genomic sequence were identical, which indicated that the liugt gene did not contain introns.
4.1i4cl gene:4-coumarate:CoA ligase (4CL, EC 6.2.1.12) is the last enzyme of the general phenylpropanoid pathway, which catalyzes the conversion of 4-coumarate and other 4-hydroxycinnamates to their corresponding CoA thiol esters. The full-length cDNA of Ii4cl was 1967 bp (GenBank accession no. GU937875) long with an ORF of 1632 bp encoding a polypeptide of 543 amino acid residues. The comparison of Ii4cl genomic DNA sequences and Ii4cl cDNA sequence revealed that the Ii4cl genomic DNA contained five introns.
5.Iicad gene:Cinnamyl alcohol dehydrogenase (CAD, EC1.1.195) catalyzes the synthesis of cinnamyl alcohols from corresponding cinnamaldehydes. The full-length cDNA of Iicad was 1402 bp (GenBank accession no. GU937874) long with an ORF of 1083 bp encoding a polypeptide of 360 amino acid residues. The comparison of Iicad genomic DNA sequences and Iicad cDNA sequence revealed that the Iicad genomic DNA contained three introns.
Five key genes involved in the lignan biosynthesis pathway were isolated from medicinal plant 1. indigotica, and their stuctural and bioinformatic analyses were carried out, which will help us to further illuminate this pathway. The research also provides a possibility to elevate the content of lignans inⅠ. indigotica by plant metabolic engineering.
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