摘要
以独行菜(Lepidium apetalum)为材料,通过分析独行菜转录组数据,设计特异性引物,克隆了独行菜肉桂酸-4-羟化酶(cinnamate-4-hydroxylase,C4H)基因的cDNA序列,命名为LaC4H,Gen Bank登录号为KX064050,并进行生物信息学分析,原核表达、纯化,组织特异性分析和诱导表达分析。结果表明:(1)LaC4H基因开放阅读框(ORF)长1 518 bp,编码505个氨基酸,其蛋白质分子质量为57.73 k D。(2)生物信息学分析显示La C4H蛋白包含细胞色素P450的保守基序和5个特征性底物结合位点,是细胞色素P450超家族成员,系统进化分析显示LaC4H蛋白与拟南芥等十字花科植物C4H蛋白同源性较高。(3)通过构建原核表达载体pET-32a-LaC4H在大肠杆菌BL21(DE3)菌株中成功表达LaC4H重组蛋白,利用Ni^(2+)亲和色谱得到了纯化的LaC4H重组蛋白。(4)荧光定量PCR结果表明LaC4H基因在茎中表达量最高,叶和花中次之,根中表达量最低。经茉莉酸甲酯(methyl jasmonate,MeJA)诱导后,叶中LaC4H表达量明显上升,诱导后48 h达到最高值,叶中LaC4H的表达量与黄酮含量之间呈正相关。这为进一步研究LaC4H基因在独行菜黄酮类化合物生物合成途径中的功能奠定基础。
Lepidium apetalum was used as an experimental material in this study. By analyzing the tran- scriptome data of L. apetalum and application of the specific primers, cDNA of cinnamate-4-hydroxylase （C4H） gene was isolated from L. apetalum and named as LaC4H （GenBank accession No. KX064050）. Meanwhile, the bioinformatic analysis, prokaryotic expression, purification, tissue-specific expression analysis and expres- sion analysis after methyl jasmonate （MeJA） treatment were carried out. The results indicated that： （1） The open reading frame （ORF） of LaC4H was 1 518 bp, which encoded a protein of 505 amino acid residues, with a predicted molecular mass of 57.73 kD. （2） Bioinformatic analysis showed that LaC4H protein contained the conserved sequences of cytochrome P450 （CYP450） and 5 substrate recognition sites （SRSs） of CYP73A1, therefore LaC4H protein was a member of CYP450 superfamily. The phylogenetic analysis indicated that LaC4H protein showed the highest homology with C4H protein from cruciferous plants （such as AtC4H fromArabidopsis thaliana）. （3） Through the construction of the prokaryotic expression vector pET-32a-LaC4H, the recombinant LaC4H protein was successfully expressed in E. coli BL21 （DE3） cells and the recombinant LaC4H protein was purified by Ni2＋ affinity chromatography. （4） Real-time PCR analysis indicated that LaC4H was expressed in a high transcript level in stems, lower levels in leaves and flowers, the lowest level in roots. After MeJA treatment, the expression level of LaC4H in leaves was increased significantly to reach the highest level at 48 h. Furthermore, the expression levels of LaC4H were positively correlated with the flavonoids contents in leaves. The results of this study provide the fundamental information on LaC4H gene in the flavonoids biosyn- thesis pathway of L. apetalum.
出处
《药学学报》
CAS
CSCD
北大核心
2017年第5期821-831,共11页
Acta Pharmaceutica Sinica
基金
国家重点基础研究发展计划"973计划"项目(2013CB531802)
河南省科技攻关计划(162102310468)
河南中医学院博士科研基金(BSJJ2011-07)
