| 摘要: |
| 【目的】分别克隆甘蓝型油菜（Brassica napus L.）和白菜型油菜（Brassica rapa L.）肉桂酰辅酶A还原酶1（cinnamoyl-CoA reductase 1，CCR1）基因，并进行生物信息学和表达模式分析。【方法】基于甘蓝型油菜和白菜型油菜转录组测序信息，分别从contig文库中获得了一个CCR1基因mRNA转录本片段，利用RACE技术分别获得一个CCR1基因的cDNA全长，对其进行生物学分析，并用实时定量PCR方法分析CCR1基因在甘蓝型和白菜型油菜开花期根、茎、叶、花中的表达差异。【结果】所克隆的甘蓝型油菜和白菜型油菜CCR1基因序列经同源序列比对分析后，将其分别命名为BnCCR1(登录号：KX138521)和BrCCR1(登录号：KX138522)。BnCCR1全长1 388 bp，开放阅读框（ORF）长为1 032 bp，编码343个氨基酸，分子质量11.56 ku，等电点4.99；BrCCR1全长1 366 bp，ORF长为1 026 bp，编码341个氨基酸，分子质量为11.36 ku，等电点5.0。2物种CCR1编码蛋白质二级结构无信号肽和跨膜结构域；亚细胞定位显示该蛋白在细胞质中存在的可能性最大。CCR蛋白多重比对分析显示，BnCCR1和BrCCR1均具有CCR蛋白典型的一个NAD(P)结合域和一个底物结合域(NWYCY)。系统进化树分析结果表明，BnCCR1和BrCCR1与同属十字花科的菘蓝、亚麻荠、拟南芥CCR形成了一个独立分支，且亲缘关系较近；其蛋白质三级结构均与矮牵牛CCR1（PDB:4r1t.1A）蛋白质三级结构相似，结构稳定。实时荧光定量PCR分析结果表明，CCR1基因在白菜型油菜和甘蓝型油菜根、茎、叶、花各器官中均有表达，其中在木质化程度较高的根和茎中表达丰度明显高于叶和花。【结论】从白菜型油菜和甘蓝型油菜中分别克隆到CCR1基因cDNA全长，该基因主要在木质化程度较高的根和茎器官中表达。 |
| 关键词: 甘蓝型油菜 白菜型油菜 肉桂酰辅酶A还原酶1 基因表达 生物信息学分析 |
| DOI： |
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| 基金项目:国家自然科学基金项目（31560362）；西藏特色农牧资源研发协同创新中心-作物研究方向（XZXTCX-2015-03，XBTSZWXK-2015-01） |
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| Cloning and expression of cinnamoyl-CoA reductase 1（CCR1） gene of Brassica napus L. and Brassica rapa L. |
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HOU Weihai,WANG Jianlin,DAN Ba,et al
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| Abstract: |
| 【Objective】The research was conducted to clone the cinnamoy-CoA reductase (CCR1) gene of Brassica napus L.and Brassica rapa L.for bioinformatics and expression analysis.【Method】Two mRNAs segments encoding cinnamoyl-CoA reductase (CCR) of Brassica napus L.and Brassica rapa L.were retrieved respectively by screening contig library of published transcriptome database,and full-length cDNAs were cloned by rapid amplification of cDNA ends (RACE),followed by bioinformatics analysis.The differences in expression in root,stem,leaf and flower of these two rapes during blooming period were analyzed by real time PCR.【Result】 The two cloned sequences were named BnCCR1 and BrCCR1 after homologous sequence alignment.BnCCR1 is a 1 388 bp full-length cDNA with a 1 032 bp open reading frame (ORF) and was predicted to encode a protein of 343 amino acids.The predicted protein would have the molecular weight of 11.56 ku and the PI of 4.99.BrCCR1 is a 1 366 bp full-length cDNA with a 1 026 bp ORF and encods a 341 amino acid polypeptide with the molecular weight of 11.36 ku and the PI of 5.0.Secondary structures of these two proteins were composed of mutiple α-helixes,β-folds and random coils,without transmembrane regions or signal peptides.The protein most likely exists in chloroplast according to subcellular localization analysis.The multiple comparison revealed that both BnCCR and BrCCR1 contained a NAD(P) binding domain and a substrate binding domain (NWYCY) typical to CCRs.Phylogenetic tree analysis showed that BnCCR1 and BrCCR1 shared a separate clade with AtCCR1,CsCCR1,and ItCCR and had a close relationship.The 3-D alignment of CCR1 of Brassica napus and Brassica rapa showed high similarity to Petunia hybrida Vilm (POB:4r1t.1A) and had a stable structure.Real-time RT PCR indicated that BnCCR1 and BrCCR1 were expressed in different tissues (root,stem,leaf,and flower) and the level was higher in the highly lignificated root and stem than in leaf and flower.【Conclusion】Two full-length cDNA sequences of CCR1 gene were isolated from Brassica napus L.and Brassica rapa L. and they were mainly expressed in highly lignificated root and stem organs. |
| Key words: Brassica napus L. Brassica rapa L. cinnamoyl-CoA reductase 1 gene expression bioinformation analysis |
