能源植物香叶树FatB基因cDNA全长的克隆及表达分析
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摘要
香叶树CLindera communis)别名香果树,属樟科山胡椒属常绿阔叶乔木或灌木,广泛分布在我国南方,可作为园林绿化树种,具有改良土壤、保持水土等生态价值,果实和种子富含油脂,是提取月桂酸,生产生物柴油的良好原料,在我国华南、西南地区有着巨大的开发和应用前景。利用分子生物学技术,挖掘香叶树果实脂肪酸生物合成相关酶基因并进行功能鉴定,有利于香叶树品种改良与利用。在此之前香叶树的研究仅限于脂肪酸成分、生物柴油特性、育苗、挥发物、生态功能、造林等基础层面,分子生物学基础研究还是空白。脂酰-酰基载体蛋白硫脂酶(fatty acyl-ACP thioesterase, FAT)作为一类植物脂肪酸合成重要调控因子,与植物的脂肪酸的成分及积累息息相关。因此,本研究利用RT-PCR和RACE技术从香叶树的种子中克隆了脂酰-酰基载体蛋白硫脂酶B型(FatB基因),并进行序列生物信息学分析,对FatB基因进行组织特异性表达分析,构建原核表达载体、真核表达载体转化大肠杆菌和拟南芥进行功能鉴定,为将来充分利用这种野生能源植物奠定基础。通过这些研究得到以下结果：
1)由于香叶树分子生物学研究的空白,且其种子中富含脂肪、多糖、多酚等次生代谢产物,RNA提取难度大,我们采用改良的CTAB法成功提取了香叶树种子RNA,可供后续分子生物学实验。
2)比对其他物种的FatB蛋白序列,在保守区设计简并引物,利用RT-PCR和RACE技术首次克隆到香叶树脂肪酸酰基载体蛋白硫酯酶B类型(FatB)基因的cDNA,全长1788bp,开放阅读框长1260bp,基因命名为LcFatB,在NCBI的注册号为：KF543781。
3)序列分析表明LcFatB基因编码419个氨基酸,氨基酸序列分子量为46.09kDa,理论等电点为6.48,LcFatB基因属于Acyl-ACP-TE家族,序列比对发现,LcFatB蛋白序列同其他物种的FatB相似性在61-73%,预测的三个保守催化活性位点为：315位的天冬酰胺、317的组氨酸和352半胱氨酸。FatB蛋白序列进化树分析表明LcFatB蛋白同加州月桂(Umbellularia californica)的UCFatB、香樟(Cinnamomum camphora)的CCFatB蛋白更相似。
4)对LcFatB基因进行组织表达分析显示,LcFatB基因在香叶树的根、叶、茎、花、种子中都有表达,但是种子中具有最高的表达水平,另外LcFatB基因在香叶树开花后30d,45d,60d,75d和90d的种子中也都被检测到,但是花后75d表达水平最高。
5) ChloroP1.1预测LcFatB蛋白有61个氨基酸长度的叶绿体转运肽,去除转运肽之后,将358个氨基酸的成熟多肽与原核表达载体pET-30a连接,转化到删除了脂酰辅酶A脱氢酶(fadE)基因的大肠杆菌表达菌株BL21(DE3),经IPTG诱导,SDS-PAGE分析发现在分子量40.5kDa处有预计大小的蛋白条带。气相色谱分析比较未转化和转化菌的脂肪酸成分发现,转化菌中C12：0占总脂肪酸含量的16.59%,是对照的3.68倍,C10：0也有较大增加,占总脂肪酸的6.28%。
6)将LcFatB基因插入到植物双元表达载体pGreen-0029的多克隆位点,构建重组植物表达载体pGreen-LcFatB,冻融法转化到根瘤农杆菌LBA4404中,花絮侵染法转化拟南芥,经过50ug/mL卡那霉素抗性筛选和PCR鉴定,证实LcFatB基因已经整合到拟南芥的基因组中,并在35S启动子的作用下能够正常的转录和表达,气相色谱分析T2代转基因拟南芥种子脂肪酸组成,发现野生型种子种不存在的C12：0、C10：0脂肪酸,C12：0含量达到2.32mg/g,比野生型高了76.33倍,C10：0含量增加到0.99mg/g。
Lindera communis has another name is Xiangguo and belonging to the family Rubiaceae, it is an evergreen broadleaf arbor or shrub, it is a fast-growing tree species which widely distributed in southern China, can be used as landscaping trees, the plant might serve as a species for conservation of soil and water as well as amelioration of soil. Its fruits and seeds contain rich oil, and they are a good raw material to extract lauric acid and produce biodiesel, it has huge prospects to development and application in China South and Southwest region. Cloning of fatty acid biosynthesis genes and functional identification of L.communis using molecular biology techniques, it help L. communis improve breeds. However, those studies have only addressed its fatty acid composition, biodiesel traits, seedling development, volatile oils, ecological functions, and afforestation, little is known about the regulatory mechanisms involved in the production of the saturated fatty acid rich oil in the developing seeds of L. communis. Molecular biology research on this species remains limited. FatB is a kind of important regulatory factors that biosynthesize fats in plant, and it is closely related to oils accumulation and ingredients. Therefore, we cloned L. communis FatB by RT-PCR and RACE, analyzed sequence structure, issue-specific expression, prokaryotic expression, and eukaryotic expression for functional identification, the main results are as follows:
1) Because L. communis seed contain high oil, polysaccharides, polyphenols and other secondary metabolites, RNA extraction is difficult, no previous experiment can be learn to extract RNA from L. communis, we have used the modified CTAB method to successfully extract L. communis seed RNA, it can be used for follow-up biological experiments.
2) Based on conserved amino acid sequences identified by the alignment of FatB proteins from other plant species, two degenerate oligonucleotide primers were designed, a full-length cDNA of L.communis FatB was cloned using RT-PCR and RACE for the first time, the nucleotide sequence of the cDNA is1788bp with an open reading frame (ORF) of1260bp, named LcFatB, NCBI's registration number is:KF543781.
3) Sequence analysis of LcFatB predicted a polypeptide of419amino acids, calculated molecular mass and predicted isoelectric point is46.09kDa and6.48respectively, LcFatB belongs to the family Acyl-ACP-TE, the deduced amino acid sequence showed61-73% identity to proteins in the FatB class of plant thioesterases, three residues (N315, H317, and C352) that are essential for TE catalytic activity are present in the mature protein, a phylogenetic tree based on the alignment of47TE sequences obtained from GenBank which showed a close relationship between LcFatB, UCFatB and CCFatB from L.communis, U. californica and C. camphora respectively.
4) Expression of the LcFatB gene in developing seeds and vegetative tissues of L.communis was analyzed by qRT-PCR, transcripts were detected in all tissues examined, expression was highest in seeds and lowest in roots, LcFatB transcript levels were higher at75d (day after flowering) DAF than other stages of seed development after flowering30,45,60and90days.
5) Predicted LcFatB protein has61amino acids in length chloroplast transit peptide using ChloroP1.1, after removing the transit peptide, the LcFatB gene was inserted into the T7-based expression vector pET-30a, the recombinant plasmid was transformed into BL21(DE3)△fadE that was deleted acyl-CoA dehydrogenase, SDS-PAGE analysis showed that a band of40.5kDa corresponding to the predicted size of mature protein. Gas chromatography analysis showed contents of the decanoic acid and lauric acid were increased significantly in transgenic bacteria, content of the lauric acid reached16.59%and is higher3.68-fold than control bacteria, content of the decanoic acid reached6.28%.
6) To further understand the metabolic function of LcFatB in plant, the LcFatB gene was inserted into a plant binary expression vector pGreen-0029to construct the recombinant expression vector pGreen-LcFatB, it was transformed into Arabidopsis thaliana via Agrohacterium-mediated transfer methods and overexpressed under the control of the cauliflower mosaic virus (CaMV)35S promoter. The transgenic plants were selected in cultures with50ug/mL kanamycin and PCR tests were conducted to confirm that the LcFatB gene had been successfully integrated into the arabidopsis genome. Gas chromatography analysis of transgenic A. thaliana seed fatty acid composition found C12:0, C10:0increased significantly, reached2.32mg/g and0.99mg/g respectively.
1)由于香叶树分子生物学研究的空白,且其种子中富含脂肪、多糖、多酚等次生代谢产物,RNA提取难度大,我们采用改良的CTAB法成功提取了香叶树种子RNA,可供后续分子生物学实验。
2)比对其他物种的FatB蛋白序列,在保守区设计简并引物,利用RT-PCR和RACE技术首次克隆到香叶树脂肪酸酰基载体蛋白硫酯酶B类型(FatB)基因的cDNA,全长1788bp,开放阅读框长1260bp,基因命名为LcFatB,在NCBI的注册号为：KF543781。
3)序列分析表明LcFatB基因编码419个氨基酸,氨基酸序列分子量为46.09kDa,理论等电点为6.48,LcFatB基因属于Acyl-ACP-TE家族,序列比对发现,LcFatB蛋白序列同其他物种的FatB相似性在61-73%,预测的三个保守催化活性位点为：315位的天冬酰胺、317的组氨酸和352半胱氨酸。FatB蛋白序列进化树分析表明LcFatB蛋白同加州月桂(Umbellularia californica)的UCFatB、香樟(Cinnamomum camphora)的CCFatB蛋白更相似。
4)对LcFatB基因进行组织表达分析显示,LcFatB基因在香叶树的根、叶、茎、花、种子中都有表达,但是种子中具有最高的表达水平,另外LcFatB基因在香叶树开花后30d,45d,60d,75d和90d的种子中也都被检测到,但是花后75d表达水平最高。
5) ChloroP1.1预测LcFatB蛋白有61个氨基酸长度的叶绿体转运肽,去除转运肽之后,将358个氨基酸的成熟多肽与原核表达载体pET-30a连接,转化到删除了脂酰辅酶A脱氢酶(fadE)基因的大肠杆菌表达菌株BL21(DE3),经IPTG诱导,SDS-PAGE分析发现在分子量40.5kDa处有预计大小的蛋白条带。气相色谱分析比较未转化和转化菌的脂肪酸成分发现,转化菌中C12：0占总脂肪酸含量的16.59%,是对照的3.68倍,C10：0也有较大增加,占总脂肪酸的6.28%。
6)将LcFatB基因插入到植物双元表达载体pGreen-0029的多克隆位点,构建重组植物表达载体pGreen-LcFatB,冻融法转化到根瘤农杆菌LBA4404中,花絮侵染法转化拟南芥,经过50ug/mL卡那霉素抗性筛选和PCR鉴定,证实LcFatB基因已经整合到拟南芥的基因组中,并在35S启动子的作用下能够正常的转录和表达,气相色谱分析T2代转基因拟南芥种子脂肪酸组成,发现野生型种子种不存在的C12：0、C10：0脂肪酸,C12：0含量达到2.32mg/g,比野生型高了76.33倍,C10：0含量增加到0.99mg/g。
Lindera communis has another name is Xiangguo and belonging to the family Rubiaceae, it is an evergreen broadleaf arbor or shrub, it is a fast-growing tree species which widely distributed in southern China, can be used as landscaping trees, the plant might serve as a species for conservation of soil and water as well as amelioration of soil. Its fruits and seeds contain rich oil, and they are a good raw material to extract lauric acid and produce biodiesel, it has huge prospects to development and application in China South and Southwest region. Cloning of fatty acid biosynthesis genes and functional identification of L.communis using molecular biology techniques, it help L. communis improve breeds. However, those studies have only addressed its fatty acid composition, biodiesel traits, seedling development, volatile oils, ecological functions, and afforestation, little is known about the regulatory mechanisms involved in the production of the saturated fatty acid rich oil in the developing seeds of L. communis. Molecular biology research on this species remains limited. FatB is a kind of important regulatory factors that biosynthesize fats in plant, and it is closely related to oils accumulation and ingredients. Therefore, we cloned L. communis FatB by RT-PCR and RACE, analyzed sequence structure, issue-specific expression, prokaryotic expression, and eukaryotic expression for functional identification, the main results are as follows:
1) Because L. communis seed contain high oil, polysaccharides, polyphenols and other secondary metabolites, RNA extraction is difficult, no previous experiment can be learn to extract RNA from L. communis, we have used the modified CTAB method to successfully extract L. communis seed RNA, it can be used for follow-up biological experiments.
2) Based on conserved amino acid sequences identified by the alignment of FatB proteins from other plant species, two degenerate oligonucleotide primers were designed, a full-length cDNA of L.communis FatB was cloned using RT-PCR and RACE for the first time, the nucleotide sequence of the cDNA is1788bp with an open reading frame (ORF) of1260bp, named LcFatB, NCBI's registration number is:KF543781.
3) Sequence analysis of LcFatB predicted a polypeptide of419amino acids, calculated molecular mass and predicted isoelectric point is46.09kDa and6.48respectively, LcFatB belongs to the family Acyl-ACP-TE, the deduced amino acid sequence showed61-73% identity to proteins in the FatB class of plant thioesterases, three residues (N315, H317, and C352) that are essential for TE catalytic activity are present in the mature protein, a phylogenetic tree based on the alignment of47TE sequences obtained from GenBank which showed a close relationship between LcFatB, UCFatB and CCFatB from L.communis, U. californica and C. camphora respectively.
4) Expression of the LcFatB gene in developing seeds and vegetative tissues of L.communis was analyzed by qRT-PCR, transcripts were detected in all tissues examined, expression was highest in seeds and lowest in roots, LcFatB transcript levels were higher at75d (day after flowering) DAF than other stages of seed development after flowering30,45,60and90days.
5) Predicted LcFatB protein has61amino acids in length chloroplast transit peptide using ChloroP1.1, after removing the transit peptide, the LcFatB gene was inserted into the T7-based expression vector pET-30a, the recombinant plasmid was transformed into BL21(DE3)△fadE that was deleted acyl-CoA dehydrogenase, SDS-PAGE analysis showed that a band of40.5kDa corresponding to the predicted size of mature protein. Gas chromatography analysis showed contents of the decanoic acid and lauric acid were increased significantly in transgenic bacteria, content of the lauric acid reached16.59%and is higher3.68-fold than control bacteria, content of the decanoic acid reached6.28%.
6) To further understand the metabolic function of LcFatB in plant, the LcFatB gene was inserted into a plant binary expression vector pGreen-0029to construct the recombinant expression vector pGreen-LcFatB, it was transformed into Arabidopsis thaliana via Agrohacterium-mediated transfer methods and overexpressed under the control of the cauliflower mosaic virus (CaMV)35S promoter. The transgenic plants were selected in cultures with50ug/mL kanamycin and PCR tests were conducted to confirm that the LcFatB gene had been successfully integrated into the arabidopsis genome. Gas chromatography analysis of transgenic A. thaliana seed fatty acid composition found C12:0, C10:0increased significantly, reached2.32mg/g and0.99mg/g respectively.
引文
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