甘蓝型油菜BnaLPAT2基因克隆与生物信息学分析及载体构建
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摘要
为进一步研究甘蓝型油菜BnaLPAT2基因,通过TAIR网站查找拟南芥AtLPAT2 (AT3G57650),在Brassica Database数据库检索到4条甘蓝型油菜LPAT2序列,据此设计引物,通过RT-PCR合成c DNA,得到4条CDS序列,其大小依次为1 173 bp、1 167 bp、1 173 bp、1 155 bp,分别命名为BnaLPAT2-1～BnaLPAT2-4。4个BnaLPAT2蛋白通过生物信息学预测分析显示:BnaLPAT2编码的氨基酸在384～390之间,理论分子量介于43 226.59～43 730.17 Da之间。甘蓝型油菜LPAT2蛋白的二级结构和跨膜区大致相同,均由LPLAT superfamily、PlsC superfamily、Acyltransf_C superfamily结构域组成,且具有较近的亲缘关系。从基因结构发现:4个BnaLPAT2均含有相同的外显子和内含子数,分别位于C08、A09、A07和A04染色体上。分析表明,克隆获得的4个甘蓝型油菜BnaLPAT2为该基因家族成员。为进一步研究甘蓝型油菜LPAT2基因的功能,我们通过双酶切技术,构建pBI121-LPAT2-napin载体,并转化根癌农杆菌GV3101。
In order to further study BnaLPAT2 in Brassica napus, the Arabidopsis thaliana AtLPAT2 gene(AT3 G57650) was searched on TAIR website and the LPAT2 sequence of Brassica napus was retrieved from the gene of AtLPAT2 and downloaded from GenBank. According to these sequences information, four pairs of primers were designed to amplify four CDS sequences. The length were 1 173 bp, 1 167 bp, 1 173 bp and 1 155 bp, they were named BnaLPAT2-1～BnaLPAT2-4. The protein encoded by BnaLPAT2 in Brassica napus was predicted by bioinformatics software. The results showed that the amino acids encoded by BnaLPAT2 was between 384 and390, and the theoretical molecular weight was between 43 226.59 and 43 730.17 Da. The secondary structure and transmembrane region of BnaL PAT2 protein in Brassica napus L. are roughly the same, containing LPLAT superfamily, PlsC superfamily and Acyltransf_C superfamily domains, and have close genetic relationship. The gene structure map shows that BnaLPAT2 contains the same exon and intron number, and spreads across four different chromosomes. These results showed that BnaLPAT2-1～4 are the four members of the BnaLPAT2 family.In order to further study the function of LPAT2 gene in Brassica napus, in addition, the BnaLPAT2 gene was successfully ligated into the plant binary expression vector p BI121 by double digestion, and transformed into GV3101.
In order to further study BnaLPAT2 in Brassica napus, the Arabidopsis thaliana AtLPAT2 gene(AT3 G57650) was searched on TAIR website and the LPAT2 sequence of Brassica napus was retrieved from the gene of AtLPAT2 and downloaded from GenBank. According to these sequences information, four pairs of primers were designed to amplify four CDS sequences. The length were 1 173 bp, 1 167 bp, 1 173 bp and 1 155 bp, they were named BnaLPAT2-1～BnaLPAT2-4. The protein encoded by BnaLPAT2 in Brassica napus was predicted by bioinformatics software. The results showed that the amino acids encoded by BnaLPAT2 was between 384 and390, and the theoretical molecular weight was between 43 226.59 and 43 730.17 Da. The secondary structure and transmembrane region of BnaL PAT2 protein in Brassica napus L. are roughly the same, containing LPLAT superfamily, PlsC superfamily and Acyltransf_C superfamily domains, and have close genetic relationship. The gene structure map shows that BnaLPAT2 contains the same exon and intron number, and spreads across four different chromosomes. These results showed that BnaLPAT2-1～4 are the four members of the BnaLPAT2 family.In order to further study the function of LPAT2 gene in Brassica napus, in addition, the BnaLPAT2 gene was successfully ligated into the plant binary expression vector p BI121 by double digestion, and transformed into GV3101.
引文
Arro yo-Caro J.M.,Chileh T.,Kazachkov M.,Zou J.,Alonso D.L.,and García-Maroto F.,2013,The multigene family of lysophosphatidate acyltransferase(LPAT)-related enzymes in Ricinus communis:cloning and molecular characterization of two LPAT genes that are expressed in Castor seeds,Plant Science,199-200:29-40
Chai G.,Bai Z.,Wei F.,King G.J.,Wang C.,Shi L.,Dong C.,Chen H.,and Liu S.,2010,Brassica GLABRA2 genes:analysis of function related to seed oil content and development of functional markers,Theoretical and Applied Genetics,120(8):1597-1610
Chen S.,Lei Y.,Xu X.,Huang J.,Jiang H.,Wang J.,Cheng Z.,Zhang J.,Song Y.,Liao B.,and Li Y.,2015,The peanut(Arachis hypogaea L.)gene AhLPAT2 increases the lipid content of transgenic Arabidopsis see ds,PLoS One,10(8):e0136170
Chen S.L.,Huang J.Q.,Lei Y.,Ren X.P.,Wen Q.G.,Chen Y.N.,Jiang H.F.,Yan L.Y.,and Liao B.S.,2012,Cloning and expression analysis of peanut lysophosphatidic acid acyltransferase gene,Zuowu Xuebao(Acta Botanica Sinica),38(2):245-255(陈四龙,黄家权,雷永,任小平,文奇根,陈玉宁,姜慧芳,晏立英,廖伯寿,2012,花生溶血磷脂酸酰基转移酶基因的克隆与表达分析,作物学报,38(2):245-255)
Howell E.C.,Kearsey M.J.,Jones G.H.,King G.J.,and Armstrong S.J.,2008,A and C genome distinction and chromosome identification in Brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization,Genetics,180(4):1849-1857
Hu Y.P.,2009,Transcript profiling,cloning and comparative functional analysis of genes involved in lipid biosynthesis from oil crops,Dissertation for Ph.D.,Biotechnology Research Institute CAASs,Supervisor:Lu C.M.,pp.1-2(胡亚平,2009,油料作物油脂合成相关基因的转录、克隆和功能比较研究,博士学位论文,中国农业科学院研究生院,导师:卢长明,pp.1-2)
Iskandarov U.,Silva J.E.,Kim H.J.,Andersson M.,Cahoon R.E.,and Mockaitis K.,2017,Specialized diacylglycerol acyltransferase contributes to extreme fatty acid content of cuphea seed oil,Plant Physiology,174(1):97-109
Ke X.,Yi Y.,and Li X.F.,2010,Ectopic expression of Crambe abyssinica lysophosphatidic acid acyltransferase in transgenic rapeseed increases its oil content,African Journal of Biotechnology,9(25):3904-3910
Kim H.U.,2005,Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase,LPAT2,is essential for female but not male gametophyte development in Arabidopsis,The Plant Cell Online,17(4):1073-1089
Ma J.J.,2015,Cloning and genetic transformation of LPAATgene in upland cotton(Gossypium hirsutum L.),Thesis for M.S.,Biotechnology Research Institute CAASs,Supervisor:Yu J.W.,pp.3-4(马建江,2015,棉花LPAAT基因的克隆与遗传转化,硕士学位论文,中国农业科学院研究生院,导师:于霁雯,pp.3-4)
Rao S.S.,and Hildebrand D.,2009,Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene,Lipids,44(10):945-951
Reynolds K.B.,Tay lor M.C.,Cullerne D.P.,Blanchard C.L.,Wood C.C.,Singh S.P.,and Petrie J.R.,2017,A reconfigured Kennedy pathway which promotes efficient accumulation of medium chain fatty acids in leaf oils.Plant Biotechnology Journal,15(11):1397-1408
Sui N.,Tian S.,Wang W.,Wang M.,and Fan H.,2017,Overexpression of glycerol-3-phosphate acyltransferase from Suaeda salsa improves salt tolerance in Arabidopsis,Frontiers in Plant Science,8:1337
Tang S.H.,2013,The roles of glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase in lipid metabolism in Brassica napus,Huazhong Agricultural University,Supervisor:Hong Y.Y.,pp.5-6(唐韶华,2013,油菜甘油磷酸酰基转移酶和溶血磷脂酸酰基转移酶基因的克隆及其在油脂代谢中的作用,硕士学位论文,华中农业大学,导师:洪月云,pp.5-6)
Taylor D.C.,and Giblin E.M.,1995,Microsomal lyso-phosphatidic acid acyltransferase from a Brassica oleracea cultivar incorporates erucic acid into the sn-2 position of seed triacyclglycerols,Plant Physiology,109(2):409-420
Wang J.,Xu R.,Wang R.,Haque M.E.,and Liu A.,2016,Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates,Bioscience Biotechnology and Biochemistry,80(6):1214-1222
Zhang J.F.,Li H.J.,Zhang X.H.,Pu X.B.,Jiang J.,Chai L.,Cui C.,and Jiang L.C.,2013,Genetic diversity and association analysis using SSR markers in Brassica napus L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(5):502-512(张锦芳,李浩杰,张雪花,蒲晓斌,蒋俊,柴靓,崔成,蒋梁材,2013,甘蓝型油菜SSR标记遗传多样性及其与农艺性状关联分析,分子植物育种,11(5):502-512)
Zhan g J.P.,Jiang M.L.,Gong Y.M.,Wan X.,Liang Z.,and Huang F.H.,2012,Effects of LPAAT gene expression on fatty acids in yeast.,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),34(5):483-488(张军平,江木兰,龚阳敏,万霞,梁焯,黄凤洪,2012,三角褐指藻LPAAT基因在酵母中表达对脂肪酸的影响,中国油料作物学报,34(5):483-488)
Zhang Y.,Li Y.C.,Mei D.S.,and Hu Q.,2007,Advances in research on oil and oil in Brassica napus,Zhiwu Xuebao(Acta Botanica Sinica),24(4):435-443(张垚,李云昌,梅德圣,胡琼,2007,油菜油脂研究进展,植物学报,24(4):435-443)
Chai G.,Bai Z.,Wei F.,King G.J.,Wang C.,Shi L.,Dong C.,Chen H.,and Liu S.,2010,Brassica GLABRA2 genes:analysis of function related to seed oil content and development of functional markers,Theoretical and Applied Genetics,120(8):1597-1610
Chen S.,Lei Y.,Xu X.,Huang J.,Jiang H.,Wang J.,Cheng Z.,Zhang J.,Song Y.,Liao B.,and Li Y.,2015,The peanut(Arachis hypogaea L.)gene AhLPAT2 increases the lipid content of transgenic Arabidopsis see ds,PLoS One,10(8):e0136170
Chen S.L.,Huang J.Q.,Lei Y.,Ren X.P.,Wen Q.G.,Chen Y.N.,Jiang H.F.,Yan L.Y.,and Liao B.S.,2012,Cloning and expression analysis of peanut lysophosphatidic acid acyltransferase gene,Zuowu Xuebao(Acta Botanica Sinica),38(2):245-255(陈四龙,黄家权,雷永,任小平,文奇根,陈玉宁,姜慧芳,晏立英,廖伯寿,2012,花生溶血磷脂酸酰基转移酶基因的克隆与表达分析,作物学报,38(2):245-255)
Howell E.C.,Kearsey M.J.,Jones G.H.,King G.J.,and Armstrong S.J.,2008,A and C genome distinction and chromosome identification in Brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization,Genetics,180(4):1849-1857
Hu Y.P.,2009,Transcript profiling,cloning and comparative functional analysis of genes involved in lipid biosynthesis from oil crops,Dissertation for Ph.D.,Biotechnology Research Institute CAASs,Supervisor:Lu C.M.,pp.1-2(胡亚平,2009,油料作物油脂合成相关基因的转录、克隆和功能比较研究,博士学位论文,中国农业科学院研究生院,导师:卢长明,pp.1-2)
Iskandarov U.,Silva J.E.,Kim H.J.,Andersson M.,Cahoon R.E.,and Mockaitis K.,2017,Specialized diacylglycerol acyltransferase contributes to extreme fatty acid content of cuphea seed oil,Plant Physiology,174(1):97-109
Ke X.,Yi Y.,and Li X.F.,2010,Ectopic expression of Crambe abyssinica lysophosphatidic acid acyltransferase in transgenic rapeseed increases its oil content,African Journal of Biotechnology,9(25):3904-3910
Kim H.U.,2005,Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase,LPAT2,is essential for female but not male gametophyte development in Arabidopsis,The Plant Cell Online,17(4):1073-1089
Ma J.J.,2015,Cloning and genetic transformation of LPAATgene in upland cotton(Gossypium hirsutum L.),Thesis for M.S.,Biotechnology Research Institute CAASs,Supervisor:Yu J.W.,pp.3-4(马建江,2015,棉花LPAAT基因的克隆与遗传转化,硕士学位论文,中国农业科学院研究生院,导师:于霁雯,pp.3-4)
Rao S.S.,and Hildebrand D.,2009,Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene,Lipids,44(10):945-951
Reynolds K.B.,Tay lor M.C.,Cullerne D.P.,Blanchard C.L.,Wood C.C.,Singh S.P.,and Petrie J.R.,2017,A reconfigured Kennedy pathway which promotes efficient accumulation of medium chain fatty acids in leaf oils.Plant Biotechnology Journal,15(11):1397-1408
Sui N.,Tian S.,Wang W.,Wang M.,and Fan H.,2017,Overexpression of glycerol-3-phosphate acyltransferase from Suaeda salsa improves salt tolerance in Arabidopsis,Frontiers in Plant Science,8:1337
Tang S.H.,2013,The roles of glycerol-3-phosphate acyltransferase and lysophosphatidic acid acyltransferase in lipid metabolism in Brassica napus,Huazhong Agricultural University,Supervisor:Hong Y.Y.,pp.5-6(唐韶华,2013,油菜甘油磷酸酰基转移酶和溶血磷脂酸酰基转移酶基因的克隆及其在油脂代谢中的作用,硕士学位论文,华中农业大学,导师:洪月云,pp.5-6)
Taylor D.C.,and Giblin E.M.,1995,Microsomal lyso-phosphatidic acid acyltransferase from a Brassica oleracea cultivar incorporates erucic acid into the sn-2 position of seed triacyclglycerols,Plant Physiology,109(2):409-420
Wang J.,Xu R.,Wang R.,Haque M.E.,and Liu A.,2016,Overexpression of ACC gene from oleaginous yeast Lipomyces starkeyi enhanced the lipid accumulation in Saccharomyces cerevisiae with increased levels of glycerol 3-phosphate substrates,Bioscience Biotechnology and Biochemistry,80(6):1214-1222
Zhang J.F.,Li H.J.,Zhang X.H.,Pu X.B.,Jiang J.,Chai L.,Cui C.,and Jiang L.C.,2013,Genetic diversity and association analysis using SSR markers in Brassica napus L.,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),11(5):502-512(张锦芳,李浩杰,张雪花,蒲晓斌,蒋俊,柴靓,崔成,蒋梁材,2013,甘蓝型油菜SSR标记遗传多样性及其与农艺性状关联分析,分子植物育种,11(5):502-512)
Zhan g J.P.,Jiang M.L.,Gong Y.M.,Wan X.,Liang Z.,and Huang F.H.,2012,Effects of LPAAT gene expression on fatty acids in yeast.,Zhongguo Youliao Zuowu Xuebao(Chinese Journal of Oil Crop Sciences),34(5):483-488(张军平,江木兰,龚阳敏,万霞,梁焯,黄凤洪,2012,三角褐指藻LPAAT基因在酵母中表达对脂肪酸的影响,中国油料作物学报,34(5):483-488)
Zhang Y.,Li Y.C.,Mei D.S.,and Hu Q.,2007,Advances in research on oil and oil in Brassica napus,Zhiwu Xuebao(Acta Botanica Sinica),24(4):435-443(张垚,李云昌,梅德圣,胡琼,2007,油菜油脂研究进展,植物学报,24(4):435-443)