甘蓝型油菜磷脂二酰甘油酰基转移酶(BnPDAT1)表达特性研究
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摘要
为探究甘蓝型油菜中BnaPDAT1基因表达特性与油脂合成之间的关系。选用2个含油量具有显著差异的甘蓝型油菜双低品系855(49. 72%)和868(35. 06%),以qRT-PCR方法检测BnaPDAT1各拷贝在两品系油菜中的表达规律,同时以薄层层析(TLC)和气相色谱(GC)检测两品系油菜中TAG的积累规律。结果表明:授粉后种子中BnaPDAT1基因及其3个拷贝表达量均呈先升高后降低的趋势,花、叶片中均有BnaPDAT1表达,三拷贝表达存在差异,但表现整体调控的特点。两品系油菜叶(Bna A10. PDAT1除外)和授粉后20 d的种子中BnaPDAT1及其3个拷贝表达量具有极显著差异,其余各时期两品系间表达差异规律不明显,BnaPDAT1在高含油量品系855中授粉后20 d表达量为全生育期最高值(11. 100 9),是868的5. 07倍;叶中表达量8. 858 6,为868的7. 34倍,表达特性与Bna C09. PDAT1相似。两品系油菜种子TAG含量变化均呈S型,授粉20 d后油脂合成进入快速增长阶段,授粉后35 d进入缓慢增长期;授粉后30 d以前TAG含量品系间差异不大,授粉后35 d开始出现差异,授粉后40 d差异进一步扩大并趋于稳定。BnaPDAT1基因表达和种子TAG含量变化没有明显的直接关系,但高含油品系中BnaPDAT1基因表达值明显高于低含油品系。
This paper aims to investigate the relationship between the expression characteristics of BnaPDAT1 gene and oil synthesis in Brassica napus L.. Two Brassica napus L. varieties,double-low strains 855( 49. 72%)and 868( 35. 06%),were selected as materials,from which 3 copies of BnaPDAT1 were detected by qRT-PCR.The regularity was expressed,and the accumulation of TAG in the two strains of rapeseed was detected by thin layer chromatography( TLC) and gas chromatography( GC). The results showed that the expression of BnaPDAT1 gene and its three copies in seeds increased first and then decreased after pollination. BnaPDAT1 was expressed in flowers and leaves,but the expression of three copies was different,but showed the characteristics of overall regulation.The expression levels of BnaPDAT1 and its three copies in leaves( except BnaA10. PDAT1) and seeds 20 days after pollination were significantly different between the two strains. The expression patterns of BnaPDAT1 and its three copies were not obvious between the two strains at the other stages. The expression level of BnaPDAT1 in the high oil line 855 reached to the highest value at 20 days after pollination( 11. 100 9),which was 5. 07 times that of line868,and the expression level in leaves was 8. 858 6,7. 34 times that of 868. The expression characteristics was similar to that of BnaC09. PDAT1. The TAG content of rapeseed seeds in both strains presented a S-type. The oil synthesis entered a rapid growth stage after 20 days of pollination,and entered a slow growth period after 35 days of pollination. The TAG content had no significant difference between the two lines at 30 days after pollination,and showed some differences after 35 days of pollination. After 40 days,the difference further expanded and stabilized.There was no significant direct relationship between BnaPDAT1 gene expression and seed TAG content,but the expression level of BnaPDAT1 gene in high oil line was significantly higher than that in low oil line.
This paper aims to investigate the relationship between the expression characteristics of BnaPDAT1 gene and oil synthesis in Brassica napus L.. Two Brassica napus L. varieties,double-low strains 855( 49. 72%)and 868( 35. 06%),were selected as materials,from which 3 copies of BnaPDAT1 were detected by qRT-PCR.The regularity was expressed,and the accumulation of TAG in the two strains of rapeseed was detected by thin layer chromatography( TLC) and gas chromatography( GC). The results showed that the expression of BnaPDAT1 gene and its three copies in seeds increased first and then decreased after pollination. BnaPDAT1 was expressed in flowers and leaves,but the expression of three copies was different,but showed the characteristics of overall regulation.The expression levels of BnaPDAT1 and its three copies in leaves( except BnaA10. PDAT1) and seeds 20 days after pollination were significantly different between the two strains. The expression patterns of BnaPDAT1 and its three copies were not obvious between the two strains at the other stages. The expression level of BnaPDAT1 in the high oil line 855 reached to the highest value at 20 days after pollination( 11. 100 9),which was 5. 07 times that of line868,and the expression level in leaves was 8. 858 6,7. 34 times that of 868. The expression characteristics was similar to that of BnaC09. PDAT1. The TAG content of rapeseed seeds in both strains presented a S-type. The oil synthesis entered a rapid growth stage after 20 days of pollination,and entered a slow growth period after 35 days of pollination. The TAG content had no significant difference between the two lines at 30 days after pollination,and showed some differences after 35 days of pollination. After 40 days,the difference further expanded and stabilized.There was no significant direct relationship between BnaPDAT1 gene expression and seed TAG content,but the expression level of BnaPDAT1 gene in high oil line was significantly higher than that in low oil line.
引文
[1] Zhang M,Fan J,Taylor D C,Ohlrogge J. DGAT1 andPDAT1 acyltransferases have overlapping functions in Ara-bidopsis triacylglycerol biosynthesis and are essential fornormal pollen and seed development[J]. Plant Cell,2009,21(12):3885-3901. doi:10. 1105/tpc. 109. 071795.
[2] Stahl U,Carlsson A S,Lenman M,Dahlqvist A,HuangB,Banas W,Banas A,Stymne S. Cloning and functionalcharacterization of a Phospholipid:Diacylglycerol acyl-transferase from Arabidopsis[J]. Plant Physiology,2004,135(3):1324-1335. doi:10. 1104/pp. 104. 044354.
[3] Mhaske M,Beljilali K,Ohlrogge J,Pollard M. Isolationand characterization of an Arabidopsis thaliana knockoutline for phospholipid:diacylglycerol transacylase gene(At5g-13640)[J]. Plant Physiol Biochem,2005,43:413-417.
[4] Fan J,Yan C,Roston R,Shanklin J,Xu C. Arabidopsislipins,PDAT1 acyltransferase,and SDP1 triacylglycerollipase synergistically direct fatty acids towardβ-oxida-tion,thereby maintaining membrane lipid homeostasis[J]. Plant Cell,2014,26(10):4119-4134. doi:10.1105/tpc. 114. 130377.
[5] Froissard M,D'andréa S,Boulard C,Chardot T. Heterol-ogous expression of At Clo1,a plant oil body protein,in-duces lipid accumulation in yeast[J]. FEMS Yeast Re-search,2009,9(3):428-438.
[6] Fan J L,Yan C S,Zhang X E,Xu C. Dual role forphospholipid:diacylglycerol acyltransferase:enhancingfatty acid synthesis and diverting fatty acids from mem-brane lipids to triacylglycerol in arabidopsis leaves[J].Plant Cell,2013,25(9):3506-3518. doi:10. 1105/tpc. 113. 117358.
[7] Pan X,Siloto R M,Wickramarathna A D,MietkiewskaE,Weselake R J. Identification of a pair of phospholip-id:diacylglycerol acyltransferases from developing flax(Linum usitatissimum L.)seed catalyzing the selectiveproduction of trilinolenin[J]. The Journal of BiologicalChemistry,2013,288(33):24173-24188. doi:10.1074/jbc. M113. 475699.
[8] Kim H U,Lee K R,Go Y S,Jung J H,Suh M C,KimJ B. Endoplasmic reticulum-located PDAT1-2 from castorbean enhances hydroxy fatty acid accumulation in trans-genic plants[J]. Plant&Cell Physiology,2011,52(6):983-993. doi:10. 1093/pcp/pcr051.
[9] Li R,Yu K,Hatanaka T,Hildebrand D F. VemoniaDGATs increase accumulation of epoxy fatty acids in oil[J]. Plant Biotechnology Journal,2010,8(2):184-195.
[10] Xu Y,Caldo K M P,Pal-Nath D,Ozga J,Lemieux MJ,Weselake R J,Chen G. Properties and Biotechnolog-ical Applications of Acyl-CoA. Diacylglycerol acyltrans-ferase and phospholipid:diacylglycerol acyltransferasefrom terrestrial plants and microalgae[J]. Lipids,2018,53(7):663-688.
[11] Yuan L,Mao X,Zhao K,Ji X,Ji C,Xue J,Li R.Characterisation of phospholipid:diacylglycerol acyl-transferases(PDATs)from Camelina sativa and theirroles in stress responses[J]. Biology Open,2017,6(7):1024-1034. doi:10. 1242/bio. 026534.
[12] Guan R,Li X,Hofvander P,Zhou X R,Wang D,Stymne S,Zhu L H. RNAi targeting putative genes inphosphatidylcholine turnover results in significant changein fatty acid composition in Crambe abyssinica seed oil[J]. Lipids,2015,50(4):407-416.
[13] Bana's W,Sanchez Garcia A,Banas'A,Banas'A,Stymne S. Activities of acyl-CoA:diacylglycerol acyl-transferase(DGAT)and phospholipid:diacylglycerolacyltransferase(PDAT)in microsomal preparations ofdeveloping sunflower and safflower seeds[J]. Planta,2013,237(6):1627-1636. doi:10. 1007/s00425-013-1870-8.
[14] Zhu Z,Yuan G,Fan X,Yang M,Yin Y,Liu J,LiuY,Cao X,Tian J,Xue S. The synchronous TAG pro-duction with the growth by the expression of chloroplastTransit peptide-fused Sc PDAT in Chlamydomonas rein-hardtii[J]. Biotechnology for Biofuels,2018,11:156.doi:10. 1186/s13068-018-1160-6.
[15]穆娇.白菜型油菜PDAT1基因的克隆与功能分析[D].杨凌:西北农林科技大学,2015.Mu J. Cloning and functional analysis of PDAT1 gene inBrassica napus L.[D]. Yang Ling:Northwest A&F U-niversity,2015.
[16]谭太龙,冯韬,罗海燕,彭烨,刘睿洋,官春云.甘蓝型油菜磷脂二酰甘油酰基转移酶(Bn PDAT1)c D-NA的克隆和功能鉴定[J].作物学报,2016,42(5):658-666.Tan T L,Feng T,Luo H Y,Peng Y,Liu R Y,Guan CY. Cloning and characterization of phospholipids:diacyl-glycerol acyltransferase(Bn PDAT1)c DNA from Brassicanapus L.[J]. Acta Agronomica Sinica,2016,42(5):658-666.
[17]陈钧辉.生物化学实验[M].北京:科学出版社,2003.Chen J H. Biochemistry experiment[M]. Beijing:Sci-ence Press,2003.
[18] Burja A M,Armenta R E,Radianingtyas H,Barrow CJ. Evaluation of fatty acid extraction methods for Thraus-tochytrium sp. ONC-T18[J]. Journal of Agriculturaland Food Chemistry,2007,55(12):4795-4801.doi:10. 1021/jf070412s.
[19]姚金保,许才康.甘蓝型黄籽油菜种子发育过程中若干特性的研究[J].中国油料,1995,17(3):4-6.Yao J B,Xu C K. Studies on some characteristics ofseed development of Brassica napus L.[J]. ChineseOilseeds,1995,17(3):4-6.
[20] Del age G,Combet C,Blanchet C,Geourjon C. AN-THEPROT:an integrated protein sequence analysis soft-ware with client/server capabilities[J]. Computers inBiology and Medicine,2001,31(4):259-267.
[21]徐赫,潘丽娟,陈娜,陈明娜,王冕,王通,禹山林,梁成伟,迟晓元.磷脂二酰甘油酰基转移酶(PDAT)基因的克隆与表达分析[J].花生学报,2018,47(4):33-40.Xu H,Pan L J,Chen N,Chen M N,Wang M,WangT,Yu S L,Liang C W,Chi X Y. Cloning and expres-sion analysis of two phospholipid:diacylglycerol acyl-transferase genes in Peanut[J]. Journal of Peanut Sci-ence,2018,47(4):33-40.
[22]赵广,宋志波,刘美兰,龙洪旭,李泽,张琳.油茶Co PDAT基因的克隆与表达分析[J].植物生理学报,2017,53(9):1619-1628. doi:CNKI:SUN:ZWSL. 0. 2017-09-006.Zhao G,Song Z B,Liu M L,Long H X,Li Z,ZhangL. Cloning and expression analysis of a phospholipid:di-acylglycerol acyltransferase(PDAT)gene in Camelliaoleifera[J]. Plant Physiology Journal 2017,53(9):1619-1628. doi:CNKI:SUN:ZWSL. 0. 2017-09-006.
[23]周雅莉,安茜,任文燕,李璐,赵静,王计平.紫苏Pf PDAT1基因序列及表达特性分析[J].山西农业大学学报(自然科学版),2018,38(12):44-49.Zhou Y L,An X,Ren W Y,Li L,Zhao J,Wang J P.Analysis of Pf PDAT1 gene sequence and expressioncharacteristics in Perilla frutescens[J]. Journal ofShanxi Agricultural University(Natural Science Edi-tion),2018,38(12):44-49.
[24] Stahl U,Carlsson A S,Lenman M,Dahlqvist A,HuangB,Banas W,Banas A,Stymne S. Cloning and func-tional characterization of a phospholipid:diacylglycerolacyltransferase from Arabidopsis[J]. Plant Physiol,2004,135(3):1324-1335.
[25]虢慧,贺慧,吴宁柔,官梅.不同关键酶基因在油菜种子发育进程中的表达[J].分子植物育种,2017,15(8):3030-3035.Guo H,He H,Wu N R,Guan M. Expression of differ-ent key enzyme gene during seeds development in rape-seed(Brassica napus L.)[J]. Molecular Plant Breed-ing,2017,15(8):3030-3035.
[2] Stahl U,Carlsson A S,Lenman M,Dahlqvist A,HuangB,Banas W,Banas A,Stymne S. Cloning and functionalcharacterization of a Phospholipid:Diacylglycerol acyl-transferase from Arabidopsis[J]. Plant Physiology,2004,135(3):1324-1335. doi:10. 1104/pp. 104. 044354.
[3] Mhaske M,Beljilali K,Ohlrogge J,Pollard M. Isolationand characterization of an Arabidopsis thaliana knockoutline for phospholipid:diacylglycerol transacylase gene(At5g-13640)[J]. Plant Physiol Biochem,2005,43:413-417.
[4] Fan J,Yan C,Roston R,Shanklin J,Xu C. Arabidopsislipins,PDAT1 acyltransferase,and SDP1 triacylglycerollipase synergistically direct fatty acids towardβ-oxida-tion,thereby maintaining membrane lipid homeostasis[J]. Plant Cell,2014,26(10):4119-4134. doi:10.1105/tpc. 114. 130377.
[5] Froissard M,D'andréa S,Boulard C,Chardot T. Heterol-ogous expression of At Clo1,a plant oil body protein,in-duces lipid accumulation in yeast[J]. FEMS Yeast Re-search,2009,9(3):428-438.
[6] Fan J L,Yan C S,Zhang X E,Xu C. Dual role forphospholipid:diacylglycerol acyltransferase:enhancingfatty acid synthesis and diverting fatty acids from mem-brane lipids to triacylglycerol in arabidopsis leaves[J].Plant Cell,2013,25(9):3506-3518. doi:10. 1105/tpc. 113. 117358.
[7] Pan X,Siloto R M,Wickramarathna A D,MietkiewskaE,Weselake R J. Identification of a pair of phospholip-id:diacylglycerol acyltransferases from developing flax(Linum usitatissimum L.)seed catalyzing the selectiveproduction of trilinolenin[J]. The Journal of BiologicalChemistry,2013,288(33):24173-24188. doi:10.1074/jbc. M113. 475699.
[8] Kim H U,Lee K R,Go Y S,Jung J H,Suh M C,KimJ B. Endoplasmic reticulum-located PDAT1-2 from castorbean enhances hydroxy fatty acid accumulation in trans-genic plants[J]. Plant&Cell Physiology,2011,52(6):983-993. doi:10. 1093/pcp/pcr051.
[9] Li R,Yu K,Hatanaka T,Hildebrand D F. VemoniaDGATs increase accumulation of epoxy fatty acids in oil[J]. Plant Biotechnology Journal,2010,8(2):184-195.
[10] Xu Y,Caldo K M P,Pal-Nath D,Ozga J,Lemieux MJ,Weselake R J,Chen G. Properties and Biotechnolog-ical Applications of Acyl-CoA. Diacylglycerol acyltrans-ferase and phospholipid:diacylglycerol acyltransferasefrom terrestrial plants and microalgae[J]. Lipids,2018,53(7):663-688.
[11] Yuan L,Mao X,Zhao K,Ji X,Ji C,Xue J,Li R.Characterisation of phospholipid:diacylglycerol acyl-transferases(PDATs)from Camelina sativa and theirroles in stress responses[J]. Biology Open,2017,6(7):1024-1034. doi:10. 1242/bio. 026534.
[12] Guan R,Li X,Hofvander P,Zhou X R,Wang D,Stymne S,Zhu L H. RNAi targeting putative genes inphosphatidylcholine turnover results in significant changein fatty acid composition in Crambe abyssinica seed oil[J]. Lipids,2015,50(4):407-416.
[13] Bana's W,Sanchez Garcia A,Banas'A,Banas'A,Stymne S. Activities of acyl-CoA:diacylglycerol acyl-transferase(DGAT)and phospholipid:diacylglycerolacyltransferase(PDAT)in microsomal preparations ofdeveloping sunflower and safflower seeds[J]. Planta,2013,237(6):1627-1636. doi:10. 1007/s00425-013-1870-8.
[14] Zhu Z,Yuan G,Fan X,Yang M,Yin Y,Liu J,LiuY,Cao X,Tian J,Xue S. The synchronous TAG pro-duction with the growth by the expression of chloroplastTransit peptide-fused Sc PDAT in Chlamydomonas rein-hardtii[J]. Biotechnology for Biofuels,2018,11:156.doi:10. 1186/s13068-018-1160-6.
[15]穆娇.白菜型油菜PDAT1基因的克隆与功能分析[D].杨凌:西北农林科技大学,2015.Mu J. Cloning and functional analysis of PDAT1 gene inBrassica napus L.[D]. Yang Ling:Northwest A&F U-niversity,2015.
[16]谭太龙,冯韬,罗海燕,彭烨,刘睿洋,官春云.甘蓝型油菜磷脂二酰甘油酰基转移酶(Bn PDAT1)c D-NA的克隆和功能鉴定[J].作物学报,2016,42(5):658-666.Tan T L,Feng T,Luo H Y,Peng Y,Liu R Y,Guan CY. Cloning and characterization of phospholipids:diacyl-glycerol acyltransferase(Bn PDAT1)c DNA from Brassicanapus L.[J]. Acta Agronomica Sinica,2016,42(5):658-666.
[17]陈钧辉.生物化学实验[M].北京:科学出版社,2003.Chen J H. Biochemistry experiment[M]. Beijing:Sci-ence Press,2003.
[18] Burja A M,Armenta R E,Radianingtyas H,Barrow CJ. Evaluation of fatty acid extraction methods for Thraus-tochytrium sp. ONC-T18[J]. Journal of Agriculturaland Food Chemistry,2007,55(12):4795-4801.doi:10. 1021/jf070412s.
[19]姚金保,许才康.甘蓝型黄籽油菜种子发育过程中若干特性的研究[J].中国油料,1995,17(3):4-6.Yao J B,Xu C K. Studies on some characteristics ofseed development of Brassica napus L.[J]. ChineseOilseeds,1995,17(3):4-6.
[20] Del age G,Combet C,Blanchet C,Geourjon C. AN-THEPROT:an integrated protein sequence analysis soft-ware with client/server capabilities[J]. Computers inBiology and Medicine,2001,31(4):259-267.
[21]徐赫,潘丽娟,陈娜,陈明娜,王冕,王通,禹山林,梁成伟,迟晓元.磷脂二酰甘油酰基转移酶(PDAT)基因的克隆与表达分析[J].花生学报,2018,47(4):33-40.Xu H,Pan L J,Chen N,Chen M N,Wang M,WangT,Yu S L,Liang C W,Chi X Y. Cloning and expres-sion analysis of two phospholipid:diacylglycerol acyl-transferase genes in Peanut[J]. Journal of Peanut Sci-ence,2018,47(4):33-40.
[22]赵广,宋志波,刘美兰,龙洪旭,李泽,张琳.油茶Co PDAT基因的克隆与表达分析[J].植物生理学报,2017,53(9):1619-1628. doi:CNKI:SUN:ZWSL. 0. 2017-09-006.Zhao G,Song Z B,Liu M L,Long H X,Li Z,ZhangL. Cloning and expression analysis of a phospholipid:di-acylglycerol acyltransferase(PDAT)gene in Camelliaoleifera[J]. Plant Physiology Journal 2017,53(9):1619-1628. doi:CNKI:SUN:ZWSL. 0. 2017-09-006.
[23]周雅莉,安茜,任文燕,李璐,赵静,王计平.紫苏Pf PDAT1基因序列及表达特性分析[J].山西农业大学学报(自然科学版),2018,38(12):44-49.Zhou Y L,An X,Ren W Y,Li L,Zhao J,Wang J P.Analysis of Pf PDAT1 gene sequence and expressioncharacteristics in Perilla frutescens[J]. Journal ofShanxi Agricultural University(Natural Science Edi-tion),2018,38(12):44-49.
[24] Stahl U,Carlsson A S,Lenman M,Dahlqvist A,HuangB,Banas W,Banas A,Stymne S. Cloning and func-tional characterization of a phospholipid:diacylglycerolacyltransferase from Arabidopsis[J]. Plant Physiol,2004,135(3):1324-1335.
[25]虢慧,贺慧,吴宁柔,官梅.不同关键酶基因在油菜种子发育进程中的表达[J].分子植物育种,2017,15(8):3030-3035.Guo H,He H,Wu N R,Guan M. Expression of differ-ent key enzyme gene during seeds development in rape-seed(Brassica napus L.)[J]. Molecular Plant Breed-ing,2017,15(8):3030-3035.
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