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作物学报 ›› 2014, Vol. 40 ›› Issue (10): 1748-1755.doi: 10.3724/SP.J.1006.2014.01748

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜LPAT4基因的克隆与表达

肖旦望1,刘聪1,胡学芳1,陈社员2,官春云2,熊兴华1,2,*   

  1. 1湖南农业大学 / 作物基因工程湖南省重点实验室, 湖南长沙 410128; 2 湖南农业大学 / 国家油料改良中心湖南分中心, 湖南长沙 410128
  • 收稿日期:2014-03-10 修回日期:2014-07-06 出版日期:2014-10-12 网络出版日期:2014-07-25
  • 通讯作者: 熊兴华, E-mail: ndxiongene@yahoo.com, Tel: 13508487613
  • 基金资助:

    本研究由国家高技术研究发展计划(863计划)项目(2012AA101107-3)和湖南农业大学作物学开发基金资助项目(ZWKF201303)资助。

Cloning and Expression Analysis of LPAT4 Gene from Brassica napus

XIAO Dan-Wang1,LIU Cong1,HU Xue-Fang1,CHEN She-Yuan2,GUAN Chun-Yun2,XIONG Xing-Hua1,2,*   

  1. 1 Crop Gene Engineering Key Laboratory of Hunan Province, Hunan Agricultural University, Changsha 410128, China; 2 Hunan Branch of National Oilseed Crops Improvement Centre, Hunan Agricultural University, Changsha 410128, China
  • Received:2014-03-10 Revised:2014-07-06 Published:2014-10-12 Published online:2014-07-25
  • Contact: 熊兴华, E-mail: ndxiongene@yahoo.com, Tel: 13508487613

摘要:

植物溶血磷脂酸酰基转移酶(lysophospholipid acid actyltransferase, LPAT)是三酰甘油生物合成过程中的一个关键酶, 在脂质的合成、种子的发育以及生物膜的流动性等方面有重要作用。本研究采用同源克隆的方法, 获得LPAT4基因的2条全长CDS序列, 长度分别为1143 bp1140 bp。生物信息学分析表明, 它们均具有LPLAT_LCLAT1样结构域, 同属于LPLAT超基因家族, 并分别被命名为BnLPAT4-1BnLPAT4-2。时空表达分析表明, 它们均为组成型表达基因, 其中BnPAT4-1在叶中的表达量最高, BnPAT4-2在胚中的表达量最高。逆境分析表明, BnLPAT4-1BnLPAT4-2NaClPEG-4000、水渍、6BAABA的胁迫下呈现出不同的表达模式。极差分析显示, ABABnLPAT4-1的表达影响较大, BnLPAT4-2的表达却对PEG4000更敏感。为进一步研究油菜BnLPAT4基因功能奠定了基础。

关键词: LPAT4, 甘蓝型油菜, 时空表达, 非生物逆境, 表达分析

Abstract:

Lysophospholipid acid actyltransferase (LPAT) is a pivotal enzyme of triacylglycerol biosynthesis, which plays a key role in lipid synthesis, development of plant seeds and bio-membrane fluidity. Through the technology of homology-based cloning, two copies of LPAT4 full-length CDS sequences (1143 bp and 1140 bp) were cloned in this study, designated as BnLPAT4-1and BnLPAT4-2, respectively. Bioinformatics analysis revealed that they shared the LPLAT_LCLAT1 like domain and belonged to the LPLAT superfamily. Temporal and spatial expression results showed that BnLPAT4-1 and BnLPAT4-2 were constitutive expression genes. Among them, the highest expression of BnLPAT4-1 was in leaf, while that ofBnLPAT4-2 was in embryo. Stresses analysis indicated that BnLPAT4-1and BnLPAT4-2 presented different expression patterns under the treatments of NaCl, PEG4000, waterlogging, 6BA and ABA. Pole difference analysis displayed that ABA had a great effect on the expression of BnLPAT4-1, while BnLPAT4-2 was more sensitive to PEG-4000.The results provided a base for the research of regulation and function of BnLPAT4 in Brassica napus.

Key words: LPAT4, Brassica Napus, Temporal and spatial expression, Abiotic stress, Expression analysis

[1]Yu W L, Ansari W, Schoepp N G, Hannon M J, Mayfield S P, Burkart M D. Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact, 2011, 10: 1–11



[2]Li Y H, Basil S, Fred B, Mats X. A, Vincent A, Philip D. B, Sébastien B, David B, Allan D, Timothy P. D, Rochus B. F, Ian A. G, Kenta K, AmélieA. K, Tony L, Jonathan E. M, Martine M, Isabel M, Ikuo N, Owen R, Lacey S, Katherine M. S, Hajime W, Ruth W, Xu C C, Rémi Z, John O. Acyl-lipidmetabolism. Am Soc Plant Biol, 2013, 11: 1–70



[3]Baud S, Dubreucq B, Miquel M, Rochat C, Lepiniec L. Storage reserve accumulation in Arabidopsis: metabolic and developmental control of seed filling. Am Soc Plant Biol, 2008, 6: 1–24



[4]Kim H U, Li Y, Huang A H. Ubiquitous and endoplasmic reticulum-located lysophosphatidyl acyltransferase, LPAT2, is essential for female but not male gametophyte development in Arabidopsis. Plant Cell, 2005, 17: 1073–1089



[5]Lopez-Villalobos A, Dodds P F, Hornung R. Changes in fatty acid composition during development of tissues of coconut (Cocos nucifera L.) embryos in the intact nut and in vitro. J Exp Bot, 2001, 52: 933–942



[6]Knutzon D S, Hayes T R, Wyrick A, Xiong H, Maelor D H, Voelker T A. Lysophosphatidic acid acyltransferase from coconut endosperm mediates the insertion of laurate at the sn-2 position of triacylglycerols in lauric rapeseed oil and can increase total laurate levels. Plant Physiol, 1999, 120: 739–746



[7]Knutzon D S, Lardizabal K D, Nelsen J S, Bleibaum J L, Davies H M, Metz J G. Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates. Plant Physiol, 1995, 109: 999–1006



[8]Bourgis F, Kader J C, Barret P, Renard M, Robinson D, Robinson C, Delseny M, Roscoe T J. A plastidial lysophosphatidic acid acyltransferase from oilseed rape. Plant Physiol, 1999, 120: 913–922



[9]Bernerth R, Frentzen M. Utilization of erucoyl-CoA by acyltransferases from developing seeds of Brassica napus (L.) involved in triacylglycerol biosynthesis. Plant Sci, 1990, 67: 21–28



[10]Taylor D C, Barton D L, Giblin E M, Mackenzie S L, Van Den Berg C, Mcvetty P. Microsomal lyso-phosphatidic acid acyltransferase from a Brassica oleracea cultivar incorporates erucic acid into the sn-2 position of seed triacylglycerols. Plant Physiol, 1995, 109: 409–420



[11]Lassner M W, Levering C K, Davies H M, Knutzon D S. Lysophosphatidic acid acyltransferase from meadowfoam mediates insertion of erucic acid at the sn-2 position of triacylglycerol in transgenic rapeseed oil. Plant Physiol, 1995, 109: 1389–1394



[12]Cao Y Z, Oo K C, Huang A H. Lysophosphatidate acyltransferase in the microsomes from maturing seeds of meadowfoam (Limnanthes alba). Plant Physiol, 1990, 94: 1199–1206



[13]Oo K C, Huang A H. Lysophosphatidate acyltransferase activities in the microsomes from palm endosperm, maize scutellum, and rapeseed cotyledon of maturing seeds. Plant Physiol, 1989, 91: 1288–1295



[14]Brown A P, Coleman J, Tommey A M, Watson M D, Slabas A R. Isolation and characterisation of a maize cDNA that complements a 1-acyl sn-glycerol-3-phosphate acyltransferase mutant of Escherichia coli and encodes a  protein which has similarities to other acyltransferases. Plant Mol Biol, 1994, 26: 211–223



[15]Ichihara K, Asahi T, Fujii S. 1-Acyl-sn-glycerol-3-phosphate acyltransferase in maturing safflower seeds and its contribution to the non-random fatty acid distribution of triacylglycerol. Eur J Biochem, 1987, 167: 339–347



[16]Griffiths G, Stobart A K, Stymne S. The acylation of sn-glycerol 3-phosphate and the metabolism of phosphatidate in microsomal preparations from the developing cotyledons of safflower (Carthamus tinctorius L.) seed. Biochem J, 1985, 230: 379–388



[17]Arroyo-Caro J M, Chileh T, Kazachkov M, Zou J, Alonso D L, Garcia-Maroto F. The multigene family of lysophosphatidate acyltransferase (LPAT)-related enzymes in Ricinuscommunis. Cloning and molecular characterization of two LPAT genes that are expressed in castor seeds. Plant Sci, 2013, 199/200: 29–40



[18]Chen S L, Huang J Q, Lei Y, Zhang Y T, Ren X P, Chen Y N, Jiang H F, Yan L Y, Li Y R, Liao B S. Identification and characterization of a gene encoding a putative lysophosphatidylacyltransferase from Arachis hypogaea. J Biosci, 2012, 37: 1029–1039



[19]Kim H U, Huang A H. Plastid lysophosphatidyl acyltransferase is essential for embryo development in Arabidopsis. Plant Physiol, 2004, 134: 1206–1216



[20]Ananda K. Ghosh N C S R. At4g24160, a Soluble acyl-coenzyme a-dependent lysophosphatidic acid acyltransferase. Plant Physiol, 2009, 151: 869–881



[21]Maisonneuve S, Bessoule J J, Lessire R, Delseny M, Roscoe T J. Expression of rapeseed microsomal lysophosphatidic acid acyltransferase isozymes  enhances seed oil content in Arabidopsis. Plant Physiol, 2010, 152: 670–684



[22]Rao S S, Hildebrand D. Changes in oil content of transgenic soybeans expressing the yeast SLC1 gene. Lipids, 2009, 44: 945–951



[23]戚维聪. 油菜发育种子中油脂积累与Kennedy途径酶活性的关系研究. 南京农业大学硕士学位论文, 江苏南京, 2008



Qi W C. Studies on correlations of developing seed lipid accumulation with Kennedy pathway enzyme activities in Brassica napus. MS Thesis of Nanjing Agriculture University, Nanjing, China, 2008 (in Chinese with English abstract)



[24]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. 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, 2012, 38: 245–255



Chen S L, Huang J Q, Lei Y, Ren X P, Wen Q G, Chen Y N, Jiang H F, Yan L Y, Liao B S. Cloning and expression analysis of lysophosphatidic acid acyltransferase (LPAT) encoding gene in peanut. Acta Agron Sin, 2012, 38: 245–255 (in Chinese with English abstract)



[25]Kim H U, Vijayan P, Carlsson A S, Barkan L, Browse J. A mutation in the LPAT1 gene suppresses the sensitivity of fab1 plants to low temperature. Plant Physiol, 2010, 153: 1135–1143



[26]Chen S L, Huang J Q, Lei Y, Zhang Y T, Ren X P, Chen Y N, Jiang H F, Yan L Y, Li Y R, Liao B S. Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea. J Biosci, 2012, 37: 1029–1039



[27]Gong Q, Li P, Ma S, Indu R S, Bohnert H J. Salinity stress adaptation competence in the extremophile Thellungiella halophila in comparison with its relative Arabidopsis thaliana. Plant J, 2005, 44: 826–839



[28]Howell E C, Kearsey M J, Jones G H, King G J, Armstrong S J. A and C genome distinction and chromosome identification in Brassica napus by sequential fluorescence in situ hybridization and genomic in situ hybridization. Genetics, 2008, 180: 1849–1857

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