DOI: 10.3724/SP.J.1006.2009.00033

Acta Agronomica Sinica (作物学报) 2009/35:1 PP.33-40

Cloning of Citrate Synthase Gene in Rapeseed (Brassica napus L.) and Its Expression under Stresses

As a key enzyme of some metabolisms, citrate synthase shows a sign for metabolism. In order to investigate the function of citrate synthase gene, the cDNA encoding citrate synthase was cloned from rapeseed leaf by RT-PCR. It was 1 659 bp long and encoded a protein with 476 amino acids. The deduced protein sequence had a mitochondrial targeting signal in N-terminal, which was very similar to the citrate synthase in Arabidopsis thaliana(96.0%). Alignment analysis showed that citrate synthase gene had high homology in plants. Under different stresses, we tested the expression of citrate synthase gene in rapeseed leaf by using semi-quantitative PCR. The expression of citrate synthase gene had no obvious change in stresses of salt, dark, high illumination, while was increased at different time in treatments of water logging, drought, IAA, and 6-BA. Interestingly the effect of ABA was contrary to that of IAA. In the treatment of sclerotium blight, the expression of citrate synthase gene was depressed. There was a saddle curve of citrate synthase gene expression in the treatment of gibberellin. The results from real-time PCR of several treatments were mainly in accord with that from semi-quantitative PCR.

Key words:Rapeseed (Brassica napus L.),Citrate synthase,Expression pattern,Stress

ReleaseDate:2014-07-21 14:30:35

[1] Wang H-Z(王汉中). Strategy for rapeseed industry development based on the analysis of rapeseed production and demand in China.Chin J Oil Crop Sci(中国油料作物学报), 2007, 29(1): 101-105(in Chinese with English abstract)

[2] O'Hara P, Slabas A R, Fawcett T. Fatty acid and lipid biosynthetic genes are expressed at constant molar ratios but different absolute levels during embryogenesis.Plant Physiol,2002,129:310-320

[3] Ohlrogge J, Pollard M, Bao X, Focke M, Girke T, Ruuska S, Mekhedov S, Benning C. Fatty acid synthesis: From CO2 to functional genomics.Biochem Soc Trans,2005,28:567-573

[4] Schnarrenberger C, Martin W. Evolution of the enzymes of the citric acid cycle and the glyoxylate cycle of higher plants: A case study of endosymbiotic gene transfer.Eur J Biochem,2002,269:868-883

[5] Morgunov I, Srere P A. Interaction between citrate synthase and malate dehydrogenase. Substrate channeling of oxaloacetate.J Biol Chem,1998,273:29540-29544

[6] de la Fuente J M, Ramirez-Rodriguez V, Cabrera-Ponce J L, Herrera-Estrella L. Aluminum tolerance in transgenic plants by alteration of citrate synthesis.Science,1997,276:1566-1568

[7] Pracharoenwattana I, Cornah J E, Smith S M. Arabidopsis peroxisomal citrate synthase is required for fatty acid respiration and seed germination.Plant Cell,2005,17:2037-2048

[8] Koyama H, Takita E, Kawamura A, Hara T, Shibata D. Over expression of mitochondrial citrate synthase gene improves the growth of carrot cells in Al-phosphate medium.Plant Cell Physiol,1999,40:482-488

[9] Koyama H, Kawamura A, Kihara T, Hara T, Takita E, Shibata D. Overexpression of mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil.Plant Cell Physiol,2000,41:1030-1037

[10] Zhang S-S(张珊珊), Ming F(明凤), Lu Q(路群). Molecular cloning and characterization of citrate synthasegene in rice (Oryza sativa).Chin J Rice Sci(中国水稻科学), 2008, 19(6): 501-505(in Chinese with English abstract)

[11] Hu L-H(胡利华), Wu H-M(吴慧敏), Zhou Z-M(周泽民). Introduction of citrate synthase gene(cs) into an elite indica rice restorer line Minghui 86 by a grobacterium-mediated method.Mol Plant Breed(分子植物育种), 2006, 4(2): 160-166(in Chinese with English abstract)

[12] Wen T(文涛), Xiong Q-E(熊庆蛾), Zeng W-G(曾伟光), Liu Y-P(刘远鹏). Changes of organic acid synthetase activity during fruit development of navel organ.Acta Hort Sin(园艺学报), 2001, 28(2):161-163 (in Chinese with English abstract)

[13] Zhang X-M(张秀梅), Du L-Q(杜丽清), Sun G-M(孙光明). Changes in organic acid concentrations and the relative enzyme activities during the development of cayenne pineapple fruit.J Fruit Sci(果树学报), 2007, 24(3): 381-338(in Chinese)

[14] Shi D-Q(石东乔), Zhou Y-H(周奕华), Chen Z-H(陈正华). Manipulation of plant fatty acid.Chin Bull Life Sci(生命科学), 2002, 14(5): 291-295(in Chinese with English abstract)

[15] Molina I, Ohlrogge J B, Pollard M. Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana. Plant J,2008,53:437-449

[16] Hua W(华玮), Li R-J(李荣俊), Liang S-P(梁述平), Lü Y-T(吕应堂). Gene expression and activity regulation of two calmodulin binding protein kinases in tobacco seedling.J Plant Physiol Mol Biol(植物生理与分子生物学学报), 2005, 31(3): 305-331(in Chinese with English abstract)

[17] Hua W, Zhang L, Liang S P, Jones R L, Lu Y T. A tobacco calcium/calmodulin-binding protein kinase functions as a negative regulator of flowering.J Biol Chem,2004,279:31483-31494

[18] Guo Y(郭彦), Yang H-S(杨洪双), Li Q-X(李清旭), Sun X-B(孙学彬). Effects of hormones on the tolerance of wild soybean Seedlings against water stress.J Henan Agric Sci(河南农业科学), 2007, (4): 37-39(in Chinese with English abstract)

[19] Li R J, Wang H Z, Mao H, Lu Y T, Hua W. Identification of differentially expressed genes in seeds of two near-isogenic (Brassica napus) lines with different oil content.Planta,2006,224:952-962

[20] Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.Nucl Acids Res,1987,15:8125-8148

[21] Attardi G, Schatz G. Biogenesis of mitochondria.Annu Rev Cell Biol,1988,4:290-333

[22] Tsuwamoto R, Fukuoka H, Takahata Y. Identification and characterization of genes expressed in early embryogenesis from microspores of Brassica napus. Planta,2007,225:641-652

[23] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-[Delta][Delta]CT method.Methods,2001,25:402-408

[24] Leland E F. Growth, seed yield, and oil content of canola grown under saline conditions.Agron J,1994,86:233-237

[25] Zhong X-H(钟雪花), Yang W-N(杨万年), Lü Y-T(吕应堂). Comparative research on some physiological characteristics of tobacco and rape under flooding stress.J Wuhan Bot Res(武汉植物学研究), 2002, 20(5): 395-398(in Chinese with English abstract)

[26] Zhu H-X(朱惠霞), Sun W-C(孙万仓), Deng B(邓斌). Study on cold hardiness and its physiological and biochemical characteristics of w inter turnip rapeseed (Brassica campetris).Acta Agric Boreali-Occident Sin(西北农业学报), 2007,16(4): 34-38(in Chinese with English abstract)

[27] Xiang J(项俊), Chen Z-B(陈兆波), Wang P(王沛), Yu L-J(余龙江), Li M-T(栗茂腾). The effect of caciz treatment on the change of drought related physiological and biochemical indexes of Brassica napus. J Huazhong Agric Univ(华中农业大学学报), 2007, 26(5): 607-611(in Chinese with English abstract)

[28] Zhao X-H(赵小虎), Chen C-L(陈翠莲), Jiao C-X(焦春香), Gan L(甘莉), Lu J-W(鲁剑巍). Physiological and biochemical reaction responses to sclerotium blight inoculation among different rapeseed varieties.J Huazhong Agric Univ(华中农业大学学报), 2006, 25(5): 488-492(in Chinese with English abstract)

[29] Yuan J(袁晶), Wang Q-M(汪俏梅), Zhang H-F(张海峰). Interactions between phytohormone signals.Chin J Cell Biol(细胞生物学杂志), 2005, 27(3): 325-328(in Chinese with English abstract)