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Acta Agron Sin ›› 2009, Vol. 35 ›› Issue (9): 1576-1583.doi: 10.3724/SP.J.1006.2009.01576


Construction of SSH Library with Different Stages of Seeds Development in Brassica napus L.

PENG Qi1,HU Yan1,DU Pei-Fen1,2,XIE Qing-Xuan1,2,RUAN Ying1,2,*,LIU Chun-Lin1*   

  1. 1Pre-State Key Laboratory for Germplasm Innovation and Resource Utilization of Crops;2College of Bio-Science and Technology,Hunan Agricultural University,Changsha,410128 China
  • Received:2009-01-02 Revised:2009-04-26 Online:2009-09-12 Published:2009-07-03
  • Contact: LIU Chun-Lin,E-mail: liucl100@126.com;RUAN Ying,E-mail: yingruan@hotmail.com; Tel: 0731-4635294


Mechanism of fatty acid metabolic is a significant research topic in rapeseed molecular breeding. There are six hundreds genes and ESTs associated with fatty acid metabolism, 14% of which are conformed to participate in acrylic-fatty acid metabolism, 86% of which are speculated on sequences similarity and conservative domain with other species. But compared to the situation in Arabidopsis thaliana, molecular regulation mechanism of fatty acid metabolism in rapeseed has been less reported. In harvested rapeseed seeds, there is difference in seed fatty acid components among different varieties or the same variety grown under different ecological conditions. To further explore the molecular mechanism of fatty acid metabolic regulation of Brassica napus L., we investigated the assimilation product transition during the seed development. The starch reached a peak content at 20 days after pollination (20DAP) and was used up quickly after 20DAP, immediately the fatty acids content rapidly increased from 30DAP to 35DAP. According to the results, 20DAP developing seeds and 35DAP developing seeds were chosen for suppression subtractive hybridization (SSH), which is an effective tool for picking out specific expression genes among different samples. Two libraries, 20DAP SSH library derived from 20DAP seed cDNA as tester and 35DAP seed cDNA as driver and 35DAP library from 20DAP seed cDNA as driver and 35DAP seed cDNA as tester were constructed. The two SSH libraries had a high quality with high suppression subtractive efficiency after tested by PCR and RT-PCR. A total of 489 clones were randomly selected from the two libraries for sequencing and 452 high quality sequences tags were obtained. Blast analysis and functional annotation showed that most of the genes in 20DAP SSH library were relative to carbohydrate metabolism, while those in 35DAP library relative to fatty acid metabolic regulation. Significantly, 5 function-unknown genes in 20DAP library and 7 in 35DAP library were found out. In summary, this work adds an extra layer of complexity to the regulation of starch-to-oil transition and at the same time the different genes, especially the function-unknown genes shed light on studies of molecular mechanism of fatty acid metabolic regulation in seeds of Brassica napus L.

Key words: Brassica napus L., Seeds development, Fatty acid, Suppression subtractive hybridization(SSH), cDNA library

[1] DiatchenkoL, LauY F, CampbellA P, ChenchikA, MoqadamF, HuangB, LukyanovS, LukyanovK, GurskayaN, SverdlovE D, SiebertP D. Suppression subtractive hybridization: A method for generating differentially regulated or tissue specific cDNA probes and libraries. Proc Natl Acad Sci USA, 1996, 93: 6025-6030

[2] Diatchenko L, Lukyanov S, Lau Y F, Siebert P D. Suppression subtractive hybridization: A versatile method for identifying differentally expressed genes. Methods Enzymol, 1999, 303: 349-380

[3] Siebert P D, Chenchik A, Kellogg D E, Lukyanov K A, Lukyanov S A. An improved PCR method for walking in uncloned genomic DNA. Nucl Acids Res, 1995, 23: 1087-1088

[4] Lukyanov K A, Launer G A, Tarabykin V S, Zaraisky A G, Lukyanov S A. Inverted terminal repeats permit the average length of amplified DNA fragments to be regulated during preparation of cDNA libraries by polymerase chain reaction. Anal Biochem, 1995, 229: 198-202

[5] Lambe T, Finlay D, Murphyand M,Martin F. Differential expression of connexin 43 in mouse mammary cells. Cell Biol Intl, 2006, 30: 472-479

[6] Sangrado-Vegas A, Lennington J B, Smith T J. Molecular cloning of an IL-8-like CXC chemokine and tissue factor in rainbow trout (Oncorhynchus mykiss) by use of suppression subtractive hybridization. Cytokine, 2002, 17: 66-70

[7] 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

[8] Li X-Y(李小艳), Zhao Y(赵云), Zhou Y-T(周云涛), Li Y-Y(李熠毅), Wang M-L(王茂林). Construction and primary analysis of suppression subtractive library of dwarf mutant ‘NDF-1’in Brassica napus. Chin J Oil Crop Sci (中国油料作物学报), 2006, 28(4): 396-402(in Chinese with English abstract)

[9] Liu Y(刘阳), Wang M-L(王茂林), Qiu F(邱峰), Zhou Y-T(周云涛), Zhao Y(赵云), Zhang F(张帆). The construction of the suppression subtractive library of the Brassica napus mutant Cr3529. J Sichuan Univ (四川大学学报), 2005, 42(5): 1029-1032(in Chinese with English abstract)

[10] Ohlrogge J B, Kuhn D N, Stumpf P K. Subcellular localization of acyl carrier protein in leaf protoplasts of Spinacia oleracea. Proc Natl Acad Sci USA, 1979, 76: 1194-1198

[11] Ohlrogge J, Browse J. Lipid biosynthesis. Plant Cell, 1995, 7: 957-970

[12] Thelen J J, Ohlrogge J B. Metabolic engineering of fatty acid biosynthesis in plants. Metab Eng, 2002, 4: 12-21

[13] Beisson F, Koo A J, Ruuska S, Schwender J, Pollard M, Thelen J J, Paddock T, Salas J J, Savage L, Milcamps A, Mhaske V B, Cho Y, Ohlrogge J B. Arabidopsis genes involved in acyl lipid metabolism: A 2003 census of the candidates, a study of the distribution of expressed sequence tags in organs, and a web-based database. Plant Physiol, 2003, 132: 681-697

[14] Huang A H C. Oleosin and oil bodies in seeds and other organs. Plant Physiol, 1996, 110: 1055-1061

[15] Murphy D J, Vance J. Mechanisms of lipid-body formation. Trends Biochem Sci, 1999, 24: 109-115

[16] 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

[17] 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

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

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

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