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

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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

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Abstract:

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

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