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作物学报 ›› 2009, Vol. 35 ›› Issue (9): 1576-1583.doi: 10.3724/SP.J.1006.2009.01576

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜种子不同发育时期SSH文库的构建

彭琦1,胡燕1,杜培粉1,2,谢青轩1,2,阮颖1,2,*,刘春林1,*   

  1. 1作物种质创新和资源利用国家重点实验室培育基地；2湖南农业大学生命科学技术学院，湖南长沙410128
  • 收稿日期:2009-01-02 修回日期:2009-04-26 出版日期:2009-09-12 网络出版日期:2009-07-03
  • 通讯作者: 刘春林，E-mail: liucl100@126.com; 阮颖，E-mail: yingruan@hotmail.com; Tel: 0731-4635294
  • 基金资助:

    本研究由国家重点基础研究发展计划（973计划）项目（206CB101603）资助。

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 Published:2009-09-12 Published online: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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被引次数

46

摘要：

利用抑制差减杂交技术构建了20 d和35 d甘蓝型油菜湘油15发育种子特异表达基因的SSH文库。随机挑取单菌落进行PCR表明，文库质量较好。在20 d和35 d SSH文库中随机挑选489个阳性克隆进行测序，获得452条高质量的表达序列标签(EST)。对序列进行Blast比对及功能注释，比较20 d和35 d SSH文库的基因表达谱，发现在20 d SSH库中参与糖代谢的基因出现频率较高，而在35 d SSH库中与脂肪酸储存有关的油体蛋白家族、与脂肪酸转运有关的柠檬酸合酶、与脂肪酸合成有关的酰基载体蛋白去饱和酶等，参与油脂代谢相关基因出现频率较高。该结果对进一步研究油菜脂肪酸代谢调控的分子机制打下了基础。

关键词: 甘蓝型油菜, 种子发育, 脂肪酸, 抑制差减杂交, cDNA文库

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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