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中国农学通报 ›› 2011, Vol. 27 ›› Issue (13): 183-187.

所属专题: 生物技术 园艺

• 林学 园艺 园林 • 上一篇    下一篇

利用抑制性消减杂交技术分离草莓抗炭疽病相关基因

苗立祥 张豫超 杨肖芳 张跃建 王汉荣 吴延军 张慧琴 蒋桂华   

  • 收稿日期:2010-11-29 修回日期:2011-01-12 出版日期:2011-06-05 发布日期:2011-06-05
  • 基金资助:

    浙江省自然科学基金;国家科技支撑计划项目;浙江省优先主题

Isolation and Cloning of cDNA Induced by Colletotrichum gloeosporioides via Suppression Subtractive Hybridization in Strawberry

  • Received:2010-11-29 Revised:2011-01-12 Online:2011-06-05 Published:2011-06-05

摘要:

利用抑制性消减杂交技术(suppression subtractive hybridization,SSH),比较接种炭疽病菌与未接菌草莓之间的基因表达差异,筛选抗草莓炭疽病菌的差异表达基因片段,为草莓抗炭疽病遗传育种提供理论依据。分别提取接种草莓与未接种草莓叶片的总RNA,分离mRNA,逆转录成cDNA,利用Rsa I进行酶切,然后以接种叶片酶切cDNA为试验方(tester),以未接菌草莓叶片酶切cDNA为驱动方(driver),构建了草莓抗炭疽病基因消减文库。菌落PCR结果表明,插入片段大部分在100~1000 bp之间,文库质量好。对文库中1500个阳性克隆进行测序,共得到了1377个有效的ESTs序列。利用NCBI数据库进行Blast同源性比较发现,这些ESTs可能与胁迫反应、信号转导、蛋白合成、蛋白相互作用、物质代谢、跨膜通道、转录因子、金属转运及未知或假定蛋白等有关,ESTs序列的获得为后续草莓抗炭疽病相关基因的克隆、表达和转基因研究提供了参考。

关键词: 甜玉米, 甜玉米

Abstract:

To uncover the mechanism of strawberry inoculated with Colletotrichum gloeosporioides and compare gene express difference of the treated and untreated leaves by using the suppression subtractive hybridization (SSH) so as to find the differential express related genes. The total RNA samples of strawberry leaves were prepared and mRNA samples were isolated. cDNA were synthesized and digested with Rsa I. One differential expressing cDNA library was constructed with the restriction enzymes cDNA of inoculated leaves as driver and the restriction enzymes cDNA of untreated as tester. PCR results suggested that the insertion segments were between 100 to 1000 bp and the library was suitable for the following work for isolation the full length of genes. 1500 clones were selected and sequenced, and 1377 unique ESTs were obtained. The ESTs were annotated by similarity to orthologs and paralogs detected with Blast in NCBI database. Some of the ESTs might be related closely with stress response, signal transduction, protein synthesis, protein-protein interaction, metabolism, transmembrane, transcription factor, metal transporter or unknown. This study lays a foundation for the further study on isolation related genes in strawberry.

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