用抑制消减杂交法分离巴西橡胶树胶乳特异表达基因

doi:10.11707/j.1001-7488.20060606

林业科学 ›› 2006, Vol. 42 ›› Issue (6): 32-36.doi: 10.11707/j.1001-7488.20060606

用抑制消减杂交法分离巴西橡胶树胶乳特异表达基因

邓柳红¹ 罗明武² 曾会才¹ 杨卫帆¹ 张春发¹

1.中国热带农业科学院热带生物技术国家重点实验室,海口571101；2.华南热带农业大学,儋州571737

收稿日期:2004-10-29 修回日期:1900-01-01 出版日期:2006-06-25 发布日期:2006-06-25

Isolation of Differentially Expressed Genes in Latex of Hevea brasiliensis by Suppressive Subtractive Hybridization

Deng Liuhong¹,Luo Mingwu²,Zeng Huicai¹,Yang Weifan¹,Zhang Chunfa¹

1.State Key Laboratory of Tropical Crop Biotechnology, Chinese Academy of Tropical Agriculture Sciences Haikou 571101;2.South China University of Tropical Agriculture Danzhou 571737

Received:2004-10-29 Revised:1900-01-01 Online:2006-06-25 Published:2006-06-25

摘要/Abstract

摘要：

采用巴西橡胶树常割树胶乳的Poly(A+)RNA为Tester,叶片Poly(A+)RNA为Driver,通过抑制消减杂交法(suppressive subtractive hybridization,SSH)构建了一个胶乳特异表达基因差减文库,通过菌落PCR及反式NorthernDot-Blot筛选鉴定阳性差异片段,获得79个胶乳特异表达阳性克隆,部分阳性克隆还通过Northern Blot杂交及RT-PCR进一步验证。随机挑选部分阳性克隆序列比较分析,结果表明有部分克隆与巴西橡胶树中已知的基因相同,而多数克隆在巴西橡胶树中是新发现,它们与橡胶生物合成、物质代谢运输、信号传导和形态建成等相关。

关键词: 巴西橡胶树, 抑制消减杂交法, 胶乳, 反式Northern Dot-Blot

Abstract:

A subtracted cDNA library was constructed via suppressive subtractive hybridization(SSH), in which the latex Poly(A+)RNA isolated from regularly tapped Hevea brasiliensis trees was utilized as tester while driver is Poly(A+)RNA derived from negative latex-producing leaf tissues. Total 79 positive clones were identified from screening of the subtracted library by PCR and Reverse Northern Dot-Blot, part of them were further verified by RT_PCR and Northern Blot.18 out of these 79 confirmed clones were randomly selected for sequencing analyses, of which the results were aligned with GenBank^? databases via BLASTN and BLASTX programs. It is shown that 5 of 18 candidates were identity with known genes in H. brasiliensis which exerted a positive role in rubber synthesis, 2 showed significant similarity to cis-prenyltransferase of H. brasiliensis, 11 were novel genes in H. brasiliensis.

Key words: Hevea brasiliensis, suppressive subtractive hybridization(SSH), latex, Reverse Northern Dot_Blot

邓柳红罗明武曾会才杨卫帆张春发. 用抑制消减杂交法分离巴西橡胶树胶乳特异表达基因[J]. 林业科学, 2006, 42(6): 32-36.

Deng Liuhong;Luo Mingwu;Zeng Huicai;Yang Weifan;Zhang Chunfa. Isolation of Differentially Expressed Genes in Latex of Hevea brasiliensis by Suppressive Subtractive Hybridization[J]. Scientia Silvae Sinicae, 2006, 42(6): 32-36.

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用抑制消减杂交法分离巴西橡胶树胶乳特异表达基因

Isolation of Differentially Expressed Genes in Latex of Hevea brasiliensis by Suppressive Subtractive Hybridization

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