Heat-inducible transgenic expression in the silkmoth Bombyx mori
Heat-inducible transgenic expression in the silkmoth Bombyx mori
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DOI：
10.1007/s00427-002-0221-8
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发表时间：
2002-04-01
影响因子：
2.4
通讯作者：
Jindra, M
中科院分区：
文献类型：
--
作者：
Uhlirov치, M;Asahina, M;Jindra, M
用新的转座子载体进行种系转化，现在可以通过非果蝇昆虫的异位蛋白表达或RNA干扰来进行基因功能的因果测试。问题仍然是如何推动转基因在体内的表达。我们利用猪Bac 3xP3-EGFP载体进行种系转化，以检测果蝇热休克HSP70启动子在活体家蚕中是否具有活性。我们克隆了家蚕核受体FTZ-F1在HSP70启动子和终止子之间的编码序列，对原载体进行了改造。获得了三个在幼虫和成虫感光器中表达Pax-6驱动的EGFP标记的独立转基因株系，获得了GFP阳性后代的可育成虫的效率高达1.7%。用反向聚合酶链式反应证实了转座子的染色体整合。转基因BinFtz-F1的热诱导在mRNA和蛋白质水平上都得到了证实。RT-PCR结果表明，果蝇热休克启动子在三个转基因株系中均有功能。尽管在25℃下有明显的基础活性，但在42℃下1h诱导了不同发育阶段和不同组织中的BmFtz-F1 mRNA。不同转基因品系的相对诱导水平不同。Northern印迹杂交仅在热休克后检测到转基因BmFtz-F1，热处理后6h仍有低水平的mRNA表达。抗BmFtz-F1抗体免疫组织化学染色显示，热休克90min后，细胞核信号明显增强。
Germline transformation with new transposon vectors now enables causal tests of gene function via ectopic protein expression or RNA interference in non-drosophilid insects. The problem remains of how to drive the transgene expression in vivo. We employed germline transformation using the piggyBac 3xP3-EGFP vector to test whether the Drosophila heat shock hsp70 promoter will be active in the live silkworm. We modified the original vector by cloning the coding sequence for Bombyx nuclear receptor Ftz-F1 between the hsp70 promoter and the terminator. Three independent transgenic lines expressing the Pax-6-driven EGFP marker in larval and adult photoreceptors were obtained with efficiencies of up to 1.7% of fertile GO adults that gave GFP-positive progeny. Chromosomal integration of the transposon was confirmed with inverse PCR. Heat induction of the transgenic BinFtz-F1 was proven at both the mRNA and protein levels. RT-PCR data showed that the Drosophila heat shock promoter was functional in all three transgenic lines. Although basal activity was apparent at 25degreesC, 1 h at 42degreesC induced BmFtz-F1 mRNA at different stages of development and in diverse tissues. The relative levels of induction differed among the transgenic lines. Northern blot hybridization detected transgenic BmFtz-F1 only after heat shock and low levels of the mRNA were still present 6 h after the heat treatment. Immunostaining of epidermis using anti-BmFtz-F1 antibody showed a clear increase of nuclear signal 90 min after a heat shock.