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热激蛋白(heat shock proteins,HSP)是生物体在不利环境条件因素刺激下应激合成的一组在进化上高度保守的蛋白质。前期转录组测序的结果发现马铃薯Favorita的小热激蛋白(small heat shock proteins,s HSPs)基因(PGSC0003DMG400009255)在接种晚疫病菌(Phytophthora infestans)24 h后表达量显著上调。因此,以马铃薯Favorita为材料,根据PGSC0003DMG400009255基因序列设计引物,并在引物5’端加上Bam HⅠ和SalⅠ酶切位点,从接种P.infestans 24小时后的Favorita的RNA中通过RT-PCR的方法获得PGSC0003DMG400009255的基因片段,并命名为s HSP-F,该基因最大开放阅读框(ORF)为594 bp,编码197个氨基酸。通过酶切连接将s HSP-F连接至表达载体p CAMBIA1301中。通过测序和酶切验证,表明s HSP-F基因成功克隆到表达载体中,该工作为进一步研究该基因的功能提供了基础。
Heat shock proteins (HSPs) are a group of evolutionarily conserved proteins that are stress-stimulated by organisms under adverse environmental conditions. The result of pre-transcriptome sequencing showed that the expression of small heat shock proteins (s HSPs) gene (PGSC0003DMG400009255) was significantly up-regulated after Phytophthora infestans inoculation. Therefore, primers were designed based on the gene sequence of PGSC0003DMG400009255 using the potato Favorita as a material, and the BamHI and SalI restriction sites were added to the 5 ’end of the primer. RT-PCR was performed from the Favorita RNA 24 hours after the inoculation of P.infestans Methods The gene fragment of PGSC0003DMG400009255 was obtained and named s HSP-F. The gene had the largest open reading frame (ORF) of 594 bp and encoded 197 amino acids. S HSP-F was ligated into the expression vector p CAMBIA1301 by restriction enzyme ligation. By sequencing and restriction enzyme digestion, the s HSP-F gene was successfully cloned into the expression vector. This work provided the basis for further study on the function of the gene.