杂交转育和累代选育对转基因家蚕外源基因整合位点及拷贝数的影响

Effect of Transgene Integration Sites and Copy Numbers on Hybrization and Breeding in Silkworm

为了解转基因家蚕在杂交转育和累代选育过程中,外源基因在基因组上的整合位点和拷贝数变化情况,通过反向PCR和southern杂交,测定外源基因在杂交一代和三代品系的拷贝数与插入位点。反向PCR结果显示,所有样品均只扩增出一条片段,且大小一致。对扩增片段进行克隆测序和比对发现,外源基因均系插入同一位点,位于24号染色体。Southern blotting试验结果表明各试验样品均得到了较好的一条杂交信号,而对照没有杂交信号,说明外源基因在各个样品中均系单拷贝插入,而且插入的位置没有随着转育和传代培养而发生变化。研究结果表明外源基因可稳定遗传,将此转基因材料与常规品种材料进行杂交,选育优良家蚕品种的方法是有效可行的。

In order to study the transformation of transgene integration sites and copy numbers in genome after hybrization and breeding,we determined the exogenous gene＇s integration sites and copy numbers in genome of hybrid offspring （F1 and F3） by inverse PCR and southern blouing.Inverse PCR results showed that,all the samples were only amplified one fragment with the same size.Then,we cloned and sequenced the amplified fragment of PCR.Result of the sequence anlignment of the amplified fragment＇ s sequence and silkworm genomic sequences showed that,exogenous gene were inserted into the same site and located on chromosome 24.All the test samples got one vivid hybridization signals by southern blotting,while there was no hybridization signal of control,which indicated that the exogenous gene in all samples were single copy and transgene integration sites remained unchanged during hybridization and breeding from generation to generation.These results indicated that exogenous gene was stably inherited,and the method of using transgenic strains and conventional strains hybridization to obtain high quality silkworm varieties was feasible and effective.