一种简便的适合抱卵孵化甲壳动物的转基因方法

A Simple and Convenient Technique of Gene Transfer for the Crustacean Animals Retaining the Fertilized Eggs for Embryogenesis

动物转基因的方法有很多,但对于许多抱卵孵化的甲壳动物,许多已有的转基因方法不可行或不易操作。在对罗氏沼虾(Macrobrachium rosenbergii)的基因改良过程中,通过精荚微注射(spermatophore-microinjection, SMI),将目的基因直接注射到交配后的雌虾胸部腹面的精荚中,再由精子介导的简便高效的转基因技术,成功地将抗冻蛋白基因导入到罗氏沼虾的胚胎中,获得了16%以上的基因转移率。这一方法的建立,为进一步开展抱卵孵化的甲壳动物的分子遗传和发育生物学的研究以及育种工作的研究奠定了基础。

Many animal transgenic approaches have been developed to introduce foreign DNA into zygotes.However, some crustacean retain their embryos developing in a clump on the abdomen,which makes it difficult to transfer genes into the fertilized eggs by traditional methods,such as microinjection and electroporation.A modified spermatophore-microinjection (SMI)method was used in the course of gene modification of giant freshwater prawn(Macrobrachium rosenbergii) which allows exogenous DNA fragments to be transferred easily and conveniently.Using this technique,we have successfully transferred the anti-freeze protein gene(sbwAFP)into the zygotes.The integration of the gene into the embryos was determined by PCR with the special primers for the exogenous gene.The highest embryonic transgenic rate is over 16%.This method is very simple,convenient and efficient for mass producing transgenic crustacean that retain embryos for developing on their bodies.