家蚕精子介导报告基因gfp和egfp转移技术的研究

Sperm-mediated Repoter Gene gfp and egfp Transfer in the Silkworm

探讨家蚕精子介导基因转移技术能否有效地将外源基因导入并整合到家蚕基因组中,使之稳定遗传.分别进行了含gfp报告基因的pG350载体和含egfp报告基因的pMD-Fib-IE-EGFP与pEGFP-N3载体的精子介导基因转移实验.结果表明,导入线性pG350载体,在14个G0代实验蛾区的DNA样品中,有5个PCR检测为阳性,阳性率为35.7%;对G1代的PCR与Southern blot检测的结果证明,导入的gfp基因存在于G1代的家蚕基因组中.导入不同浓度或构型的pMD-Fib-IE-EGFP与pEGFP-N3载体时,对G0代的PCR检测结果表明,导入线性pMD-Fib-IE-EGFP组的PCR阳性率为61.2%,而导入环状质粒组的PCR阳性率为57.1%;导入环状pEGFP-N3组的PCR阳性率为46.2%;对部分PCR阳性蛾区进行Southern blot检测结果表明,导入的egfp基因整合在G0代家蚕基因组中.本研究的结果表明,家蚕精子介导基因转移能够将外源基因有效地导入、整合于家蚕基因组,并可遗传至G1代.

In this study, the effiency of sperm-mediated gene transfer was analyzed for foreign gene introduced into and intergrated in the silkworm＇s genome and inherited by the offspring. Different concentration or conformation vector DNA including gfp or egfp report gene were used in the gene transfer experiment on silkworm. The results showed that 5 DNA samples extracted from 14 Go generation spawns were positive by PCR （positive proportion 35.7%） after introduction linearized pG350 with the concentration 2. 0 rng/mL into moth,and the results of PCR and Southern blot detection of G1 generation indicated that the gfp gene was intergrated in the silkworm＇s genome. The positive proportion of introduction linearized or circular pMD-Fib-IE-EGFP was 61.2% and 57.1% by PCR respectively. When the circular pEGFP-N3 was transferred with the concentration 1. 2 mg/mL, the positive proportion was 46.2% by PCR. The hybridization results indicated that the egfp gene had intergrated in the genome of Go generation silkworm. From this study,the conclusion was drawn that the sperm-mediated gene transfer technology could introduce foreign gene into silkworm,and the foreign gene also could intergrated in the genome and transmitted to G1 generation.