| Silkworm, Bpmbyx mori, ;an organism which can transform protein efficiently, has the advantages of short life circle, high rate of reproduction and mass raising and so on. Silkindustry has a long history of more than 5000 years in China. As a model organism, the silkworm's basic and molecular biological research have been obtained great proceedings, and the groundwork of transgehic research is gradually becoming perfect. Transgenic research of silkworm plays an irriportant role in basic theory research, special Tace breeding and bioengineering pharmaceutics. In the respect of basic '.theory research, because foreign gene can be replicated, integrated and expressed in the host and is under the control of genetic background of th^ receptor genome, transgene itself should be an ideal functional marker. It is very valuable in the study of, the regulation mechanism of gene expression. For example, the .spatiotemporal specific cis-regulation element and their remote action, trans-acting factors and their interactions, gene cooperative expression, the feedback regulation of the complex promoter-enhancer-foreign gene, and the information of the extra-chromosome recombination are almost from the model transgenic animal. In the respect of race improvement, some characters of silkworm can be improved by transgenes, such as disease resistance, sex control, improvement of silk quality and so on. In addition, transgenic silkworm can be utilized as bioreactors to ppQdttGp,engineered proteins, such as antihemop$ilic factor andinterteukin.Transgenic mammals can be produced by normal path: zygote microinjection, cell culture, transplantation of zygote and developing mature individuals. However, transgenic silkworm can only be produced by introducing foreign gene into the silkworm egg. Because of the richness of yolk and hardness of chorion, it is difficult for foreign gene to penetrate hard4chorion and introduce into the cell nucleus. In addition, at present the integrating vector is not ideal enough, even though the foreign gene is transferred into the fertilized egg of silkworm, it is still difficult to integrate into silkworm genome stably. All of the above restricts the development of silkworm transgenic research.Aiming at the above problems, we experimented on sperm-mediated transformation method and the foreign gene targeting in fib-H by homologous recombination. The research approached the superiority of sperm-mediated transformation method, the maintenance and insertion of the foreign gene in the host cells, the dynamic process of the homologous recombination, and the feasibility of mitochondrial DNA as transgenic vector. The main experimental results were summarized as the follows:1. Construction of the homologous arms directing the homologous recombinationFor the sake of seeking for the more efficient integrating strategy to make the foreign gene insert in the specific site and don't destroy the host expressional and regulatory elements such as promoters and introns, we experimented targeting the Bombyx mori fibroin heavy-chain (fib-H) exon II with the green fluorescent protein (GFP) gene by homologous recombination technology. The fib-H exon II appears to present a very highly repel itive and GC-rich core flanked by nonrepetitive 5' and 3' ends. This repetitive core is composed of alternate arrays of 12 repetitive and 11 amorphous domains. To amplify the repetitive region, a new rnulti-step amplification with different primer PCR reaction system was set up. in which the buffer's pH was adjusted, pfu Taq DNA polymerase and Klenow fragment were combined, the organic reagent methylamine was added to uncoil the secondary structure. Cloning the amplified product into pUC19, the homologous arms pool was constructed.To compensate the descent of pH for the heat-up, exalting the Tris-HCl buffer's pH can avoid the abscission of the purine and make the long sequence PCR go on smoothly.The GC-rich template's secondary structure is complex and stable. The organic reagent methyiamine contributes to unco...
|
PDF Full Text Request