Study On Genetical Manipulation Technique And Its Application In Silkworm, Bombyx Mori.

The silkworm, Bombyx mori, is a domestic insect that has been continuously selected to improve silk production in sericulture for several thousand years. There are more and more molecular biological studies on this insect for its economic importance and as a typical model for Lepidopteran. Due to its large-size and high protein synthesis ability as well as the expediency in mass culture of the silkworm, this insect was considered as a good candidate for producing recombinant proteins. Both germline transformation and baculovirus based bioreactor had been successfully explored for this purpose. However, the genetical manipulation techniques for silkworm lags behind those for some other organisms and need to be further improved. Here on one hand, electroporation, a method to introduce foreign genes into silkworm eggs, was firstly systematically analyzed and optimized. And two sets of plasmid vectors for silkworm transformation were tested after introduced into silkworm eggs by electroporation. On the other hand, a purification friendly bioreactor on silk gland specific secretory expression of foreign gene in silkworm Bombyx mori with rAcMNPV system was built based on the facts that some silkworm strain is susceptible to rAcMNPV. In the first part of this thesis, it was shown that when introduced into silkworm eggs by electroporation, the foreign gene in the newly hatched and 3rd instar larva DNA can be detected by PCR and PCR product Southern blotting, respectively. The amount of foreign gene in 3rd instar larva DNA was about 1/100 of that in newly hatched larva DNA. The efficiency of introducing foreign gene into silkworm eggs was voltage dependent and showed high difference between the tested silkworm strains. The choice of methods and time for detection of foreign gene for different purposes and electroporation parameters for foreign gene indroduction into eggs of different silkworm strains should based on these results. In virtue of the large quantity processing ability of the electroporation method, we used it asforeign gene introduction method for silkworm transgenic vector test. When piggyBac transposon system was used, it was found that in vitro transcribed transposase mRNA facilitated transposition to take place earlier and NLS could result in higher transposition probability and earlier transposition as well. This means the effiency for piggyBac systems to transpose foreign gene into silkworm genome could be hightened, which will benefit the present silkworm transgenis status characterised with low positive rates. In order to see the effect of length of homologous sequences on the recombination probability in silkworm, linearized vectors containing varied length of flanking homologous arms around a reporter gene were transferred into silkworm eggs by electroporation. The one with 2.6 kb total arm length gave higher G1 positive ratio than the other two with total arm length of 1.5 kb and 800 bp respectively, which means the longer homologous arm length should be prefered in plasmid construction. The second part of this thesis is based on the previous results of our laboratory, which shown that there are some silkworm strain susceptible to rAcMNPV. Here, To evaluate the possibility of establishing an in vivo baculovirus expression system in a silk gland specific secretory way, the recombinant Autographa californicanucleopolyhedrovirus (Acserpegfpâ–³EGT) carrying the reporter gene egfp downstream of the PCR cloned silkworm ser1 promoter and signal peptide coding sequence was generated. The purified recombinant baculovirus Acserpegfpâ–³EGT was injected into the haemocoel of newly ecdysed 5th instar silkworm larvae at the amount of 106 pfu per larva. At 5 days post injection, green fluorescence derived from EGFP could be observed with fluorescent microscope in only the silk gland but not other tissues after dissection of the silkworm. By making an opening on the silk gland wall, green fluorescence could be observed in the outflow of silk gland indicating the secretion of EGFP and the effectiveness of ser 1 signal peptide. Western blotting assay confirmed that EGFP exists in the water-soluble part of cocoon silk too. We also established a simple protocol to purify EGFP from the secreted silk proteins. From the above results, it was concluded that rAcMNPV system not only can be used for constructing silkworm based bioreactor, but also can be utilized as gene introduction vector for in vivo analysis of the tissue specific gene transcription regulation, expression and secretion in silworm. The reason for rAcMNPV, other than rBmNPV, was chosen in this study was that rAcMNPV system is a commercialized system with routing operation methods that can be easily carried out in normal laboratory. Comparing with...