Gene origination is a basic process during the species evolution. How genes originate and how new genes function and contribute to the better survival of the speecies keep attracting biologist. In order to understand the original of new genes in D. menlanogaster, it seems to be an appealing task to tranfer the new genes into other related species, such as D. yakuba. However there was no such transformation system in our lab. We only have P element transgenic system which works only in D. williston and D. melanogaster. So it is emergent to develop a new platform wich could work in other fuit fly species. We have chosen minos transposon as our tool to set up our new transgenic platform. Minos transposon element originated from Drosophila Hydei is about 1.8kb length harboring two inverted repeats and a transposase gene. The mechanism of the Minos transposition is 刢ut-past. just the same as other type II transposon. Minos works in insect species like silk worm, beetles and mosquito. Minos was widely used as a transgenic tool in insects and mammal cell lines and it could be an ideal candidate genetic tool for us to finish the transgenic works in other fruit fly species.
In our experiment we used Minos transgenic system to set up our new platform in the fruit fly D. melanogaster and D . yakuba. We used a Minos vector plasmid with the enhanced green fluorescent protein (eGFP) gene as a selection marker fused with 3xP3 promoter (an eye specific expressed promoter) and a helper plasmid with the Minos transposase gene controlled by the hsp70 promoter. By microinjection of the two plasmids DNA into fruit fly embryos and 37. induced expression of transposase, the minos moved in the germ line cell of the embryos. We screened transgenic flies in the following generation by eGFP eye
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marker. We got flies with the eyes expressed the eGFP both in D. yakuba and D. melanogaster and confirmed them by amplification of a minos specific fragment . To our knowledge this is the first report showing that Minos succeeds in mediating the transformation of D. yakuba. And the success use of Minos in D. yakuba made us convenience to finish our further job in the D. yakuba which proceeded our investigation in the origin of new genes in D. melanogaster and the other fruit fly species. Rice (Oryza sativa L.) is one of the most important cereal species of the world, supplying more than 50% calories for the world population. It has the smallest genome in monocot plants and. It is a wonderful model organism full of genome data resources and gene mutation database. The development of plant transgenic techniques improves the rice gene function investigation. Agrobacterium tumefaciens has been routinely utilized in rice gene transfer.The T-DNA of Agrobacterium transfer DNA fragments of interest into the rice and integrate into the genome of rice. The wild type T-DNA sited in a plasmid in A. tumefaciens with other virus genes which help the transposition of T-DNA into the plant. Various efforts has been done to facilitate the rice transformation. The T-DNA separated into a new plasmid with high quality of cloning, selection, reproduction and transformation. This improvement brings a new rice transformation system 搕he binary vector system?which means both T-DNA and Ti plasmids are cooperated to finish transgenic work.
Here we referred to the expression data of 揜ice Annotation Project database?and chosen several candidates high expression genes. promoter region. We clone them into the reconstructed T-DNA plasmids and obtained 45 candidate promoter vectors. We used Yundao2 (a variety of rice) to induce callus for the transformation of the promoter T-DNA vectors. We chosen GUS gene as the select marker for the efficiency of candidate promoters. By GUS staining of the transformed callus, we
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select promoters which were efficient and tissue specific promoted for our future use in the rice transgenic experiments.
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