【作者】 王娜；

【导师】 程家安； 蒋明星；

【作者基本信息】 浙江大学 ， 环境生物学， 2010， 博士

【摘要】 遗传转化技术已在生物基因功能分析、生理生化过程研究以及生物反应器开发等方面大量应用,但同时还存在许多问题,包括外源基因向基因组的整合效率较低,转基因有时不能按预定的方式表达,转入基因后有可能引起生物本身的功能基因发生突变等。要解决这些问题,提高遗传转化的效率,并提高整合的定向性(Site-specific integration)是关键。家蚕Bombyx mori L.是重要的经济昆虫,同时也是昆虫基因功能和生命过程研究的模式昆虫。近十多年来,人们对其开展了大量的遗传转化研究,获得了很多的家蚕转基因品系,但对其定向遗传转化的报道还很少。为此,本实验在家蚕细胞中进行了定向遗传转化的初步研究,以期为提高家蚕遗传转化效率、提高遗传转化的定向性提供一些借鉴。主要结果如下：1.采用双荧光素酶报告基因检测(Dual-Luciferase Reporter Assay)技术,比较了热激蛋白启动子(hsp70和hsp82)、家蚕肌动蛋白启动子(A3)、多聚泛素(polyubiquitin)启动子(PUB)、a微管蛋白启动子(α-tub)、丝素轻链启动子(Fib-L)、人工合成启动子3×P3及苜蓿丫纹夜蛾多角体病毒(AcNPV)增强子-启动子组合(hr5-IE1)8种启动子在家蚕细胞BmN内的活性。结果显示hr5-IE1活性最强,A3次之,其余启动子活性均较弱。构建含有hr5-IE1启动子和piggyBac的转座酶编码区的质粒作为辅助质粒,与EGFP载体质粒一起转染家蚕细胞后,实现了EGFP基因整合到细胞基因组中。2.采用质粒间转座分析(Interplasmid transposition assay)方法,研究了酵母转录激活蛋白Ga14的DNA结合区(DNA-binding domain, DBD)序列和UAS组成的系统(简称Ga14-UAS系统)在提高家蚕遗传转化定向性中的作用。将三个质粒,即表达Ga14-piggyBac融合转座酶的辅助质粒,含有Kan抗性基因和E. coli ori因子的供体质粒,及含有UAS定向序列的受体质粒,共转染家蚕卵巢细胞系(Bm-12)和果蝇细胞系(S2),结果发现,在Ga14-piggyBac融合体作用下,两种细胞中的转座效率分别比对照提高了3.8倍和4.5倍,而且插入位置趋向于少数几个位点。3. LexA是生物体内一种重要的阻遏蛋白,其结合的DNA位点(Binding sites,BS)具明显的序列特征。我们依据LexA与其BS序列结合的原理,构建了可表达LexA-piggyBac融合转座酶的辅助质粒,以及含LexA BS序列的受体质粒pGDV1-Lex-BS。将这两个质粒和供体质粒pB[KOα]一起转染家蚕Bm-12细胞,结果发现,与对照相比转化效率提高了9.0倍,但转座并不集中在一些特定的位点。与前述Ga14-piggyBac融合转座酶相比,在Bm-12细胞系中,LexA-piggyBac介导的遗传转化效率要高一些,但对转化的定向能力较差。4.在家蚕细胞内建立了一个基因组中插入EGFP-Lex-BS片段的细胞系。用于今后LexA-piggyBac融合转座酶在提高转座效率、定向能力等方面的进一步研究。根据本研究结果,针对目前家蚕遗传转化效率较低、转基因的整合缺乏定向性的不足,可选择采用hr5-IE1作为piggyBac转座酶表达的启动子,以提高该酶的表达水平,从而提高转化效率；也可采用piggyBac和Ga14-DBD或LexA融合后所形成的转座酶来提高转化效率；另外,Ga14-piggyBac融合酶还可考虑用于定向遗传转化的研究。这些结果,对提高当前的家蚕定向遗传转化水平有一定的指导意义。更多还原

【Abstract】 The genetic transformation technology has been used widely in researches involving gene functions, physiological and biochemical processes, and bioreactor development. However, several problems exist despite of this success, including low integration efficiency of transgenic genes, low expression levels and somehow unpredictable expression patterns of transgenes, and mutation of inherent genes due to the random integration of transgens. One of approaches to resolve these problems is to increase the efficiency of transformation and the level of site-specific integration.The silkworm, Bombyx mori L., is one of the most economic insects and a model for gene function and biological research in insects. So far there have been a large number of reports targeting the genetic transformation of this insect, with very few of them addressing the site-specific transformation. In this study, we conducted some assasys in the silkworm cells, with the purpose of founding some systems that have the potential of realizing site-specifc integrations in this insect.The major results are as follows:1. To find a promoter that can be used to strongly drive expression of piggyBac transposase and thereby increase the transposition efficiceny, eight promoters were compared in cultured silkworm cells (BmN) as to their transcriptional activity. These promoters were hsp70, hsp82, actin3 (A3), polyubiquitin (PUB), a-tubulin, fibroin-L, artificial promoter 3×P3 and immediately early 1 gene promoter flanked by the hr5 enhancer element (hr5-IE1). The results showed that hr5-IE1 displayed the highest transcriptional activity, followed by A3, while the activities of the other six promoters were relatively low. When transfected with an EGFP vector, and a piggyBac helper plasmid in which the expression of piggyBac transposase was driven by hr5-IE1, the EGFP cassette was successfully integrated into the genome of BmN cells.2. By using interplasmid transposition assays, the efficiency of applying a chimeric Gd14-piggyBac transposase to achieve site-specific integration onto a DNA target plasmid was evaluated within silkworm Bm-12 and fruitfly Drosophila S2 cells. The Gal4-piggyBac transposase has a Ga14 DNA binding domain (DBD), and the target plasmid has upstream activating sequences (UAS) to which the Gal4 DBD can bind with high affinity. The results indicated that, in the Bm-12 and S2 cells, the transpositional activity of Ga14-piggyBac transposase was observed to be 3.8- and 4.5-times higher, respectively, compared to the controls absent with Ga14-UAS interaction. Moreover, the Ga14-piggyBac transposase tended to direct the integration of piggyBac element to certain sites of the target plasmid, although the target-directing specificity was not as high as expected.3. The chimeric LexA-piggyBac transposase was evaluated regarding its capacity of increasing transformation efficiceny and site-specific interaction level in the silkworm cell. This fused transposase was supposed to be able to bind a binding-site (BS) sequence which was located on a plasmid DNA molecule to be targeted during interplasmid transpositions. The results showed that, in the Bm-12 cells, the transpositional activity of LexA-piggyBac transposase was 9.0-times higher than the transposase absent with LexA-BS interaction. However, the integrations on the target molecule appeared to be random.4. A silkworm cell line was established which was genetically transformation with EGFP-LexA-BS fragment. This cell line can be used in future studies to further evaluate the capacity of LexA-piggyBac transposase in increasing transformation efficiency and site-specific integration level.The above results suggest that the promoter-enhancer hr5-IE1 has the potential of serving as a sound element in future piggyBac-based transgenic research of Bombyx mori with the capability of increasing transformation efficiency. The chimeric transposases, Ga14-piggyBac and LexA-piggyBac may also serve as approach to increase transformation efficiency. Moreover, the Gd14-piggyBac transposase has the potential of realizing site-directed transgenesis in this insect.更多还原