【作者】 曾凡锁；

【导师】 詹亚光；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2005， 硕士

【摘要】 本研究分析了由农杆菌介导法获得的转基因白桦的细胞学水平变异情况,同时用RAPD标记方法对转基因白桦在分子水平的变异情况进行了分析,并根据多态性指数与其他转基因植物的变异作了比较研究。本研究还进一步深入研究外源基因在转基因白桦中的整合特性及对转录表达的影响、目的基因和标记基因(及报告基因gus)的整合与表达的相关性和稳定性。结果如下: 1.采用常规SDS方法、改良SDS法、常规CTAB法、高盐CTAB法和改良的CTAB法对富含多糖的白桦成熟叶片DNA进行提取,并对得到的DNA进行电泳分析和含量测定,结果表明改良的CTAB法所提取的DNA纯度高,质量较好,用限制性内切酶酶切后条带均一,证明该方法适于提取白桦成熟叶片的基因组DNA,能满足进一步分析的要求。 2.应用细胞学方法分析了由农杆菌介导法获得的转基因白桦的细胞学变异情况,结果表明转基因白桦的染色体变异频率为78.5%,远远高于非转基因白桦的变异频率(15.3%),且变异以非整倍体占多数。同时用RAPD标记方法研究了转基因白桦在DNA水平的变异情况,结果显示在转基因苗群体中,14个引物共检测到90个位点,其中多态位点57个,占63.33%;腋芽增殖组培苗群体中检测到的90个位点中,14个为多态位点,占全部位点的15.6%。按检测到的多态位点比率排序,二者顺序为:转基因苗TR>腋芽增殖组培苗CK。转基因苗TR与腋芽增殖组培苗CK间的遗传距离为0.6502,相似系数为0.5219。由此可以看出两群体间有一定程度的变异产生。这些都说明了转基因白桦产生的DNA变异显著高于组培苗对照。DNA多态性指数为31.67,并与其它转基因植物的变异情况作了比较研究。 3.跟踪研究30个转基因白桦无性系中的目的基因整合的稳定性,结果表明目的基因在三年中均稳定存在。这30株转基因白桦中有28株的gus基因能够稳定表达,占总数的93.3%。 4.利用HpaI单酶切转基因白桦基因组DNA,通过Southern杂交确定目的基因在转基因抗虫白桦中的拷贝数(或整合位点数)。结果显示,在供试的21株转基因白桦中拷贝数较低介于1-8个之间。其中有四株为单拷贝整合,一株为两拷贝,四株为三拷贝,其余为五至八拷贝。bt基因在单拷贝和多拷贝植株间均有转录水平沉默发生,这说明外源基因的转录水平沉默与其拷贝数之间没有必然联系。且bt表达具有一定的时序性,即从5月至8月表达量逐渐降低。 5.在20株转基因白桦中gus、nptⅡ、bt三个基因均已整合,三个基因的共整合频率达100%。在10个无性系中只有6个无性系的gus、nptⅡ、bt三个基因共表达。更多还原

【Abstract】 In this study the occurrence of cytological variation in transgenic birch plants produced by infecting calli with Agrobacterium tumefaciens has been studied. And genetic variation between transgenic plant population and control population has been analyzed by RAPD.The comparison of Polymorphism index produced in previous analyses of transgenic plants and transgenic birch obtained by infecting calli with Agrobacterium tumefaciens has also been analyzed. This study also analysed the influence of different integration patterns on the expression of exogenous gene.1. Genomic DNA was extracted from mature birch leaves in which polysaccharide is very rich by five different methods of SDS, improved SDS, CTAB, CTAB （with high salt）, and improved CTAB. The DNA was analyzed by agarose gel electrophoresis, concentration test, rations of D₂₆₀/D₂₈₀ and digestion of restriction enzyme. The results show that the DNA sample obtained by improved CTAB method had high concentration DNA and low protein and polysaccharide content. So the improved CTAB method is the best method for DNA extraction from mature birch leaves.2. The occurrence of somaclonal variation in transgenic birch plants produced by infecting calli with Agrobacterium tumefaciens has been verified by cytological and RAPD analysis. The cytological analysis of transgenic plants showed that the frequency of non-diplod is 78.5%, and the non-transgenic control is 15.3%. In this study 14 arbitrary primers were used to analyze genetic variation between transgenic plant population and control population. 90 loci were detected in total.. In transgenic plant population the percentage of polymorphic loci was 63.33%. The percentage of polymorphic loci was 15.6% in control population. The genetic distance was 0.6502 and the genetic identify was 0.5219 between the transgenic plant population and control population. The results showed that genetic variation in transgenic plant population was much higher than that in control population. The RAPD result indicated the transgenic plants’ PI was 31.67. The comparison of data produced in previous analyses of transgenic poplar obtained by infecting calli with Agrobacterium tumefaciens （PI is 57.82） showed that transgenic birch is characterized by fewer genomic changes.3. Among 30 transgenic plants bt gene integration was stabile in three years. And the expression of gus gene was stabile among 28 out of these plants in three years.4. In order to determine the copy number single restriction enzyme HpaI was used to digest the genome DNA. Southern blot analysis of the transgenic birch plants indicates that the Bt gene has been integrated into the genome of the birch. Among the 21 transgenic birch plants, the copy number varies from one to eight. The results of RT-PCR analysis show that bt gene silence in translation level occurs not only in single copy but also in multi-copy plants. So no absolute correlation was found between the numbers of transgene copies and the gene expression. The results of RT-PCR analysis also show that the expression of bt gene at mRNAlevel is different at different developmental stages. Gus gene and npt II was 100%. Bt gene, gus gene and npt II gene was co-expression in six from ten clone.更多还原