摘要：早期、无损伤、易于操作的阳性芽检测技术,对提高柑橘遗传转化效率是十分有利的。本文设计和构建了nptⅡ：：mgfp5融合基因,通过锦橙上胚轴转化体系研究其在柑橘遗传转化中的作用。nptⅡ：：mgfp5融合基因全长1 578 bp,编码525个氨基酸,作为表达载体p1301NG的选择标记和报告基因。通过农杆菌介导法转化锦橙上胚轴,再生培养1周后用体式荧光显微镜观察外植体的再生情况。在再生培养第9天时即观察到GFP荧光再生芽的形成,再生培养后10~15 d是GFP再生芽形成的高峰期。GUS染色和PCR分子检测结果证明,GFP荧光检测结果是真实可靠的。研究结果表明：nptⅡ：：mgfp5融合基因能够在再生培养早期筛选到阳性转化芽,有助于提高阳性转化芽的存活率,提高柑橘遗传转化效率。

摘要：为了获得CsLOB1启动子较大片段删除的突变体,利用CRISPR/Cas9技术对CsLOB1启动子进行多位点编辑。通过在CsLOB1启动子的EBEPthA4区域及其上、下游设计不同的靶标位点,构建了2个植物表达载体pCas9CsLOB1:2sites和pCas9CsLOB1:3sites,分别对CsLOB1启动子同时进行2个位点和3个位点的编辑。测序结果表明pCas9CsLOB1:2sites和pCas9CsLOB1:3sites的基因编辑效率分别为64.7%和80.0%,突变体植株在2个sgRNA之间发生了DNA片段的删除。进一步的分析发现,不同的sgRNA具有不同的突变效率,其差异是由于不同的sgRNA对CsLOB1-识别和结合能力的差异造成的。本结果表明对CsLOB1启动子进行多位点编辑可以获得删除较大DNA片段的突变体。

摘要：[Objective] This study aimed to establish a multiplex PCR system for de- tecting transgenic ingredients from Citrus. [Method] Based on the pBI121 plasmid sequences published in GenBank and actin gene sequence of Citrus, the primers specific to CaMV35S promoter, NOS promoter, NOS terminator and actin gene were designed, to establish a multiple PCR system which could detect four types of sequences. In addition, orthogonal tests were performed to determine the optimal concentrations of all the components in PCR reaction system, as well as the optimal PCR cycle parameters. [Result] The optimal PCR reaction system should contain 2.5μl of 10xPCR buffer, 2.0μl of MgCI2 （25 mmol/L）, 2.0 μl of dNTP mixture （2.5 mmol/L of each dNTP）, 1.0 μl of actin gene primers （10μmol/L）, 1.0μl of 35S promoter primers （10 μmol/L）, 1.5 μl of NOS promoter primers （10 μmol/L） and 0.5 μl of NOS terminator primers （10μmol/L）, 0.1 μg of template DNA, 1.25 U of Taq DNA polymerase; ddH20 was added to the total reaction system of 25μl. The PCR reaction program consisted of pre-denaturing at 94℃ for 5 min; 31 cycles of denaturing at 94℃ for 30 s, annealing at 64.1℃ for 45 s and extension at 72℃ for 50 s; final extension at 72℃ for 10 min. The reaction system optimized with the orthogonal tests could detect as less as 0.1% transgenic component in the tested samples. [Conclusion] The MPCR detection system established in this study can meet the requirements in theory for detecting the genetically modified ingredients in Citrus or the deep-processed products.

摘要：[目的]建立柑橘转基因成分的多重PCR检测体系。[方法]根据GenBank中pBI121质粒序列和柑橘(Citrus.)Actin基因序列,分别设计CaMV35S启动子、NOS启动子、NOS终止子特异引物和Actin基因的特异引物,建立能同时检测出4种序列的多重PCR检测体系,同时通过正交试验确定该体系的最佳引物浓度和比例及PCR反应体系中各因素的浓度及反应程序,并对该方法的灵敏度进行验证。[结果]试验得到的最佳MPCR反应体系为:10×buffer 2.5μl,25 mmol/L MgCl22.0μl;dNTP Mixture(2.5 mmol/L each)2.0μl,10μmol/L的Actin基因、35S启动子、NOS启动子、NOS终止子引物分别加入1.0、1.0、1.5、0.5μl,模板DNA 0.1μg,Taq DNA聚合酶1.25U,加ddH2O至25μl。PCR反应程序为:94℃预变性5 min;94℃30 s,64.1℃45 s,72℃50 s,31个循环;72℃10 min。试验中,经正交优化后的4重PCR反应灵敏度达0.1%。[结论]该研究建立的MPCR检测体系,理论上已能满足柑橘或其深加工产品的转基因成分检测。