利用酵母双杂交技术筛选油菜FAD2的互作蛋白

利用酵母双杂交技术筛选油菜FAD2的互作蛋白(含选题审批表,任务书,开题报告,中期检查表,毕业论文8200字)
摘 要：【目的】 利用酵母双杂交技术（two-yeast hybrid）鉴定甘蓝型油菜（Brassica napus）中脂肪酸脱氢酶（FAD2）与未知功能的78号基因（编号为No.T35-78基因，编号来自http://genes.mit.edu/GENSCAN.htmL,目前相关信息较少）编码产物是否发生相互作用。【方法】 利用实验室提供的空质粒pGADT-7、pGBK-7分别与FAD2及78号基因cDNA片段连接得到重组质粒pGADT-7-FAD2（prey vector）与pGBKT-7-78(bait vector)，运用LiAc/PEG/ssDNA介导法将空质粒分别与重组猎物载体(prey vector)及诱饵载体(bait vector)两两组合共转化(cotransformation)感受态酵母PJ69-4A，再分别观察转化子在SC/-leu-trp以及SC/-leu-trp-his+3-AT固体培养基上的生长情况。【结论】转化后的酵母菌PJ69-4A均可在SC/-leu-trp上生长，而涂布接种至SC/-leu-trp-his+3-AT的培养基上时则不生长。由此得出结论，在油菜细胞内，FAD2与78号基因表达产物可能不发生相互识别或者不存在直接作用。
关键词：酵母双杂交；甘蓝型油菜；脂肪酸脱氢酶；78号基因；互作；LiAc/PEG/ssDNA介导法转化

Screening for Proteins Interacting with FAD2 in Brassica napus by Two-Yeast Hybrid
Abstract: [Objective] Use two-yeast hybrid to determine whether FAD2(fatty acid desaturase 2) interacts with the product encoded by a DNA fragment named NO.78(Numbered as No.T35-78 ,from http://genes.mit.edu/GENSCAN.htmL), which is still partly unknown .[Methods] Conducting a sery of double cotransformations on yeast PJ69-4A via LiAc/PEG/ssDNA-mediated method with empty plasmids including pGADT-7 and pGBKT-7 respectively in combination with FAD2 cDNA-inserted plasmid pGADT-7-FAD2(prey vector) and Gene No.78 cDNA–inserted plasmid pGBKT-7-78(bait vector),followed by observation on the growth of transformed yeast on SC/-leu-trp and SC/-leu-trp-his containing 3-AT of vaerity in concentration . [Conclusion] All the four batches of cotransformation resulted in well growing yeast colonies on SC/-leu-trp while no colonies couLd be detected when reinocuLated by streaking on SC/-leu-trp-his plates. Deducted from the result ,in Brassica napus cells,there is probably neither direct mutual recognition nor interaction between them .
Key words: two-yeast hybrid;Brassica napus;fatty acid desaturase;Gene NO.78;interaction;LiAc/PEG/ssDNA mediated transformation
 

目 录  
摘要……………………………………………………………………………1
关键词…………………………………………………………………………1
Abstract ………………………………………………………………………………1
Keywords ……………………………………………………………………………2
1前言……………………………………………………………………………2
1.1引言…………………………………………………………………………3
1.2原理…………………………………………………………………………2
1.3 实验流程  …………………………………………………………………5
1.4目的与研究意义……………………………………………………………6
2 材料与方法…………………………………………………………………………6
2.1材料…………………………………………………………………………6
    2.1.1 菌株、载体…………………………………………………………6
2.1.2 主要试剂……………………………………………………………6
2.1.3 主要仪器设备………………………………………………………7
2.2方法…………………………………………………………………………9
    2.2.1酵母菌株的活化与扩大培养 ……………………………………9
2.2.2 感受态细胞的制备与共转化………………………………………9
3 结果与分析 ………………………………………………………………………… 10
3.1 PJ69-4A菌株活化……………………………………………………… 10
3.2 SC/-leu-trp二缺培养基上长出转化子……………………………… 11
3.3 SC/-leu-trp-his+3-AT上筛选转化子…………………………………11
4.讨论 ……………………………………………………………………………………12
参考文献………………………………………………………………………………… 14
致谢…………………………………………………………………………………………15