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摘要：菊花CmBBX24基因是B-Box家族中的成员，具有调控菊花开花时间和非生物胁迫抗性的作用，除此之外，还影响赤霉素的生物合成。然而，与之相互作用的蛋白知之甚少。本实验在之前用酵母双杂交的方法，从菊花‘神马’的花芽分化的cDNA文库中筛选出与CmBBX24互作蛋白CmBBX22的基础上，构建pGBKT7-CmBBX24诱饵表达载体，与pGADT7-CmBBX22共转化到酵母Y2H中，通过在SD/-Trp/-Leu/-His/-Ade 四缺培养基上报告基因表达的筛选，证明CmBBX24和CmBBX22存在互作。并通过基因枪法轰击洋葱表皮细胞，得知CmBBX24和CmBBX22表达的蛋白均定位在细胞核上。本实验为研究CmBBX22菊花的生物学功能奠定了基础。37293
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毕业论文关键词：酵母双杂交；菊花‘神马’；CmBBX24；CmBBX22；蛋白质相互作用
The interaction of Chrysanthemum CmBBX22 and CmBBX24 by Yeast Two hybrid System
Abstract：Chrysanthemum CmBBX24 gene is a member of the B-Box family，which can regulate chrysanthemum flowering time and abiotic stress resistance，in addition，it can also affect the biosynthesis of gibberellins. However，little is known with the interaction of the protein. On the basis of screening with yeast two hybrid from the chrysanthemum ‘Jinba’s cDNA Library of flower bud differentiation，the interacting protein of CmBBX22 was found，we constructed pGBKT7-CmBBX24 bait plasmid and pGADT7-CmBBX22 prey plasmid to be co-transformed into yeast Y2H on the media SD /-Trp/-Leu/-His/-Ade media，which is to prove the interaction between CmBBX24 and CmBBX22. And the subcellular localization of the 2 proteins in onion epidermal cells by Biolistic Bombardment indicated the expression of CmBBX24 and CmBBX22 protein are localized in the nucleus. This study lays the foundation for the study of biological function of CmBBX22 transgenic chrysanthemum.
Key words: yeast two hybrid；Chrysanthemum；CmBBX24；CmBBX22；protein interaction
目 录
摘要 3
关键词 3
Abstract 3
Key words 3
引言 3
1 材料与方法 4
1.1 材料 4
1.2 方法 5
1.2.1 获得CmBBX22全长 5
1.2.2 构建CmBBX22的AD载体 5
1.2.3 构建CmBBX24及CmBBX24N的BD载体 7
1.2.4 构建pMDC43-CmBBX24和pMDC43-CmBBX22载体 8
1.2.5 亚细胞定位 8
1.2.6 转录激活活性分析 8
1.2.7 互作验证 9
2 结果与分析 9
2.1 CmBBX22全长获得 9
2.2 构建CmBBX22-AD载体 9
2.3 构建CmBBX24及CmBBX24N-BD载体 11
2.4 构建pMDC43-CmBBX22和pMDC43-CmBBX24载体 12
2.5 亚细胞定位 13
2.6 转录激活活性分析 13
2.7 互作验证 14
3 讨论 15
致谢 16
参考文献 16
图1 CmBBX22高保真PCR电泳结果9
图2 LA Taq PCR加酶切位点结果.10
图3 双酶切（BamH I&Not I）10
图4 重组质粒菌落PCR扩增结果10
图5 LA Taq PCR加酶切位点结果.11
图6 双酶切（EcoR I&Sal I）.11
图7 重组质粒菌落PCR扩增结果11
图8 重组质粒菌落PCR扩增结果12
图9 pMDC43-CmBBX22重组质粒菌落PCR扩增结果12
图10 pMDC43-CmBBX24重组质粒菌落PCR扩增结果12
图11 亚细胞定位13
图12 转录激活活性分析14