【作者】 黄胤怡；

【导师】 沈明山； 陈亮；

【作者基本信息】 厦门大学 ， 细胞生物学， 2002， 硕士

【摘要】 查尔酮合酶(Chalcone synthase，CHS)是类黄酮合成途径中的一个关键酶。CHS表达量的增加或减少都可能导致植物花色的变异。本实验利用RT-PCR方法克隆查尔酮合酶基因(CHS)的cDNA，通过两步的克隆，构建在植物中表达CHS的植物双元表达载体p1301BΩC。并以农杆菌介导将CHS导入茑萝中，初步建立了茑萝的遗传转化系统，为今后茑萝遗传改良的进一步研究奠定基础。主要结果如下：(1) 以即将开放的中国水仙的花蕾为材料，采用异硫氰酸胍-苯酚-氯仿法提取RNA，反转录成cDNA后用特异引物PCR扩增出1． 2kb左右的片段。核酸序列分析表明，该片段的编码区长1167bp，编码389个氨基酸。与已知的植物CHS核苷酸同源率均达80％以上，氨基酸同源率高达85％。实验结果证明该片段即为中国水仙的CHS cDNA。(2) 为了能在在植物中表达(CHS，通过一步的中间克隆，将CHS连上Camv35s启动子和nos终止子。进一步将重组片段插入植物表达载体pCAMBIA1301，酶切及测序结果都证明植物双元表达载体p1301BΩC构建获得成功。(3) 建立了茑萝的基因转化受体系统。在没有前人的工作基础上，摸索了茑萝脱分化和分化的条件。实验认为茑萝子叶愈伤组织诱导培养基为MS+6-BA 0． 5 mg／L+2,4-D 2 mg／L，胚轴愈伤组织诱导培养基为MS+6-BA 1mg/L+NAA 0． 5 mg／L。愈伤组织分化培养基为MS+6-BA 2 mg/L。(4) 初步建立了农杆菌介导的茑萝遗传转化体系。实验结果表明叶盘法不适合茑萝的遗传转化。而GUS检测表明茑萝的疏松愈伤组织有较高的遗传转化率。为了进一步验证茑萝的转化，进行了分子检测实验。以载体T-DNA上的一段DNA设计一个引物，以CHS上的一段DNA为另一个引物，进行PCR检测。同时，还以DIG标记的Camv 35s上的一段为探针进行Southern点杂交和PCR-Southern杂交。二者都进一步证实了CHS基因已整合到茑萝基因组中。更多还原

【Abstract】 Chalcone Synthase (CHS) is a key enzyme in the biosynthesis of all classes of flavonoids. Increase or decrease its expression in plant would change flower color. We cloned the cDNA sequence of CHS gene from Narcissus by RT-PCR and constructed plant expression vector p1301BC by one median clone. At the same time, we transfered CHS to Quamocliy pennata by Agrobacterium mediated and established genetic transformation system of Quamocliy pennata through the guide of GUS reporter gene. The main results are as below.1. We extracted RNA from Narcissus, cloned the cDNA sequence of CHS gene by RT-PCR and analyzed the coding sequence of gene. The result demonstrates that the sequence of the coding region is 1167bp,encodes a protein of 389 amino acid. The homology between Quamocliy pennata CHS’s cDNA and other plant’s is above 80%, 85% between amino acid.2. In order to express CHS in plant, we constructed plant expression vector p1301B Q C. First added Camv 35s promoter and nos terminator to CHS by one median clone. And then ligated the whole fragment into plant expression vectorpCAMBIA-1301. The result of identification of restriction Sites of p1301B Q C and the sequence examination showed that the construction was successful.3. Tissue culture procedure in the Quamocliy pennata genetic transformation was developed. Cotyledon produce callus in MS+6-BA 0.5 mg/L+254-D 2 mg/L and hypocotyl produce it in MS+6-BA 1 mg/L+NAA 0.5 mg/L. Callus initiate in MS+6-BA 2 mg/L.4. Established efficient transgenic technology of Quamocliy pennata. Gus examinaton showed that the rate of transgene was higher. And PCR analysis ,PCR-Southem and Southern analysis all showed that CHS was intigrated into Quamocliy pennata chromosome successfully.更多还原