A Artemisia squalene synthase gene and its cDNA cloning method, a Artemisia squalene synthase gene and its cDNA cloning method, DNA extraction, according to the plant DNA Extraction Kit manual extraction of Artemisia annua DNA. RNA extraction, PCR amplification with DNA extracted from Artemisia as template, SSPri5 and SSPri3 as primers, amplified by Takara One Shot LA PCR Mix amplification conditions were as follows: 94 C, 1min, 94 C, 57 C, 30s, 1min, 5min, 72 C, 30 circular, 72 C, 4 C, 10min RTPCR amplification with artesunate extracted RNA as template, DH5 alpha transformation competent cells, coated plate, blue white screening recombinants. The results were identified by PCR amplification and EcoR I enzyme digestion. PCR amplification was carried out by Promega Tf1 enzyme. The reaction conditions were: 94 C, 1min to 94 C, 30s, 57 C, 1min, 72 C, 5min, 30 cycles to 72 C, 10min to 4 C. Finally, using the T7 promoter sequencing primers in ABI Prism310DNA sequencer.
【技术实现步骤摘要】
一种青蒿鲨烯合酶基因及其cDNA的克隆方法
本专利技术涉及一种青蒿鲨烯合酶基因及其cDNA的克隆方法，具体地说是以一种青蒿鲨烯合酶基因及其cDNA的克隆方法。
技术介绍
目前公知的青蒿素是我国自主研发的抗疟原虫中药有效单体，对氯奎抗性疟原虫有效，现已成为全球抗疟药的希望。青蒿素是青蒿经类异戊二烯次生代谢途径所形成的倍半萜衍生物。利用基因工程对青蒿进行基因敲变（knockdown）或敲除（knockout）已成为青蒿遗传改良的两大方向。
技术实现思路
诊断标准：研究对象：青蒿（ArtemisiaannuaL．）采自我国海南省三亚市（由四川引种）。试剂；植物DNA提取试剂盒（E．Z．N．A．PlantDNAKit）、植物RNA提取试剂盒（E．Z．N．A．PlantRNAKit）均购自Omega公司；质粒DNA纯化试剂盒（ConcertRapidPlasmidMiniprepSystem）、DNA片段回收试剂盒（ConcertRapidGelExtractionSystem）均购自LifeTechnologies公司；聚合酶链反应（PCR）扩增试剂盒（OneShotLAPC...
【技术保护点】
一种青蒿鲨烯合酶基因及其cDNA的克隆方法， PCR 扩增以提取的青蒿DNA为模板，SSPri5、SSPri3为上下游引物，用Takara公司的One Shot LA PCR Mix扩增，扩增条件为： 94℃，1min→94℃，30s，57℃，1min，72℃，5min，30个循环→72℃，10min→4℃RTPCR扩增以提取的青蒿RNA为模板，转化DH5α感受态细胞，涂板，蓝白斑筛选重组子;分别经PCR扩增和EcoRⅠ酶切进行鉴定;PCR扩增采用Promega公司的Tf1酶，反应条件为：94℃，1min→94℃，30s，57℃，1min，72℃，5min，30个循环→72...
【技术特征摘要】
1.一种青蒿鲨烯合酶基因及其cDNA的克隆方法，PCR扩增以提取的青蒿DNA为模板，SSPri5、SSPri3为上下游引物，用Takara公司的OneShotLAPCRMix扩增，扩增条件为：94℃，1min→94℃，30s，57℃，1min，72℃，5min，30个循环→72℃，10min→4℃RTPCR扩增以提取的青蒿RNA为模板，转化DH5α感受态细胞，涂板，蓝白斑筛选重组子;分别经PCR扩增和EcoRⅠ酶切进行鉴定;PCR扩增采用Promega公司的Tf1酶，反应条件为：94℃，1min→94℃，30s，57℃，1min，72℃，5min，30个循环→72℃，10min→4℃;最后在ABIPrism310DNA测序仪上用T7启动子引物测序。2.根据权利要求1所述的方法，其特征是：扩增条件为：94℃，1m...
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