CAJ | 학술논문

从NCBI的dbEST数据库中下载假臭草同族植物的EST序列，经EST序列处理及SSR位点查询，首次设计了27对假臭草EST-SSRs引物，以海南省文昌市假臭草 DNA为模板，采用单因素和L16(45)正交试验对其EST-SSRs PCR反应体系进行优化。建立了假臭草EST-SSRs最佳20μLPCR反应体系： Mg2+浓度为2.75 mmol/L，模板DNA用量为35 ng， Tag DNA聚合酶为2.25 U， dNTPs浓度为0.3 mmol/L，引物浓度为0.6μmmol/L。并用14个不同来源的假臭草样本对其体系进行验证实验，结果均能获得稳定的、清晰明亮的扩增谱带。假臭草EST-SSRs 标记的开发为深入研究其遗传多样性提供了基础。
종NCBI적dbEST수거고중하재가취초동족식물적EST서렬，경EST서렬처리급SSR위점사순，수차설계료27대가취초EST-SSRs인물，이해남성문창시가취초 DNA위모판，채용단인소화L16(45)정교시험대기EST-SSRs PCR반응체계진행우화。건립료가취초EST-SSRs최가20μLPCR반응체계： Mg2+농도위2.75 mmol/L，모판DNA용량위35 ng， Tag DNA취합매위2.25 U， dNTPs농도위0.3 mmol/L，인물농도위0.6μmmol/L。병용14개불동래원적가취초양본대기체계진행험증실험，결과균능획득은정적、청석명량적확증보대。가취초EST-SSRs 표기적개발위심입연구기유전다양성제공료기출。
In this study, we download ESTs form dbEST database and first designed 27 primer pairs for Praxelis clematidea after EST pretreatment and SSR locus identification. The sample were collected from Wenchang City in Hainan province as DNA template, and the optimization and establishment of EST-derived SSR-PCR reaction system for Praxelis clematidea was carried out by single factor test and orthogonal experiment design of L16(45). The result was indicated that the optimal EST-SSRs PCR reaction system (20μL) was contained 2.75 mmol/L Mg2+, 35 ng DNA template, 2.25 U Tag DNA polymerase, 0.3 mmol/L dNTPs and 0.6 μmol/L primer. This system was tested by 14 Praxelis clematidea from different places, which stable and clear polymorphic bands were acquired. Accordingly, the development of EST-SSRs can be used for the genetic diversity research of Praxelis clematidea.