摘要
针对柑橘老熟组织中富含多糖、多酚、色素和其它次生代谢产物的特点,以柑橘老叶老皮为试材,在传统CTAB提取法的基础上通过添加增效剂并优化配方,建立了一种快速、简便的提取植物老熟组织及其内部病原总核酸的CTAB-Triton提取法。新的提取液对细胞的裂解能力更强,且解决了传统CTAB溶液因低温析出而致DNA得率损失的弊端。比较了该法和微量葡聚糖柱纯化法制备模板时韧皮部杆菌的检出率,并用该法制备模板,分析了韧皮部杆菌在柚子叶片中的分布情况,及对其它几种柑橘病原菌进行PCR/RT-PCR检测。结果表明,CTAB-Triton方法能有效提取柑橘老熟叶片的DNA,所得DNA质量高,产量比商品化试剂盒大,提高了韧皮部杆菌的检出率,且可用于柑橘其他病原真菌、细菌,以及DNA或RNA病毒病害的PCR/RT-PCR检测,也可用于定量PCR检测和病原种群多态性分析。
The presence of high concentrations of polysaccharides,polyphenols,pigment,and other secondary metabolites in mature citrus leaves is big challenging to obtain high quality DNA/RNA for citrus disease research. In this study,a reliable,fast and enhanced total nucleic acid extraction protocol(CTAB-Triton),modified from the cetyltrimethylammonium bromide(CTAB)method,was established for DNA extraction of mature citrus tissues. The novel extraction buffer was superior to traditional buffer in cell lysis,and avoided low yield due to CTAB-DNA co-precipitation when temperature was lower than 15 ℃. Meanwhile,good-quality DNA and higher DNA yield were obtained through this extraction method,comparing with the commercial DNA isolation kits. The infection rate of‘Candidatus Liberibacter asiaticus'(CLas)extracted by CTAB-Triton and Sephadex G-50 extraction method was compared,CTAB-Triton method significantly improved the detection rate. SYBR Green-based quantitative PCR(q PCR)assay was performed to compare the concentration of CLas within different parts of pummelo leaves. This new method was also suitable for the extraction of citrus fungi,bacteria,and DNA/RNA viral diseases for the pathogen detection and genetic diversity analysis.
出处
《园艺学报》
CAS
CSCD
北大核心
2014年第11期2342-2352,共11页
Acta Horticulturae Sinica
基金
公益性行业(农业)科研专项(201003067
2010-2014)
重庆市自然科学基金项目(cstc2012jj A80025)
重庆市两江学者项目(2013)
关键词
柑橘
韧皮部杆菌
柑橘黄龙病
DNA提取
柑橘病害检测
citrus
‘Candidatus Liberibacter asiaticus'
huanglongbing
DNA extraction
citrus disease detection
