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作 者:魏巍[1] 许艳丽[1] 朱琳[2] 董楠[1] 韩晓增[1] Li S
机构地区:[1]中国科学院东北地理与农业生态研究所中国科学院黑土区农业生态重点实验室,黑龙江哈尔滨150081 [2]东京大学生物工学专攻,日本东京1138657 [3]美国农业部农业研究局作物遗传研究中心,美国stoneville38776
出 处:《土壤与作物》2013年第1期53-58,共6页Soils and Crops
基 金:中国科学院知识创新工程重大项目(KSCX1-YW-09-09);国家"十一五"科技支撑计划(2006BAD21B01-15)
摘 要:基于木霉菌属真菌内转录间隔区(ITS)序列,设计特异性引物DG和DT应用于土壤木霉菌属基因组DNA的实时荧光定量检测,并建立相应的实时荧光定量PCR体系。应用该反应体系对黑土区长期无施肥(NF)、长期施用N、P化肥(NP)以及长期配施N、P化肥和有机肥(NPM)3种方式下大豆田土壤木霉菌基因组DNA进行绝对定量检测。结果表明,引物DG和DT对木霉菌属真菌有较好的特异性;实时荧光定量PCR反应标准曲线相关系数R2=0.993,斜率为-0.2829,而熔点曲线无杂峰;在大豆生育时期的苗期,NF、NP及NPM措施下每克干土中木霉菌基因组DNA含量分别为0.4ng、0.8ng和1.2ng,且NPM措施含量显著高于NF及NP措施(P<0.05)。图4,参17。Base on the internal transcribed spacers (ITS) sequences of Trichoderma spp.,a pairs of specificity primers(DG and DT)were designed for the real-time quantitative PCR assay of soil Trichoderma. The optimal reaction system of real-time quantitative PCR was established. The masses of genomic DNA of Trichoderma spp. in soybean filed were quantified by using this reaction system,and the genomic DNA was extracted from three long-term fertilization management; no fertilization(NF),chemical fertilization applied with nitrogen and phosphorus(NP)and chemical N,P combined with pig manure(NPM). Primers DG and DT possessed a good specificity to the amplification of Trichoderma spp..The correlation coefficient(R2) and slope of the equation from the standard curve were 0.993 and -0.282 9 respectively,and the melting curve only showed a single peak. At seedling stage of soybean growth stage,the mass of genomic DNA of Trichoderma spp. in NF,NP and NPM treatments were 0.4 ng,0.8 ng and 1.2 ng per gram of soil,respectively. The mass in NPM measure was higher than that in NF and NP measure significantly (P<0.05).
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