棉花根际土壤古菌实时荧光定量PCR技术的建立及时空分布特征

Establishment and Spatiotemporal Distribution Characteristics of Rhizosphere Soil Archaea of Cotton by Real-time Fluorescent TaqMan-quantitative PCR

【目的】建立快速检测新疆棉花不同生育期、不同地区的根际土壤古菌的数量的实时荧光定量PCR技术,并分析时空分布特征。【方法】运用TaqMan探针根据古菌16S rRNA基因特异性引物对棉花总DNA进行扩增,纯化实时荧光定量PCR产物得到基因序列进行MiSeq测序并构建标准曲线,分析不同生育期及不同地区的棉花根际土壤古菌数量的动态变化以及群落丰度比较。【结果】阿拉尔地区棉花黄萎病病株根际土壤古菌的数量在4个生育时期呈现下降趋势,在棉花吐絮期降至最低1.11×10^(4) copies/g(FRW);哈密、石河子、图木舒克和乌苏土壤古菌数量从棉花苗期到花铃期逐渐减少,在吐絮期升高达到最大,其中哈密吐絮期古菌数量最高达1.75×10^(6) copies/g(FRW);精河和库尔勒棉花根际土壤古菌数量波动明显,呈现先升后降再升趋势,在吐絮期最大。对检测棉花根际古菌群落进行丰度比较Soil_Crenarchaeotic_Group_SCG>Thermoplasmata>Unclassified_p_Thaumarchaeota>Marine_Group_I>Unclassified_k_norank_d_Archaea>Halobacteria>others。SCG纲在不同生育时期和地区均占绝对优势,蕾期为79.94%,哈密为88.84%。【结论】运用TaqMan探针法检测到古菌数量。在不同生育时期棉花病株根际土壤古菌数量较高,其中吐絮期棉花根际土壤古菌数量最高;在不同植棉地区,哈密棉花根际土壤古菌数量最高。不同生育时期和地区的棉花根际土壤古菌群落组成及丰度差异显著,地区土壤环境差别很大。

【Objective】To analyze the temporal and spatial distribution characteristics by using TaqMan probe to establish a real-time fluorescence quantitative PCR technology for rapid detection of the number of Archaea in rhizosphere soil of different growth stages of cotton in Xinjiang.【Methods】The total DNA of cotton was amplified by 16S rRNA gene specific primers,and the gene sequence obtained by real-time fluorescent quantitative PCR was sequenced by MiSeq,and then the standard curve was constructed.The dynamic changes of the number of Archaea in cotton rhizosphere soil and the comparison of community abundance in different growth periods and different regions were analyzed.【Results】The number of soil Archaea in the rhizosphere of cotton verticillium wilt in Alar region showed a downward trend in four growth stages,the lowest was 1.11×10^(4) copies/g(FRW)in cotton boll opening stage,The number of soil Archaea in Hami,Shihezi,Tumushuke and Wusu decreased from cotton seeding stage to flower stage,and increased to the maximum at boll opening stage,among which the highest number of Archaea in Hami was 1.75×10^(6) copies/g(FRW).In Jinghe and Korla cotton rhizosphere,the number of soil Archaea fluctuated obviously,showing a trend of first increasing,then decreasing,and then increasing,among which the largest appeared in the boll opening stage.Comparison of abundances of rhizosphere archaea communities in cotton found that Thermoplasmata>Unclassified_p_Thaumarchaeota>Marine_Group_I>Unclassified_k_norank_d_Archaea>Halobacteria>others.SCG was dominant in different growth stages and regions,79.94%in bud stage and 88.84%in Hami.【Conclusion】The number of Archaea can be detected successfully by TaqMan probe method.At different growth stages,the number of cotton rhizosphere Archaea was relatively high,among which the number of cotton rhizosphere Archaea was the highest at the bolling stage,and the number of Hami cotton rhizosphere Archaea was the highest in different cotton planting areas.There were significant differences in the composition and abundance of soil Archaea community in cotton rhizosphere in different growth stages and regions,which indicated that the soil environment in the region was quite different.