新疆棉花根际细菌TaqMan探针实时荧光定量 PCR的建立及时空动态分析

【目的】 建立快速检测棉花根际细菌的TaqMan探针实时荧光定量PCR,对新疆棉花不同生育时期根际细菌数量进行检测以及时空动态分析。【方法】 根据棉花根际细菌16S rDNA基因序列,设计、合成特异性引物和TagMan探针,以构建的重组质粒作为阳性标准品,建立标准曲线,并对实际样品进行检测。【结果】 新疆棉花不同生育时期根际细菌数量变化不尽相同:库尔勒、阿拉尔和哈密的棉花根际细菌数量变化趋势在苗期至花期之间相同,而花期至絮期之间变化各不相同;石河子、乌苏和图木舒克的棉花在整个生育时期内根际细菌数量变化趋势基本相同;精河的棉花根际细菌从苗期开始缓慢增加,蕾期至花期之间变化不大,花期之后快速增加。新疆不同采样地点棉花根际细菌数量变化也不尽相同:根际细菌数量变化趋势是东疆、南疆、北疆依次减少。其中,棉花根际细菌数量最多的是东疆地区的哈密,吐絮期达到1.7×10⁷ copies/g(FRW),最少的是北疆地区的精河,苗期仅为8.5×10⁴ copies/g(FRW)。【结论】 建立的TaqMan探针实时荧光定量PCR方法可以快速检测棉花根际细菌的数量,新疆棉花根际细菌数量在时间和空间上变化不尽相同,其中,根际细菌数量最多的是哈密的吐絮期,最少的是精河的苗期。

Establishment and Spatiotemporal Dynamic Analysis of Rhizosphere Bacteria of Cotton in Xingjiang using Real-time Fluorescent TaqMan-quantitative PCR

【Objective】 In order to determine the total amount of rhizosphere bacteria of cotton during different growth periods and study the spatiotemporal dynamics, a rapid real-time fluorescent TaqMan-quantitative PCR technique was established.【Method】 A set of primers and TaqMan probe specific for rhizosphere bacteria of cotton were designed according to the conserved region of 16S rDNA gene, and the recombinant plasmid were constructed as a standard curve, and practical soil samples were detected using the technique.【Result】 The number of rhizosphere bacteria in the rhizosphere of Xinjiang cotton varied in different growth periods. The number of inter - rhizosphere bacteria in Korla , Alaer and Hami were the same between the seedling stage and the flowering period, while there was a difference between flower stage and boll opening stage; The trend of rhizosphere bacteria in the whole growth period of cotton in Shihezi, Wusu and Tumu Shuker were basically the same; The rhizosphere bacteria began to increase slowly from seedling stage in Jinghe, and there was little change between bud stage and flower stage, but it increased rapidly from boll opening stage. The number of total rhizosphere bacteria of cotton showed different trends in different sampling sites of cotton in Xinjiang. The spatial variation trend of number of rhizosphere bacteria was decreasing in the Eastern, Southern, Northern Xinjiang. Among them, the largest number of rhizosphere bacteria was in Hami in Eastern Xingjiang, and reached 1.7×10⁷ copies/g (FRW) in boll opening stage, the least was only 8.5×10⁴ copies/g(FRW) in seedling stage in Jinghe in northern Xingjiang.【Conclusion】 The method of real-time fluorescent TaqMan-quantitative PCR can be used to rapidly detect the number of total rhizosphere bacteria of cotton, and when using this method we find that the number of total rhizosphere bacteria showed different trends in different growth periods and sampling sites of cotton in Xinjiang. The largest number of rhizosphere bacteria was found in Hami in boll opening stage, the least was detected in seedling stage in Jinghe County.