两个柑橘品种内生细菌富集方法的筛选  

Screening of Enrichment Methods of Endophytic Bacteria From Newhall Navel Orange and Satsuma Mandarin

作　　者：吴思梦[1] 刘冰[1] 蒋军喜[1] 周英[1] 鄢明峰 

机构地区：[1]江西农业大学农学院,江西南昌330045

出　　处：《核农学报》2018年第2期266-273,共8页Journal of Nuclear Agricultural Sciences

基　　金：国家自然科学基金项目(31460139)

摘　　要：为探究不同预处理对柑橘内生细菌的富集效果,本研究以脐橙和蜜柑的叶片与果皮为材料,通过对其内生细菌基因组DNA提取和Illumina测序,比较分析了3种富集内生细菌的方法。结果表明,与对照相比,3种方法均能有效富集内生细菌。样品组织经A处理(1.5%离析酶R-10+1.5%纤维素酶R-10+8.55%蔗糖)、B处理(1.5%离析酶R-10+1.5%纤维素酶R-10+12.8%甘露醇+0.12%MES+0.36%CaCl_2·2H_2O+0.011%NaH_2PO_4)和C处理(8.55%蔗糖)处理后,植物叶绿体DNA占宏基因组DNA的比例均有所降低,其中脐橙叶片和蜜柑果皮经过A处理效果最佳,分别为7.34%、74.89%;脐橙果皮和蜜柑叶片经过B处理效果最佳,分别为36.07%、9.65%;与对照相比,经3种处理能增加OTU分类单元数量,增加变形菌门和厚壁菌门细菌的含量,可获得更多内生细菌种类。由此可知,3种方法在内生细菌富集中均有一定的效果,其中A和B处理最佳,使内生细菌能够更好地被富集,有效减少了柑橘叶绿体DNA的干扰。本试验结果为柑橘及其他植物内生细菌的富集方法和相关研究提供了理论参考。In order to explore the enrichment effect of different methods on citrus endophytic bacteria,the leaves and peels of Newhall Navel orange and Satsuma Mandarin were used as test materials,three methods for enriching the endophytic bacteria were compareed after extracting the macro genomicDNA of plant tissue and detecting the sequence on the Illumina Hi Seq platform. The result showed that compared with the untreated control,three treatments can effectively enrich the endophytic bacteria. When the sample tissues were pre-treated with solution A（ 1. 5% Macerozyme R-10 ＋ 1. 5% cellulase R-10 ＋ 8. 55% sucrose）,solution B（ 1. 5% Macerozyme R-10 ＋ 1. 5% cellulose R-10 ＋ 12. 8% mannitol ＋ 0. 12% MES ＋ 0. 36% CaCl_2·2 H_2O ＋ 0. 011% NaH_2PO_4） and solution C（ 8. 55% sucrose） respectively,the proportion of chloroplast DNA to endophytic bacterial genomicDNA decreased,and the lowest chloroplast DNA proportion was obtained in the sample tissues treated with solution A or B. The chloroplast DNA proportion in endophytic bacterial genomicDNA of NNO leave and SM peel treated with solution A were 7. 34% and 74. 89% respectively,while that of NNO peel and SM leaves treated with Solution B were 36. 07% and 9. 65% respectively. Compared to the control,the number of OTU taxa and the amount of Proteobacteria and Firmicutes from the samples treated with three solutions increased,and more types of endogenous bacteria were identified. Three methods are effective at a certain level for the endogenous bacterial enrichment among which solution A or B treatment was the best,the endophytic bacteria were able to be enriched effectively and the interference of plant chloroplast decreased when the genomicDNA of bacteria from citrus was extracted. The results can provide reference for the research of the endophytic bacteria of citrus and other plant.

关 键 词：温州蜜柑 纽荷尔脐橙 内生细菌 富集方法 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]