【摘要】：新疆土壤由于環(huán)境特殊,人為干擾較少,擁有巨大的微生物資源寶庫,但是目前通過傳統(tǒng)培養(yǎng)方法能分離到的生境中的菌種不到總量的0.01%-10%,大部分微生物資源因?yàn)樘幵诨畹牟豢膳囵B(yǎng)(viable but non-culturable,VBNC)狀態(tài)而未被人們認(rèn)識,更無法被我們所用。VBNC狀態(tài)菌作為自然界中未能開發(fā)利用的巨大微生物資源,新的微生物資源的鑒定及微生物環(huán)境功能的研究值得引起關(guān)注。本課題旨在基于復(fù)蘇促進(jìn)因子(1resuscitation promoting factor, Rpf),采用MPN(most probable number)培養(yǎng)體系,對新疆土壤樣品進(jìn)行VBNC資源菌的復(fù)蘇可培養(yǎng)化研究。共獲得并分離新疆土壤菌株60株,其中VBNC狀態(tài)菌48株,構(gòu)建了一個(gè)小型的VBNC資源菌庫,豐富了實(shí)驗(yàn)室的菌種資源。所分離VBNC菌株分屬于16個(gè)屬：Bacillus, Brevibacillus, Staphylococcus, Brevibacterium, Arthrobacter, Kocuria, Microbacterium, Cellulosimicrobium, Rhodococcus, Streptomyces, Enterobacter, Escherichia, Brevundimonas, Pseudomonas, Ensifer 和 Herbaspirillum。體現(xiàn)了新疆土壤Rpf復(fù)蘇可培養(yǎng)化的VBNC菌具有分布廣,種類多的特性。其中以Bacillus屬菌數(shù)最多,占分離VBNC菌株總數(shù)的40%；放線菌屬種類最多,包括Streptomyces, Microbacterium等7個(gè)菌屬。經(jīng)過對分離菌株的初步分類學(xué)鑒定,共獲得14株潛在的新種。通過形態(tài)學(xué)、細(xì)胞化學(xué)和分子等多相分類學(xué)手段對其中一株Arthrobacter屬的菌株DSXY973完成了菌株系統(tǒng)發(fā)育定位和描述工作。本研究表明Rpf不僅對高GC革蘭氏陽性菌有良好的復(fù)蘇促進(jìn)作用,對低GC革蘭氏陽性菌也具有很好的復(fù)蘇促進(jìn)效果,其中Bacillus屬對Rpf最為敏感,另外,Rpf對一部分革蘭氏陰性菌也有一定的復(fù)蘇促進(jìn)效果。本研究為Rpf對環(huán)境中VBNC狀態(tài)資源菌的復(fù)蘇分離提供了科學(xué)依據(jù),為新菌種資源的開發(fā)和后續(xù)應(yīng)用研究提供了新的思路和途徑。
[Abstract]:Because of the special environment and less human disturbance, Xinjiang soil has a huge treasure trove of microbial resources, but the bacteria species in the habitats that can be separated by traditional culture methods are less than 0.01-10% of the total amount. The majority of microbial resources are not known because they are in a living, uncultivated (viable but non-culturable,VBNC state, and they are not used by us as a huge microbial resource that cannot be exploited and utilized in nature. The identification of new microbial resources and the study of microbial environmental function deserve attention. The purpose of this study was to study the resuscitation and culturability of VBNC resource bacteria in Xinjiang soil samples based on 1resuscitation promoting factor, Rpf), (MPN (most probable number) culture system). A total of 60 strains of soil strains were obtained and isolated from Xinjiang, of which 48 strains were VBNC status bacteria. A small library of VBNC resources was constructed, which enriched the resources of bacteria in the laboratory. The isolated VBNC strains belong to 16 genera: Bacillus, Brevibacillus, Staphylococcus, Brevibacterium, Arthrobacter, Kocuria, Microbacterium, Cellulosimicrobium, Rhodococcus, Streptomyces, Enterobacter, Escherichia, Brevundimonas, Pseudomonas, Ensifer and Herbaspirillum.. The results showed that the VBNC bacteria which could be cultivated by soil Rpf resuscitation in Xinjiang had the characteristics of wide distribution and variety. Among them, the Bacillus genus was the largest, accounting for 40% of the total isolated VBNC strains, and the actinomycetes genus was the most, including 7 genera such as Streptomyces, Microbacterium. Through the preliminary taxonomic identification of the isolated strains, a total of 14 potential new species were obtained. The phylogenetic localization and description of one of the strains of Arthrobacter genus DSXY973 were carried out by means of morphological, cytochemistry and molecular multiphasic taxonomy. The results showed that Rpf not only promoted the recovery of Gram-positive bacteria with high GC, but also promoted the resuscitation of Gram-positive bacteria with low GC. Bacillus genus was the most sensitive to Rpf. Rpf can also promote the recovery of some Gram-negative bacteria. This study provides a scientific basis for the recovery and isolation of VBNC resource bacteria in the environment by Rpf, and provides new ideas and approaches for the development and application of new strain resources.
【學(xué)位授予單位】：浙江師范大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：S154.3

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本文編號：2347411