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采用研磨法分离纯化猪笼草各组织器官中的内生菌,并采用水解圈法和摇瓶发酵法分别进行初筛与复筛,对筛选出的菌株进行16SrDNA分析鉴定。结果表明:在猪笼草叶片、捕虫囊、根和茎等4种器官中共分离出25株内生菌;进一步从中筛选出2株可产胞外蛋白酶的细菌A3、L5,其中菌株A3的产酶能力较高,水解圈/菌落直径比(D/d)值为6.5,发酵液的蛋白酶活力为17.58U/mL;菌株L5的D/d值为3.0,发酵液的蛋白酶活力为15.77U/mL;16SrDNA鉴定结果表明,菌株A3、L5与芽孢杆菌属成员具有99%的同源性,其中A3是枯草芽孢杆菌(Ba-cillus subtilis),L5可能是其的新变种或新亚种。
Endophytic bacteria in tissues and organs of Nepenthes were isolated and purified by grinding method. The primary and secondary screening were carried out by hydrolytic circle method and shake flask fermentation method respectively. The 16SrDNA was identified by the selected strains. The results showed that 25 strains of endophytes were isolated from four kinds of organs, including Nepenthes, pitta, roots and stems. Two strains of bacteria A3 and L5 producing extracellular proteases were screened out, of which strain A3 The enzyme activity was higher, the value of hydrolytic circle / colony diameter ratio (D / d) was 6.5 and the protease activity of fermentation broth was 17.58U / mL. The D / d value of strain L5 was 3.0 and the protease activity of fermentation broth was 15.77U / mL. The results of 16S rDNA identification showed that the strains A3 and L5 shared 99% homology with members of the genus Bacillus, of which A3 was a member of the genus Ba-cillus subtilis and L5 was probably a new variant or a new subspecies thereof.