【作者】 薛爱琴；

【导师】 胡秀芳；

【作者基本信息】 浙江理工大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 胶质类芽孢杆菌具有溶磷解钾固氮等多种功能,广泛用于农业、废水处理及生物冶金等领域。为了了解影响胶质类芽孢杆菌功能发挥的关键因素及确定筛选高效菌株的生物指标,本研究对27株胶质类芽孢杆菌和1株土壤类芽孢杆菌的表型和遗传多样性进行了分析。首先,采用ERIC-PCR对28个菌株进行了遗传多样性分析,并对多糖合成关键基因-糖基转移酶家族I（GTFs I）在不同菌株中的存在情况进行了检测。ERIC-PCR获得稳定而丰富的条带,每株胶质类芽孢杆菌扩增得到7^-14条条带,其中6个条带为多个菌株共有;胶质类芽孢杆菌与土壤类芽孢杆菌VKPM B-7517的ERIC扩增条带相似性极低。聚类分析表明,在70%-90%的相似度水平,25个菌株与对照菌株胶质类芽孢杆菌VKPM B-7519聚为一群,该群又分为4个簇,菌株YC12单独为1个群,与RAPD-PCR、gyr B分类结果相似。但ERIC-PCR的条带多样性与稳定性强于RAPD,表明ERIC-PCR具有较高的分辨力,可有效用于种内菌株的鉴定。另外,从11条特异性的RAPD扩增条带中,克隆得到4个功能性基因序列,其中有1个多糖合成关键基因-糖基转移酶家族I基因（960 bp）,该基因分布于26株胶质类芽孢杆菌中,但菌株YC12和土壤类芽孢杆菌未扩增到该基因。ERCI-PCR和糖基转移酶家族I基因分析均证明菌株YC12与其它胶质类芽孢杆菌存在异质性,值得进一步研究。其次,分析了胶质类芽孢杆菌的产酸产多糖及溶磷解钾固氮等功能多样性。结果,胶质类芽孢杆菌的产糖量为2.04^-13.12 mg·ml^-1,其中12个菌株的多糖产量高于9 mg·ml^-1,菌株CGMCC 1.2326、KNP413、CGMCC 1.3714和VKPM B-7517的多糖产量较低。菌株的荚膜面积、菌落直径、发酵液的粘度也呈现多态性,无氮培养基上培养5天菌株的荚膜面积为200.07-857.23μm2,菌落直径为2.00-5.72 mm,发酵液粘度为45.40 -8.77mPa.s。相关性分析表明,多糖产量与荚膜面积、菌落直径和发酵液粘度具有较高的相关性,相关系数分别为0.714、0.824和0.923。产酸（用pH的降低量表征）分析表明,胶质类芽孢杆菌明显产酸,使发酵液pH值降低1.21-2.62,其中菌株SCS3、SCS10、YC8、YC15、YC17、YC1、YC12、YC13、KNP414和VKPM B-7519产酸能力较强。功能多样性分析显示,胶质类芽孢杆菌的溶磷解钾固氮能力存在多样性。胶质类芽孢杆菌使发酵液中的可溶性P和K分别增加0.05^-102.72 mg·l^-1和0.02-27.92 mg·l^-1。其中释磷能力最强的是菌株YC17(102.72 mg·l^-1),其次是菌株SCS17、YC15、SCS16,它们的释磷量分别是96.09 mg·l^-1、84.95 mg·l^-1、79.49 mg·l^-1;释钾能力最强的菌株是SCS10,其次是KNP414、YC9、YC15。相关性分析表明,产酸与溶磷解钾能力存在显著的正相关,相关系数分别为0.777和0.778,产多糖能力与溶磷解钾的相关系数是0.426和0.562,因此产酸产多糖能力可作为溶磷解钾功能的生物指标。凯氏定氮检测发现,胶质类芽孢杆菌在无氮培养过程中,可使总氮量增加0.47^-18.28 mg·l^-1,表明这些菌株具有固氮能力,其中固氮能力最强的是菌株VKPM B-7519(18.28 mg·l^-1),其次是NYY2、YC17、YC15、YC10和SCS10,其固氮量分别为8.15 mg·l^-1、8.04 mg·l^-1、7.64 mg·l^-1、7.52 mg·l^-1、7.08 mg·l^-1。固氮能力和pH值降低量的相关系数为0.395,与产多糖量的相关系数为0.290,相关性较低。胶质类芽孢杆菌的遗传多样性和表型多样性具有一定的相关性。依据ERIC和RAPD构建的聚类图分为两组,每组又分为两个分支:一个分支的菌株多糖产量较高,另一个分支的菌株多糖产量较低。多糖产量与遗传多样性具有较强的相关性。同样,产酸与遗传多样性之间也存在类似的相关性。总之,本论文证实了胶质类芽孢杆菌的表型特征和遗传特性存在明显的多样性。酸与多糖产量与遗传特性之间具有较好的关联性,荚膜面积、菌落直径、发酵液粘度与多糖产量具有较高的相关性,可作为大规模菌株筛选的有效生物学指标。更多还原

【Abstract】 Paenibacillus mucilaginosus, a potassium- and phosphate-dissolving and nitrogen-fixing bacterium in soil, has been widely used as biological fertilizer in China. To study the vital factor affecting the function of P. mucilaginosus, 27 P. mucilaginosus strains from different origins and their diversities in phenotype, genotype were investigated in this study.Firstly, the ERIC-PCR was carried and the glycosyltransferase group I gene（GTFs I）, playing a vital role in polysaccharide biosynthesis, was cloned in genotypic diversity analysis. In the ERIC-PCR test, the P. mucilaginosus strains produced rich and stable bands, which was different from that of strain P. edaphicus VKPM B-7517. Each strain produced 7^-14 bands, of which 6 were shared by many P. mucilaginosus strains. The ERIC pattern was analyzed with the NTSYS-pc software package, and the dendrogram showed that the P. mucilaginosus strains were grouped into 4 clusters according to their origins. Strain YC12 was located in one group by itself. The RAPD-PCR and gyr B gene gave the similar results. However, the numbers of diversity bands present in the ERIC-PCR fingerprintings were greater than that of RAPD-PCR analysis. This result was coincident with great distinguish ability of ERIC-PCR, which can be used to identify intraspecific species.Four functional gene fragments were cloned from 11 special RAPD amplification bands, which included the gene of GTFs I （960 bp）. GTFs I was positively amplified from 26 P. mucilaginosus strains but not from strains YC12 and P. edaphicus VKPM B-7517. The results of ERIC-PCR and GTFs I gene analysis indicated that strain YC12 was different from other P. mucilaginosus strains, which need further research.Secondly, the phenotypic characteristics of Paenibacillus strains, including the sizes of capsules and colonies, productions of polysaccharides and acid, P and K dissolution, and nitrogen fixation capability, were investigated. The Paenibacillus strains produced 2.04^-13.12 mg·ml^-1 polysaccharide production. Twelve of the strains had a polysaccharide production greater than 9 mg·ml^-1, however, strains CGMCC 1.2326, KNP413,CGMCC 1.3714 and VKPM B-7517 produced only 2.04-4.72 mg·ml^-1 polysaccharide. The sizes of the capsules and colonies cultured on the nitrogen free medium for 5 d were 200.07-857.23μm2 and 2.00-5.72 mm, respectively. The fermented liquid viscosity varied between 45.40-8.77 mPa.s. Therefore, the correlations between polysaccharide levels and colony and capsule sizes, and the fermented liquid viscosity were calculated, and the correlation coefficients were 0.824, 0.714, 0.923 respectively. The acid production ability was different noticeably between the strains, which could be demonstrated from the pH decrease of 1.21-2.62 in the zymotic fluid. Strains SCS3, SCS10, YC8, YC15, YC17, YC1, YC12, YC13, KNP414 and VKPM B-7519 appeared to produce more acid.The capacities of P, and K-dissolution and N-fixation were also various between the strains. the concentration of soluble P and K increased by 0.05^-102.72 mg·l^-1 and 0.02-27.92 mg·l^-1, respectively. The maximum increase of P was observed in strain YC17 (102.72 mg·l^-1), followed by strains YC15, SCS16, SCS17, the concentration of soluble P increased were 96.09 mg·l^-1,84.95 mg·l^-1,79.49 mg·l^-1 respectively. The maximum increase of soluble K was observed in strain SCS10(27.92 mg·l^-1), followed by strains KNP414, YC9, YC15. A positive correlation was obtained between acid production and solubilized P and K, with correlation coefficients of 0.777 and 0.778, respectively. The correlation coefficient between polysaccharide production and solubilized P and K were 0.462 and 0.526, respectively. Obviously, both acid and polysaccharide could be the biomarker of P and K dissolution. All the strains appeared to fix nitrogen, and the maximum N-fixing was observed in strain VKPM B-7519 (18.28 mg·l^-1), followed by strains NYY2, YC17, YC15, YC10 and SCS10 with nitrogen increment of 8.15 mg·l^-1, 8.04 mg·l^-1, 7.64 mg·l^-1, 7.52 mg·l^-1 and 7.08 mg·l^-1, respectively. The correlation coefficients between N and polysaccharides and acid production were 0.290 and 0.395, respectively.Interestingly correlation analysis revealed a clear relationship between polysaccharide diversity and molecular （gyrB gene and RAPD） diversity. In the case of polysaccharide diversity, each of the two groups in the phylogenetic trees based on ERIC and RAPD was divided into two branches. One branch contained the strains with higher polysaccharide production, and the other branch contained the strains with lower polysaccharide production. In the case of acid production, a similar relationship can be found. The relationship between molecular markers and phenotypic traits is accordingly apparent.In conclusion, there were large diversities observed in the phenotype and genetic traits of P. mucilaginosus strains. The diversity of genotypic showed good correspondence with that of acid and polysaccharide productions. Colony, capsule sizes, and the fermented liquid viscosity may be the biomarker of polysaccharide production and thus could be used conveniently during preparatory isolation of functional strains on a large scale.更多还原