【作者】 乌日娜；

【导师】 张和平；

【作者基本信息】 内蒙古农业大学 ， 畜产品安全生产， 2009， 博士

【摘要】 乳酸菌广泛存在于动物、植物及自然环境中,是同人类生活和生产密切相关的重要微生物之一。Lactobacillus casei Zhang是内蒙古农业大学“乳品生物技术与工程”教育部重点实验室筛选自内蒙地区、蒙古国和新疆地区牧民家庭以传统方法制作的自然发酵酸马奶中,研究表明,该菌株具有许多重要益生性状和功能,尤其是对酸和胆盐具有较强的耐受性,是乳酸菌研究和益生菌开发利用的理想菌种。此项研究从蛋白质组的水平,对益生菌L. casei Zhang在胆盐和酸胁迫下蛋白质差异表达及其分子机制进行了探讨。将蛋白质组学技术与基因组学、代谢组学、传统生物学技术等相结合,为研究乳酸菌对环境胁迫的反应、耐受和适应机制,为益生菌分离筛选、开发推广,提供了较为全面系统的研究思路。研究利用液氮研磨法成功分离提取了L. casei Zhang菌体蛋白质,系统优化了干酪乳杆菌蛋白质组双向凝胶电泳技术(2-DE),使之有效地用于干酪乳杆菌双向凝胶电泳图谱的构建。利用上述方法和技术,研究首先建立了L. casei Zhang和对比菌株L. casei XM2-1在pI 4-7范围内不同生长阶段的全蛋白质表达图谱,并且比较了L. casei Zhang在胆盐和酸胁迫下差异表达蛋白质图谱。经过比较分析,研究利用质谱检测技术和生物信息学技术对其中差异表达蛋白质进行了鉴定、功能分类和代谢途径分析等。同时,利用实时荧光定量PCR技术对某些差异表达蛋白质的相应基因进行了mRNA水平的分析与验证。结果表明:经过对L. casei Zhang在对数生长期与稳定期双向凝胶电泳图谱分析显示,利用优化的蛋白质组双向凝胶电泳技术可以分离到L. casei Zhang约500个蛋白点,通过基质辅助激光解吸电离飞行时间质谱(MALDI-TOF/MS)鉴定出110种蛋白质,并对蛋白质功能、密码子偏好性、蛋白疏水性等进行了分析,从而构建了L. casei Zhang的参考双向凝胶电泳图谱。进一步比较分析L. casei Zhang对数生长期与稳定期的2-DE图谱表明,47种蛋白质在L. casei Zhang不同生长阶段的表达变化明显,其中33种蛋白质在稳定期时的蛋白表达量比对数生长期增强至少2.5倍,包括Dnak、GroEL、LuxS、PK和GalU等19种蛋白质表达量增强在3.0倍以上;此外,6种蛋白质表达量在稳定期下降2.5倍以上;2种蛋白质只在对数生长中期表达;8种蛋白质只发现于稳定期图谱中。根据功能分类,这些差异蛋白质主要为应激蛋白及代谢相关蛋白,可能与L. casei Zhang稳定期对环境变化具有较强耐受性有密切关系。同时,研究利用实时荧光定量PCR技术对其中的8个差异蛋白质,包括DnaK、GroEL、Hsp20-1、Hsp20-2、Galactose mutarotase related enzyme、Beta subunit of pyruvate dehydrogenase complex E1 componen、UDP-glucose pyrophosphorylase和Esterase C的相应基因进行了mRNA水平的动态变化分析,显示不同的基因具有的不同表达模式。而对比菌株L. casei XM2-1与L. casei Zhang的双向凝胶电泳图谱非常相似,说明了它们是同一种属,但是存在菌株间差异表达蛋白质。L. casei XM2-1在对数生长期与稳定期双向凝胶电泳图谱分析表明,有16种蛋白质表达变化明显,其中12种蛋白质在稳定期表达强度增强2倍以上,而有2种蛋白质表达量在稳定期下降。利用双向凝胶电泳技术和质谱技术,研究从蛋白质组的水平对L. casei Zhang在胆盐胁迫下的蛋白质差异表达情况及其胆盐耐受机制进行了初步探索。结果表明,与不含胆盐正常生长至对数生长中期的菌体相比,L. casei Zhang在含有1.5%胆盐的培养基中生长至对数生长期中期,有26种蛋白质表达发生了变化,其中包括应激蛋白、糖代谢蛋白、肽酶、氨基酸代谢蛋白、转录蛋白、核糖体蛋白、核酸代谢蛋白和脂肪代谢蛋白等,说明L. casei Zhang应对胆盐应激具有复杂的调控机制,不是一种或几种蛋白质发生作用。经过实时荧光定量PCR验证和短时间胆盐胁迫蛋白质组研究,其中与应激作用、维持细胞膜稳定性及抗氧化等机制相关联的蛋白质可能在L. casei Zhang耐胆盐中起到非常重要的作用。此外,在研究中还发现,GroEL、DnaK、UspA、GalU、NagA、LuxS、EF-Tu和30S ribosomal protein S2等蛋白质在L. casei Zhang稳定期蛋白质组比较中表达量也增强,也许暗示胆盐与酸应激对干酪乳杆菌有某些相似的胁迫作用,从而诱导了共同的应激机制。但是,胆盐和酸胁迫这两种不同的环境压力也可诱导L. casei Zhang产生不同的特异性表达差异蛋白质,也表明了不同胁迫可能对L. casei Zhang产生不同的影响。L. casei Zhang和对比菌株L. casei XM2-1酸胁迫下生长蛋白质变化结果显示,在不同pH值条件下生长时,分别有15种和14种蛋白质表达发生了变化,主要包括应激蛋白、脂酶、翻译因子等。两株菌在不同pH值下生长虽然有相似的蛋白差异点,但也有个别差异蛋白质表现出菌株依赖性,可能是不同菌株在酸胁迫下特异性表达的差异蛋白质。利用双向电泳技术和质谱技术,对酸和胆盐环境胁迫下干酪乳杆菌L. casei Zhang差异蛋白质组进行了比较研究,不仅有利于从分子水平上揭示其耐受和适应酸和胆盐胁迫的机制,还可以为L. casei Zhang的安全性和其他益生特性研究,提供理论依据,从而为开发、利用和推广我国具有自主知识产权的益生菌干酪乳杆菌L. casei Zhang奠定基础。更多还原

【Abstract】 Lactobacillus casei Zhang, isolated from traditional home-made koumiss in Inner Mongolia of China, was considered as a new probiotic bacterium by probiotic selection tests, especially L. casei Zhang was able to anti-acid and bile salts.In this study, a proteomic research was carried out to identify and characterize proteins expressed by L. casei Zhang under acid or bile salts stress after optimizing the two-dimensional gel electrophoresis （2-DE） conditions.Cytosolic proteins of the strain cultivated in MRS broth in the exponential phase and stationary phase were resolved by two-dimensional gel electrophoresis （2-DE） using pI 4-7 linear gradients. The number of protein spots quantified from the gels was 487±21 （exponential phase） and 494±13 （stationary phase） respectively, among which a total of 110 proteins were identified by MALDI-TOF/MS and/or MALDI-TOF/TOF according to significant growth phase-related differences or high expression intensity proteins. Accompanied with analysis of the COG, CAI, GRAVY value of each protein identified to the whole set of genes encoding proteins of L. casei Zhang, the study provided a very first insight into the profile of protein expression as a reference map of L. casei.Forty-seven spots of L. casei Zhang were also found in the study showing statistically significant differences between exponential phase and stationary phase. Thirty-three of them increased at least 2.5-fold in the stationary phase in comparison with the exponential phase, and including nineteen protein spots （e.g., Hsp20, Dnak, GroEL, LuxS, PK, GalU） whose intensity up-shifted above 3.0-fold. The transcriptional profiles were conducted to confirm several important differential expression proteins by using real-time qRT-PCR. The analysis suggests that the differentially expressed proteins were mainly categorized as stress response proteins and key components of central and intermediary metabolism, implicating these proteins might play potential important role for the adaptation to the surroundings, especially the accumulation of lactic acid in the course of growth, and the physiological processes in bacteria cell. The furter proteomic analysis of L. casei XM2-1 in exponential phase and stationary phase found that sixteen spots were in the study showing statistically significant differences, including 12 proteins up-shifted and 2 proteins down-regulated. From one point of view, the similarity of 2-DE maps between L. casei XM2-1 and L. casei Zhang proved they are belonging to the species of L. casei. From another point, there was particularly differential expression proteins depended on the individual strain.Proteome of L. casei Zhang was studied to achieve a better understanding of the ability to anti-bile salts stress. The protein patterns obtained under growing with 1.5 % or bile salts free condition were compared by 2-DE. Twenty-six proteins were identified to display clearly differential expression, and function annotation demonstrated that they mainly belonged to stress response proteins, carbohydrates metabolism, maintaining membrane stability, amino acid metabolism and anti-oxidative stress response, translation, ribosomal protein, nucleotide metabolism, fat acid metabolism and unknown function. The findings indicated that the response of L. casei Zhang to bile salts stress involves a complex physiological mechanism. From the results of further proteomic study, which the L. casei Zhang instantaneous exposing in 1.0 % oxgall for 30min under changeless of pH condition, the conserved hypothetical protein LCAZH₀885, cysteine synthase, acetyltransferase, aminopeptidase C, and dioxygenase, which deduced by long time bile salts stress, were also found changing expression. Moreover, transcriptional expression of some genes was validated by using the real-time qRT-PCR. On these results, the analysis presumes that proteins involved in the stress response, maintaining cell membrane stability, and anti-oxidative stress response may be playing an important role for bile salts tolerance in L. casei strains.To identify proteins that are differentially expressed by two strains of L. casei Zhang and L. casei XM2-1 growing under conditions of low pH, whole cellular proteins extracted from bacteria grown in batch culture at pH 7.0 or 5.5 were analyzed by 2-DE electrophoresis. Fifteen and forteen proteins, including stess proteins, esterase, and translation factor, etc., altered expression at low pH respectively in L. casei Zhang and L. casei XM2-1. Tht futher analysis shows that there were similar deduced proteins by the acid stress in both L. casei strains, and also had the special expression proteins depended on each strain.更多还原