福建省常见尸食性蝇类的COⅠ及16S rDNA序列鉴定

目的利用线粒体细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunitⅠ,COⅠ)以及16S核糖体脱氧核糖核酸(ribosomal deoxyribonucleic acid,rDNA)基因片段序列对福建省常见尸食性蝇类种属进行分子鉴定,探讨两种遗传标记的鉴别效力。方法收集福建9个地区案件现场捕获的常见尸食性蝇类样本22只,经形态学鉴定后提取DNA,扩增COⅠ及16S rDNA基因片段,测序后上传GeneBank数据库,使用BLAST、MEGA 10.0软件进行序列比对、同源性分析以及种内和种间遗传距离分析,并采用非加权组平均法(unweighted pair-group method with arithmetic means,UPGMA)分别构建福建省常见尸食性蝇类COⅠ及16S rDNA基因片段序列的系统发育树。结果经形态学鉴定,共收集到3科5属6种常见尸食性蝇类。基因序列分析结果显示,16S rDNA基因片段序列的种间遗传距离均数为1.8%~8.9%,种内遗传距离均数为0.0%~2.4%;COⅠ基因片段序列的种间遗传距离均数为7.2%~13.6%,种内遗传距离均数为0.0%~6.3%。结论COⅠ和16S rDNA基因片段序列均能够对不同蝇种的种属进行鉴别,16S rDNA对于福建省常见丽蝇科尸食性蝇类的种属鉴别价值更高。

牦牛肠道与粪便乳酸菌的分离鉴定及PCR-16 S rDNA鉴定

以取自四川省不同地区的牦牛粪便、肠道内容物为材料,用MRS琼脂双层培养基进行厌氧培养,分离到50株乳酸菌,经生化鉴定为嗜热链球菌(2株)、乳酸乳球菌(1株)、保加利亚乳杆菌(5株)、嗜粪乳杆菌(10株)、嗜酸乳杆菌(8株)、乳酸乳杆菌(9株)、肠乳杆菌(10株)、弯曲乳杆菌(5株)。采用乳酸菌16 S rDNA通用引物,对分离的8种菌的16 S rDNA一段可变区序列进行扩增,均得到大小约470 bp的产物;扩增产物经纯化、测序后与GenBank中标准菌株的核甘酸序列比较,同源性均大于97．5％,同源性分析与生化试验的结果是一致的。证实,牦牛肠道和粪便的乳酸菌较为丰富,且乳杆菌的数量较多,这可能与牦牛复杂的生长环境有关。

Isolation and Identification of High-Quality Lactic Acid Bacteria in Forage Corn

[ Objective] To screen suitable lactic acid bacterium strains from forage corn which can be used as silage additives. [ Method] The lactic acid bacterium strains were isolated by inoculation on MRS solid medium containing calcium carbonate, and they were preliminarily identified through morphological, physiological and biochemical experiments. The acid production efficiency was determined. Twelve strains having strong acid-pro- duction ability were selected, and their salt tolerance and acid tolerance were detected. The sequences of their 16 S rDNA were also analyzed. [ Result] A total of 44 lactic acid bacterium strains were isolated from the forage com. As evidenced by the physiological and biochemical experi- ments, the twelve strains having strong acid-production ability belonged to Leuconostoc, Lactobacillus and Enterococcus, respectively, and they had strong salt tolerance and acid tolerance. According to the sequences of 16 S rDNA, A4, B9, B11, B12 and B14 were Lactobacillus plantarum; A1, A2., A7, A11 and B8 were Leuconostoc mesenteroides dextran subspecies; and AB and A9 were Enterococcus hirae. [ Conclusion ] The lactic acid bacterium strains with strong acid-production ability isolated from forage corn can be developed into silage additives.

Effect of Lycopus lucidus Turcz.supplementation on gut microflora and short chain fatty acid composition in Crj:CD-1 mice

We investigated the diversity and composition of microflora in feces of Lycopus lucidus Turcz.-fed mice.In addition,we evaluated the production of major cytokines(Interleukin-6 and-10)which are related to inflammation and fatty acid composition of several tissues.16S ribosomal DNA sequencing-based microbiome taxonomic profiling analysis was performed utilizing the EzBioCloud data base.Male mice fed on L.lucidus showed a significantly reduced number of lactic acid bacteria and coliform in the feces compared with the control group(p<0.05).16S rDNA sequencing analysis of fecal samples showed that L.lucidus supplementation decreased the community of harmful microflora(Enterobacteriaceae including Escherichia coli and Bacteroides sp.)in feces compared with the control group(p<0.05).There were no significant differences in mRNA expression of cytokine IL-6 and IL-10 between the control and L.lucidus fed groups.The fecal fatty acid composition in the L.lucidus group had percentages of 4:0,6:0,8:0 and 10:0 in the intestine but those short chain fatty acids were not detected in the control group.Our results showed that L.lucidus supplementation influenced gut environment by decreasing harmful microflora and increased the percentages of several short fatty acids.

饲料玉米中优质乳酸菌的分离和鉴定

[目的]从饲料玉米中筛选适合做青贮饲料添加剂的高效乳酸菌。[方法]用MRS+CaCO3固体培养基筛选乳酸菌,经形态学和生理生化试验进行初步鉴定,测定其产酸效率;挑选出12株产酸率强的乳酸菌进行耐盐和耐酸能力的测定,并对其进行16 S rDNA鉴定。[结果]从饲料玉米中共分离得到44株乳酸菌,12株产酸能力强的菌株经生理生化试验鉴定分别属于明串珠菌属、乳杆菌属和肠球菌属,且12株菌株都具有良好的耐盐、耐酸能力。经16 S rDNA鉴定:A4、B9、B11、B12和B14为植物乳杆菌;A1、A2、A7、A11和B8为肠膜明串珠菌葡聚糖亚种;A8和A9为兼性肠球菌。[结论]从饲料玉米中筛选出具有较强产酸能力的乳酸菌,作为青贮饲料添加剂。

Characteristics of dehalogenase from bacteria isolated from the Gut of Pond-reared Rohu (<i>Labeo rohita</i>) Juveniles in Myanmar

Unwarranted accumulation of halogenated compounds in the rivers and streams has in recent years emerged due to the widespread use agricultural pesticides. The presence of these halogenated compounds in the water does not only suppress the immune system of fish but adversely induce serious morbidity and mortality among cultured stocks. Importantly, gradual accumulation of these compounds in the system of cultured and wild freshwater fish species cultured in ponds and floating net-cages in dams and rivers, respectively, poses some risks to humans, the end users. In this study, we attempted to isolate bacteria from the gut of pond-reared rohu (Labeo rohita) in Myanmar, screened the isolated bacteria for dehalogenase gene using molecular technique and tested the ability of these bacteria to degrade halogenated compounds in vitro. The eight bacterial strains studied were identified as Enterobacter mori strain MK- 121001, Enterobacter cloacae strains MK121003, MK-121004, MK121010, Ralstonia solanacearum strain 121002, Acinetobacter baumannii strain MK121007, Chromobacterium violaceum strain MK121009 and Pantoea vagans strain 121011. Only three bacterial strains (MK121002, MK121007 and MK121009) were capable of degrading 2,2-dichloropropionic acid (2,2-DCP) as the sole carbon source up to a final substrate concentration of 20 mM. Their mean growth doubling time ranging from 6-23 hours with the maximum of chloride ion released of 85%. PCR amplifica- tion with oligonucleotide primers designed from group I dehalogenase revealed the presence of deha- logenase genes in all isolates suggesting dehalogenase gene in strains 121001, 121003, 121004, 121010 and 121011 were silenced. In contrast, group II dehalogenase primers did not show any PCR amplification. These results suggest that MK121002, MK121007 and MK121009 only encode a group I dehalogenase and its non-stereoselectivity is in agreement with previoulsly described group I haloacid dehalogenase. The partial gene sequences were blasted but no significant sequence identity was observed. Therefore, it suggest the 2-haloacid dehalogenase of MK121002, MK12-1007 and MK121009 might be a novel group I 2-haloacid dehalogenase. The results indicated a broad distribution of dehalogenation genes in many micro- bial genomes that harbor dehalogenase(s) due to the exposure of the microorganisms to the naturally occurring or man-made halogenated compounds in the environmental systems. So far, microorganisms capable of producing dehalogenases were mainly isolated from soil and scarcely from aquatic animals and their environments. To the authors’ knowledge, this is the first report on the isolation of dehalogenase-producing bacteria from the gut of pond-reared freshwater fish, Labeo rohita, in Myanmar.

Microbial diversity in lake sediments detected by PCR-DGGE

In this study,PCR-denaturing gradient gel electrophoresis(DGGE)was applied to analyze the microbial communities in lake sediments from Lake Xuanwu,Lake Mochou in Nanjing and Lake Taihu in Wuxi.Sediment samples from seven locations in three lakes were collected and their genomic DNAs were extracted.The DNA yields of the sediments of Lake Xuanwu and Lake Mochou were high(10 mg/g),while that of sediments in Lake Taihu was relatively low.After DNA purification,the 16S rDNA genes(V3 to V5 region)were amplified and the amplified DNA fragments were separated by parallel DGGE.The DGGE profiles showed that there were five common bands in all the lake sediment samples indicating that there were similarities among the populations of microorganisms in all the lake sediments.The DGGE profiles of Lake Xuanwu and Lake Mochou were similar and about 20 types of micro-organisms were identified in the sediment samples of both lakes.These results suggest that the sediment samples of these two city lakes(Xuanwu,Mochou)have similar microbial communities.However,the DGGE profiles of sediment samples in Lake Taihu were significantly different from these two lakes.Furthermore,the DGGE profiles of sediment samples in different locations in Lake Taihu were also different,suggesting that the microbial communities in Lake Taihu are more diversified than those in Lake Xuanwu and Lake Mochou.The differences in microbial diversity may be caused by the different environmental conditions,such as redox potential,pH,and the concentrations of organic matters.Seven major bands of 16S rDNA genes fragments from the DGGE profiles of sediment samples were further reamplified and sequenced.The results of sequencing analysis indicate that five sequences shared 99%-100%homology with known sequences(Bacillus and Brevibacillus,uncultured bacteria),while the other two sequences shared 93%-96%homology with known sequences(Acinetobacter,and Bacillus).The study shows that the PCR-DGGE technique combined with sequence analysis is a feasible and efficient method for the determination of microbial communities in sediment samples.