2种玉米青贮饲料青贮过程中主要微生物的变化规律研究

旨在研究玉米青贮过程中各主要微生物的数量变化及变化趋势.做去棒揉丝和带穗切段瓶制青贮,取青贮前玉米青贮原料及在装瓶后第0.5、1、2、3、4、5、6、7、9、11、13、15、20、25、30、35、40、45、50天的青贮样品,用无菌水浸泡,然后用选择性培养基培养其中的主要微生物,包括乳酸菌、酵母菌和霉菌,最后进行计数.结果表明,在玉米青贮密封后60 d内,水分含量变化很小.pH在第2天下降到4以下,然后稳定在3.5左右,带穗切段玉米青贮pH低于去棒揉丝玉米青贮.乳酸菌数量剧增,在第6、7天时达到最高峰,为109数量级,之后数量缓慢下降,于第15～20天时稳定在107数量级,去棒揉丝玉米青贮的乳酸菌数量在第7天达到最高峰,带穗切段玉米青贮则在第6天出现最高峰,其稍高于去棒揉丝玉米青贮.酵母菌在青贮初期数量有些波动,出现最高峰到达107数量级后数量随时间的延长而减少,去棒揉丝玉米青贮时酵母菌数量迅速下降,第40天后没有再检测到酵母菌的存在,而带穗切段玉米在青贮12 h之内数量迅速增加,之后缓慢下降,第50天后没有再检测到酵母菌的存在.霉菌因密封后氧气缺乏而数量很快减少,最晚在第11天左右便检测不到,去棒揉丝玉米青贮中霉菌数量下降较迅速,第4天之后便检测不到霉菌存在,而带穗切段玉米青贮则比较平缓,直到第11天之后便检测不到霉菌存在.综上所述,在玉米青贮过程中主要微生物随时间大致呈现逐步减少的趋势；对玉米秸秆进行纵切揉丝处理有利于玉米青贮饲料的制作.     

Influence of soil factors on the soil enzyme inhibition by Cd

A comparative study was conducted on the toxicity of Cd to alkaline phosphatase activity (ALP) and dehydrogenase activity (DHA) in 18 top soils with contrasting soil properties representative of 14 major soil types in China. Soil pH and carbonate content, soil organic matter, and cation exchange capacity (CEC) largely affected the Cd toxicity on two enzyme activities; with the soil pH having only minor effect on the median ecological dose values based on total Cd concentrations (ED_{50 T}). The values of ED_{50 T}/ED_{50 W} (based on water-soluble Cd content) of alkaline phosphatase and dehydrogenase were strongly influenced by pH and CEC contents, which explained up to 71% of the variation for alkaline phosphatase, 82% of the variation for dehydrogenase, and also were significantly correlated with the parameter K_F derived from Freundlich adsorption isotherms. This study suggests that the values of ED_{50 T}/ED_{50 W} could be useful to evaluate the buffer capacity of soils which protects soil enzymes from harmful effects of heavy metal.     

Screening and Analysis of Proteins Interacting with TaPDK from Physiological Male Sterility Induced by CHA in Wheat

To further research the regulatory network of pyruvate dehydrogenase kinase (designated as TaPDK) in physiological male-sterility (PHYMS) of wheat induced by chemical hybridizing agent (CHA) SQ-1, an anther cDNA library was constructed, and the proteins interacting with TaPDK were screened via yeast two-hybrid technique. Subsequently, a few candidate proteins in nucleotide expression levels were detected by real-time quantitative PCR. Yeast-two hybrid screening was performed by mating yeast strain Y2HGold containing BD-TaPDK bait plasmid with yeast strain Y187 including anther cDNA library plasmid. Diploid yeast cells were plated on synthetic dropout nutrient medium (SD/-Ade/-His/-Leu/-Trp) (QDO), and further were incubated on QDO medium containing AbA and X-α-Gal. The interactions between TaPDK and the proteins obtained from positive colonies were further confirmed by co-transformation validation. After plasmids DNA were extracted from blue colonies and sequenced, the sequences results were analyzed by bioinformatic methods. Finally, 24 colonies were obtained, including eight genes, namely non-specific lipid-transfer protein precursor (TanLTP), polyubiquitin (TaPUbi), glyceraldehyde-3-phosphate dehydrogenase, proliferating cell nuclear antigen (TaPCNA), CBS domain containing protein (TaCBS), actin, guanine nucleotide-binding protein beta subunit, chalcone synthase, and three new genes with unknown function. The results of quantitative RT-PCR showed that the expression levels of TanLTP, TaPUbi, and TaPCNA were obviously up-regulated in PHYMS anther, and TaCBS expression was only increased at the tricellular stage in PHYMS anther compared with in fertile lines. Whereas, the expression of TaPDK was obviously down-regulated in PHYMS lines. Collectively, these datas indicated that the majority of candidate proteins might be related to pollen abortion in PHYMS lines, which further suggested that TaPDK plays multiple roles in pollen development, besides participating in regulating pyruvate dehydrogenase complex activity.     

微透析法获取鹅掌柴嫩茎质外体汁液的研究

该文研究的重点是获取质外体汁液。假设微透析技术可以在植物上得到质外体汁液,并以盆栽鹅掌柴为材料,在活体嫩茎插入微透析探针后的不同时间段内分别收集透析液,测定其中的离子浓度和苹果酸脱氢酶活性加以验证。结果表明:在探针插入120 min后,收集的透析液中Na+、K+和Ca2+浓度逐渐趋于平缓,并且苹果酸脱氢酶的活性消失,从而证明120 min后得到的微透析液,没有受到破损细胞液的污染,是纯净的鹅掌柴嫩茎质外体汁液，从而为活体、方便、快捷地获取植物质外体汁液提供新的方法。      

Molecular cloning,characterization and nutritional regulation of key enzymes required for the effective utilization of marine wax esters by Atlantic salmon (Salmo salar L.)

Previous studies had shown that wax ester‐rich lipid extracted from calanoid copepods could be a useful alternative to fish oil as a provider of long‐chain n‐3 polyunsaturated fatty acids in diets for use in salmon aquaculture. Effective utilization of wax ester requires digestion and metabolism in the intestine with the fatty alcohol component being oxidized to fatty acid in intestinal cells through the combined activities of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH). We studied wax ester utilization in Atlantic salmon using a candidate gene approach, focusing on ADH and ALDH as sequence information was available for these genes, including fish sequences, facilitating isolation of the cDNAs. Here, we report on the isolation and cloning of full‐length cDNAs for ADH3 and ALDH3a2 genes from salmon intestinal tissue. Functional characterization by heterologous expression in the yeast, Saccharomyces cerevisiae, showed the products of these cDNAs had long‐chain ADH and ALDH enzyme activities. Thus, ADH3 was capable of oxidizing long‐chain fatty alcohol, and ALDH3a2 was capable of oxidizing long‐chain fatty aldehyde to the corresponding fatty acid. The genes were highly expressed in intestinal tissue, particularly pyloric caeca, but their expression was not increased in salmon fed dietary copepod oil in comparison to fish fed fish oil.     

双向电泳结合蛋白质印迹技术分析猪囊尾蚴乳酸脱氢酶

为了研究猪囊尾蚴乳酸脱氢酶(LDH)的表达,试验选择四川省雅江县呷拉乡猪带绦虫病患者,给其口服槟榔-南瓜子驱虫,收集、制备虫卵悬液(8万个/mL),再给3头20日龄三元杂交乳猪猪灌胃虫卵悬液,每头1 mL,40 d后收集、制备猪囊尾蚴蛋白,进行双向电泳(2-DE)分析,将凝胶蛋白斑点转移至聚偏氟乙烯膜(PVDF膜),用自制的大鼠抗猪带绦虫LDH血清作为一抗、健康大鼠血清作为阴性对照进行蛋白质印迹(Western-blotting)分析。结果表明:双向电泳凝胶共检测到(207±9)个蛋白质斑点,相对分子质量(Mr)为14 400～94 000,等电点(pI)为3.0～10.0;试验组特异性抗原抗体阳性杂交斑点为1个,阴性对照未见阳性杂交斑点;将Western-blotting检测的抗原抗体阳性杂交斑点与原双向电泳凝胶斑点进行比对,找到对应蛋白斑点,经ImageMaster 2D Platinum 5.0软件分析后初步确定该蛋白斑点的pI/Mr为7.03/35 368,与猪带绦虫LDH的pI/Mr理论推导值接近,说明猪囊尾蚴表达LDH。     

1株利用乳酸的猪源丁酸梭菌的分离鉴定及基因组序列分析

【目的】 研究1株利用乳酸的猪源丁酸梭菌的基因组信息。【方法】 采用厌氧培养法, 通过乳酸分解培养基(LADM)初筛、梭菌增殖培养基(RCM)复筛, 从饲喂乳酸菌发酵饲料的健康仔猪肠道内容物中分离利用乳酸快、丁酸转化率高的菌株, 对所筛选的菌株进行形态鉴定、16S rDNA序列分析、乳酸转化特性试验、基因组重测序和框架图测序, 并对其编码蛋白进行功能注释。【结果】 分离得到1株分离菌LY33, 基于形态和16S rDNA序列分析鉴定为丁酸梭菌。菌株LY33对乳酸具有较好的利用能力, 在生长第6天可将66 mmol的乳酸基本转化完全, 生成17 mmol左右的丁酸。LY33菌株的重测序表明, 其编码基因整体变异较小, 与参考基因组相比, LY33菌株全基因组杂合比率为0.022‰, 单核苷酸多态性(SNP)位点变异总数21 590个(占整个基因组0.4666%), 插入缺失(InDel)突变总和594个(占0.0128%), 不存在拷贝数变异(CNV), 结构变异(SV)注释的变异总数103个(占0.0003%)。突变基因主要为与菌株生长、能量代谢调节、维生素合成(特别是生物素)有关的基因, 分布于2条染色体的多条序列上。LY33菌株框架图测序及其编码基因蛋白的功能注释表明, 其基因组序列长度4 627 127 bp, GC含量28.60%, 含有4 171个基因, 编码区总长度占比84.12%, 参与了糖代谢(carbohydrate metabolism)、膜转运(membrane transport)和氨基酸代谢(amino acid metabolism)等多种代谢途径转化过程, 基因组中抗大环内酯类药物、抗杆菌肽、VanI糖肽抗性基因个数较多。【结论】 本研究从饲喂4%乳酸菌发酵饲料的健康仔猪肠道内容物中分离到1株利用乳酸快、丁酸转化率高的LY33菌株, 经鉴定为丁酸梭菌, 其具有良好的应用前景, 可作为一种新的微生物资源用于微生态制剂产品。     

乳酸菌固态混合发酵研究

以玉米粉、豆粕、麦麸为基质,以保加利亚乳杆菌、嗜酸乳杆菌、嗜热链球菌为发酵菌种,采用固态发酵技术,以活菌数为指标,通过单因素和L9(34)正交试验确定了三种菌混合发酵的最佳条件,并对其发酵产物的常规营养成分进行分析测定。结果表明:固态基质中玉米粉:豆粕:麦麸=1:1:1、培养基初始含水量80%p、H值6.3、接种量为10%、三种菌接种比例为1:1:1、发酵温度40℃时的发酵效果最好。在此条件下,保加利亚乳杆菌数为3.0×109 CFU/g,嗜酸乳杆菌数为4.6×109 CFU/g,嗜热链球菌数为5.8×109 CFU/g,发酵产物粗蛋白质、粗脂肪和氨基酸态氮含量分别是发酵前的1.16、1.12和6.94倍。为开发一种新型生物饲料打下基础。     

不同油脂对瘤胃微生物体外产气及其区系动态变化的影响

本试验利用3头瘤胃瘘管山羊提供瘤胃液,以淀粉、纤维素、酪蛋白为底物进行体外培养,研究花生油、菜油、玉米油和豆油等对瘤胃发酵产气及微生物活力状况的影响。结果表明:培养液36 h总产气量在20.61～39.67mL,除花生油组与对照组差异不显著外(P>0.05),其他油脂组都显著低于对照组(P<0.05);随着培养时间的延长各组产气量呈现波动变化。培养液总脱氢酶以豆油组最高,显著高于对照组;并依次显著高于玉米油、花生油、菜籽油等组(P<0.05)。另外,培养液原虫DNA、细菌DNA、微生物DNA、原虫/细菌区系比例的均值与对照组,以及油脂组间差异都不显著(P>0.05),但随着培养时间的延长,微生物DNA在各时间点都以豆油与玉米油组的较高;原虫DNA一般在16 h达到最高,而细菌DNA则在8 h或16 h最高,并显著高于1 h或4 h的量(P<0.05、P<0.01),且各个试验组在培养过程中随时间的动态变化模式也不尽相同。总体看来,不同油脂对瘤胃微生物体外产气及区系动态变化的影响不同。     

乳酸菌富铁的研究

为了使铁离子转化为易被动物体吸收的微量元素,以防治仔猪因缺铁而造成的贫血等病症,本研究用乳酸菌富集铁元素。首先测得乳酸菌在硫酸亚铁和有机铁2种来源的铁元素中所能正常生长的最高铁离子浓度,然后加入适宜浓度的铁离子培养乳酸菌至最高活菌数。菌液离心后冲洗3次并烘干,用原子分光光度法测定乳酸菌干细胞内铁离子的含量,比较乳酸菌对2种形态铁元素的富集能力。最后做饲养试验。结果表明:乳酸菌对有机铁的耐受质量浓度(0.5 g/L)比硫酸亚铁(0.1 g/L)高;乳酸菌对有机铁的富集能力(1.590 mg/g)比硫酸亚铁(0.024mg/g)强。含有机铁的乳酸菌制剂对降低断奶仔猪死亡率有显著效果。