宏基因组学及其在瘤胃微生物中的应用研究进展

宏基因组学是研究生态群体基因功能的一门崭新的学科。它通过免培方法获得微生物的纯培养,主要技术包括DNA的提取、文库的构建和目标基因克隆的筛选,可用于发现新基因、开发新的生物活性物质、研究群落中微生物多样性等方面。文章介绍了宏基因组学的基本方法,并对瘤胃微生物宏基因组学的应用现状进行了综述。

宏基因组学用于瘤胃微生物代谢的研究进展

瘤胃微生物生态群落是一个非常复杂的生物体系,有着巨大的基因资源以及丰富的生态资源。宏基因组学通过免培养技术,研究生境中全部微小生物遗传物质的总和,在研究瘤胃微生物代谢和开发瘤胃微生物资源上展示出强劲的优势。该技术的应用有望解决反刍动物营养研究中诸多热点或难点问题。为此,本文介绍了宏基因组文库构建和筛选流程,详细讨论了流程中目的基因/基因组富集方法、载体选择原则和样品核酸提取等方法,并对瘤胃宏基因组学的应用现状进行了综述。

瘤胃微生态系统中纤毛虫的生物学意义

作为瘤胃生态系统中微生物群体之一的瘤胃纤毛虫 ,其种群构成、种群关系变化及其与宿主间的复杂关系是当代瘤胃消化代谢研究的一个热点。瘤胃纤毛虫在瘤胃微生态系统中具有重要的生物学意义 ,它对稳定瘤胃内环境 (pH值、发酵类型、甲烷生成、氨氮浓度 ) ,帮助纤维素、蛋白质消化及防止宿主中毒 (硝酸盐、亚硝酸盐、酸 )具有十分重要的意义。然而 ,有一部分学者主张全部去纤毛虫 ,以达到提高动物生产力的目的 ,而且证明在某些日粮条件下瘤胃去原虫确实提高了绵羊出生率及羊毛生长速度。实际上 ,纤毛虫的去留 ,不能机械行事 ,要根据不同的日粮类型。

复合微生态制剂对哺乳期犊牛生长性能及瘤胃微生态区系的影响

本试验旨在探讨复合微生态制剂对哺乳期犊牛生长性能及瘤胃微生态区系的影响。选取3日龄健康荷斯坦母犊牛21头,随机分成3组,每组7头,单栏饲养。空白对照组(C组)犊牛饲喂基础饲粮,植物乳酸杆菌+枯草芽孢杆菌组(ZK组)每日每头犊牛饲喂基础饲粮+5 g植物乳杆菌+5 g枯草芽孢杆菌,植物乳杆菌+枯草芽孢杆菌+低聚果糖组(ZKT组)每日每头犊牛饲喂基础饲粮+5 g植物乳杆菌+5 g枯草芽孢杆菌+5 g低聚果糖。ZK组和ZKT组犊牛均在饲喂基础饲粮1 h后将相应的复合微生态制剂溶解在温水中,用奶瓶灌入。预试期7 d,正试期50 d。结果表明:各组之间平均日增重和平均日采食量无显著差异(P>0.05)。微生物群落Alpha多样性分析表明,各组之间可见物种数量、Chao1指数、ACE指数、Shannon指数及Simpson指数无显著差异(P>0.05);Beta多样性分析表明,ZKT组基于非加权距离主成分分析聚类较好,C组与ZK组相对分散,说明组间群落结构差异较大。以非加权距离矩阵做UPGMA聚类分析,聚类树不能将C组有效区分,ZK组聚类较好,ZKT组聚类最好,说明复合微生态制剂的添加可影响犊牛微生态区系。瘤胃内容物细菌菌落组成分析发现,犊牛瘤胃内容物细菌优势菌门为拟杆菌门、厚壁菌门、放线菌门等,优势菌属为欧陆森氏菌属、未鉴定普雷沃氏菌科、醋香肠菌属等。Tax4Fun功能预测显示,ZK组的基因表达丰度在细胞过程、环境信息处理和未鉴定功能3个一级功能层较C组增高,ZK组的基因表达丰度在遗传信息处理、感染性疾病、氨基酸代谢、其他氨基酸代谢、碳水化合物代谢5个二级功能层比ZKT组、C组升高。综上所述,复合微生态制剂对哺乳期犊牛生长性能和瘤胃菌群丰富度及多样性无显著影响;而ZKT组哺乳期犊牛断奶前的瘤胃微生态区系明显改变,ZK组在功能预测上要好于ZKT组。

日粮精粗比对奶牛瘤胃微生态发酵活动及产物的影响

奶牛瘤胃就像一个大发酵罐,在日粮消化过程中起着重要作用,是一个由细菌、原虫和真菌组成的独特的微生物生态系统。而瘤胃微生物的种类及其数量关系影响奶牛的各种营养物质的消化利用情况,但是瘤胃微生物种类变化也受日粮精粗比的影响。因此,研究高精料和低精料日粮对奶牛瘤胃微生态发酵活动及产物的影响,为高效利用饲料及维持奶牛健康提供理论基础。

热喷麦秸营养补添效果的研究

选用装配瘤胃和十二指肠瘘管的成年杂种羯羊九只,分三组进行试验:1.热喷麦秸组:该组试羊除每日每只给饲400克基础混合精料外,自由采食热喷麦秸,2.热喷麦秸营养补添组:该组试羊饲喂方式基本上和热喷麦秸组相似,所不同的是除基础混合精料和热喷麦秸外,每日每只试羊另外补饲50克鱼粉和0.75千克青草;3.非热喷组:该组试羊每日每只除给饲400克基础混合精料外,另外自由采食未经热喷处理的麦秸。所用的基础混合精料的组成(%)为:玉米60.0;麸皮22.5;大豆饼15.0和混合矿物质2.5。试验总共进行118天。试验期间除观察羊毛生长和增重指标外,主要是对各组试羊的瘤胃微生态环境进行了检测。检测指标包括有瘤胃内48小时干物质消失率;粗蛋白降解率;瘤胃液的 PH 值,氨 N 水平和挥发性脂肪酸(VFA)的数量和比例;瘤胃食糜的 N/S 比;微生物蛋白产量;瘤胃和十二指肠食糜流通量;进入十二指肠的氨基酸数量以及采食量等多项指标。试验结果表明,热喷麦秸补添鱼粉和青饲后,降低了瘤胃内乙酸的比例,提高了丙酸的比例,NGR 指标也趋于改善;瘤胃和十二指肠食糜流通量增加;进入小肠的氨基酸数量增多;采食量明显提高;同时羊毛生长和增重指标也要比其他两组高。

热喷麦秸营养补添效果的研究

选用装配瘤胃和十二指肠瘘管的成年杂种揭羊9只，分3组进行试验：①热喷麦秸组：该组试羊除每日每只给饲400g基础混合材料外，自由采食热喷麦秸；②热喷麦秸营养补添组：该组试羊饲喂方式基本上和热喷麦秸组相似，所不同的是除基础混合糖料和热喷麦秸外，每日每只试羊另外补饲50g鱼粉和0．75kg青草；③非热喷组：该组试羊每日每只除给饲400g基础混合格料外，另外自由采食未经然喷处理的麦秸。所用的基础混合材料的组成（％）为：玉米60．0；铁皮22．5；大豆饼15．0和混合矿物质2．5。试验总共进行118天。试验期间除观察羊毛生长和增重指标外，主要是对各组试羊的疙胃微生态环境进行了检测。检测指标包括有周胃内48小时干物质消失率；粗蛋白降解率；瘤胃液的PH值，氨N水平和挥发性脂肪酸（VFA）的数量和比例；瘤胃食糜的N／S比；微生物蛋白产量；瘤胃和十二指肠食糜流通量；进入十二指肠的氨基酸数量以及采食量等多项指标。试验结果表明，热喷麦秸补添鱼粉和青饲后，降低了瘤胃内乙酸的比例，提高了丙酸的比例，NGR指标也趋于改善；瘤胃和十二指肠食糜流通量增加；进入小肠的氨基酸数量增多；采食量明显提高；同时羊毛生长和增重指标也要比其它两组高。

Biological Characteristics and Rumen Microorganisms of Gayal(Bos frontalis)in Yunnan Province

[Objective]The paper was to provide reference for further study and development of gayal（Bos frontalis）.[Method]According to the research status at home and abroad,the biological characteristics and rumen microorganisms of gayal in Yunnan Province was studied.[Result]Gayal in Yunnan had typical body form and very good meat production performance,its muscle fiber diameter was significantly less than other kinds of cattle;the water holding ratio,muscle tenderness and muscle succulency were significantly higher than others;its amount,shape and structure of chromosome were different from yellow cattle（Bos taurus）and wild cattle（Bos gaurus）,and the amounts of those chromosomes（2n）were 58,60 and 56,respectively.It could create hybrid with yellow cattle;the gayal＇s special diet was bamboo,its in vitro dry matter digestibility（IVDMD）on various crude forage was significantly higher than yellow cattle in Yunnan;the viable bacteria and cellulolytic bacteria in rumen were 4.51×109 and 1.63×109 CFU/ml,which was significantly higher than yellow cattle in Yunnan,its dominant bacteria in rumen mainly was cellulolytic bacteria.[Conclusion]Gayal not only had high academic value,but also had a great development value.

Comparison of gene expression profiles between primary tumor and metastatic lesions in gastric cancer patients using laser microdissection and cDNA microarray

AIM. To study the differential gene expression profiles of target cells in primary gastric cancer and its metastatic lymph nodes using laser microdissection （LMD） in combination with cDNA microarray. METHODS： Normal gastric tissue samples from 30 healthy individuals, 36 cancer tissue samples from primary gastric carcinoma and lymph node metastasis tissue samples from 58 patients during gastric cancer resection were obtained using LMD in combination with cDNA microarray independently. After P27-based amplification, aRNA from 36 of 58 patients （group 1） with lymph node metastasis and metastatic tissue specimens from the remaining 22 patients （group 2） were applied to cDNA microarray. Semiquantitative reverse transcriptase polymerase chain reaction （RT-PCR） and imrnunohistochemical assay verified the results of microarray in group 2 and further identified genes differentially expressed in the progression of gastric cancer. RESULTS： The expression of 10 genes was up-regulated while the expression of 15 genes was down-regulated in 22 gastric carcinoma samples compared with that of genes in the normal controls. The results were confirmed at the level of mRNA and protein, and suggested that four genes （OPCML, RNASE1, YES1 and ACK1） could play a key role in the tumorigenesis and metastasis of gastric cancer. The expression pattern of 3 genes （OPCML, RNASE1 and YES1） was similar to tumor suppressor genes. For example, the expression level of these genes was the highest in normal gastric epithelium, which was decreased in primary carcinoma, and further decreased in metastatic lymph nodes. On the contrary, the expression pattern of gene ACK1 was similar to that of oncogene. Four genes were further identified as differentially expressed genes in the majority of the cases in the progression of gastric cancer. CONCLUSION： LMD in combination with cDNA microaro ray provides a unique support foe the identification of early expression profiles of differential genes and the expression pattern of 3 genes （OPCML, RNASE1 and YES1） associated with the progression of gastric cancer. Further study is needed to reveal the molecular mechanism of lymph node metastasis in patients with gastric cancer.

Expression of COX-2, PCNA, Ki-67 and p53 in gastrointestinal stromal tumors and its relationship with histopathological parameters

AIM: To investigate the expression of Cyclooxygenase-2 (COX-2), proliferating cell nuclear antigen (PCNA), Ki-67 and p53 in gastrointestinal stromal tumors (GISTs) and its relationship with histopathological parameters. METHODS: Twenty-five GISTs were examined by light microscopy and immunohistochemistry. c-kit, CD34, SMA, S-100 protein, COX-2, PCNA, Ki-67 and p53 were detected immunohistochemically and the relationship was evaluated among histopathologic parameters such as mitotic index (MI), tumor grade, tumor size, COX-2, PCNA, Ki-67 and p53. RESULTS: COX-2 protein expression was found in 19 of 25 (76%) of the tumors, and expression was noted in the cytoplasm of the tumor cells. p53 was significantly related to MI and tumor grade but no relationship was found between COX-2, proliferation markers and MI, tumor grade and tumor size. CONCLUSION: COX-2 is expressed in most GISTs and it may play an important role in the proliferation and progression of these tumors or a useful marker to identify GIST. Although immunohistochemical assessment of p53 can be used for distinguishing the risk groups of GISTs, tumor size and mitotic rate should be considered at the same time.

Effect of 2-(8-hydroxy-6-methoxy-1-oxo-1H-2-benzopyran-3-yl) propionic acid in combination with carboplatin on gastric carcinoma growth in vivo

AIM： To investigate the effects of 2-（8-hydroxy-6- methoxy-l-oxo-lH-2-benzopyran-3-yl） propionic acid （NM-3） alone and in combination with carboplatin on tumor growth and apoptosis in mouse models of human gastric cancer constructed by subcutaneous implantation of histologically intact tumor tissue. METHODS： Human gastric cancer SGC-7901 tissues were implanted into the dorsal subcutis of nude mice. One week after tumors reached to a volume of 50-100 mm3 for around 1 wk, these mice were randomly divided into 8 groups （n = 10）. NM-3 was injected peritoneally at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg every other day for 5 wk, combined with carboplatin （5 mg/kg） every third day for 4 wk. As controls of combined treatment, another 4 groups of mice were injected with either NM-3 at 10 mg/kg, 20 mg/kg or 40 mg/kg, or with carboplatin alone （5 mg/kg）. The control mice received normal saline. Tumor weight, tumor growth inhibition （TGI）, and intratumoral microvessel density （MVD） were evaluated. Apoptosis of human gastric cancer was detected by TUNEL method and flow o/tometry analysis, respectively. RESULTS： The mean tumor volume （692.40 ± 58.43 mm3, 548.30 ± 66.02 mm3, 382.13 ± 43.52 mm3） after treatment with carboplatin combined NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg was lower than that after treatment with either NM-3 at the dose of 10 mg/kg, 20 mg/kg or 40 mg/kg or with carboplatin alone. Compared with the normal saline group, NM-3 administered at 10 mg/kg, 20 mg/kg or 40 mg/kg significantly reduced the tumor weight in these groups （P 〈 0.05）. Carboplatin used alone at 5 mg/kg showed minimal effects. But NM-3 in combination with carboplatin had greater effects of tumor weight than either NM-3 or carboplatin alone. NM-3 alone at the dose 10 mg/kg or in combination with carboplatin had no obvious effects on body changes. Two mice died of diarrhea in each of the two groups treated with 40 mg/kg NM-3 or with 40 mg/kg NM-3 in combination with carboplatin. A significant increase in apoptosis was observed in the NM-3 treated groups, and the effect was more significant in the groups treated with carboplatin in combination with NM-3 at 10 mg/kg, 20 mg/kg and 40 mg/kg, than in the control group. The induction of apoptosis was positively associated with the dose of NM-3. NM-3 significantly reduced the neomicrovascular formation of gastric cancer. The MVD was lower in the groups treated with NM-3 or with NM-3 in combination with carboplatin than in the group treated with carboplatin or in the normal saline group （P 〈 0.05）. CONCLUSION： The results suggest that the inhibitory effect of NM-3 on gastric cancer growth is mediated through decreased angiogenesis and the increased induction of apoptosis. Furthermore, NM-3 alone at the dose of 10 mg/kg or in combination with carboplatin has no obvious effects on body changes, indicating that NM-3 in combination with carboplatin may be effective in the treatment of gastric cancer. The toxicity of NM-3 needs further studies.

The Human Microbiota in Health and Disease

Trillions of microbes have evolved with and continue to live on and within human beings. A variety of environmental factors can affect intestinal microbial imbalance, which has a close relationship with human health and disease. Here, we focus on the interactions between the human microbiota and the host in order to provide an overview of the microbial role in basic biological processes and in the development and progression of major human diseases such as infectious diseases, liver diseases, gastrointestinal cancers, metabolic diseases, respiratory diseases, mental or psychological diseases, and autoimmune diseases. We also review important advances in techniques associated with microbial research, such as DNA sequencing, metabonomics, and proteomics combined with computation-based bioinformatics.Current research on the human microbiota has become much more sophisticated and more comprehensive.Therefore, we propose that research should focus on the host-microbe interaction and on causeeffect mechanisms, which could pave the way to an understanding of the role of gut microbiota in health and disease, and provide new therapeutic targets and treatment approaches in clinical practice.

Effects of Rumen-protected Methionine on Rumen Fermentation, lactation Performance and Plasma Characteristics in Dairy Cows

The objective was to evaluate effects of rumen-protected methionine （RP-Met） supplementation on rumen fermentation, lactation performance and plasma characteristics in dairy cows. Twenty-four multiparous （2.2 - 0.40 parity） Holstein dairy cows, averaged 620 ± 12.3 kg of BW, 68 ± 2.5 day in milk and daily milk production averaged 26.0 ±0.3 kg/cow were used in a replicated 4 × 4 Latin square experiment. The treatments were： control （without RP-Met）, LRP-Met, MRP-Met and HRP-Met with 20, 40 and 60 g RP-Met per cow per day, respectively. RP-Met was hand-mixed into the top one-third of the daily ration. Experimental periods were 30 days with 15 d of adaptation and 15 d of sampling. Dry matter （DM） intake and milk yields were not affected （P 〉 0.05） with increasing RP-Met supplementation. Yields of 4%FCM, ECM, milk fat and milk protein were higher （P 〈 0.03） for RP-Met supplementation than control and were quadratically （P 〈 0.05） changed due to the higher percentage of milk fat and protein for RP-Met supplementation than control （P 〈 0.03）. Ruminal pH tended to be lower （P = 0.071） for RP-Met supplementation than control, whereas total VFA concentration tended to be higher （P = 0.086） for RP-Met supplementation than control. Ratio of acetate to propionate decreased linearly （P = 0.001） from 4.01 to 3.57 as RP-Met supplementation increased due to the increase in propionate production. Digestibilities of DM, OM, CP, NDF and ADF in the total tract were higher （P 〈 0.05） for RP-Met supplementation than control and were quadratically （P 〈 0.05） increased with increasing RP-Met supplementation. Plasma concentrations of glucose not affected （P 〉 0.05） with RP-Met supplementation, Plasma concentrations of non-esterified fatty acids （NEFA） were significantly lower （P = 0.017） for RP-Met supplementation than control and were linearly （P = 0.011） changed. Plasma concentrations of beta-hydroxybutyrate tended to be lower （P = 0.068） for RP-Met supplementation than control and were linearly （P = 0.001） changed. The present results indicate that supplementation of diet with RP-Met improved the content of milk fat and protein, rumen fermentation and feed digestion, decreased plasma concentrations of NEFA. It was suggested that the RP-Met stimulated the digestive microorganisms or enzymes in a dose-dependent manner. In the experimental conditions of this trial, the optimum RP-Met dose was about 25 g RP-Met per cow per day.

改善教学方式 提高教学效果——浅谈养殖专业课程的教学改革

职高养殖专业课具有实用性强,涉及面广,理论密切联系生产实际的特点。而职高学生由于缺乏相应的感性认识,总是死记硬背,生搬硬套。学起来感到枯燥乏味。实践证明,传统的教学模式已不适应现有专业课的教学,传统的教学模式中教师在课堂上组织的再好,讲解的再生动,也只是在黑板上插秧,在屋子里造车。只会越来越糟,不能充分发挥学生的主动参与性。那么如何调动学生的学习积极性,提高养殖专业课的教学效果呢?笔者从改革教学方法入手,对课堂教学、实验等教学环节进行了一些有益的探索。

Improvement effect of acupuncture on locomotor function in Parkinson disease via regulating gut microbiota and inhibiting inflammatory factorrelease

Objective To observe the effect of acupuncture on the intestinal flora in Parkinson disease(PD)model mice and explore the mechanism of acupuncture in improving the locomotor function in PD.Methods Thirty-two C57BL/6 mice were randomly divided into a control group,a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)group,a MPTP+acupuncture group(MPTP+A),and a MPTP+madopar group(MPTP+M),with 8 mice in each group.Except for the control group,the other groups were intraperitoneally injected[25 mg/(kg·bw)]with MPTP to establish PD mouse models.After successful modeling,the MPTP group received no intervention,the MPTP+A received acupuncture at Tianshu(ST25),Guanyuan(CV4),and Zusanli(ST36),and the MPTP+M was given madopar[125 mg/(kg·bw)]by intragastric gavage.After consecutive 10-day interventions,the intestinal function and behaviors of the mice were detected.The 16S rRNA gene sequence was used to analyze the composition of fecal intestinal flora in each group.Real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)and enzyme-linked immunosorbent assay were used to detect the expression levels of inflammatory cytokines in the brain and serum.The expression levels of tyrosine hydroxylase(TH)andα-synuclein in the substantia nigra(SN)were detected by immunohistochemical staining.Toll-like receptor(TLR)2 and lipopolysaccharide receptor CD14(CD14)in the SN were determined by RT-qPCR.Myeloid differentiation factor(MyD)88,nuclear factor kappa-B(NF-κB)and Akt1 in the SN were detected by Western blotting.Results After the intervention,compared with the control group,the intestinal motility,fecal water content,and the expression of TH in the SN were significantly decreased in the MPTP group(P<0.05),along with an increasedα-synuclein expression(P<0.05).Additionally,the results of the fecal microflora test showed that the alpha diversity of the MPTP decreased,and the levels of inflammatory cytokines[tumor necrosis factor(TNF)-α,inducible nitric oxide synthase(iNOS),interleukin(IL)-1β,and IL-6]in the serum and SN,and the expression of NF-κB in the SN were significantly increased(P<0.05).Compared with the MPTP group,acupuncture intervention significantly enhanced the autonomous horizontal movement and coordination ability of PD mice(P<0.05);acupuncture and madopar interventions significantly reduced the levels ofα-synuclein,inflammatory cytokines(TNF-α,iNOS,IL-1β,and IL-6)in the serum and SN,and the NF-κB expression in the SN,along with significantly increased alpha diversity richness index(P<0.05).In addition,the relative abundance of Bacteroides increased significantly in the MPTP+A(P<0.05),while the relative abundance of Firmicutes and Cyanobacteria decreased significantly(P<0.05).Conclusion Acupuncture intervention can improve locomotor function,reduceα-synuclein aggregation and inflammatory factors expression,and increase the Akt signaling pathway in PD mice.In addition,acupuncture intervention can benignly regulate the intestinal flora of PD mice.Therefore,it suggests that acupuncture intervention can protect PD model mice probably by regulating intestinal flora and activating Akt signaling pathway.