猪附红细胞体16S rRNA基因的序列测定和系统进化分析

从确诊为猪附红细胞体感染的猪场，无菌采集血样，抽提猪附红细胞体基因组DNA，采用真细菌的通用引物进行16S rRNA基因扩增，对扩增产物进行克隆和测序。从3个地理位置不同的猪场均成功地扩增出长度为1469bp的核苷酸序列。系统进化分析表明，3个猪场样品所测序列一致性达99．52％以上，具有相同的基因型，但与国外报道的猪附红细胞体Illinois株同源性为95％，属于同一基因群，但基因型不同；所有种类的附红细胞体和血巴尔通氏体组成同一进化分支，这类血营养菌与支原体科，支原体属的病原最靠近(75％)，而与立克次氏体目的病原较远(70％)。上述研究证实，广东所流行的猪附红细胞体是一种新基因型的猪附红细胞体，建议命名为猪附红细胞体广东株型；为反映进化关系，猪附红细胞体和其它血营养菌应划归于支原体科的支原体属。     

毒害艾美耳球虫(Eimeria necatrix)广东株微线蛋白-2基因在大肠杆菌的表达

根据克隆的毒害艾美耳球虫(Eimeria necatrix)广东株微线蛋白-2基因(EnMIC-2(Gd))的cDNA序列设计特异性引物，用PCR方法扩增其阅读框架(ORF)后，克隆至质粒表达载体pET-32a( )，成功构建了重组表达质粒pET-32a( )-EnMIC-2。用CaCl2法将其转化至宿主细菌E.cliBL21(DE3)，并用IPTG成功诱导了EnMIC-2重组抗原在大肠杆菌表达。表达的重组蛋白约占菌体总蛋白的10．8％，其相对分子质量约为55000。重组蛋白经SDS-AGE分析后，用E．necatrix(广东株)感染鸡的高免血清进行Western Blotting分析，结果为阳性。     

广西猪瘟流行毒株全基因组的克隆与序列分析

参考已发表的猪瘟病毒(CSFV)Shimen株、HCLV株、Paderborn株的核苷酸序列，设计并合成11对引物。应用RT—PcR技术，成功地分11个片段扩增了CSFV广西流行毒株GXWZ02的全基因组，将这11个基因片段克隆，并测定了其核苷酸序列。应用计算机生物软件Vector NT1将11个基因片段进行拼接。确认CSFV GXWZ02株全基因组序列的长度为12296个核苷酸(GenBank收录号AY367767)。将GXXZ02株与国内外已发表的Slhimen、HCLV、39、Brescia、Eystrup、Glentorf、Alfort187、Pader190rn、CS、ALD、GPE、P97、LPC毒株进行全基因组核苷酸序列和推定的氨基酸序列比较，核苷酸同源性分别为85．4％、84．6％、88．7％、85．7％、85．7％、85．3％、85．6％、95．3％、85．7％、85．7％85．4％、83．4％、84．3％，推定的氨基酸同源性分别为92．6％、91．7％、94．2％、93．1％、92．9％、92．3％、93．0％、97．7％、92．6％、93．0％、92．6％、90．5％、90．6％。GXWZ02与Shimen、HCLV的全基因组序列及推定的氨基酸序列有明显差异，表明GXWZ02株在遗传性和抗原性上发生了较明显的变化。系统发育树分析，所比较的14株CSFV被分为2个群：GXWZ02、Paderborn和39这3个毒株被归为群Ⅰ，其余的11个毒株被归为群Ⅱ，GXWZ02与Paderborn的亲缘关系最接近。将GXXZ02与Shimen、HCLV基因组中单个基因分别进行核苷酸及推定的氨基酸序列同源性比较，结果显示，基因E^ms、E2、P7、NS2、NS5A的遗传变异程度大。而NS3、NS4A、NS4B的遗传性则相对保守。     

羊附红细胞体病PCR检测方法的建立

根据羊附红细胞体的16S rRNA基因参考序列，设计1对特异性引物．建立了检测羊附红细胞体的PCR技术。用本方法从感染血样中特异扩增出1条预期大小为1169bp的片段。该方法灵敏、快速、特异性高．可用于羊附红细胞体病的早期快速诊断和流行病学调查。     

三峡库区消落带不同海拔狗牙根草地土壤微生物生物量碳氮磷含量特征

为探究库区消落带优势草本植物狗牙根人工植被恢复后土壤质量及肥力的变化,选择三峡库区消落带150, 160和170 m海拔人工构建狗牙根植被土壤为研究对象,并以裸地作为对照,测定土壤微生物生物量碳、氮、磷和相关理化性质。结果表明:(1)各海拔狗牙根微生物生物量均显著高于裸地,表明狗牙根人工植被构建对土壤微生物恢复具有重要意义。(2)土壤微生物生物量碳、氮含量160 m高程显著高于150和170 m高程,但土壤微生物生物量磷(SMBP)却显著低于150和170 m高程,需要特别注意P元素的下移,加强水体P含量的检测。(3)土壤微生物生物量碳氮比和碳磷比变化范围分别为5.93~15.62和7.11~19.99,土壤微生物生物量碳、氮、磷占土壤有机碳、全氮、全磷百分比的范围分别为0.68%~2.57%、0.68%~3.33%、1.95%~5.23%。狗牙根土壤微生物生物量碳氮比和碳磷比显著低于裸地,表明狗牙根土壤有效氮、有效磷高于裸地;土壤微生物生物量碳、氮、磷占土壤有机碳、全氮、全磷百分比显著高于裸地,表明狗牙根土壤营养元素周转速率快于裸地。(4)微生物生物量碳、氮、磷与土壤有机碳、全氮和土壤含水率有显著或极显著相关性,与土壤pH值呈不同程度的负相关。因此,在三峡库区消落带进行狗牙根草地恢复重建能显著提高土壤微生物生物量及土壤质量,对加强三峡库岸生态系统的稳定性具有重要意义。     

施氮对狼尾草在南方贫瘠旱坡地生长、能源品质及氮肥利用率的影响

为探究狼尾草作为生物质能源原材料在南方贫瘠旱坡地生长对氮素的响应,研究4个施氮水平(0、112.5、225.0和337.5 kg N·hm^-2)对能源型狼尾草桂能草1号农艺性状、产量、SPAD值、生物质能源品质及氮肥利用率的影响。结果表明,施用氮肥可促进狼尾草横、纵向生长,随施氮量的增加狼尾草的株高、茎粗和单位面积有效茎数逐步提高,但叶片叶绿素含量和鲜、干草产量的提高程度在施氮量超过225.0 kg N·hm^-2后,有饱和趋势。施用氮肥还可明显提高狼尾草纤维素含量、半纤维素含量、热值和折合标准煤产量,显著降低干物质、木质素和灰分含量,总体改善了狼尾草的能源利用品质;但施氮量达337.5 kg N·hm^-2时,狼尾草热值下降,且对折合标准煤产量的增加作用不明显,降低了狼尾草的能源利用品质。此外,在一定施氮量范围内(112.5~225.0 kg N·hm^-2),狼尾草氮肥利用效率并未随施氮量的增加而显著降低,但施氮量达337.5 kg N·hm^-2时,氮肥利用率显著降低。因此,狼尾草在南方贫瘠旱坡地种植,为获得理想的生物质能源材料需及时补充氮素营养,但施氮量不宜超过225.0 kg N·hm^-2。     

AtSOS基因在紫花苜蓿中的表达及其耐盐性研究

本研究采用基因工程技术改良紫花苜蓿耐盐性,通过种植转基因耐盐紫花苜蓿达到改良土壤的目的。以紫花苜蓿子叶节为外植体,通过农杆菌介导法将来源于拟南芥的AtSOS1-AtSOS2-AtSOS3多基因表达载体导入阿尔冈金紫花苜蓿中,经PCR检测、抗除草剂筛选和RT-PCR鉴定,获得了能稳定表达的转基因株系。以转基因的紫花苜蓿和野生型紫花苜蓿为材料进行盐处理,每个处理重复3次,测定其生理生化指标、株高、Na⁺和K⁺含量、细胞膜透性、叶绿素含量。结果显示,在不同盐浓度处理下,所有植株的株高均有所增长,但在100和200 mmol·L^-1的NaCl处理下,转基因植株的长势显著高于野生型植株;随着处理时间的增加,所有植株的叶绿素含量均呈先上升后下降的趋势,且野生型植株叶绿素含量均低于转基因植株;在100和200 mmol·L^-1的NaCl处理下,转基因植株的细胞膜透性、超氧化物歧化酶活性和脯氨酸含量的增加量均小于野生型植株,而过氧化物酶、过氧化氢酶活性和可溶性糖含量的增加量均大于野生型;各植株中丙二醛含量均下降,且野生型植株下降的更为明显;盐处理后,转基因植株根系中Na⁺的积累比野生型植株少,而K⁺的吸收多于野生型植株。转AtSOS基因的紫花苜蓿通过发挥AtSOS途径的作用,促进了植物体将细胞内的Na⁺外排,从而减轻盐胁迫对植物体的伤害,提高了转基因植株的耐盐性。     

Inhibitory effect of AuNPs-Endostar on melanoma lung metastasis in mice

AIM: To observe the influence of gold nanoparticles combined with Endostar (AuNPs-Endostar) on the melanoma lung metastasis of mice and the underlying mechanism. METHODS: C57BL/6 mice (n=24) were selected for constructing the model of spontaneous lung metastasis of melanoma B16-F10 cells. Subsequently, the mice were randomly divided into Endostar group, AuNPs group, AuNPs-Endostar group and model group. After the formation of melanoma, the mice in each group were injected with different drugs through tail vein for 0.1 mL daily. After 9 d, the mice were narcotized for cutting the tumors in situ. After the operation, they were raised for 2 weeks before killed for obtaining the lung tissues to observe the situation of the metastasis. HE staining was utilized for observing the necrosis status of the tumors in situ, while immunostaining was applied for testing the expression of CD31, carbonic anhydrase-IX (CA-IX), vimentin and zonula occludens-1 (ZO-1) in the tumors. RESULTS: Compared with model group, the pulmonary metastasis in the groups with medical treatment was obviously reduced. In AuNPs-Endostar group, the metastasis inhibition rate was the highest, and the tumor necrosis was also decreased obviously, with the significant reduction of CD31, CA-IX and vimentin expression in the tumors and significant increase in ZO-1 expression. CONCLUSION: Compared with using Endostar or AuNPs alone, the combination of AuNPs with Endostar significantly improves the curative effect of inhibiting the pulmonary metastasis of melanoma in the mice. The mechanism may be related to reducing the tumor angiogenesis, norma-lizing the blood vessels and improving tumor hypoxia, thus inhibiting the tumor epithelial-mesenchymal transition, increasing the tight junctions between tumor cells and decreasing the invasiveness.     

Effect of Different Concentrate-roughage Ratios on Rumen Degradability of Cassava Stems and Leaves Total Mixed Ration in Goat

The rumen degradations of TMR were studied in this experiment,in which cassava stems and leaves were used as roughage and formulated with concentrate in different proportions.The purpose of this study was to formulate the available TMR for finishing goat,and provide a theoretical basis for the applications of cassava stems and leaves in goat production.Six Hainan Black goats with permanent rumen fistulas were selected,the rumen degradabilities of DM,CP,NDF and ADF of TMR with 6 different concentrate to forage ratio（2:8,3:7,4:6,5:5,6:4 and 7:3）were tested using nylon bag method.The results showed that,the DM effective degradabilities at the ratio of 4:6,5:5,6:4,7:3 were significantly higher than that of 2:8 and 3:7（P<0.05).CP effective degradabilities at the ratio of 2:8,3:7,4:6 were significantly higher than that of 5:5,6:4 and 7:3（P<0.05).The NDF effective degradation at the ratio of 5:5 was significantly higher than that in the other groups（P<0.05).The ADF degradation at the ratio of 3:7 was significantly higher than that of 4:6,5:5, 6:4 and 7:3（P<0.05).The results indicated that,the nutrient degradation in rumen were higher when the concentrate to forage（cassava stems and leaves）ratio in TMR were 3:7,4:6 and 5:5,and the recommended ratio range were 4:6 to 5:5 in production practice.