利用16S rRNA全长序列鉴定植物乳杆菌

从标明含有嗜酸乳杆菌的活菌胶囊中分离到一株乳杆菌Lal菌株，利用16S rRNA全序列分析法和传统分类法对Lal菌株进行分类鉴定表明：Lal菌株属于植物乳杆菌种新株系，该菌株现在已经被中国科学院北京微生物所菌种保藏中心收录保存，编号为ASl．2986。     

16S rRNA在兽医病原菌分类鉴定中的应用

介绍16S rRNA基因的特点，阐述其用于病原菌分类鉴定的基本原理，综述其在兽医病原菌分类鉴定中的应用。     

Protein expression of cyclin D2 and p16 in proliferation and differentiation of cultured cardiac myocytes

AIM:To investigate the protein expression of cyclin D₂ and p16 in proliferation and differentiation of cultured cardiac myocytes.METHODS:One-day-old Sparague-Dawley rats were used. Cardiac myocytes(CM) were collected by a trypsin-dispersal method and cultured. Cell growth line and fluorescence activated cell sorting (FACS) were used to investigate the proliferation of CM. Ultra-thin sections were made to observe the ultrastructure of CM under transmission electron microscope. The expression of cyclin D₂ and p16 in CM were measured using immunocytochemistry and image analysis.RESULTS:①Results of cell growth line and FACS analysis showed that cultured CM could proliferate in the first 3 cultured days, but the ability decreased quickly, concomitant with differentiation. CM was obseved quiescent in cell cycle three days later. The ultrastructure of CM showed the large amount of myofilaments and mitochondrion. ②The protein expression of cyclin D₂ in 3,4,5 day CM group was 0.89 times(P＜0.05),0.80 times (P＜0.05) and 0.56 times (P＜0.01) of that in 1 day group, respectively. The expression of p16 in CM was increased during the culture process, 2,3,4,5 day group were 1.63 times, 1.72 times, 1.99 times and 2.84 times (P＜0.01) of that in 1 day group, respectively.CONCLUSION:Cultured neonatal rat cardiac myocytes could proliferate during the first 3 days after incubation, but the ability of proliferation decreased, from the fourth day, concomitant with differentiation. Cyclin D₂ and p16 play the key roles in CM postnatal development. Downregulation of cyclin D₂ and upregulation of p16 may induce CM differentiation.     

Molecular Identification of a New Member of the Clover Proliferation Phytoplasma Group (16SrVI) Associated with Centaurea Solstitialis Virescence in Italy

In the United States, yellow starthistle (Centaurea solstitialis) is an annual invasive weed with Mediterranean origins. Malformed plants displaying witches' broom, fasciations, abortion of buds and flower virescence symptoms were observed in central Italy. Attempts to transmit the causal agent from the natural yellow starthistle host to periwinkle by grafting, resulted in typical symptoms of a phytoplasma, i.e. yellowing and shortening of internodes. The detection of phytoplasmas was obtained from both symptomatic yellow starthistle and periwinkle by the specific amplification of their 16S-23S rRNA genes. PCR amplification of extracted DNA from symptomatic plant samples gave a product of expected size. Asymptomatic plants did not give positive results. An amplicon obtained by direct PCR with universal primers P1/P7 was cloned and sequenced. The homology search using CLUSTALW program showed more than 99% similarity with Illinois elm yellows (ILEY) phytoplasma from Illinois (United States) and 97% with Brinjal little leaf (BLL) phytoplasma from India. Digestion of the nested-PCR products with restriction enzymes led to restriction fragment length polymorphism patterns referable to those described for phytoplasmas belonging to the clover proliferation (16S-VI) group. Since this is a previously undescribed disease, the name Centaurea solstitialis virescence has been tentatively assigned to it. This is a new phytoplasma with closest relationships to ILEY and BLL, but distinguishable from them on the basis of 16S rDNA homology, the different associated plant hosts and their geographical origin.     

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。     

规模化畜禽养殖场粪便中多重耐药菌分离鉴定及其耐药特征

为了解禽畜粪便中多重耐药菌的污染特征,本研究对鸡粪、牛粪、猪粪和有机肥这4种不同样品中多重耐药菌进行了计数分析,并对不同来源的多重耐药菌株进行了分离和纯化,进一步开展了基于16S rDNA序列比对的分子生物学鉴定以确定其种属地位,以及基于药敏试验的耐药性特征分析。结果表明:不同养殖动物粪便中四环素、恩诺沙星、磺胺甲恶唑和泰乐菌素4种抗生素多重耐药菌的绝对数量和相对数量排序均为鸡粪>猪粪>牛粪,粪源有机肥中可培养的多重耐药菌的绝对数量和相对数量在堆肥后均有所下降。通过对耐药菌株鉴定和分类学分析,发现禽畜粪便中多耐药菌的菌门集中分布在Proteobacteria、Firmicutes和Actinobacteria,多重耐药菌的优势菌属为Escherichia、Corynebacterium、Kurthia;而有机肥样品中多重耐药菌的优势菌属为Staphylococcus、Glutamicibacter。菌株的药敏试验表明,3类动物粪污中的多重耐药菌都对红霉素、四环素有着较高的耐药率,均高于80%,而对阿米卡星这种抗生素表现出较好的敏感性,其耐药率低于30%。通过对Ⅰ、Ⅱ类整合子基因盒的扩增,发现PCR产物的大小从0.8 kb到1.8 kb不等,基因盒ad A2、dfr A17、dfr A1及sat2在养殖场粪污中检出率均较高。     

Identification of a phytoplasma associated with pomegranate little leaf disease in Iran

During 2012–2014 surveys for the presence of phytoplasma diseases in Fars province (Iran), pomegranate little leaf symptoms were observed in several orchards in Khafr and Neyriz areas. Samples collected from symptomatic plants positively reacted in nested PCR assays using P1/P7 followed by R16F2n/R16R2 primer pairs producing the expected 1,250 bp DNA fragments. Real and virtual RFLP analysis showed that the sequences of phytoplasma strains from Khafr and Neyriz (KPLL and NPLL strains, respectively) were identical to each other and belong to 16SrII phytoplasma group, subgroup D. Phylogenetic analysis of the R16F2n⁄R16R2 DNA region confirmed that KPLL and NPLL phytoplasmas were enclosed in the same clade as other 16SrII-D subgroup phytoplasmas. This is the first reported occurrence of a 16SrII phytoplasma infecting pomegranate trees.     

文心兰HSP70基因的克隆及表达分析

为研究文心兰热激蛋白70基因（命名为OnHSP70）的分子特性及表达特点,以文心兰‘柠檬绿’（Oncidium hybridum ‘Honey Angel’）为材料,采用RT-PCR技术克隆OnHSP70,利用生物信息学方法分析其分子特性,通过qRT-PCR技术分析其在不同组织及不同非生物胁迫处理下的表达特点。生物信息学分析表明：该基因开放阅读框为1944 bp,编码647个氨基酸,翻译的蛋白为稳定蛋白,属于HSP70超家族,其蛋白二级结构由41.11%的α-螺旋、17.77%的延伸链、7.73%的β-转角和33.38%的无规卷曲组成,具有2个高度保守的功能域。聚类分析表明：该蛋白与铁皮石斛和深圳拟兰的HSP70亲缘关系较近。qRT-PCR分析表明：文心兰HSP70在4种不同组织器官中具有表达差异性,在花中的表达量最高;高温处理后基因的表达量明显上升;40 ℃高温胁迫4 h时表达量达峰值;茉莉酸甲酯及水杨酸处理时表达量呈现下调响应。本研究为后期该基因功能研究及提高文心兰对高温的适应性提供理论基础。