Studies on rickettsia-like organism (RLO)disease of tropical marine pearl oyster——Epidemiological investigation of RLO disease in larvae populations of maricultured Pinctada maxima

The epidemiological investigations on the disease and death in mature eggs, embryonic de- velopmental periods and larvae populations [including oocytes, fertilized ovum, early embryonic phase larvae (6 h), D-shaped phase larvae (24 h), early umbo phase larvae, umbo phase larvae, post um- bo phase larvae and eyespot phase larvae] in the Luhuitou Pearl Oyster Mariculture Farm of Sanya City, Hainan Province in April 1995 showed that there were two peaks of mortality rates which occurred in early umbo phase or umbo phase and post umbo phase (Figs 3-5 and Table 1) respectively from pre- embryonic developmental periods to larvae phases. It indicated that the onset and death of Pinctada maxima larvae populations followed a pattem of outbreak. Between the prevalence, intensity of RLO infection and the mortality rates of larvae populations were of obvious positively correlations. Generally, every peak of RLO infection is always follwed by a peak of mortality rate of larvae hosts, and the mor- tality rates of larvae hosts declined with the decreased RLO infection (see Figs 3-5 ). Under the trans- mission electron microscopy (TEM), no rickettsia-like organisms were discovered in oocytes, fertilized ovum and early embryonic phase larvae (6 h). The RLO inclusions occurred first in the D-shaped phase larvae (24 h) under histological examination. Absences of RLO in transmission electron exami- nation of oocytes of RLO-infected adult females, ferilized ovum and early embryonic phase larvae (6 h) indicated that RLO may not be transmitted transovarially. But RLO for host infection may be trans- mitted by contact transmission since RLO inclusions were first identified regularly in D-shaped phase larvae (24 h), while these D-shaped phase larvae were still unable to take food during hatching 24 hours. In addition, the result of epidemiological investigation showed that no odservable death occurred in D-shaped larvae populations, but early obvious death occurred in larvae populations in the seventh day after fertilization, in a rate of about 21. 8%. It indicated that there was a incubation peried from RLO infection for host (D-shaped phase larvae) to host onset and death occurred clinically.

Studies on rickettsia-like organism (RLO) disease of tropical marine pearl oyster-Epidemiological investigation of RLO disease in juvenile populations of maricultured Pinctada maxima

The death which occurred in juvenile population was a severe problem in the course ofPinctada maxima artificial culture. With the methods of field investigation, histological study and statistic analyses, the epidemiological study was carried out on the disease and death in juvenile populations of Pinctada maxima in the Xinying Pearl Oyster Mariculture Farm of Lingao County (for the A, B and C batches of cultured juveniles hanged in the sea) and the Xincun Pearl Oyster Mariculture Farm of Lingshui County (for the D batch of cultured juvenile hanged in the pond), Hainan Province from November 1993 to April 1995.The results show that the deaths which occurred in juvenile populations of Pinctada maxima presented an outbreak pattern. The peak of mortality rates, in general, occurred in 4-to 6-month old pearl oyster juveniles, and the mortality rates gradully declined with the extention of pearl oyster age after cul-turing 8 months. The correlation between the mortality rates of juvenile populations and mean body lengths of juvenile populations show that the mortality rate become higher under 4 cm of mean body lengths of juvenile population and become obvious declined over 5 cm of mean body lengths. The peak of mortality rate occurred in 1-3 cm of mean body lengths. The results of histological observation showed that rickettsia-like organism inclusions were common, histological widespread infective agent among every batches (A, B, C and D) of cultured juvenile populations. The mean severity indices (SI) of rickettsia-like organism (RLO) infection were positively correlated with mortality rates of juvenile populations. After or within every peaks of RLO infection were all accompanied with the peaks of mortality rates of host populations and the mortality rates declined with decreased RLO infection. So the evidences of histological observation and epidemiology in this study indicated that rickettsia-like organism (RLO) may be as an important pathogenic organism of disease and death of cultured Pinctada maxima. In addition, a few ciliates were discovered only in part of batch A of cultured juvenile population. The mortality rates in juvenile populations were not correlated with the weekly mean temperature and salinity of sea water.

Rickettsia-like organism infection associated with mass mortalities of blood clam, Tegillarca granosa, in the Yueqing Bay in China

A series of mass mortalities of the cultured blood clam, Tegillarca granosa, occurred in the Yueqing Bay of China from 2005 to 2009. An obligate intracellular prokaryote, designated as rickettsia-like organism （RLO）, was frequently found in the moribund or dead blood clam sample during ultra- structural examination. These organisms were usually round, ellipsoid or occasionally dumbbell- shaped, ranged from approximately 0.28 to 0.71 #m in size and had a trilaminar cell wall. Two reproductive modes of organisms, transverse binary fission and budding, were observed. The or- ganisms were able to form eosinophilic inclusions. Most inclusions were found within epithelial and connective tissues of the mantle, gills and digestive tube. The biological and morphological char- acteristics indicate that these organisms may belong to the family Rickettsiaceae. RLOs exhibited significant pathogenicity. Cytopathological examinations revealed extensive necrosis and destruc- tion in the infected cell. The degree of tissue destruction was positively related to the number of RLO inclusions in the tissues, and the cytopathological effects were positively related to the number of intracellular RLO. RLOs and their inclusions were discovered throughout different disease areas and in different time periods. The infection intensity of the RLOs was positively correlated with the mortality rate of clams. Therefore, RLO infection might be associated with mass mortalities of cultured blood clams in the Yueqing Bay.

三叶青两个受体蛋白基因的克隆及功能分析

植物细胞受体类蛋白是一类重要的结构蛋白,在环境信号感知及细胞间信息传递中起重要作用。本研究克隆了三叶青(Tetrastigma hemsleyanum Diels et Gilg)的两个受体蛋白基因ThRLP1与ThRLK1,并对其进行了生物信息学分析、器官特异性表达分析和在块根发育不同时期的表达分析,以明确两基因与三叶青芽发育和块根形成的相关性。生物信息学分析表明,所克隆的两个基因中一个为细胞外受体类蛋白基因(ThR-LP1),其氨基酸序列具有特征的亮氨酸重复序列,亚细胞定位推测在细胞壁;另一个为质膜受体类蛋白激酶基因(ThRLK1),推测氨基酸序列中具有特征的丝氨酸/苏氨酸激酶的催化结构域,亚细胞定位推测在细胞核。器官特异性表达分析发现ThRLP1在块根中的表达量显著低于其他器官,ThRLK1在块根中的表达量显著低于枝蔓但高于其他器官,两个基因的表达水平并没有显示出与芽发育有相关性;块根发育不同时期表达量分析表明,ThRLP1在块根初始膨大期的表达量显著低于其他时期,ThRLK1表达量随块根发育进程而提高,ThR-LK1基因的表达与块根发育进程呈现出一定的正相关性。所克隆的两个受体蛋白可通过大肠杆菌原核表达体系正确表达出相应蛋白。本研究可为进一步明确受体类蛋白激酶基因在三叶青块根发育分子网络中的功能提供初步的参考依据。

用离散量预测原核生物蛋白质的亚细胞位置

基于不同亚细胞位置中蛋白质的氨基酸组成及序列信息不同这一观点,以单个氨基酸含量及两两组合氨基酸含量为信息构成离散源,分别计算了原核生物蛋白质三类亚细胞位置的标准离散量D(Xe),D(Xp),D(Xc).利用离散增量的概念预测蛋白质的亚细胞位置,它是由这个蛋白质的离散量D(X)与三个标准离散量D(Xe),D(Xp),D(Xc)之间离散增量的最小值所决定的.采用Self-consistency检验和Jack-knife检验方法,给出了选择五组不同信息作为离散源中参数时的预测结果.与现有的方法比较,发现用Jack-knife检验法预测extracellular类蛋白质时,给出的离散量方法能够给出最好的预测性能,结果也表明提取更多有效的序列信息是提高预测精度的关键.

中国对虾类淋巴组织培养中细胞异常的病理研究

在对养殖中国对虾和海捕中国对虾的类淋巴 器官进行组织培养时，发现迁出的细胞空泡化、细胞内颗粒较多。培养一周后由养殖中国对 虾类淋巴组织迁出的细胞开始大量脱落，而由海捕中国对虾类淋巴组织培养出的细胞可生长 30d左右。对未经培养的类淋巴组织及培养的类淋巴组织进行透射电镜观察，发现在养殖中 国对虾类淋巴组织的细胞中除了有球形病毒及球状和多面体状病毒包涵体外，在细胞质中还 含有大量类立克次氏体样原核生物，而且每个细胞中的类立克次氏体均由一单层膜包在一起 ；在海捕中国对虾类淋巴组织的细胞中也发现有球形病毒及球状病毒包涵体，另外还在少量 细胞中发现有类立克次氏体样原核生物存在。感染病毒的组织细胞呈现空泡化、核膜肿胀破 裂、核质固缩、高尔基体肿胀、线粒体嵴消失等病理变化。感染有类立克次氏体样原核生物 的细胞不同程度地呈现出细胞器消失、核染色质固缩等病理结构。

Biological Concept of Bacterial Pathogenicity (Theoretical Review)

Biological nature of the bacterial pathogenicity phenomenon is based on the interaction of prokaryotic and eukaryotic organisms. The phenomenon is the poly-functional biological potency of germs that are realized by factors (determinants) of pathogenicity. Some fundamental biological functions are responsible for bacterial pathogenicity in a multi-cellular host organism: the adhesive function, the function of invasion and penetration into the cell, the function of evasion of host defense, and the damage function. The action of adhesion, invasion and evasionis directed to towards establishing an ecological niche in multi-cellular host while the aim of the damaging function is destruction of the environment.

青藏高原湖泊沉积物中有机碳组分构成及其对微生物群落结构的影响

【目的】探究湖泊沉积物中有机碳组分构成及其对湖泊微生物群落结构的影响。【方法】本研究采集了青藏高原29个湖泊共81个沉积物样品,通过硫酸水解法分析样品中易降解和难降解有机碳含量及其与环境变量之间的相关性;同时使用高通量测序技术分析样品中原核微生物和真菌群落的多样性和组成及其与易降解和难降解有机碳含量的相关关系。【结果】本研究的青藏高原湖泊沉积物中易降解有机碳Ⅰ(labile organic carbon Ⅰ, LOCⅠ)、易降解有机碳Ⅱ(labile organic carbon Ⅱ, LOCⅡ)和难降解有机碳(recalcitrant organic carbon, ROC)的含量分别为0.03–29.62 mg/g、0.02–23.38 mg/g和0.64–75.72 mg/g,ROC是沉积物有机碳的主要组分(占比为54.97%±19.50%)。LOCⅠ含量与海拔、总氮、总磷、钙离子、活性钙和活性铁含量显著相关(P<0.05);LOCⅡ含量与总氮、钙离子和活性钙含量显著相关;而ROC含量与海拔、总氮、总磷、钙离子和活性钙含量显著相关。其中,钙离子和活性钙的浓度与3种有机碳组分的含量均存在显著正相关,暗示有机质与钙结合的方式可能是青藏高原湖泊沉积物有机碳保存的重要机制。LOCⅠ、LOCⅡ和ROC含量与沉积物中原核微生物群落α多样性均呈显著正相关;ROC含量与真菌群落α多样性呈显著正相关。LOCⅠ含量与α变形杆菌(Alphaproteobacteria)、放线菌(Actinobacteria)、厌氧绳菌(Anaerolineae)、海藻球形菌(Phycisphaerae)和红嗜热菌(Rhodothermia)的相对丰度显著相关;LOCⅡ含量与Anaerolineae、γ变形菌(Gammaproteobacteria)和Phycisphaerae相对丰度显著相关;ROC含量与Actinobacteria和Anaerolineae的相对丰度显著相关。LOCⅠ含量是影响原核微生物和真菌群落组成的显著环境因子,其解释量分别为1.1%和0.3%。环境因素解释了样品间原核微生物群落7.2%和真菌群落3.9%的变化,而空间因子解释了原核微生物群落14.6%和真菌群落6.4%的变化。随机过程对原核微生物和真菌群落组成的贡献分别为50%和47%。【结论】本研究暗示湖泊沉积物有机碳的保存受到铁、钙元素化学保护机制的影响;有机碳组分含量显著影响原核微生物和真菌群落多样性和组成,但其贡献值较低;环境理化参数、空间因子和生态随机过程对湖泊沉积物原核微生物和真菌群落组成具有重要影响。本研究加深了我们对湖泊沉积物有机碳组分构成及其影响微生物群落组成的认识。

典型管控有机污染场地土壤剖面微生物群落分布特征及与环境因子关系解析

有机污染场地是目前全球重要的土壤环境污染研究热点,然而,目前针对有机污染场地中的多种有机污染物影响下的土壤微生物分布特征研究尚少。利用高通量测序技术,研究了典型有机污染场地中原核微生物的多样性和垂向分布特征、物种之间的共现模式,并探讨了物种组成与环境因子之间的关系。结果表明:有机污染场地中有机污染物会降低土壤原核微生物多样性并促使群落结构垂向趋同化。场地土壤原核微生物主要由变形菌门(Proteobacteria),绿弯菌门(Chloroflexi),厚壁菌门(Firmicutes),拟杆菌门(Bacteroidetes),放线菌门(Actinobacteria)和Patescibacteria组成。共存网络分析结果表明:物种之间正相关关系(合作关系)占主导地位,在抵抗外界有机污染压力时起关键作用。此外,物种-环境因子网络分析表明:有机污染场地中2种高浓度有机污染物对土壤原核微生物群落类群有重要影响。

腐生葡萄球菌M36耐有机溶剂脂肪酶基因的克隆与原核表达

脂肪酶是重要的工业用酶,在食品加工、生物柴油的合成等领域具有广泛的应用。但是在应用中有机溶剂对脂肪酶具有一定的毒性,因此获得耐有机溶剂的脂肪酶基因并实现高效表达是脂肪酶规模化应用的前提。本研究应用PCR技术首次从耐有机溶剂脂肪酶产生菌腐生葡萄球菌M36基因组DNA中扩增得到脂肪酶Ⅲ基因lip3(GenBank AccessionNo.FJ979867),其编码区长度为741bp,编码247个氨基酸,推测蛋白分子量大小为31.6kD。它与腐生葡萄球菌lip3推测的基因(GenBank AccessionNo.AP008934)只有83%的同源性。将该基因与大肠杆菌表达载体pET-DsbA连接,转化大肠杆菌EscherichiacoliBL21(DE3)获得重组菌株BL21(DE3)/pET-DsbA-lip3,在pH8、25oC条件下,OD600为1.0时用0.4mmol/LIPTG诱导12h酶活达到25.8U/mL。重组酶在甲醇、正己烷、异辛烷、正庚烷等有机溶剂中具有较好的耐性。lip3基因的克隆及在大肠杆菌中有效表达的研究为进一步进行基因工程改造和脂肪酶应用奠定了基础。

牙龈卟啉单胞菌Hgp44基因的克隆表达与纯化

目的克隆表达牙龈卟啉单胞菌（Porphyromonas gingivalis，Pg）牙龈素中黏附素区域的Hgp44基因，并纯化目的蛋白。方法通过聚合酶链反应（PCR）和基因重组技术，克隆得到PgATCC33277的Hgp44基因，插入到克隆载体pMD18-T中并测序鉴定。经过酶切后将目的基因片段与表达载体pET22b相连，构建出表达质粒pET22b—Hgp44。将重组质粒转化到感受态细胞BL21（DE3）中，用异丙基-β-D-硫代半乳糖苷（isopropyl—β-D—thiogalactoside，IPTG）诱导表达融合蛋白，通过SDS聚丙烯酰胺凝胶电泳及蛋白质印迹法进行分析。用固定化金属亲和层析法对重组蛋白进行分离纯化。结果目的基因片段约为1100bp，与预期大小相符，测序结果与GenBank中ATCC33277国际标准菌株的RgpA的等位基因序列U15282-致。IPTG诱导后的菌体经SDS聚丙烯酰胺凝胶电泳后形成一个以包涵体形式存在的44000的融合蛋白。蛋白质印迹法检测证实其具有免疫原性。用镍离子金属螯合层析柱纯化出了目的蛋白，最后得到约3．5mg／L的目的蛋白。结论成功克隆表达了PgHgp44基因，并纯化出了目的蛋白。

原核生物RNA聚合酶辅助因子σ因子的研究进展

在原核生物中,σ因子是RNA聚合酶的辅助因子,被用来识别启动子中的功能序列,在基因的转录调控中发挥着重要的功能。目前原核模式生物中的可替换型σ因子大部分已被发现,其中大肠杆菌有4种,枯草芽胞杆菌有18种。从σ因子的分类、应答机制、识别元件、结构特征等方面进行了归纳。将为系统的理解原核生物σ因子的功能,及其在基因表达调控中的贡献等方面提供帮助。