Molecular analysis of the genus Asparagus based on matK sequences and its application to identify A. racemosus, a medicinally phytoestrogenic species

The plant Asparagus racemosus is one of the most widely used sources of phytoestrogens because of its high content of the steroidal saponins, shatavarins I-IV, in roots. The dry root of A. racemosus, known as "Rak-Sam-Sip" in Thai, is one of the most popular herbal medicines, used as an anti-inflammatory, an aphrodisiac and a galactagogue. Recently, the interest in plant-derived estrogens has increased tremendously, making A. racemosus particularly important and a possible target for fraudulent labeling. However, the identification of A. racemosus is generally difficult due to its similar morphology to other Asparagus spp. Thus, accurate authentication of A. racemosus is essential. In this study, 1557-bp nucleotide sequences of the maturase K (matK) gene of eight Asparagus taxa were analyzed. A phylogenetic relationship based on the matK gene was also constructed. Ten polymorphic sites of nucleotide substitutions were found within the matK sequences. A. racemosus showed different nucleotide substitutions to the other species. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the matK gene was developed to discriminate A. racemosus from others. Only the 650-bp PCR product from A. racemosus could be digested with BssKI into two fragments of 397 and 253-bp while the products of other species remained undigested. Ten commercially crude drugs were analyzed and revealed that eight samples were derived from A. racemosus while two samples of that were not. Thus, the PCR-RFLP analysis of matK gene was shown to be an effective method for authentication of the medicinally phytoestrogenic species, A. racemosus.     

Application of Molecular Marking Technologies in Heredity Breeding of Eucalyptus

Two clonal trial stands of Chinese Fir （Cunninghamia lanceolata） were used in this study, one was 19-year-old stand which included 38 clones, and the other was 17-year-old stand including 102 clones.The statistical analyses showed that there were very significant genetic variations in height, DBH,volume and ratio of heartwood(R_hw),wood basic density（ρ_b ） of the clones in the two stands. The repeatability of clones was in median to high level,and the genetic CV was different over the all five traits.There were very significant phenotypic and genetic correlations among height,DBH and volume,and negative correlations among growth, R_hw andρ_b.The selection method experiment indicated that index selection could improve volume, R_hw andρ_b,showing synthetically superior selection effects compared to any individual trait selection methods.     

菊花绿变病植原体在中国的发生及分子鉴定

对内蒙古农业大学校园内表现花器绿变症状的菊花样品进行采集和DNA提取,应用植原体16S rRNA基因和rp基因的引物进行巢式PCR扩增,从感病样品中分别扩增得到了长度均约为1.2 kb的片段。序列一致性分析表明,菊花绿变植原体16S rRNA基因与翠菊黄化植原体匈牙利风信子株系(GenBank登录号MN080271)、印度玉米株系(KY565571)、印度繁缕株系(KC623537)和印度马铃薯株系(KC312703)的核酸一致性最高,为99.9%,rp基因序列与翠菊黄化植原体立陶宛洋葱株系(GU228514)的核酸一致性最高,为99.8%。基于16S rRNA基因和rp基因构建系统进化树时发现,菊花绿变植原体均与16SrI-B亚组成员聚为一起。16S rRNA基因相似性系数分析表明,菊花绿变植原体与洋葱黄化植原体(AP006628)的相似性系数最高为1.00,洋葱黄化植原体(AP006628)在分类上属于16SrI-B亚组。因此,我们可以确定该菊花绿变植原体属于16SrI-B亚组。这是我国首次报道菊花绿变病的发生。     

Prototheca zopfii genotype 2: the causative agent of bovine protothecal mastitis?

In order to clarify the epidemiology of bovine protothecal mastitis, 30 Prototheca zopfii mastitis isolates were genetically investigated. Based on the 18S rDNA, which allows a differentiation of the former species P. zopfii in two distinct P. zopfii genotypes and Prototheca blaschkeae sp. nov., newly developed genotype-specific PCR-assays as well as RFLP-assays were applied.

All mastitis isolates investigated could be assigned to P. zopfii genotype 2 suggesting that this genotype is the aetiological agent of bovine Prototheca mastitis.     

Population differentiation in the banana leaf spot pathogen Mycosphaerella musicola, examined at a global scale

Single-copy restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of the global population of Mycosphaerella musicola , the cause of Sigatoka (yellow Sigatoka) disease of banana. The isolates of M. musicola examined were grouped into four geographic populations representing Africa, Latin America and the Caribbean, Australia and Indonesia. Moderate levels of genetic diversity were observed for most of the populations ( H  = 0·22–0·44). The greatest genetic diversity was found in the Indonesian population ( H =  0·44). Genotypic diversity was close to 50% in all populations. Population differentiation tests showed that the geographic populations of Africa, Latin America and the Caribbean, Australia and Indonesia were genetically different populations. Using F _ST tests, very high levels of genetic differentiation were detected between all the population pairs ( F _ST > 0·40), with the exception of the Africa and Latin America-Caribbean population pair. These two populations differed by only 3% ( F _ST = 0·03), and were significantly different ( P  < 0·05) from all other population pairs. The high level of genetic diversity detected in Indonesia in comparison to the other populations provides some support for the theory that M. musicola originated in South-east Asia and that M. musicola populations in other regions were founded by isolates from the South-east Asian region. The results also suggest the migration of M. musicola between Africa and the Latin America-Caribbean region.     

piggyBac转座酶的瞬时表达对莱茵衣藻体内piggyBac转座子的作用

piggyBac转座系统作为一种遗传修饰的工具,在很多生物体如哺乳动物、昆虫、酵母中已经得到了广泛应用.然而,目前piggyBac转座系统在绿藻一莱茵衣藻(Chlamydomonas reinhardti)中是否有活性很少有入研究.本研究构建含有一个拷贝或多个拷贝并且能够稳定表达piggyBac转座元件的莱茵衣藻藻株.然后在含有转座元件的藻株中瞬时表达普通的piggyBac转座酶和密码子优化后的piggyBac转座酶,通过限制性片段长度多态性(restriction fragment length polymorphism,RFLP)分析piggyBac转座元件的转座现象.转座酶转入莱茵衣藻细胞内一周和两周之后与转入之前的RFLP图谱对比,有很明显的差异.转入转座酶一周后的TR1藻株原有的1.5kb大小的片段消失,同时出现了3条新的片段:2.8、4.2和6.5 kb.两周之后,这3条片段又发生了变化,表明转座现象存在并且持续发生.转入密码子优化后转座酶一周后的TR3藻株中,1.5、3和4kb大小的片段没有发生改变,但是6.5 kb大小的片段消失了,与此同时新出现了一个2.8 kb大小的片段.两周之后,TR3的限制性片段多态性图谱并没有发生变化.为了检测转座酶是否成功转入细胞中,我们在新的接头序列两侧进行侧翼PCR,同时对PCR产物进行序列比对分析,结果显示,piggyBac转座子偶尔会在非常规性位点5’-TTT-3’和5’-ACGCAG-3’处发生剪切,而常规性剪切位点发生在5’-TTAA-3’处.研究结果表明,piggyBac转座酶对莱茵衣藻体内piggyBac转座子具有转座作用,piggyBac转座系统可以用于莱茵衣藻的遗传学修饰.     

大黄鱼冰鲜杂鱼饲料中细菌多样性研究

为研究大黄鱼（Larimichthys crocea）冰鲜杂鱼饲料中细菌的多样性,用细菌通用引物构建了细菌16S rRNA基因克隆文库。从文库中随机挑选125个克隆子进行限制性片段长度多态性（ RFLP）分析,得到82个不同的RFLP 带型。对部分代表性克隆子进行测序,得到23条有效序列。序列同源性分析和系统进化分析结果表明,大黄鱼冰鲜杂鱼饲料中细菌主要分为3大类群,其中γ-变形菌纲（γ-Proteobacteria）细菌占据明显地优势地位,约占所分析克隆子数的84.4％;其次是黄杆菌纲（Flavobacteria）细菌,约占10.9％;还有少量的梭杆菌纲（Fusobac-teria）细菌,约占4.7％。γ-变形菌纲细菌中以嗜冷杆菌（Psychrobacter sp.）最多,其次是发光杆菌（Photobacteri-um）。     

Isolation and characterization of novel soilborne lytic bacteriophages infecting Dickeya spp. biovar 3 (‘D. solani’)

Nine bacteriophages infecting Dickeya spp. biovar 3 (‘Dickeya solani’) were isolated from soil samples collected in different regions in Poland. The phages have a typical morphology of the members of the order Caudovirales, family Myoviridae, with a head diameter of c. 90–100 nm and tail length of c. 120–140 nm. In host range experiments, phage ?D5 expressed the broadest host range, infecting members of all Dickeya spp., and phage ?D7 showed the narrowest host range, infecting isolates of Dickeya dadantii and ‘D. solani’ only. None of the phages was able to infect Pectobacterium spp. isolates. All phages were prone to inactivation by pH 2, temperature of 85°C and by UV illumination for 10 min (50 mJ cm^?2). Additionally, phages ?D1, ?D10 and ?D11 were inactivated by 5 m NaCl and phage ?D2 was inactivated by chloroform. Phages ?D1, ?D5, ?D7 and ?D10 were characterized for optimal multiplicity of infection and the rate of adsorption to the bacterial cells. The latent period was 30 min for ?D1, 40 min for ?D5, 20–30 min for ?D7 and 40 min for ?D10. The estimated burst size was c. 100 plaque‐forming units per infected cell. The bacteriophages were able to completely stop the growth of ‘D. solani’ in vitro and to protect potato tuber tissue from maceration caused by the bacteria. The potential use of bacteriophages for the biocontrol of biovar 3 Dickeya spp. in potato is discussed.     

贵州地方猪品种HSL基因多态性与屠宰性状的关联性研究

本研究旨在分析HSL基因多态性与地方猪品种瘦肉率之间的相关性。采用RFLP方法,以二元杂交猪（长白猪&#215;大约克猪）为对照,对贵州地方猪品种糯谷猪和萝卜猪的HSL基因进行多态性分析。结果表明,贵州地方猪品种HSL基因外显子1中存在BsaH I酶切片段长度多态性,从3个猪群体中都检测到AA、AB和BB基因型,克隆测序后证实,A等位基因于第442 bp处发生了单碱基突变（G→A）,导致氨基酸Val→Ile替换。地方猪品种以A等位基因占优势,杂交猪以B等位基因占优势。将猪胴体性状与HSL基因型之间进行关联分析,结果显示大部分年龄段的猪品种中,BB、AB和AA 3种基因型的瘦肉率和眼肌面积均表现出从高到低的变化趋势,而背膘厚则完全相反。结果提示,HSL外显子1的B等位基因具有增加胴体瘦肉率和眼肌面积、降低背膘厚的遗传效应,可作为贵州地方猪品种瘦肉率的分子标记。