小麦线粒体DNA的高效提取方法

以小麦黄化苗为材料, 通过简单差速离心、DNaseⅠ处理得到无核DNA杂质的线粒体, 用SDS和蛋白酶K裂解线粒体, 经酚/氯仿抽提除去蛋白, 并用RNase A消化而得到单纯线粒体DNA(mtDNA)。对所提取的mtDNA进行紫外吸收光度分析, A₂₆₀/A₂₈₀ 平均为1.92, A260/A230 平均为2.09, 平均每克黄化苗可提取mtDNA 26.85 mg; 并对mtDNA进行琼脂糖凝胶电泳和RAPD扩增, 均得到清晰的电泳图谱。结果表明: 此提取方法得到的mtDNA, 不但产率高、结构完整, 而且能有效去除核DNA、RNA和蛋白质等杂质, 获得高质量的mtDNA用于PCR反应和各种遗传学分析。研究还发现, 通过调整线粒体裂解温度(先50℃裂解1 h, 再37℃裂解1 h), 亦可大幅度提高mtDNA的产率。     

质粒pCAMBIA1301的检测

用引物延伸芯片法实现对转基因水稻中 生物芯片技术是生物技术和微制造技术的融合, 已广泛用于生命科学的研究及实践、医学科研及临床、药物设计、环境保护、农业、军事等各个领域。而基因芯片是生物芯片中的一种,是指将大量基因探针分子固定于支持物上,然后与标记的样品进行杂交,所以一次可对大量核酸分子进行检测分析,从而解决了传统核酸印迹杂交技术操作复杂、自动化程度低、检测目的分子数量少、效率低等不足。文章探讨了用基因芯片这一新的检测手段对转基因植物的初步检测,采用一种新的反应机制－引物延伸芯片法（arrayed primer extension）,实现了样品扩增和杂交的一步化,而在传统的基因芯片检测中要需要两步来完成,从而为目前基因芯片中大片段样品的检测提供了一种可能性。 Abstract: Biochip technology which had emerged from the fusion of biotechnology and micro/nanofabrication technology at the end of 1980s has been widely used in life science ,medicine,clinical diagnosis,durg design,agricμLture,envioment pretection and strategics. DNA microarray (also call gene chip,DNA chip),one kind of biochips,is small chip containing many oligonucleotide probe .It can hybridize with labelled sample which makes it possible to detect large numbers of oligonucleotides at one time.So DNA microarray can overcome the disadvantage of traditional hybridization technology such as complexity,low automatization,poor efficiency and amount of detcting molecμLes. This paper describes a new method to detect transgenic plant with gene chip.We have developed a novel arrayed-primer extension technique. It combines hybridization and PCR at one step, while two separate steps are needed in the ordinary DNA microarray, therefore our method provide a feasibility to detect long DNA fragment .     

环境因子对新疆鹅喉羚群体遗传多样性的影响

环境因素在物种进化和遗传变异过程中起着非常重要的作用。为探讨新疆鹅喉羚遗传多样性与环境因子的关系,本研究采用聚合酶链式反应（PCR）和直接测序的方法,测定了新疆鹅喉羚11个群体84份样本的线粒体DNA Cyt b基因（1140 bp）和D-loop区（1100 bp）序列,分析各群体遗传多样性及环境因子对遗传多样性的影响。结果显示新疆鹅喉羚具有较高的单倍型多样性,较低的核苷酸多样性,表明其遗传多样性处于较低水平。环境因子与群体遗传多样性的相关性分析结果表明,海拔、年均降水量、年均气温、人口数量是影响新疆鹅喉羚遗传多样性的主要环境因子,其中海拔是最关键的环境因子。本研究结论为新疆鹅喉羚群体有效合理的保护与管理提供理论依据。     

小鼠基因组研究进展

小鼠基因组研究进展李善如１,２王冬平１陈永福２（１．军事医学科学院实验动物中心,北京１０００７１）（２．中国农业大学生物学院,北京１０００９４）ＴｈｅＤｅｖｅｌｏｐｍｅｎｔｏｆＭｏｕｓｅＧｅｎｏｍｅＲｅｓｅａｒｃｈＬＩＳｈａｎｒｕ１,２ＷＡＮＧＤｏｎ...     

Potyvirus属成员基因组全序列的简并引物PCR和RACE扩增方法

Based on multi-alignment of complete polyprotein amino acid sequences of genus Potyvirus,five degenerated primers were designedThey were Sprimer(5′-GGX AAY AAY AGY GGX CAZ CC-3′),pNIa(+)(5′-TNY TGG AAM CAY TGG AT-3′),pCI2(+)(5′-GCX ACX AAX ATX ATX GAX AA-3′),pCI1(+)(5′-GTX GGX TCX GGX AAX TCX AC-3′)and pHC(+)(5′-TGY GAY AAY CAZ TTX GA-3′)(X=A,T,C or G:Y=T or C;Z=A or G;N=A or T;M=A,T or G)Using degenerated PCR and modified RACE methods,a protocol for determination of complete genome sequence of potyviruses was established and proved to be successful on five potyviruses     

分子时代的白僵菌研究

白僵菌属是全球分布的最常见的虫生真菌。自上世纪90年代以来,有关白僵菌的研究进入分子时代,各方面的研究都有快速甚至是突破性的进展。作为复合种的球孢白僵菌和布氏白僵菌已被分割,而且还有其他新种被陆续命名,有多种有性型被发现;在查明一些致病基因及其作用机制后,通过转基因改良菌株工作取得突破性进展;分子生态学的研究解决了与白僵菌在农林生态系中的流行有关的一些难题。文中就白僵菌的分类学与系统演化、生物学、分子致病机制与基因工程,以及生态学与流行病学等方面的研究进展进行了综述。     

北部湾涠洲岛水域发现上颌和须板异常的布氏鲸:对保护的启示

2018年初以来,北部湾涠洲岛附近出现了布氏鲸（Balaenoptera edeni）的活动。一头上颌与须板异常的小布氏鲸个体引发热议,其视频在网络上广泛流传。我们在船只调查时,目击该个体10次,其以单独活动为主（90%）,主要出现在涠洲岛到斜阳岛之间的水域,最小凸多边形家域面积为14km2,核心家域面积为166.9km²。然而,在 2019年3月30日我们发现该个体已死亡漂浮在海面,根据尸体腐烂状况来推测,该个体的死亡时间大约为3~5日,死亡原因不明。根据照片和现场解剖分析,推测该小布氏鲸的上颌和鲸须异常可能是被渔网或绳索缠绕导致的。由于无法从外形上确认属于哪一个亚种,因此我们测定了该个体的线粒体DNA D-loop（mitochondrial DNA, mtDNA）和细胞色素b（cytochrome b, Cyt b）基因,分别得到909bp和395bp的序列,经比对和系统发育重建,发现该个体属于近岸分布的小布氏鲸亚种（Eden’s whale, B. e. edeni）。由于小布氏鲸具有一定季节迁移特性,我们无法判断造成其上颌伤害的渔网或绳索是否在中国水域。尽管如此,仍然建议当地部门应加强宣传,减少渔网等海洋垃圾的丢弃和排放,为小布氏鲸营造一个安全的栖息环境。     

重庆市26个南亚果实蝇种群mtDNA 16S rRNA基因部分序列及其系统进化

对重庆市26个南亚果实蝇Bactrocera(Zeugodacus)tau(Walker)种群线粒体16S rRNA基因进行测序,获得长约350bp片段的序列。对获得的序列分析表明,A,T,C,G平均含量分别为35·0%,41·3%,7·2%,16·5%,其中保守位点数342个,变异位点数5个,简约信息位点2个,自裔位点2个,所有碱基转换总数为136,替换总数为50。利用MEGA2·1软件重建系统发生树,发现其中21个南亚果实蝇种群未出现分化,另外有5个南亚果实蝇种群出现了分化,但遗传分化程度小。     

Genetic consequences of Pleistocene fragmentation: Isolation, drift, and loss of diversity in rock iguanas (Cyclura)

Pleistocene fragmentation of the Great BahamaBank resulted in one large and several smallpopulations of rock iguanas (Cycluracychlura). We explore patterns of geneticvariation within and among these islandpopulations using mitochondrial sequence data(partial ND4 to tRNA^Leu) in combinationwith eight polymorphic microsatellite loci (2to 10 alleles). Genetic data support twophylogeographically distinct groups, AndrosIsland and the Exuma cays. This resultconflicts with current subspecific taxonomy inwhich three subspecies are described. Analysesof allelic data indicate that most islandpopulations are currently demographicallyindependent. Pairwise Fst values between eightisland populations range from 0.18 to 0.63, and6 of 135 individuals are misassigned in anassignment test. Population-genetic diversityis characterized using standard measures suchas number of alleles and heterozygosity (H) inaddition to a normalized Shannon-Weaver indexof diversity (D). We find genetic diversity inthe Andros Island population comparable to thatin other non-piscine animals (avg. # ofalleles = 5, avg. H = 0.56, avg. D = 0.66) while inthe Exuma cays populations these measures aremuch lower (avg. # of alleles = 2.75–1.625, avg.H = 0.43–0.17, avg. D = 0.45–0.18). These dataare used to discuss conservation managementstrategies, including prioritization andtranslocation.     

Development of specific PCR primers for the detection of Rhizoctonia solani AG 2-2 LP from the leaf sheaths exhibiting large-patch symptom on zoysia grass

Detection of Rhizoctonia solani AG 2-2 LP isolates causing large-patch disease on zoysia grass was done using polymerase chain reaction (PCR). Specific primers were designed based on an amplified region using random amplified polymorphic DNA (RAPD)-PCR. Fifteen primers and three cultural types of R. solani AG 2-2 (types IIIB, IV and LP) were used for RAPD-PCR. The banding patterns by RAPD-PCR showed that the three cultural types were clearly distinguishable. A dendrogram constructed from the results of RAPD-PCR showed that the three cultural types of AG 2-2 clustered separately. The sequence of one PCR-amplified region which appeared only in LP isolates using primer A09 was selected for designing specific primers. Primer pair A091-F/R gave a single product from pure fungal DNA of LP isolates but not from those of the other two types (IIIB and IV), R. solani AG 1, 2-1, 2-3, 2-tulip, 3-10 and BI isolates and other turfgrass fungal pathogens. Primer pair A091-F/R also gave a single product from diseased leaf sheaths and this product was in accordance with those of pure fungal DNA of LP isolates. Primer pair A091-F/R did not yield PCR product from healthy leaf sheaths. The frequencies of detection of LP isolates from leaf sheaths of zoysia grass using PCR with primer pair A091-F/R were higher than those of the conventional isolation technique. These results showed that the PCR-based technique using specific primers A091-F/R is useful for the rapid detection of LP isolates from leaf sheaths of zoysia grass exhibiting large-patch symptoms.