百草枯对人肝HepG2细胞生长和周期的影响

为全面理解百草枯(PQ)对人肝的毒性效应,寻找新的方法增强肝对PQ的解毒能力,本文采用显微观察和流式细胞术分析了PQ处理24h对人肝HepG2细胞生长和周期的影响.结果显示,7.21mg/L的PQ处理即可抑制部分HepG2细胞的生长,23.36mg/L的PQ可导致HepG2细胞周期停滞,S期细胞明显减少、G1和G2期细胞明显增多,且细胞生长抑制和周期受阻程度与PQ处理浓度呈正相关.该结果表明,PQ对HepG2细胞的生长和周期具有明显阻滞作用,生长因子类或促增殖类药物可能有助于增强人肝对PQ的解毒能力.     

构建重组质粒的二步PCR方法

建立一种简单的重组质粒构建方法——二步PCR,不需要限制性内切酶对基因和载体进行酶切.第一步常规PCR中,用正向引物(IF)和反向杂合引物(IR)扩增黑曲霉木聚糖酶Xyn基因,IF与基因特异性匹配,IR引物3’端与基因序列匹配、5’端含有19 bp p ET20b一端正链序列;第二步反向PCR中,以扩增的Xyn基因作为正向引物,其3’端含有19 bp与环状p ET20b模板退火、并在高保真Q5 DNA聚合酶作用下延伸,与模板另一端互补的反向引物VR协同作用,重组质粒通过PCR扩增出来.反向PCR产物经T4 DNA连接酶环化,并经Dpn I限制性内切酶消解处理,可以直接转化BL21(DE3)感受态细胞.用这种方法将622、639、1125、1209 bp大小的基因重组p ET20b中,显示出有较广泛的普适性.     

花椰菜自交不亲和性的分子标记研究

运用随机扩增多态性DNA(RAPD)和inter-简单重复序列(ISSR)等分子标记技术,分别对5组花椰菜自交不亲和系和对应的自交亲和系的基因组进行指纹差异分析.采用10个10 mer随机引物和5个ISSR引物进行PCR扩增,结果表明,扩增片段分子量在0.3～5 kb之间,指纹图谱的稳定性和重复性较好.经过3次以上重复发现,ISSR4引物扩增图谱中,自交亲和系比不亲和系多扩增出3 800 bp片段;ISSR6引物扩增图谱中,除第3组外,不亲和系比亲和系多扩增出1 100 bp片段.结果表明,花椰菜自交不亲和系与自交系基因组DNA之间存在差异,扩增出的差异片段与花椰菜自交不亲和性相关,并可作为自交不亲和系和自交亲和系育种筛选的分子标记.初步探讨了花椰菜自交不亲和性的遗传机制及RAPD和ISSR技术在自交不亲和系选育过程中的应用前景.     

牛病毒性腹泻病毒GSTZ株E1基因原核表达载体的构建及表达

为研究牛病毒性腹泻病毒的囊膜糖蛋白E1的抗原性,参考了Oregon C24V BVDV毒株的基因组序列(AF091605.1),并设计一对特异引物(上游引物5’-CCC GGA TCC ATG GCC TCT CCC TAC TGT-3’,下游引物5’-GGG CTC GAG TTA CCC TTG TGC TCC TGT T-3’),利用RT-PCR从病毒基因组中扩增E1基因609 bp全长片段,测序结果表明,与C24V株核苷酸同源性达100%.扩增产物经BamHI和XhoI双酶切,亚克隆至原核表达载体,双酶切鉴定表明成功构建了重组表达载体pET-32a(+)-E1.重组表达载体转化BL21(DE3)宿主菌,IPTG诱导表达,经SDSPAGE和Western-blot检测,成功诱导表达出25ku E1蛋白.实验结果为进一步研究BVDVE1蛋白的功能提供了参考.     

盐藻Psy基因保守序列的克隆及同源性分析

文中根据两种藻——Dunaliella bardawil和Haematococcus pluvialis的Psy cDNA保守序列以及6种植物——Arabidopsis thaliana，Oryza sative，Tagetes erecta，Lycopersicon esculentum，Capsicum annuum和Daucus carota的Psy基因氨基酸保守序列设计了上游引物5’-GAGTACGCCAAGACCTTCTA-3’和下游引物5’-GTTGTCATAGTCATTCTTCTC-3’．以盐藻基因组为模板、以46～35℃每循环递减0．5℃的退火温度扩增获得的大小约为2490bp的片段，经NCBI／BlastP同源性分析推测其为Psy基因的部分片段．     

花椰菜自交不亲和性的分子标记研究

运用随机扩增多态性DNA(RAPD)和inter-简单重复序列(ISSR)等分子标记技术。分别对5组花椰菜自交不亲和系和对应的自交亲和系的基因组进行指纹差异分析．采用10个10mer随机引物和5个ISSR引物进行PCR扩增。结果表明．扩增片段分子量在0．3～5kb之间，指纹图谱的稳定性和重复性较好．经过3次以上重复发现，ISSR4引物扩增图谱中。自交亲和系比不亲和系多扩增出3800bp片段；ISSR6引物扩增图谱中。除第3组外，不亲和系比亲和系多扩增出1100bp片段．结果表明．花椰菜自交不亲和系与自交系基因组DNA之间存在差异．扩增出的差异片段与花椰菜自交不亲和性相关。并可作为自交不亲和系和自交亲和系育种筛选的分子标记．初步探讨了花椰菜自交不亲和性的遗传机制及RAPD和ISSR技术在自交不亲和系选育过程中的应用前景．     

常规PCR与RACE结合法快速从cDNA文库中克隆基因

首先根据Fe(Ⅱ)-转运蛋白基因家族的保守区设计同源的常规PCR引物(F1，R1)，扩增出490bp的特异片段。然后根据RACE法原理，巧妙地设计3′RACE(F2)和5′RACE(R3)特异性引物，使两者既能分别与文库载体上的筛选扩增引物配对扩增cDNA 3′和5′末端序列，又能使扩增出的3′RACE和5′RACE产物序列有重叠部分；另外设计引物时还兼顾到使F2和R3也能配对，从而能对3′RACE和5′RACE扩增产物进行双特异性引物常规PCR鉴定。该方案不但能够直接根据序列鉴定3′RACE和5′RACE产物是否为同一基因序列，还能快捷地用常规PCR鉴定3′RACE和5′RACE扩增产物是否为特异性扩增，避免了对非特异性扩增产物的克隆测序等繁琐的鉴定过程，优化了基因克隆策略。最后根据拼读出的全长序列设计引物(F4，R4)扩增出完整编码区。应用该方法成功地从小金海棠缺铁处理的根系cDNA文库中克隆了Fe(Ⅱ)-转运蛋白基因的cDNA。说明该方案是一种从cDNA文库中克隆目的基因的快捷优化方法。     

运用RAPD技术检测Cu2+对草鱼基因组DNA的影响

对草鱼腹腔注射了CuSO4,抽取注射前后其血液以提取基因组DNA。选用10个随机引物对草鱼基因组DNA进行了RAPD扩增,结果表明:5个引物能产生1～5条扩增带,扩增产物分子大小在200～3500bp之间,其中引物S15和S16能检出用CuSO4染毒前后草鱼基因组DNA的差异。CuSO4可使草鱼基因组DNA发生改变,应对其使用加以限制。     

不同作物间共用SSR引物的初步研究

根据相关报道,利用互联网在GENE BANK中查询油菜的简单重复序列及其两端序列来设计引物,并合成了2对甘蓝型油菜的SSR引物（Bn92A,Bn72A),通过所建立的降温PCR（Touchdown PCR)方法,对来自禾本科,十字花科,芸香科,锦葵科等12种经济作物的DNA进行PCR扩增。结果显示,PCR扩增产物条带清晰。所有12份材料用2对SSR引物扩增出的主带片段大小一致,分别为150bp(Bn92A),250bp(Bn72A）。这与文献报道在甘蓝型油菜中SSR扩增出的片段大小完全一致。本研究说明,各物种基因组间存在着相当大的相似性,这2对甘蓝型油菜的SSR引物可在其它经济作物分子标记的基因组比较作图应用。     

藏羚羊(AAC)_n微卫星富集文库构建与引物筛选

基于磁珠富集法,利用生物素标记的寡核苷酸探针(AAC)8从藏羚羊(Tibetan antelope)基因组酶切的300~1000bp片段中筛选微卫星位点,纯化的富集片段连接到pCR@2.1载体和转化到Trans5α感受态细胞.随机挑取180个白斑克隆,PCR检测到条带数大于或等于2的阳性克隆为56个,阳性率为31.1%.对阳性克隆测序,获得43条含微卫星座位的序列,微卫星重复次数在5~20次之间.舍弃不适宜设计引物的微卫星座位,试验设计并合成了20对微卫星引物;以3个样点(青海、新疆、西藏)的24个藏羚羊基因组为模板进行PCR扩增,获得8对可得到稳定扩增产物的微卫星引物,其中4对引物的扩增产物具有多态性,座位分别为AAC050、AAC056、AAC165和AAC477.上述引物可用于藏羚羊及近缘物种的遗传多样性、种群遗传结构等研究.     

Construction of single-chromosome DNA library fromLilium regale Wilson

A protocol of simple rapid microdissection of single-chromosome, amplification and cloning of its DNA fromLilium regale Wilson is described. Single-chromosome, microdissected by micromanipulator, was put into a 0.5 mL Eppendorf tube and digested with Sau3A, and then the Sau3A linker adaptors were ligated to the ends of DNA fragments. After 2 rounds of PCR amplification with one chain of linker adaptor as primer, the PCR products thus obtained have a length of 300–2500 base pairs (bp) with predominant fragments at about 1000 bp. Southern blot analysis confirmed that the PCR products originated from the genome ofLilium regale Wilson. By cloning the amplification products from the second round of PCR, single-chromosome DNA library was constructed, in which about as many as 100000 recombinant clones were produced. A total number of 84 clones were analysed, and it was revealed that the inserts ranged in size from 300 to 1800 bp, with an average of780 bp. Compared with the methods described in other literature, this protocol, eliminating the need for enzymatic digestion and ligating micromanipulation of chromosomal DNA in nanoliter volumes, permits the efficient amplification of single chromosome (not tens of chromosomes as reported before) and the fragments (780 bp in average) cloned in this study are longer than those reported before (650 bp in average).     

Cloning and sequence analysis of a gene encoding polygalacturonase-inhibiting protein from cotton

Polygalacturonase-inhibiting proteins (PGIP) play important roles in plant defense of pathogen, especially fungi. A pair of degenerated primers is designed based on the conserved sequence of 20 other known pgip genes and used to amplify Gossypium barbadense cultivation 7124 cDNA library by touch-down PCR. A 561 bp internal fragment of the pgip gene is obtained and used to design the primers for rapid amplification of cDNA ends. A composite pgip gene sequence is constructed from the products of 5′ and 3′ RACE, which are 666 bp and 906 bp respectively. Analysis of nucleic acid sequence shows 69.2% and 68.7% similarity to Citrus and Poncirus pgip genes, respectively. Its open reading frame of the gene encodes a polypeptide of 330 amino acids, in which 10 leucine-rich repeats arrange tandemly. A new set of primers is designed to the 5′ and 3′ ends of the gene, which allows amplification of the full-length gene from the cotton cDNA library. Genomic DNA analysis reveals that this gene has no intron.     

浅蛤属(Macridiscus)物种特异性线粒体DNA分子标记及其长度多态性和个体内异质性

浅蛤属(Macridiscus)包括3个形态非常相似的物种,即黑浅蛤(M .melanaegis)、等边浅蛤(M .multifarius)和半步目浅蛤(M .semicancellata),其中前二者为同域分布.根据形态学特征,浅蛤属物种较难区分.本研究以改进的CTAB法提取浅蛤个体基因组DNA 后,用引物Macr_CO1_F16834和Cytb_Macr_R2PCR 配对进行聚合酶链式反应(PCR),扩增线粒体DNA控制区至细胞色素b基因(Cytb)的DNA 片段并进行序列测定,结果发现在黑浅蛤控制区偏3′端,存在该种特有的长度为363bp的数目可变串联重复序列(variablenumberoftandemrepeat,VNTR),种内因该VNTR出现1~3次不等的串联拷贝,而表现出广泛的长度多态性和个体内线粒体异质性现象,这是目前在动物线粒体控制区中发现的最大的VNTR;在半步目浅蛤的控制区的偏3′端,则同时存在14和17bp的2个较短的VNTRs,该物种也因这2个VNTRs的拷贝数目不同,而存在广泛的长度多态性;而等边浅蛤的该段序列长度保守,没有与其他物种共享的VNTR.以该段序列作为分子标记,运用PCR技术能够快速准确地识别浅蛤属物种.      

家兔RAPD图谱及双引物的运用研究

运用单引物获得了较理想的五个家兔个体的ＲＡＰＤ图谱，并探讨了双引物的运用。结果表明：ＲＡＰＤ可用于家兔品种（系）鉴定，具有较大运用潜力。双引物能比单引物扩增出更多的具有多态性的条带，双引物的运用是提高多态性的有效而且很经济的手段。     

Microdissection of chromosome 7B of common wheat and cloning of low-copy specific DNA sequences

The 7B chromosome of common wheat was microdissected from pollen mother cells of the 7B monosomic line of common wheat cv. Chinese Spring (CS). After proteinase K and DNA topoisomerase Ⅰ treatments, the isolated chromosomes were subjected to 1-3 rounds of DOP-PCR amplification, which produced continuous DNA fragments ranging from 150 to 700 bp. Ge-nomic Southern hybridization confirmed that the PCR products were originated from the wheat genome. Cloning of portion ( > 200 bp) of the 3rd round DOP-PCR products (50 μL) could generate about 20 000 recombinant clones. Characterization of 50 randomly chosen clones indicated that 21 clones produced discrete PCR products with the size of 240-600 bp. Dot-blot hybridization showed that among the 21 clones, 11 ( ～ 55%) were of low-copy nature while 10 ( ～ 45%) were repetitive. Southern hybridization with the complete set of the CS "nullisomic-tetrasomic (NT)" lines demonstrated that all the 6 low-copy clones were specific to either chromosome 7B or the 7th homoeologous group, whereas the 3 arbitrarily chosen repetitive clones were non-specific, disperse sequences.     

Chromosome microdissection by laser microbeam, chromosomal fragment isolation and amplificationin vitro in barley (Hordeum vulgare L.)

A method for microdissection, isolation and amplification of plant chromosomal fragments using laser microbeam and a glass microneedle was established. Firstly, 7H chromosome of barley (Hordeum vulgare L.) was dissected by Nd: YAG laserbeam with suitable parameters and the fragment comprising a satellite was isolated with a glass microneedle which was fixed on a micromanipulator. Then, the chromosomal fragment DNA was amplified by LA_PCR (linker adaptor PCR) for two rounds. The size of the DNA fragments of PCR products varied from 500-3 000 bp and the PCR products originated from the genome of barley were verified by Southern hybridization. Compared with previous reports, there are some advantages in this research. The performance is easier, the dissection is more precise and the cost is low. It also permits efficient amplification with only one single chromosome fragment. Laser microbeam_glass microneedle method may be useful in the microdissection of special chromosome regions, especially in plants with middle or small chromosomes.     

Cloning and characterization of vernalization-related gene (ver203F) cDNA 3' end

Based on the cDNA fragment sequence of vernalization-related gene verc203 cloned by differential screening in our lab, the 5' primer has been designed. The cDNA 3' end of ver203 gene (1 197 bp) has been cloned by the RACE method. And it is identified by Northern blotting that its expression is special in vernalization treatment. After comparing the sequence in the nucleotide sequence databases of Genbank, EMBL and DDBJ, the gene has homology with Hordeum vulgare jesmonate-induced protein gene. It is suggested that this gene might be related to the signal transduction mediated by jamonate.     

Detection of Grass Carp Hemorrhage Virus （GCHV） from Vietnam and Comparison with GCHV Strain from China

Grass carp plays an important role in small-scale aquaculture in Vietnam. However, a severe disease, known in Vietnam as “Red Spot Disease“, is causing significant economic loss in grass carp aquaculture. In this study, the tissue samples isolated from the grass carp with Red Spot Disease in Vietnam are investigated and eomparied with the control GCHV isolated in China by experimental infection, culture cell infection, serological cross reactivity, and RT-PCR amplifica-tion. Infected grass carp exhibits hemorrhage symptoms about 5 days after experimental injection with GCHV-V (Vietnam) strain. The symptoms and lethality induced by the GCHV-V strain are identical to that induced by the Chinese GCHV-9014 strain. The Chinese GCHV-873 strain in-duces typical cytopathogenic effects in 4 cell lines, such as CIK, CAB, FHM and GCO, from the6 fish cell lines examined. No cytopathogenic effects are observed in all the 6 examined cell lines,including CAB, FHM, CIK, EPC, CCO and G(X), infected by the GCHV-V strain and GCHV-9014 strain. Immunodiffusion assays demonstrate an obvious cross-reactivity among three GCHVstrains. Precipitin lines are clearly observed not only between the anti-GCHV-873 serum and thetwo strains GCHV-873 and GCHV-9014, but also between the anti-GCHV-873 serum and the GCHV-V strain. GCHV can be detected by immunodiffusion assays after three generations of blind propagations in the cell lines inoculated by GCHV-V strain. This implicates that GCHV-Vviruses have been replicated and amplified despite there being no cytopathogenic eifects observed in these examined cell lines. Three genome segments of GCHV, including S8, S9 and S10, are amplified by three sets of PCR primers designed according to the segment sequences published re-cently. The Q8fp and Q8rp primer set specific for genome segment S8 amplifies a 955 bp frag-ment from the extracted sample of diseased fish with Red Spot Disease, and the fragment size is i-dentical to that amplified by the same primer set from control GCHV-873 strain. Simultaneously,the Q9fp and Q9rp primer set specific for genome segment S9 generates a same 635 bp product,and the Q10fp and Q10rp primer set specific for genome segment S10 produces a same 697 bp fragment from both template samples of diseased fish with Red Spot Disease and control GCHV-873 strain. The RT-PCR amplification and corresponding size comparison data indicate that the three GCHV-V genome segments extracted from the diseased grass carp with Red Spot Disease in Vietnam should be identical to that in control GCHV-873 strain from China. The data confirm that the causative agent of grass carp Red Spot Disease in Vietnam is a virus, and the virus is closely similar to GCHV strain in China.     

RAPD技术在罗汉松性别辨别中的应用

部分植物（包括不少濒危物种）是雌雄异株，且在非花期仅凭形态学特征难以辨别其性别；这给品种改良、植物保护工作都带来了麻烦，以罗汉松（Podocarpus macrophyllus(Thunb.)D.Don)、短叶罗汉松（Podocar、pus macrophyllus Var.maki Endl）为具体实验材料，就RAPD技术在植物性别判断方面进行了尝试，在筛选了340种随机引物后，发现其中23种引物的产物有良性的多态性；最终筛选到了可以扩增出与性别相关的特异性条带的引物P20，在这基础上对表现出多态性的RAPD产物进行了UPGMA聚类分析，观察到各样本在遗传多态性上，性别间的差异大于变种与原种间的差异，同时还对单引物、双引物的RAPD结果进行了对比。