杉木单染色体的显微分离及体外扩增

染色体微分离及体外扩增是传统细胞生物学和现代分子生物学相结合而发展起来的一项新技术.自1981年Scalenghe等[1]首次在果蝇唾腺染色体上取得成功后,该技术很快在人及动物分子遗传学研究中得到广泛应用,并取得很多重要的结果.     

PCR法克隆紫穗槐4-香豆酸:CoA连接酶全长cDNA

4 香豆酸 :CoA连接酶 (4CL)是木质素生物合成过程中重要的酶 .该文利用简并寡核苷酸PCR法结合cDNA末端快速扩增PCR法直接获得了紫穗槐 4CLAcDNA全长序列 ,避免了复杂的构建、筛选cDNA文库的过程 .先用简并PCR得到了 4CLA1片段 ,又据此片段设计反向嵌套引物 ,用RACE方法获得未知的 5′和 3′端序列 .所获得的全长 4CLAcDNA ,编码 5 40个氨基酸 .氨基酸序列同源性分析表明4CLA是典型的 4CL蛋白 ,含有预计的AMP binding位点、催化反应区和保守的Cys .     

Relationship between three novel SNPs of BRCA1 and canine mammary tumors

The BRCA1 gene plays an important role in the development of human breast cancer, and recent research indicated that genetic variations of BRCA1 are also related to canine mammary tumors (CMTs). Here, using rapid amplification of cDNA ends (RACE), we cloned the 5′- and 3′-UTRs of BRCA1. By direct sequencing of the flanking sequences of the 5′- and 3′-UTRs of BRCA1, three previously unreported single-nucleotide polymorphisms (SNPs) were identified, two (−1228T >C, −1173C >T) in the putative promoter regions and one non-synonymous SNP (63449G >A) in exon 23. Compared with 16 normal samples, the sequences from 34 CMTs suggested that SNP (−1173C >T) was associated with the development of CMTs (odds ratio (OR)=2.57, 95% confidence interval (CI): 1.07–6.15).     

土壤微生物总 DNA 提取及其 PCR 优化

采用3种不同土壤微生物总 DNA 提取方法对广东省乐昌杨东山十二度水自然保护区的样地土壤进行比较研究,进一步通过 PCR 扩增,研究牛血清白蛋白(BSA)对整个 PCR 扩增过程的影响。结果表明：试剂盒提取法相对于其他两种提取方法更加方便有效,当土壤样品量比较大时,直接提取法是最经济有效的方法;而在土壤微生物的 PCR 扩增实验中,加入2μl 的 BSA,可以有效地抑制腐殖酸等对后续 PCR 扩增的影响。     

RNAi转基因大豆品系‘B5C9123-5’的RPA可视化检测技术

为开发RNAi转基因作物的田间可视化鉴定方法,以DNA嵌合染料SYBR Green I为材料,采用设置重组酶聚合酶扩增(Recombinase Polymerase Amplification,RPA)特异性引物的手段,建立了一种快速、低成本、可视化的RPA用于转基因大豆‘B5C9123-5’的检测。结果表明,在靶标不存在时,RPA体系中游离的SYBR Green I染料使溶液呈现较弱的黄色荧光。在靶标存在的情况下,RPA扩增产生的双链DNA与SYBR Green I结合导致溶液从黄色转变为绿色并使荧光迅速增强。通过对RPA扩增时间和温度进行优化,阳性结果可在20 min内用肉眼鉴别,检测限为1.00 ng/μL。通过设置不同RPA引物,该方法可用于现场快速检测多种RNAi转基因作物。     

漾濞泡核桃ISSR-PCR反应体系的建立和优化

以漾濞泡核桃优株为研究对象,研究其ISSR-PCR反应体系中的5个影响因子(模板DNA浓度、Taq DNA聚合酶浓度、引物浓度、dNTPs浓度、Mg2+浓度)对PCR扩增效果的影响,从而建立起漾濞泡核桃的最佳ISSR-PCR反应体系。结果表明:模板DNA最佳浓度为50 ng/(25μL),Taq聚合酶浓度为0.75U/(25μL),引物浓度为0.7mmol/L,dNTPs浓度为0.20mmol/L,Mg2+浓度为2.0mmol/L,利用该最佳体系对试验中所选取的样本进行扩增反应。建立了适宜于漾濞泡核桃的ISSR-PCR扩增的最佳体系,该体系的建立为利用ISSR分子标记技术对漾濞泡核桃进行种质资源研究奠定了基础。     

猪多杀性巴氏杆菌环介导等温扩增检测方法的建立

为建立猪多杀性巴氏杆菌(Pm)的快速检测方法,本研究以Pm的PlpB基因序列为靶基因设计特异性引物,建立了Pm特异性的环介导等温扩增(LAMP)快速检测方法.经反应条件优化,确定反应条件为63℃,20 min.检测结果显示,建立的LAMP检测方法具有良好的特异性,灵敏度为25 cfu/mL;与大肠杆菌、副猪嗜血杆菌、胸膜肺炎放线杆菌、沙门氏菌无任何交叉反应.采用建立的LAMP方法与常规PCR方法对临床54份分离样品进行检测,检测结果为31份为阳性,23份为阴性,二者符合率为100％.该方法的建立为Pm在临床上的快速检测提供了一种新的技术手段.     

泥鳅MSAP技术反应体系的建立及优化

为获得不同倍性泥鳅基因组DNA甲基化水平及模式,以泥鳅Misgurnus anguillicaudatus为研究对象,利用正交试验建立了甲基化敏感扩增多态性( MSAP)方法的反应体系,并利用该方法对二倍体泥鳅鳍基因组DNA进行了MSAP分析。结果表明：最佳双酶切反应体系为800 ng的泥鳅基因组DNA,用EcoR I、 Hpa II和Msp I各10 U,在37℃下反应8 h后即可酶切;最佳预扩增反应体系为模板4μL、预扩增引物0·8μL、0·2 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U;最佳选择性扩增反应体系为预扩增产物稀释20倍的模板2μL、引物1·5μL、0·375 mmol/L dNTPs、1·5 mmol/L Mg2+和 Taq 1 U;二倍体泥鳅全甲基化率分别为24·1%、20·8%、22·3%,半甲基化率分别41·4%、20·8%、33·3%,总甲基化率分别为65·5%、41·6%、55·6%。研究表明,该反应体系稳定、可靠、重复性好,为MSAP技术在多倍体泥鳅相关研究中的应用奠定了基础。     

Characterization of the DNA nucleotide sequences in the genome of red sea bream iridoviruses isolated in Korea

The nucleotide sequences of DNA fragments amplified by polymerase chain reaction (PCR) from four different genomic regions of nine red sea bream iridoviruses (RSIVs) isolated from different species of fish, different areas and in different years in Korea were compared with the reported reference sequences. One isolate, RSIV Namhae, showed 100% homology to the reference sequences, while the other eight isolates, which appeared to contain identical nucleotide sequences, showed 96.6–98.9% homology with reference sequences depending upon the target regions of PCR gene amplification. However, differences in nucleotide sequences were not apparent between the RSIVs isolated in different locations, in different years or in different host species. We also cloned and sequenced the 3′ end flanking region (K1) of the DNA polymerase (DPOL) gene using the cassette ligation-mediated PCR method. This sequence was 4436-bp long and possessed two open reading frames (ORF-1 and ORF-2) oriented in opposite directions. The putative proteins encoded by these two ORFs could not be characterized by comparison with the proteins of other species in the data banks. The presence of the ribonucleotide reductase small subunit (RNRS) gene at the 3′ end of the K1 region allowed us to determine that these two genes, RNRS and DPOL, are separated 5508 bp and oriented in the same direction in the genome of RSIV. Moreover, it is of interest that a PstI-restriction fragment, of which the sequence but not the location within the RSIV genome had previously been reported, is located at nucleotide positions from 1096 to 2054, extending from within the ORF-1 region, spanning the intervening sequence between ORF-1 and ORF-2, and extending into the ORF-2 region. Various repeating sequences up to 86 bp were present at the 3′ ends of ORFs, especially within the nucleotide sequences at the 3′ terminus of ORF-2. No similarities were detected when the DNA sequences of the K1 region were compared to the DNA sequences of a repetitive element in the genome of other iridoviruses.