Genomic and cDNA microsatellites from apricot (Prunus armeniaca L.)

We developed primers for the amplification of 24 polymorphic nuclear microsatellites in apricot (Prunus armeniaca L.). Thirteen loci originated from three genomic libraries enriched for TC, TG and AAG motifs. Eight loci were developed from three fruit EST (Expressed‐Sequence‐Tag) libraries and three from a leaf cDNA microsatellite‐enriched library. There were up to nine alleles per polymorphic locus in 12 different cultivars. No difference in allele numbers were shown between cDNA and genomic‐source loci. Mean expected heterozygosity was 0.65 (range: 0.15–0.87). Mendelian segregation was confirmed for all loci. These markers should be helpful for diversity studies, genome mapping and cultivar identification in apricot and related species.     

毛足棒角蝗基因组DNA文库的构建

纯化毛足棒角蝗 (Dasyhippusbarbipes)虫体组织的高分子量基因组DNA ,经限制性内切酶Sau3AI部分消化后 ,10 %～ 4 0 %蔗糖密度梯度离心分离 10～ 2 0kb的DNA片段。以λEMBL3为克隆载体 ,与 10～ 2 0kb的DNA片段进行连接和包装 ,构建了毛足棒角蝗的基因组DNA文库。经测定该文库的滴度是 2× 10 5pfu/mL ,根据计算 ,99%的毛足棒角蝗的基因包含在该基因组文库中。     

灰树花总DNA的制备及基因组文库的构建A

灰树花是一种珍贵的药用真菌,因为多糖含量较高,较难获得高质量的总DNA,本文提出了一种制备高质量灰树花总DNA及构建灰树花基因组文库的方法。该方法制备的灰树花总DNA,经Sau3AI酶切后,用于构建基因组文库,可得到2×105个转化子/50mg,平均插入片段为14kb。本研究为下一步克隆灰树花中的基因以及进行其他分子生物学研究奠定了基础。Abstract： Grifola frondosa, is a valuable medicinal fungus. High quality total genomic DNA is difficult to prepare due to its high polysaccharide content. A method for the preparation of Grifola frondosa total genomic DNA and construction of Grifola frondosa, genomic library is described. Genomic DNA prepared by this method is digested by Sau3A I restriction enzyme. Constructed genomic library give a titer of 2×105 transformants/50mg , with a average insert size of 14kb. This has paved way for the cloning of other Grifola frondosa genes and molecular biology studies.     

雨生红球藻基因组文库的构建及鉴定

本研究以雨生红球藻34-1n为材料,提取其基因组DNA,利用限制性内切酶Sau3AⅠ对基因组DNA进行酶解,回收6~8kb的基因组DNA片段,并浓缩至200ng/μL。该片段与经BamH Ⅰ酶切和去磷酸化处理后的pUC18载体连接,然后电击转化到受体菌Escherichia.coli DH5α中,获得雨生红球藻34-1n的基因组文库。该文库的平均插入片段长度约为6.5kb,获得6×105个克隆数。通过PCR筛选,由雨生红球藻基因组文库中获得含bkt1序列的单克隆菌,与β-胡萝卜素氧化酶序列(GenBank:DQ086233.1)进行比对,结果表明bkt1基因组序列含有6个外显子。本研究为进一步鉴定雨生红球藻相关基因提供了一个文库平台。     

盐藻基因组DNA文库的构建(英文)

以ＬａｍｂｄａＦＩＸ○ＲⅡ为载体,构建了盐藻（Ｄｕｎａｌｉｅｌｌａｓａｌｉｎａ）的基因组文库。该文库包含了１．１×１０６个重组子,插入片段平均大小为１８ｋｂ左右,含插入片段的频率为１００％。该文库的容量约为盐藻单倍体基因组的２００倍。     

构建丝状真菌大片段基因组文库载体DNA的制备

载体DNA的制备是构建大片段基因组文库的关键步骤之一,高质量载体DNA受到酶切、脱磷等因素的影响,以载体pBHYG为材料,优化了限制性内切酶胁HindⅢ酶切和小牛肠碱性磷酸酶(CLAP)脱磷的作用条件,并在T4连接酶作用下自连,通过胶回收纯化制备了可用于进一步构建大片段基因组文库的线性载体DNA。     

红脐鳞地衣基因组文库的构建

地衣是真菌和一种或多种光合微生物形成的稳定的共生联合体 ,既是先锋生物 ,又是敏感生物。环境的变化及生境的片断化 ,使得许多地衣种类处于濒危状态。保护珍稀濒危地衣物种的方法包括地衣体的移植 ,地衣中菌藻的分离培养及基因组文库的构建等。本研究用改进的CTAB方法提取基因组总DNA ,以Lamb daGEM 11为载体 ,构建了红脐鳞 (Rhizoplacachrysoleuca)的基因组文库 ,文库中同时含有该地衣共生菌与共生藻的DNA。该文库包含 8.5× 10 5个重组子 ,插入片段的平均大小为 19kb。文库的容量约为红脐鳞单倍体基因组的 10 0倍。该基因组文库的构建为保护稀有与濒危地衣物种提供了一个新的途径 ,并可进一步开展有关地衣的分子操作研究 ,如地衣冰核蛋白的异源表达等。     

Molecular phylogeny of date palm (Phoenix dactylifera L.) cultivars from Saudi Arabia by DNA fingerprinting

Genetic diversity among 13 different cultivars of date palm (Phoenix dactylifera L.) of Saudi Arabia was studied using random amplified polymorphic DNA (RAPD) markers. The screening of 140 RAPD primers allowed selection of 37 primers which revealed polymorphism, and the results were reproducible. All 13 genotypes were distinguishable by their unique banding patterns produced by 37 selected primers. Cluster analysis by the unweighted paired group method of arithmetic mean (UPGMA) showed two main clusters. Cluster A consisted of five cultivars (Shehel, Om-Kobar, Ajwa, Om-Hammam and Bareem) with 0.59–0.89 Nei and Li's coefficient in the similarity matrix. Cluster B consisted of seven cultivars (Rabeeha, Shishi, Nabtet Saif, Sugai, Sukkary Asfar, Sukkary Hamra and Nabtet Sultan) with a 0.66–0.85 Nei and Li's similarity range. Om-Hammam and Bareem were the two most closely related cultivars among the 13 cultivars with the highest value in the similarity matrix for Nei and Li's coefficient (0.89). Ajwa was closely related with Om-Hammam and Bareem with the second highest value in the similarity matrix (0.86). Sukkary Hamra and Nabtet Sultan were also closely related, with the third highest value in the similarity matrix (0.85). The cultivar Barny did not belong to any of the cluster groups. It was 34% genetically similar to the rest of the 12 cultivars. The average similarity among the 13 cultivars was more than 50%. As expected, most of the cultivars have a narrow genetic base. The results of the analysis can be used for the selection of possible parents to generate a mapping population. The variation detected among the closely related genotypes indicates the efficiency of RAPD markers over the morphological and isozyme markers for the identification and construction of genetic linkage maps.Communicated by H.F. Linskens     

野生大豆基因文库的构建

以氯化铯密度梯度离心法纯化噬菌体λEMBL4,将纯化的EMBL4 DNA用BamH1/SalI双酶切制成载体。用CTAB(十六烷基三甲基溴化铵)法提取野生大豆(种名待定)大分子DNA,Sau3A部分酶解,从琼脂糖凝胶中回收10—22kb“目的”DNA片段,与载体连接,体外包装成重组噬菌体。所得重组子值为8×10(?)pfu(噬菌斑形成单位),达到了构建野生大豆基因文库要求的理论值。以栽培大豆7S贮藏蛋白a′-cDNA作探针,用噬菌斑原位杂交法从文库中筛选出一个阳性克隆。     

从构建的小鼠gDNA文库中筛选Sry基因

从Ｃ５７ＢＬ／６Ｊ公鼠脾脏组织抽提大分子ＤＮＡ,进行ＭｂｏＩ部分酶切,低熔点琼脂糖凝胶分离、回收酶切后所需长度的ＤＮＡ片段并与λＧＥＭ○Ｒ－１１ＢａｍＨＩ臂进行连接和λ噬菌体体外包装反应,构建成Ｃ５７公鼠基因组λ噬菌体文库,对实验中遇到的技术问题进行了探讨,比较了两种浓度连接产物的包装效率,并成功地从该文库中筛选到含Ｓｒｙ基因的单克隆     

小麦—簇毛表6VS／6AL易位系可转化人工染色体（TAC）文库的构建

可转化人工染色体（Ｔｒａｎｓｆｏｒｍａｔｉｏｎ ｃｏｍｐｅｔｅｎｔ Ａｒｔｉｆｉｃｉａｌ Ｃｈｒｏｍｏｓｏｍｅ,ＴＡＣ）是具有克隆和转移大片段基因能力的新型载体,是植被基因克隆和转化的有效工具。为了克隆泪科抗白粉病基因和其它基因,本研究用ＴＣＡ载体ｐＹＬＴＡＣ１７构建了带有抗白粉病基因Ｐｍ２１的小麦＝簇毛麦６ＶＳ／６ＡＬ易位系的基因组ＤＮＡ文库。该文库包含２１０万个克隆平均插入征段３５ｌｂ,相当于     

龙葵抗阿特拉津生物型叶绿体基因文库的建立

用对阿特拉津(Atrazine)除草剂抗性的龙葵生物型B_(12)株系作材料,制备叶绿体DNA。B_(12)株ctDNA(叶绿体DNA)经BamHI酶解,在0.7％琼脂糖凝胶电泳上呈现24条带,其中最大的片段为18.6kb,最小的片段为1kb。用pBR322作为载体,构建B_(12)株ctDNA BamHI片段文库。通过与探针的分子杂交,从中筛选出含有编码叶绿体32kd蛋白质的阿特拉津抗性基因的克隆pSB135和含有ATP合酶α亚单位基因的克隆pSB132。     

荷斯坦奶牛瘤胃微生物元基因组Fosmid文库的构建与分析

采用包埋法提取荷斯坦奶牛瘤胃微生物大片段总DNA,纯化后脉冲场电泳回收大小为36～48 kb,与pcc2FOS vector连接,转染至大肠埃希菌EPI 300宿主细胞,构建瘤胃微生物Fosmid基因组文库.对文库进行鉴定,该文库平均插入片段大小约35 kb,共保存30 000个克隆,空载体率小于2%,库容达1 050 Mb.     

Assessment of genetic fidelity of micropropagated date palm (Phoenix dactylifera L.) plants by RAPD and ISSR markers assay

RAPD (Random Amplified Polymorphic DNA) and ISSR (Inter-Simple Sequence Repeats) markers assay were employed to validate the genetic stability of date palm (Phoenix dactylifera L.) plants multiplied through somatic embryogenesis with upto forty two in vitro subcultures. Out of the 160 RAPD and 21 ISSR primers screened, 30 RAPD and 12 ISSR primers produced a total of 347 (246 RAPDs + 101 ISSRs) clear, distinct and reproducible amplicons, which were monomorphic across all micropropagated plants (27) studied. Thus, a total 8592 bands (number of plants analysed x number of amplicons with all the primers) were generated which exhibited homogeneous banding patterns with both RAPD and ISSR markers. These results indicate that the micropropagation protocol developed by us for rapid in vitro multiplication is appropriate and suitable for clonal propagation of date palm and corroborated the fact that somatic embryogenesis can also be used as one of the safest modes for production of true-to-type plants.     

Molecular diversity in Ligurian local races of common bean (Phaseolus vulgaris L.)

Abstract

Eight varieties of Ligurian common beans (Phaseolus vulgaris L.) were analysed using molecular approaches. Results were compared with two commercial cultivars (‘Cannellino’ and ‘Borlotto’). Data suggest that all Ligurian bean varieties have a low genetic variability and are very close to the commercial varieties. In particular, the three ‘Bianco’ varieties showed a molecular affinity, probably due to their common genomic origin.     

伪狂犬病毒闽A株基因文库的构建及物理图谱分析

本文报道以质粒ｐＢＲ３２２作载体,用鸟枪法克隆出了ＰＲＶ闽Ａ株除ＢａｍＨＩ－１,２外的所有酶切片段,构建了ＰＲＶ闽Ａ株基因文库,并以克隆出的ＢａｍＨＩ片段用光生物素标记作探针,应用分子杂交法确定了ＰＲＶ闽Ａ株绝大部分限制性内切酶位点的位置。     

Random amplified polymorphic DNA (RAPD) analysis in Indian mung bean (Vigna radiata (L.) Wilczek) cultivars

Greengram [Vigna radiata (L.) Wilczek], also known as mung bean, widely cultivated in a large number of countries, is an important pulse crop of Asia and is considered one of the ancestral species of the genus Vigna. Since yields of greengram have remained low across subtropical and tropical Asia, it is important to estimate genetic diversity in existing cultivars in order to see if the lack of genetic variability might be a constraining factor. In this study, 32 Indian cultivars of greengram were subjected to random amplified polymorphic DNA (RAPD) analysis using 21 decamer primers. A total of 267 amplification products were formed at an average of 12.71 per primer with an overall polymorphism of 64%. The extent of polymorphism was moderate to low. Jaccard similarity coefficient values ranged from 0.65 to 0.92. The cluster analysis resulted in mainly three clusters revealing greater homology between cultivars released from the same source. The results of principal components analysis also substantiated this conclusion. The close genetic similarity between the cultivars could be explained due to the high degree of commonness in their pedigrees. The narrow genetic base of the greengram cultivars revealed in the present analysis emphasises the need to exploit the large germplasm collections having diverse morphoagronomic traits in cultivar improvement programs. This revised version was published online in July 2006 with corrections to the Cover Date.     

随机十肽库的构建及血管形成素结合肽的筛选

将合成的含有随机序列（NNK）₁₀的寡核苷酸片段,克隆入噬菌体呈现载体噬菌粒pCANTAB5E的SfiⅠ,NotⅠ位点,即cpⅢ蛋白信号肽与成熟肽之间,电转化E.coli TG1,构建了噬菌体表面呈现的十肽库,实际库容为3.53×10⁷,插入率为66.7%.经辅助噬菌体M13KO7超感染后,获得滴度为4.8 ×10¹¹ pfu/ml的噬菌体上清.经过两轮panning筛选和富集,从构建的随机十肽库中筛选到26个具有血管形成素结合活性的重组噬菌体克隆,对其中12个阳性噬菌体克隆的短肽序列进行了分析,ELISA检测结果显示12个阳性噬菌体克隆都能够与血管形成素特异性结合.