Construction and characterization of a BAC library of soybean

We have constructed a soybean (Glycine max (L.) Merrill) bacterial artificial chromosome (BAC) library from green leaf protoplasts of the cultivar, Misuzudaizu. The library contains 53,760 clones with an average insert size of 116 kb. About 2.9% chloroplast DNA origin was revealed by PCR and colony hybridization. Apart from 2.8% clones having no insert, this library represents 5.2 genome equivalents. With this genome coverage, the probability of having any DNA sequence represented in the library is higher than 99.5%. Three-dimensional pools of the BAC library in combination with the use of a high efficiency genome scanning (HEGS) electrophoresis facilitate rapid and efficient PCR-based screening. An average of five positive clones were identified after screening the BAC library with SSR and STS markers. BAC-end walking was performed for three SSR associated BACs. This library will provide a good resource for positional cloning of agronomically and biologically important QTL genes that Misuzudaizu possesses.     

一种水载型有机木材防腐剂的制备及其性能研究

为了制取不含金属的水载型有机木材防腐剂,选择十二烷基二甲基苄基氯化铵(BAC)、多菌灵和吡虫啉作为木材防腐剂的组分,以5%BAC、2%多菌灵、1%吡虫啉和10%乳化剂的配方,在乳化时间30 min、乳化温度70℃、搅拌速度1 000r/min的条件下进行乳化,得到稳定的水载型有机木材防腐剂.对木材防腐剂进行防腐、防霉实...     

苏云金芽胞杆菌质粒p26复制功能区的克隆

利用特异引物从基因组BAC文库筛选到1个35 kb的片段,对该片段测序比对分析发现可能含有p26的复制区.为了验证该片段中是否含有p26的复制区,首先,将大肠杆菌和苏云金芽胞杆菌穿梭载体pHT304用EcoR I酶切切掉苏云金芽胞杆菌复制子后自连,获得复制子克隆载体pHT304E;随后,将12 kbEcoR I片段亚克...     

大豆细胞质雄性不育系配套保持系线粒体基因组BAC文库的构建

以大豆细胞质雄性不育系配套保持系材料(JLCMSl)的黄化苗为材料,纯化得到线粒体.低熔点琼脂糖包埋线粒体成胶块,消化裂解后用HindⅢ部分酶切,通过脉冲场电泳回收40～60kb酶切片段.将回收产物连接到载体pIndigo-BAC 5,通过电击转化大肠杆菌DH10B感受态细胞,获得的BAC文库包含2 000个单克隆,平...     

大白菜抗霜霉病同源基因的筛选与亚克隆文库的构建

根据植物抗霜霉病基因保守区设计简并引物,利用三维PCR方法对大白菜(Brassica campestris ssp.pekiensis)‘85-1'BAC文库进行筛选,从19 200个BAC克隆中筛选到含有目的基因的10个阳性克隆.利用HindⅡ对其中的94一G-17克隆进行酶切,回收1～5 kb的DNA片段并连接到载...     

普通小麦品种小偃54中α/β-醇溶蛋白编码基因的克隆与序列分析

醇溶蛋白是小麦籽粒贮藏蛋白的重要组分,其组成与含量对小麦加工品质具有重要影响。本研究建立了利用PCR从普通小麦基因组BAC文库中筛选含有α/β-醇溶蛋白基因序列BAC克隆的方法,并获得9个不同的含有α/β-醇溶蛋白基因的BAC克隆。从其中鉴定出17个α/β-醇溶蛋白基因,其编码区序列长度为852~957 bp。12个序列在编码区内存在提前终止密码子,推测为假基因。其他5个成员(Gli-Xy54-1、Gli-Xy54-2、Gli-Xy54-3、Gli-Xy54-7和Gli-Xy54-13)分别编码291、310、311、287和317个氨基酸残基,都具有α/β-醇溶蛋白一级结构的典型特征。根据推导的氨基酸序列中乳糜泻病诱发因子的分布情况及多聚谷氨酰胺重复区的长度差异,推测Gli-Xy54-7可能定位于6A染色体,Gli-Xy54-2、Gli-Xy54-3和Gli-Xy54-13可能定位于6B染色体,Gli-Xy54-1可能定位于6D染色体。基因聚类分析支持了上述推论。这是第一次从普通小麦中筛选到包含α/β-醇溶蛋白基因的BAC克隆,并从中得到目标基因全长,对进一步研究普通小麦基因组中α/β-醇溶蛋白编码基因的组成、表达与功能有较好的参考价值。     

Preparation of high molecular weight （HMW） genomic DNA from tea plant （Camellia sinensis） for BAC library construction

A bacterial artificial chromosome （BAC） library is an invaluable resource tool to initiate tea plant genomics research, and the preparation of high molecular weight （HMW） genomic DNA is a crucial first step for constructing a BAC Library. In order to construct a BAC library for enhancing tea plant genomics research, a new method for the preparation of tea pant high molecular weight （HMW） genomic DNA must be developed due to young tea plant leaves and shoots are notably rich in both tea polyphenols and tea polysaccharides. In this paper, a modified method for preparing high quality tea plant HMW genomi~ DNA was optimized, and the quality of tea plant genomic DNA was evaluated. The results were as follows： Critical indicators of HMW DNA preparation were the appearance of the smooth nuclei in solution （as opposed to sticky-gummy） before agarose plug solidification, non-dark colored nuclei plugs after lysis with an SDS/proteinase K solution, and the quality and quantity of HMW DNA fragments after restriction enzyme digestion. Importantly, 1% dissolved PVP-40 and 1% un-dissolved PVP-40 during the nuclei extraction steps, in conjunction with the removal of PVP-40 from the plug washing and nuclei lysis steps, were critical for achieving HWM tea plant DNA suitable for BAC library construction. Additionally, a third PFGE fraction selection step to eliminate contaminating small DNA fragments. The modifications provided parameters that may have prevented deleterious interactions from tea polyphenols and tea polysaccharides. The HMW genomic DNA produced by this new modified method has been used to successfully construct a large-insert tea plant BAC library, and thus may be suitable for BAC library construction from other plant species that contain similarly interfering compounds.     

猪Myl1基因启动子克隆及多态性研究

肌球蛋白轻链1基因(Myl1)编码的不同剪切体(主要是MLC1f和MLC3f)与哺乳动物骨骼肌肌纤维类型相关.通过猪BAC文库筛选及引物步行法测序获得Myl1基因的启动子序列,采用PCR-RFLP技术分析蓝塘猪、大花白猪、大白猪、长白猪和杜洛克猪5个品种Myl1基因启动子区的多态性.结果表明,Myl1基因-678C/T位点在蓝塘和大花白等本地猪种中多态性丰富,而在杜洛克、长白和大白等外来猪种中几乎只有CC基因型.     

半滑舌鳎含sox9基因BAC克隆的筛选及BAC-FISH定位

以本实验室构建的半滑舌鳎BAC文库为基础,通过对其进行有序混合,构建了两步PCR筛选体系;对性别决定相关的sox9基因的序列设计特异性引物,筛选获得阳性单一BAC克隆。利用BAC-FISH技术将包含sox9基因的BAC克隆定位在半滑舌鳎染色体上。结果显示,sox9基因在雌、雄半滑舌鳎的一对常染色体的长臂上分别存在两个杂交信号位点,信号稳定且特异。研究证实了该文库筛选体系的有效性;首次实现了对性别相关的sox9基因在半滑舌鳎染色体上的定位,其结果为揭示sox9基因参与鱼类性别控制的机制提供了重要基础。     

细菌人工染色体(BAC)文库在畜禽遗传资源保存中的应用

作者介绍了国际畜禽遗传资源保存的研究现状与趋势,分析了细菌人工染色体(bacterial artificial chromosome,BAC)文库的发展和应用状况,阐述了BAC文库应用于畜禽遗传资源保存的特有优势,提出了建立BAC文库将成为畜禽遗传资源保存的一种重要补充形式,有利于提升畜禽遗传资源保存的安全性和长久性,也为今后进一步研究畜禽品种的特定性状提供了无可替代的材料,对实现全方位保存畜禽遗传资源具有重要的实用价值和推动作用。