Chlex-100法快速提取甜瓜白粉病菌基因组DNA

现以白粉病菌分生孢子为材料,用Chlex-100法快速提取白粉病菌基因组DNA,并以此为模板PCR扩增出5.8S-ITS rDNA区段,以建立快速提取甜瓜白粉病菌基因组DNA的方法。结果表明:Chlex-100法是快速提取白粉病菌基因组DNA的高效方法。     

基于纤维素膜吸附的黄瓜基因组DNA快速提取方法

为快速获取与鉴定黄瓜基因组DNA,建立一种基于纤维素膜吸附的黄瓜基因组DNA快速提取方法,以市售华北型黄瓜为材料,制备纤维素膜片,筛选黄瓜裂解试剂,优化提取反应条件,建立了包括黄瓜裂解、膜片吸附、膜片清洗、核酸洗脱等步骤的黄瓜基因组DNA提取方法,可以在5 min内从黄瓜果实中提取基因组DNA用于PCR扩增。黄瓜果实的表皮、表层果肉和中心果肉都可以实现基因组DNA提取和PCR扩增,最少可以从13 mg黄瓜果实中提取并检测到黄瓜管家基因Act1片断。利用膜吸附DNA提取技术实现了黄瓜基因组DNA的快速提取,为黄瓜基因研究与快速鉴定提供了一种简便方法。     

扩展青霉基因组DNA五种提取方法比较

为寻找一种适合于扩展青霉基因组DNA的提取方法,比较了固体培养、液体静置培养和液体振荡培养3种培养方式的提取效果,分别采用氯化苄法、蜗牛酶法、CTAB法、SDS法及异硫氰酸胍法提取扩展青霉菌基因组DNA,经DNA质量浓度测定及电泳分析,比较5种方法的差异,并利用扩展青霉的多聚半乳糖醛酸酶(Polygalacturonase)基因内一段保守序列,设计PCR引物,扩增大小为288 bp的特异目的基因,检测其灵敏性.结果表明:固体培养的方式较适合扩展青霉基因组DNA的提取,另外5种提取方法均能提取出扩展青霉菌基因组DNA,扩增出目的条带,其中氯化苄法和蜗牛酶法所提DNA的纯度均较好,但蜗牛酶法所提DNA量较少.因此,氯化苄法是5种提取方法中最可靠的DNA提取方法,所提DNA浓度高、纯度好、结果稳定,可作为PCR反应的模板进行特异性扩增,且该方法的最低检测限大约为1.01×10-1μg/mL.     

氯化苄法提取植物内生放线菌基因组DNA

以植物内生放线菌菌株GL0806123为试材,用氯化苄快速提取植物内生放线菌菌株GL0806123基因组DNA。建立用氯化苄提取植物内生放线菌基因组DNA的方法。结果表明:用氯化苄法能够成功提取植物内生放线菌菌株GL0806123基因组DNA,以此为模板PCR扩增出16S rDNA区段,测序后可用于菌种鉴定。氯化苄法是一种快速提取植物内生放线菌基因组DNA的方法。     

茭白黑粉菌及茭白植株中DNA的提取方法

研究了茭白黑粉菌及茭白植株中基因组DNA的高效提取方法。采用改进的CTAB（十六烷基三甲基溴化铵）法提取DNA,根据琼脂糖凝胶电泳检测、A260/A280的测定和PCR扩增结果表明,用改进的CTAB法提取茭白黑粉菌及茭白植株基因组DNA,所得DNA无论是纯度还是完整性都比较好,适合于酶切和PCR扩增,可以用于分子生物学研究。     

甜菜干种子DNA提取的不同方法比较

分别采用SDS法、CTAB法及碱裂解法对甜菜干种子基因组DNA进行提取．并利用1％琼脂糖判断DNA的纯度,λDNA判断DNA的含量,结果表明,SDS法和CTAB法提取的甜菜基因组DNA含量较高,提取的DNA总量接近,碱裂解法提取的DNA含量较低。3种方法提取的DNA均能够满足SSR—PCR的要求,并且扩增务带没有差异,由于碱裂解法提取DNA快速和简单,因此适合大群体DNA的提取及对模板要求不高的PCR反应中,而SDS法及CTAB法适合一次性大量提取甜菜基因组DNA以及对于DNA纯度要求较高的PCR反应中。     

节瓜基因组DNA提取方法比较研究

以节瓜的成熟干种子、子叶和不同发育阶段的真叶为材料,选用尿素提取法、高盐低pH值法、SDS法和CTAB法等4种DNA提取方法进行比较。结果表明:不同节瓜材料均可以提取到基因组DNA,其中子叶和第1片真叶的提取效果最好;尿素提取法提取种子DNA最为简便快捷,CTAB法提取叶片能得到纯度较高的基因组DNA。经PCR检验,干种子和子叶提取的基因组DNA都可用于PCR扩增。     

柑橘苗期SSR鉴定分析体系的优化及初步应用

以柑橘幼苗微量叶片（3～5mg）为材料,改进柑橘基因组DNA提取方法,并对3种PAGE凝胶浓度和3种银染色方法进行了筛选和优化,建立了一种经济,高效的柑橘苗期SSR分析体系。试验结果表明：该微量叶片提取方法需材少,用时短,所提取基因组DNA质量好、纯度高,完全适用于SSR—PCR扩增反应。     

不同方法提取野生荔枝基因组DNA效果的比较

以广东、广西地区的3个野生龙眼的幼嫩叶片为试材,采用SDS法、常规CTAB法和改良2× CTAB法提取荔枝叶片基因组DNA,比较研究从富含多酚、多糖和色素等次生代谢物质的野生荔枝叶片中获得高质量DNA的提取方法.结果表明:采用改良的2×CTAB法提取的荔枝叶片基因组DNA纯度最高、质量最佳,可直接用于荔枝叶绿体DNA trnL-F基因的PCR扩增分析,且扩增后条带清晰.表明改良的2×CTAB方法获得的DNA纯度和产率较高,符合进行野生分子标记的实验要求.     

杏仁基因组DNA微量提取的改良方法

基于报道的大豆干种子提取方法,对高脂肪的杏仁进行了基因组DNA提取方法的改良研究,并以提取的基因组为模板进行了PCR扩增验证。结果表明:改良的SDS法提取的杏仁基因组DNA具有纯度和浓度高、条带清晰、降解少等优点,并在高倍稀释条件下仍能获得有效扩增。     

马铃薯抗晚疫病基因R8、RB和抗病毒病基因Rx1、Ryadg的多重PCR检测

在218份马铃薯材料中筛查晚疫病持久抗性基因RB和R8标记的基础上,进一步筛查了马铃薯X病毒(PVX)和马铃薯Y病毒(PVY)极端抗性基因Rx1和Ry_adg的标记。利用筛查出的含有Rx1和RB标记的‘德薯3号’和含有Ry_adg和R8标记的P182马铃薯资源材料混合DNA为模板,选择已报道的Rx1和Ry_adg的标记引物,并根据RB和R8序列设计特异引物,同时设计马铃薯GBSS基因特异引物为内参监控PCR反应体系有效扩增,对多重PCR延伸温度、引物组合浓度等进行优化,建立了同时检测4个抗病基因的多重PCR方法。应用该方法对选育品系样品进行筛查,鉴定到聚合上述4个抗性基因的材料4份。本研究中建立的多重PCR检测体系用于分子标记辅助选择,为马铃薯晚疫病和病毒病多抗基因聚合育种提供了简单、高效的技术方法并为多抗性基因聚合新品种选育创制新资源。     

安徽乌菜DNA提取与InDel引物筛选

利用改良的SDS法提取了153个品种的安徽乌菜DNA,并进行2次重复,均获得了较理想的DNA产物。通过试验确定了安徽乌菜InDel-PCR的10μL最佳反应体系:1×PCR Buffer 1μL,dNTP 0.2μL,引物1μL,Taq E 0.1μL,DNA 1μL,ddH2O 6.7μL;最佳PCR扩增反应程序:94℃预变性5 min;94℃变性30 s,55℃退火40 s,72℃延伸40 s,共36个循环,最后72℃延伸10 min,10℃保存。从供试的66对InDel引物中筛选出31对适合安徽乌菜、条带清晰、重复性高、多态性好的引物,为安徽乌菜品种鉴定、遗传多样性分析等奠定了基础。     

Development of DNA markers for hybrid identification in Leucadendron (proteaceae)

A rapid and reliable method to accurately identify hybrids at an early age is essential to the success of Leucadendron breeding programs because identification based on morphology can be difficult or impossible when the seedlings are young. DNA based PCR-RFLP and random amplified microsatellite polymorphism (RAMP) markers were developed for this purpose. Unexpected non-parental fragments appeared during the PCR-RFLP analysis of the nuclear ITS region of L. uliginosum 05 × L. procerum 04 hybrids. Mixing DNA from both parents in a single PCR also produced the non-parental fragment, suggesting that PCR recombination had introduced a novel restriction site into the products from the hybrids. Sequencing of individual amplified ITS products from the hybrids confirmed this conclusion. To avoid this complication, RAMP markers were developed for accurate hybrid identification in Leucadendron. RAMP analysis generated a considerable number of polymorphic products, and showed more discrimination in identifying Leucadendron hybrids than did PCR-RFLP.     

萝卜Ogura-CMS育性恢复基因Rfo的高通量SNP标记开发及其应用

利用萝卜Ogura-CMS保持系和恢复系育性恢复基因Rfo的序列,开发了基于高通量竞争性等位基因特异性PCR(KASP)分型技术的SNP标记:Rfo-SNP1。利用该标记对24个萝卜品种的304个单株进行了育性恢复基因Rfo的基因型鉴定,其中289株获得基因分型结果,占整个群体的95.07%。从分型结果看,育性恢复基因Rfo的基因型为rfrf的有110株(占36.18%),基因型为Rf Rf和Rfrf的分别为156株(占51.32%)和23株(占7.57%)。在12个品种中获得基因型为rfrf的单株,可作为保持系并用于不育系转育。利用PCR-RFLP标记和回交群体单株育性鉴定,进一步证实Rfo-SNP1标记不但能够实现高通量检测,而且能够实现萝卜Ogura-CMS育性恢复基因Rfo基因型的精准鉴定。目前利用该方法已成功转育了6套稳定遗传的雄性不育系及保持系。     

Functional characterization of watermelon (Citrullus lanatus L.) EST–SSR by gel electrophoresis and high resolution melting analysis

The present work was conducted to characterize the functionality of 257 watermelon EST–SSR primer pairs for their PCR amplification and polymorphisms. EST–SSR markers were tested on DNA sample panels of six watermelon cultigens and two related species of Citrullus lanatus var. citroides and Citrullus colocynthis based on agarose gel electrophoresis and high resolution melting (HRM) analysis. Successful PCRs were shown for 240 primer pairs (79%), and 173 primer pairs (67%) were polymorphic in a watermelon DNA sample panel on agarose gel electrophoresis. In addition, HRM analysis of 24 EST–SSR markers that were monomorphic on agarose gel separation identified an additional 19 polymorphic markers, indicating that HRM is an efficient tool for the rapid screening of sequence variations and allele discrimination. In the assessment of genetic relationships, six watermelon cultivars were closely related together (0.91–0.97) and demonstrated a narrow genetic base in the watermelon genetic pool. A high level of genetic dissimilarity (0.36–0.97) was shown between watermelon species and other related species. Marker transferability to melon species (Cucumis melo L.) was examined by cross-species PCR amplification and genetic diversity assessment in eight melon cultigens. Of the 257 EST–SSR primer pairs, 79 (32.9%) showed successful PCR amplification from melon DNA samples. A dendrogram of the genetic relationship based on 22 EST–SSR markers showed a clear classification of melon genotypes in accordance to fruit characteristics. The EST–SSR markers characterized in this study will contribute to diverse genomic investigations and breeding efforts, including comparative genome mapping, marker-assisted selection, and DNA fingerprinting for genetic diversity and cultivar identification in many cucurbit crops.     

云南出口新鲜松茸产地属性DNA指纹图谱构建

收集19个出口松茸主产地的97个样本,使用逆转录因子的DNA分子标签对样本基因组进行PCR扩增;为尽可能区分原产地,对某些产地的样本增加先使用限制性内切酶处理基因组DNA,再次使用逆转录分子标签的步骤。采用全自动DNA分析系统获得一系列DNA指纹图谱。通过比较分析找到同一地理居群共有的DNA指纹图谱特征以及酶切前后的图谱差异。这些指纹图谱特征与松茸原产地间存在显著相关性。     

甜菜EST-SSR引物的开发与应用

利用NCBI公共数据库现有的甜菜（Beta vulgaris L.）表达序列标签（expressed sequence tags,EST）数据信息,开发了甜菜EST-SSR标记。在所有的29830条甜菜EST序列中共确认得到20109条非冗余EST序列,总长为11287.6kb。在含有微卫星重复的6951条EST序列中按照SSR引物设计要求,最终获得了2845个EST-SSR,平均每3.96 kb含有1个SSR。EST-SSR的分布频率和特征分析表明,A/T单碱基重复最多,其次是AAG/CTT三核苷酸重复,AG/CT二核苷酸重复,ACCTCC/AGGTGG等六核苷酸重复最少。随机合成了100对SSR引物,并分别选用6个甜菜品种进行多态性检验,将其按遗传相似性分为两组,多态信息含量（polymorphism information content,PIC）平均值为0.47。本研究证实这种全新的开发甜菜SSR标记的方法具有高效、多态性较高的特点,在甜菜遗传多样性分析、功能基因定位、遗传图谱构建以及比较基因组等研究方面有广阔的利用前景。     

分子生物学技术在菇类研究中的应用

本文报道了应用ＲＦＬＰｓ和ＲＡＰＤ技术分析香菇类缘关系，鉴别品系及分析木耳杂交后人攻原体质体融合子的结果，发现供试的３２个香菇菌株的总ＤＮＡ限制性内切酶片段长度存在多态性；木耳杂交后代、融合子及其亲株间ＤＮＡ ＰＣＲ扩增产物谱带型出现差别，这充分说明ＲＦＬＰｓ和ＲＡＰＤ技术在分析遗传变异、鉴别品系、鉴定杂种和融合子研究中的重要作用。     

Quick detection of sex determining region protein gene in mouse fetuses

AIM: To detect quickly the Y-chromosome specific sex determining region protein (Sry) gene in mouse fetuses on embryonic day 14.5 with a PCR method. METHODS: We designed specific primers with the OLIGO 5. 0 software. Templates were prepared in 30 minutes by the following way. About 1 mg embryonic tissue but not fetal liver was suspended, and treated with 200μL of lysis buffer, consisting of PCR buffer containing 20 mg/L proteinase K, 0. 5% NP-40, and 0.05% Tween 40, at 60°C for 15 minutes, heated for 5 minutes at 100 °C, 10μL was used as template. The PCR react ion was performed in 50μL, using two sets of primers specific for Sry gene (chromosome Y) and IL-3 gene (chromosome 11) . PCR conditions and cycle numbers were optimized. The assessment of the results was done by electrophoresis in 3% agarose run at high voltage. The specificity of the method was conf irmed by fluorescent in situ hybridization (FISH) using a specific male probe on embryonic tissue cells. RESULTS: Electrophoresis showed that PCR product of male control DNA consisted of a 649 bp product representing the IL-3 gene and a 444 bp product representing the Y-specific Sry gene, female control DNA only one 649 bp product. Fetuses with two bands matching those as seen inmale control DNA are the presumpt ive male fetuses. Fetuses, only the IL-3-associated 649 bp band, are the presumptive female fetuses. These were confirmed by FISH. The ent ire procedure took <3. 5 h. CONCLUSION: The established PCR assay offers a quick, simple, accurate, and sensitive detection of sex determining region protein gene in mouse fetuses. This method allowed the preparation and culture of pure male and female hematopoietic stem cells from fetal tissue.