直接从人工老化的菜心干种子中提取基因组DNA用于RAPD分析

利用 4 0℃、1 0 0 %RH对菜心种子进行人工加速老化处理获得了不同活力的种子批 ,利用平衡酚_氯仿法直接从人工老化的菜心干种子中提取基因组DNA ,并对提取的基因组DNA进行了RAPD扩增。结果表明 ,所提取的基因组DNA量多 ,而且比较整齐一致。引物S2 0 8扩增所获得的基因组DNA指纹图谱上的DNA带清晰、明亮 ,从而表明利用本方法从人工老化菜心干种子中直接提取的基因组DNA完全可以用于RAPD分析。     

向日葵种子不同部位微量提取DNA用于PCR的研究

探索了从向日葵成熟种子的单粒种子、1/2种子、1/4种子、1/8种子中提取DNA的方法,结果表明,无论是从单粒种子、1/2种子、1/4种子还是1/8种子都能提取质量较好的DNA,对所有提取的DNA,进行RAPD分析,都能得到扩增产物,1/8种子提取的DNA量可保证用于2次PCR扩增。从种子的不同部位提取的DNA与从幼苗中提取的DNA用于RAPD分析,所得结果一致。说明直接从微量向日葵种子提取DNA应用于分子检测是可行的,但直接从向日葵果皮中提取DNA的技术有待进一步研究。     

用于分析土壤微生物结构变化规律的变性梯度凝胶电泳条件研究

研究确定土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为进一步研究分析土壤中微生物结构变化规律提供理论依据。土壤微生物基因组DNA提取采用直接法和间接法进行比较; PCR扩增条件调整扩增体系、DGGE电泳条件调整变性剂范围,并对其结果进行比较分析。通过对DGGE电泳相关条件的研究,结果显示,土壤中粗基因组DNA采用直接法提取,然后进行纯化; PCR扩增体系中加入BSA,DGGE电泳系统组成中变性剂浓度范围为35%～55%。确定了土壤微生物基因组DNA提取方法、PCR扩增条件、DGGE电泳条件,为后续的相关研究提供理论依据。     

利用不同方法从深加工牦牛肉产品中提取基因组DNA效果的比较

为了从深加工牦牛肉产品中获得片段较大、数量较多的基因组DNA,以用于分子追溯的检测,尝试并系统地比较了异硫氰酸胍法、酚-氯仿抽提法和试剂盒提取法的提取效果。通过定量分析、凝胶电泳和PCR片段大小梯度扩增等手段,对利用3种方法提取的基因组DNA的数量和质量进行了比较。结果显示,利用酚-氯仿抽提法提取的基因组DNA质量不佳,异硫氰酸胍法和试剂盒提取法的提取效果相当,PCR可以扩增出730 bp左右的核外基因细胞色素b,而核内基因只能扩增出300bp左右的核基因DNA片段(内乳铁蛋白基因,271 bp;BoLA-DRB3基因,302 bp)。综合考虑提取时间、费用、数量和质量等各方面的因素,认为异硫氰酸胍法操作简便、快速、廉价、有效,是较好的从动物深加工肉产品中提取基因组DNA的方法。     

石斛干品基因组DNA的提取与RAPD分析

市场中药干品的药性差异一直是影响中药标准化的瓶颈,而检测技术相对落后是导致这一现象的主要原因。DNA分子水平检测的困难是药材干品的基因组DNA难以提取。本文以铁皮石斛(Dendrobium candidum)干茎为材料,采用了四种方法从干品石斛中提取基因组DNA。结果表明,采用改良的CTAB法可从石斛干品尤其是干茎皮中提取质量较高的基因组DNA,其分子量大于23kb,以此DNA为模板进行不同引物的PCR扩增可获得清晰的RAPD条带。该研究初步建立了石斛干品合适的RAPD技术体系。     

黑翅土白蚁基因组DNA提取方法的优化

目的 为快速地提取到质量较好的黑翅土白蚁基因组DNA进行白蚁种群多样性的研究,对基因组DNA提取方法进行了比较与改进.方法 先初步采取CTAB法与蛋白酶K法对黑翅土白蚁基因组DNA的提取方法进行比较,再利用正交设计法对蛋白酶K法中裂解液、蛋白酶、RNA酶及作用时间4个因素进行优化.结果 蛋白酶K法获得的基因组DNA的质量与产量稍优于CTAB法;较佳的提取步骤组合为:裂解液150 μL,蛋白酶K 6μL,作用时间1h,RNA酶可不添加.结论 采用优化后的方法获得的基因组DNA为模板进行PCR扩增,得到了清晰、稳定的扩增谱带,完全可用于相关后续实验.     

紫杉醇产生菌的细胞总核酸提取

树状多节孢（Nodulisporium sylviforme)为中国新记录属种,首次发现它可产生特效抗癌药物紫杉醇,国内外尚未见其DNA提取等方面的研究报道。为了获得高质量的DNA,利用单因素试验对其细胞总核酸的提取方法进行了探索。找到了一套适合紫杉醇产生菌DNA的提取较为简便易行可靠的方法。用此法直接从紫杉醇产生菌中提取DNA,并将所提取DNA进行随机引物扩增（RAPD）,得到了较清晰的扩增图谱。     

不同预处理方法影响昆虫干标本DNA提取效果

为从昆虫干标本中获取高质量DNA,以利于后期PCR扩增目的基因,本研究对DNA提取前昆虫干标本的预处理方法进行探讨,分别测定其纯度和质量,并用不同引物进行了扩增效果对比。结果显示:经过预处理的样品DNA平均含量均高于CK的52.283 ng/滋L,最高为0.9%Na Cl处理3 h,平均含量达122.632 ng/滋L;米象4种预处理方法提取的DNA利用引物Lco1490/Hco2198及UEA7/UEA10均能扩增出650 bp长度条带,仅有0.9%Na Cl溶液浸泡3 h处理的米象可通过引物J173/J1331扩增出1 000 bp的长片段条带。研究表明,经过预处理后,一定程度上减少了基因组DNA的断链,主要对样品DNA的质量浓度影响显著;以9%Na Cl溶液3 h预处理后的昆虫干标本,利用磁珠法所提DNA的质量及PCR扩增效果最佳。     

三种提取苹果绵蚜基因组DNA方法的比较

参考国内外昆虫基因组DNA提取的常用方法,选取KAc法、氯仿-异戊醇法和盐析法3种方法对苹果绵蚜(Eriosoma lanigerum)基因组DNA进行提取.通过基因组DNA直接琼脂糖凝胶电泳、SSR引物扩增产物的琼脂糖凝胶电泳和聚丙烯酰胺凝胶电泳检测,对3种方法所提基因组DNA的质量进行了比较,并综合分析了提取所需时间及所用试剂的毒性大小等.结果表明,盐析法操作程序较简便快捷,节时省工,可得较好质量的DNA样本,且对试验操作人员无伤害,是一种值得推广应用的基因组DNA提取方法.     

煤地质环境微生物总基因组DNA提取方法的优化

高质量的DNA是进行分子生物学研究的基础。通过对传统DNA抽提方法(酚-氯仿法)进行改进,并以Rhodococcus sp.R04和煤粉为实验材料对细胞破碎条件进行优化,建立了一种可用于煤地质环境微生物基因组DNA高效提取的改良方法。以改良法和商业试剂盒提取煤地质环境微生物基因组DNA,通过琼脂糖凝胶电泳、细菌及古菌特异性片段的PCR扩增来评价所提取DNA的质量。改良法和试剂盒法均能获得煤地质环境微生物基因组DNA,并能用于多种特异性PCR扩增。与试剂盒提取的DNA相比,改良法获得的DNA片段主带明显,约占总DNA含量的50%,分子量大小接近23 kb,并且提取量大,约为试剂盒的5-10倍。同时,能用于如DNA文库构建和宏基因组测序等。此外,改良法所用试剂普通,价格便宜,提取的煤地质环境微生物基因组DNA质量较高,适于实验室和科学研究。     

大白菜杂交种'冠春'杂交率的RAPD分析

从春大白菜品种‘冠春’及其亲本中提取基因组DNA,用320个随机引物进行RAPD扩增,从中筛选出5 个可将亲本和子代区分的引物S4、S47、S73、S134和S194。S4产生父本特征带S4-370;S47和S134产生母本特征带S47-700 和S134-1200;S73和S494产生亲本互补的特征带S73-660、S73-730和S494-400、S494-1770,上述谱带均在子代中出现。以这5个引物产生的特征谱带建立杂交种‘冠春’及其亲本的RAPD特异指纹。通过对134个‘冠春’的种子进行纯度鉴定,结果表明2个父本和4个母本与大田检测结果完全一致。进一步验证了4种鉴定大白菜杂交种方法的可行性。     

RAPD analysis of seed purity in a commercial hybrid cabbage (Brassica oleracea var. capitata) cultivar.

The use of random amplified polymorphic DNA (RAPD) markers for evaluating seed purity in a commercial F1-hybrid cabbage (Brassica oleracea var. capitata) cultivar is demonstrated. Genomic DNA isolated from single ungerminated seed was found to be suitable for RAPD analysis. DNA from F1-hybrid and its parental lines was subjected to RAPD screening with 36 random decamer arbitrary primers. A total of 241 scorable products were observed with 54 (22%) being polymorphic. The RAPD data showed that the parental lines of this commercial cabbage cultivar were not very closely related. Two primers were chosen for purity testing of the F1-hybrid seeds. The sib (inbred seed; seed from self-pollination of parental lines) contamination results obtained by RAPD analysis were comparable to the commonly used grow-out trial and isozyme analysis, hence showing that RAPD analysis can be used for seed purity testing of commercial hybrid cabbage seeds.     

两株假单胞菌菌株的RAPD分析及分子生物学鉴定

根据DNA随机扩增多态性(RandomAmplifiedPolymorphicDNA,简RAPD)分子标记技术设计鉴别引物,建立一种快速、准确检测病人体内新发现的假单胞菌菌株的分子生物学方法.采用RAPD分析方法对该菌种的对照菌株AcinetobactercalcoaceticusKHW14(简称A.calcoaceticusKHW14)和新分离的菌株Acinetobactercalcoaceticus(简称A.calcoaceticus)进行指纹分析,依据两菌株的差异序列设计两对引物,并建立最佳的PCR扩增体系,产物经1.2%琼脂糖凝胶电泳得菌株特异性电泳图谱.此图谱可作为鉴定两菌株的标准图谱,RAPD分析方法具有良好的重复性,同时也进一步验证了两菌株的同源性.     

Optimization of DNA extraction from seeds and leaf tissues of Chrysanthemum (Chrysanthemum indicum) for polymerase chain reaction

Chrysanthemums constitute approximately 30 species of perennial flowering plants, belonging to the family Asteraceae, native to Asia and Northeastern Europe. Chrysanthemum is a natural cosmetic additive extracted from Chinese herb by modern biochemical technology. It has the properties of anti-bacterial, anti-viral, reducing (detoxification) and anti-inflammation. It possesses antioxidant characteristics, which could assist in minimizing free-radical induced damage. Therefore, it is widely used in skin and hair care products. Chemical composition of this herbal remedy includes kikkanols, sesquiterpenes, flavonoids, various essential oils containing camphor, cineole, sabinol, borneole and other elements that interfere with DNA, causing erroneous or no PCR products. In the present study, testing and modification of various standard protocols for isolation of high-quality DNA from leaf tissues and seeds of C. indicum was done. It was observed that the DNA obtained from seeds and leaf tissues with a modified cetyltrimethylammonium bromide buffer protocol was of good quality, with no colored pigments and contaminants. Also, DNA could be extracted from leaf tissues without using liquid nitrogen. Quality of DNA extracted from seeds was much better as compared to that extracted from leaf tissues. The extracted DNA was successfully amplified by PCR using arbitrary RAPD primers. The same protocol will probably be useful for extraction of high-molecular weight DNA from other plant materials containing large amounts of secondary metabolites and essential oils.     

Stability of RAPD fingerprints in potato: Effect of source tissue and primers

Variations in random amplified polymorphic DNA (RAPD) profiles from leaf, stem, root, and tuber tissues were observed in case of two glasshouse grown potato cultivars using 40 decamer primers suggesting possible danger of cultivar misidentification. Genomic DNA extracted from the above four tissues of four in vitro grown potato cultivars, however, produced more uniform RAPD fingerprints. A significant effect of random primers on fingerprint uniformity was observed in case of both glasshouse and in vitro grown samples. A new concept of stability index for random primers based on homogeneity of RAPD profiles obtained from different tissues of a single plant have been introduced. It is concluded that RAPD analysis of genomic DNA extracted from any tissue of in vitro grown potato plants using 14 selected decamer primers could be used to develop RAPD fingerprints for identification of Indian potato cultivars.     

白菜与甘蓝之间体细胞杂交种获得与遗传特性鉴定

为拓宽白菜育种的基因资源,改良白菜品质,以白菜(Brassica campestris,2n=20,AA)和甘蓝(B.oleracea L.var.capitata,2n=18,CC)的子叶和下胚轴为材料分离、制备原生质体。采用40%聚乙二醇(Polyethylene glycol,PEG)进行原生质体融合。融合细胞在以0.3 mol/L蔗糖、0.3 mol/L葡萄糖为渗透稳定剂,附加1.0 mg/L 2,4-D+0.5 mg/L 6-苄氨基嘌呤(6-BA)+0.1 mg/L 1-萘乙酸(NAA)+1.0 mg/L激动素(Kinetin,Kin)的改良K8p培养基中培养并诱导细胞分裂。小愈伤组织经增殖培养后在MS+0.2 mg/L玉米素(Zeatin,ZEA)+1 mg/L 6-BA+0.5 mg/L Kin+0.4 mg/L NAA的固体分化培养基上诱导出不定芽。30 d后再转入MS基本培养基,获得完整的再生植株。将生根的植株转移到花盆,并对其杂种性质进行形态学、细胞学和分子生物学鉴定。结果表明,经细胞融合分裂出的320个愈伤组织中,获得了35棵再生植株,其再生率达10.94%。形态学观察显示,绝大多数再生植株的叶面积较大,株型和叶型为两种杂交亲本的中间型,部分植株的叶片浓绿、肥厚。染色体计数结果显示,36.4%的再生植株染色体数为2n=38;36.4%的再生植株的染色体数为2n=58 60;27.2%的再生植株的染色体数为2n=70 76,超过两个融合亲本的染色体数的总和。流式细胞仪测定DNA含量显示,再生植株DNA含量变化比较大,其结果与染色体鉴定结果相吻合。随机扩增多态性DNA(Randomamplified polymorphic DNA,RAPD)和基因组原位杂交(Genomic in situ hybridization,GISH)分析结果证明再生植株具有双亲基因组。体细胞杂种花粉育性比较低,杂交、回交后其育性逐渐获得恢复,与白菜回交后代逐渐恢复了育性。通过体细胞杂交和回交、杂交获得了形态变化广泛的个体,为白菜的品种育种提供多样的种质资源。     

Molecular Marker Analysis of Differentially Aged Seeds of Soybean and Safflower

Storage of seeds for extended periods causes a number of degradative changes related to the aging process such as decreased seedling vigor and reduced germination. In this study, molecular markers were used to study the aging process in seeds of two different plants species. Seeds of three differentially aged seed groups, including control (un-aged), naturally aged, and accelerated aging, from soybean (Glycine max) and safflower (Carthamus tinctorius) were evaluated for genetic variability using random amplification of polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), and simple sequence repeat (SSR) markers. For both plant species, naturally aged and accelerated aged groups clustered together with RAPD markers, whereas control and naturally aged seeds showed similarity in both AFLP and SSR profiles. Based on these findings, it can be concluded that observed changes in DNA profiles of seeds from different aged groups did not contribute to accumulation of genetic variations of the same magnitude. Therefore, seed of similar viability must be selected for molecular marker analysis for plant variety protection, among other comparative studies.     

菲律宾蛤仔基因组DNA的提取及其RAPD分析

对菲律宾蛤仔(Ruditapes philippinarum)的三种组织,即性腺、斧足和内脏团的基因组DNA提取方法进行了研究,并对所提取的DNA作RAPD分析。结果表明性腺的DNA的得率明显比斧足和内脏团高。并且探讨了用于对基因组DNA进行RAPD研究的4种引物及最佳PCR条件,得到了较好的RAPD结果。     

白菜的EST标记及其对油菜的通用性

根据白菜的表达序列标签,设计了28对引物。在对引物、dNTP、MgCl2的浓度及退火温度等参数进行测试后,建立了合适的PCR反应体系。在此反应体系下,以构建EST的白菜自交系A的DNA为模板,对设计的引物进行了筛选,发现有18对引物能对白菜DNA扩增出产物。用筛选出来的引物分别对17个白菜类品种进行PCR扩增,用琼脂糖凝胶电泳分析其产物的多态性,发现10对引物有多态性,这占了筛选引物的55.6%。为检测白菜EST标记的通用性,进一步利用设计的引物对不同油菜品种的DNA进行PCR扩增。在检测的28对引物中,共有24对引物能扩增出产物,占引物总数的85.7%,显示多态性的引物为18对,占引物总数的64.3%.。在对白菜DNA能扩增出产物的18对引物中,对油菜完全可用,且有13对引物产生多态性。而在那些对白菜未扩增出产物的10对引物中,也有6对能扩增出产物,其中5对显示多态性。文章研究结果证明,通过EST建立分子标记是可行的,而且这种标记对近缘物种是可通用的。