一种棉花线粒体DNA的提取方法

线粒体是重要的细胞器,它有自身的基因组。其基因组DNA与细胞核基因组DNA相比,含量较低。棉花当中富含棉酚、丹宁等物质,这对提取DNA有很大的影响。因此我们根据棉花自身的特点,找到了一种提取棉花线粒体DNA经济有效的方法,其质量可以满足限制性酶切、PCR、分子杂交等实验的要求。     

一种改进的动物线粒体DNA提取方法

线粒体DNA(mtDNA)已被广泛用于动物群体遗传学和进化生物学的研究,并取得了许多有意义的结果。有效的mtDNA提取方法无疑是开展这方面研究的前提。关于动物mtDNA的提取方法,国内外已有不少报导。概括起来,可分为:1)氯化铯超速离心法,2)柱层析法,3)DNase法,4)碱变性法。本文报道了一种改进的碱变性提取法,与其它方法相比,具有应用范围广、简便、经济等优点。     

一种改进的禽类线粒体DNA提取方法

介绍一种碱变性法从禽类脏器中提取大量线粒体DNA,比较了从不同脏器提取mtDNA的难易程度和得率。结果表明：肝脏、脾脏的匀桨操作易于心脏,且肝脏的得率高于心脏,更高于脾脏;同时对影响mtDNA产量和质量的匀桨速度和次数,差速离心速度,溶液Ⅱ中SDS含量及溶液pH值等因素进行了初步分析和探讨。在借鉴前人研究基础上对禽类脏器mtDNA提取方法上进行了改进和优化,建立了一种从禽类脏器中有效制备mtDNA的方法。结果表明,这种方法得到的mtDNA可以满足限制性酶切实验的要求。     

动物线粒体DNA提取简易流程及其优化

目的:研究动物线粒体DNA(mtDNA)提取的简易流程,以提取到纯度高、结构完整的动物mtDNA。方法:采用SDS碱变性法,从动物的肝脏和性腺等组织中提取mtDNA,并增加了DNaseⅠ、RNase消化步骤,以除去核DNA及RNA的污染;用紫外分光光度计分析mtDNA的纯度和得率。结果:mtDNA的得率为0.5～0.8μg/g,D260nm/D280nm值为1.77～1.82,能满足对mtDNA进行RFLP分析、RAPD分析、测序分析等的要求。结论:改进的动物mtDNA提取方法简便可行,值得推广。     

南方鲇肝脏线粒体DNA的简便分离纯化方法

摸索出可消除南方鲇肝脏ｍｔＤＮＡ制备中出现的白色干扰物的方法,采用该方法处理后的ｍｔＤＮＡ可用于限制性分析。     

线粒体DNA提取方法的比较

目的：将提取线粒体DNA的碱变性法、Triton法、改进高盐沉淀法加以比较,以得到最方便快速提取线粒体DNA的方法。方法：分离Wistar大鼠小肠上皮细胞,用3种方法提取线粒体DNA,紫外分光光度法定量。用琼脂糖凝胶电泳和线粒体ATPase 8亚基基因PCR扩增产物鉴定所提取的线粒体DNA。结果：改进高盐沉淀法线粒体DNA量最多,Triton法最少。OD260／OD280均在1．78-l.85间。将改进高盐沉淀法提取线粒体DNA用于PCR扩增,测定出了线粒体DNA ATPase 8亚基基因序列。结论：改进高盐沉淀法提取线粒体DNA具有操作简单,产量多的优点,该法所提取mtDNA可用于mtDNA测序。     

一种从毛发中提取DNA 的简易方法

用PCR 缓冲液及蛋白酶K 在PCR仪上对单支毛囊进行消化,　可获得用于PCR反应的足量的DNA。还对几种从毛发中提取DNA的不同方法进行了比较,并对从毛干中提取线粒体DNA进行了讨论。     

提取线粒体DNA方法的比较及较纯线粒体基因获取方法的确立

目的:对2种常用的线粒体DNA提取方法进行比较,分析提取物中核DNA的存在情况,同时建立新检测方法降低线粒体假基因干扰。方法:以仅在核DNA中存在的基因β-actin作为核DNA存在的标定基因,通过PCR方法扩增线粒体DNA上的一段基因MTND5-2,并以β-actin做参比,比较常用的2种提取线粒体DNA方法的优劣,即碱法Ⅰ(先提取完整线粒体后从中获得线粒体DNA)和碱法Ⅱ(根据线粒体DNA与核DNA的结构差异,从中获得双链环状的线粒体DNA)。结果:2种线粒体DNA提取方法并不能获得仅含线粒体DNA的纯提取物,碱法Ⅰ获得的线粒体DNA纯度相对较高;以碱法Ⅰ提取物为模板进行PCR,可获得更多较纯的线粒体目的基因。结论:碱法Ⅰ较碱法Ⅱ可获得更纯的线粒体来源的目的基因;新建方法可获得较纯的线粒体基因,且是一种简单、方便、经济的方法。     

一种改良的植物DNA提取方法

植物组织中含有大量多糖、多酚、酯类等次生代谢产物, 要从中提取高质量的DNA比较困难。针对这一情况, 该文提出一种改良CTAB植物DNA提取方法(mCTAB), 并以10种常见植物为实验材料, 与4种常用的植物DNA提取试剂盒作对比。结果表明, mCTAB法提取的DNA产率高且质量好, PCR扩增成功率也较高, 而提取成本显著低于DNA提取试剂盒, 可有效用于植物DNA条形码等研究的植物DNA提取。     

一种提取质粒DNA的改良方法

本文详细介绍了一种改良碱裂解法提取质粒ＤＮＡ的方法,该法采用ＮＨ４Ａｃ代替苯酚和氯份的抽提过程,得率高,质量好,完全达到了分子生物学常规实验的要求,如酶切、连接、转化大肠杆菌、ＰＣＲ等,甚至用于序列测定和植物遗传转化,该法重复性好,操作简单、实用．     

大豆重复序列的克隆,特性分析及在染色体上的定位

从大豆栽培品种Ｕｎｉｏｎ（Ｇ．ｍａｘ）基因组ｐＵＣ１８质粒文库中,以基因组ＤＮＡ为探针,筛选出一个重复序列家族。序列分析表明,此重复序列的重复单位为９１ｂｐ,拷贝数约为１０￣５,其序列约占基因组ＤＮＡ的０．９％。基因组ＤＮＡ不同限制酶片段Ｓｏｕｔｈｅｒｎ杂交分析和染色体原位杂交分析表明此重复序列主要以串联方式集中分布在Ｍ２和Ｍ１１号染色体的臂上,而另外一些则散布于整个Ｍ１２和Ｓｍ７号染色体上。以该序列为探针片大豆属不同亚属１３个种的１８个品系的Ｓｏｕｔｈｅｒｎ杂交结果表明,此重复序列为Ｓｏｊａ亚属所特有。这一Ｓｏｉａ亚属特异重复序列的发现,从另一个角度支持应把Ｓｏｊａ亚属的３个种Ｇ．ｓｏｊａ、Ｇ．ｇｒａｃｉｌｌｉｓ、Ｇ．ｍａｘ划分为一个种的观点。     

Effects of temperature on germination and mitochondrial dehydrogenases in two soybean (Glycine max) cultivars

The effects of eight germination temperatures from 10°C to 35°C on germination and dehydrogenase activities of two soybean (Glycine max [L.] Merr.) cultivars were investigated after 48 h of seedling growth. Axis fresh weights of cv. Chippewa increased as germination temperature increased from 10°C to 35°C. In contrast, axis fresh weights for the cv. Wells increased more slowly with increasing temperature and reached a maximum at c. 25°C. In general, in vitro activities of glutamate dehydrogenase (GDH), NADP-isocitrate dehydrogenase (NADP-ICDH), and malate dehydrogenase (MDH) from the axes of cv. Chippewa correlated well with increases in axis fresh weights. GDH and MDH activities from axes of the cv. Wells also reflected increases in axis fresh weights although the correlation was not as evident as for the cv. Chippewa. NADP-ICDH activity from ‘Wells’ axes was highest at 35°C even though germination was poor at this temperature. GDH and MDH activities from cotyledons of both cultivars were not correlated with axis weight increases. No GDH activity was detected in ‘Wells’ cotyledons from seeds germinated at 35°C.     

NaCl胁迫下野生和栽培大豆幼苗体内离子的再转运

采用NaCl根际处理和叶面饲喂^22Na方法,研究了野生大豆(Glycine soja)——耐盐的BB52、盐敏感的N23232和栽培大豆(Glycine max)——较耐盐的Lee68幼苗在盐胁迫及解除过程中对Na^ 、Cl^-的吸收和再转运。结果表明,在NaCl根际处理12h过程中,BB52和Lee68幼苗根对Na^ 、Cl^-吸收和向茎、叶的运输逐渐增加,10h时趋于稳定,Na^ 、Cl^-含量高低顺序是根＞茎＞叶。但N23232的Na^ 、Cl^-含量则是茎＞根＞叶。在用NaCl对根处理10h后再解除NaCl处理的0～36h内,BB52吸收的Na^ 、Cl^-较多地留于根部或转运至根茎过渡区,叶中较少。N23232吸收的Na^ 较多地转运至茎部,而Cl^-含量在幼苗各部分无差异。叶片饲喂^22Na 10h后,BB52吸收^22Na较N23232多,并较多地向根部运输。从离子再转运角度讨论了BB52的耐盐性。     

高质量提取大豆叶片总RNA的改良方法

豆科植物总RNA的提取相对比较困难,大豆叶片富含多糖、蛋白质,传统方法提取效果均不理想.在总RNA的提取方法Trizol法和热酚-氯化锂沉淀法的基础上,针对大豆叶片的特殊性,做了适当的改良,从而获得了完整性好,高纯度的大豆叶片总RNA,并成功进行了基因片段的RT-PCR实验,结果证实所获得的RNA完全可以适应后续实验要求.     

Coumarin effects on Glycine max hypocotyl explants

Coumarin induced root formation and stimulated fresh weight production in hypocotyl explants of Glycine max L. cultured in vitro. All stimulatory effects caused by coumarin were induced within a relatively narrow range of concentrations between 1–500 μM, yielding optimum dose response curves. When coumarin was combined with kinetin fresh weight increased considerably, at optimum concentrations to a level almost as high as that obtained with NAA (10 μM) and kinetin (10 μM). Root formation was almost completely inhibited when kinetin was added in combination with coumarin. NAA + coumarin had small stimulatory effects on fresh weight. but were inhibitory in root formation. The frequency of rooting per explant, texture and pigmentation were also affected by different treatments.     

毛豆中腈菌唑残留量的气相色谱法测定

采用气相色谱法定量分析毛豆上腈菌唑的微量残留量。样品经乙腈提取,液液分配,中性氧化铝柱层析净化后,以气相色谱电子俘获检测器法(GC-ECD)测定,DB-1701毛细管柱、氮气为载气,柱温150℃20℃/min 260℃(10 min),气化室温度240℃,检测器温度300℃,外标法定量。该方法快速、准确,在0.05~2.00 mg/L范围内线性相关系数r²=0.9998,平均回收率91.1%~99.0%,变异系数1.22%~2.94%,最小检测量1.0×10^-12 g,最低检出浓度5.0×10^-4 mg/kg。     

缺铁敏感度不同的大豆品种对缺铁的适应机制

与供铁处理相比,对缺铁敏感的大豆品种“哈８３”幼苗在缺铁胁迫条件下根际没有酸化现象,根系对Ｆｅ（Ⅲ）的还原能力也没有明显增强。但抗缺铁的大豆品种“８７０１”幼苗根际则严重酸化,根系对Ｆｅ（Ⅲ）的还原能力显著增强;加入能抑制根系Ｈ＋－ＡＴＰ酶活性、减弱根际酸化作用的Ｈ＋－ＡＴＰ酶抑制剂正钒酸钠会降低根系对Ｆｅ（Ⅲ）的还原能力;说明根际酸化与根系还原Ｆｅ（Ⅲ）能力相互联系,初步证实根细胞原生质膜Ｈ＋－ＡＴＰ酶和缺铁诱导的还原酶相互偶联的假说。     

Plant Regeneration from Protoplasts of Soybean (Glycine max L.)

Protoplasts were isolated enzymatically from immature cotyledons of soybean. The protoplasts divided to form calli in the K8P liquid medium. The calli further grew to 2–3 mm on the solid K8 medium and were transferred onto the MSB medium (MS minerals+B5 organic components+0.5–1.0 mg/l 2,4-D+0.2–0.5 mg/l BA) to obtain compact and nodular calli. Shoot formation was initiated on M1 medium (MSB medium with 0.15 mg/1 NAA, and BA, KT and ZT, 0.5 mg/l of each, 500 mg/1 CH). Differentiation frequency was 13.6–24.2%. Plants have been regenerated from protoplasts of immature cotyledons in 2 cultivars, and normal pods were obtained from them.     

Composite and clinal distribution of Glycine soja in Japan revealed by RFLP analysis of mitochondrial DNA

 Wild soybean (Glycine soja Sieb. et Zucc.), regarded as the progenitor of cultivated soybean [G. max (L.) Merr.], is widely distributed in East Asia. We have collected 1097 G. soja plants from all over Japan and analyzed restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA) in them. Based on the RFLPs detected by gel-blot analysis, using coxII and atp6 as probes, the collected plants were divided into 18 groups. Five mtDNA types accounted for 94% of the plants examined. The geographic distribution of mtDNA types revealed that, in many regions, wild soybeans grown in Japan consisted of a mixture of plants with different types of mtDNA, occasionally even within sites. Some of the mtDNA types showed marked geographic clines among the regions. Additionally, some wild soybeans possessed mtDNA types that were identical to those widely detected in cultivated soybeans. Our results suggest that the analysis of mtDNA could resolve the maternal lineage among plants of the genus Glycine subgenus Soja. Received: 16 June 1997/Accepted: 5 August 1997     

大豆血红蛋白基因lba转化根瘤菌工程菌株的构建

以土著大豆根瘤菌接种大豆幼苗45 d后获得的根瘤为材料,提取其总RNA并反转录成cDNA,采用同源序列克隆法扩增大豆血红蛋白基因lba编码区序列。利用DNA重组技术,将lba基因连到lac启动子的下游,利用带有发光酶标记基因luxAB的质粒载体pTR102构建表达载体pTR-Plac-lba。采用三亲本杂交的方式,将表达载体pTR-Plac-lba及作为对照的空载体pTR102分别转化土著大豆根瘤菌,获得根瘤菌工程菌株SFH(pTR-Plac-lba)和SFH(pTR102)。盆栽试验发现,接种SFH(pTR-Plac-lba)的大豆植株各生理指标明显高于接种SFH(pTR102)、土著根瘤菌以及未接菌的大豆植株各生理指标。试验证明,导入大豆血红蛋白基因lba的根瘤菌工程菌株SFH(pTR-Plac-lba)对于提高大豆根瘤的固氮酶活性,增加大豆产量起到显著效果。