植物总基因组DNA提取纯化方法综述

归纳植物DNA提取各个环节,同时对DNA提取过程中的常见问题、原因及应对策略作简要的分析。

水稻花粉管通道法导入含Xa21总基因的研究简报

采用花粉管通道法，将含Ｘａ２１基因（广谱抗白叶枯病基因）的水稻（ｏｒｙｚａｓａｚｔｉｖａ）品种１１８８的总ＤＮＡ及ＰＢＩ１２１质粒ＤＮＡ导入丙１０６７、宁６７、繁７、宁波２号，２９３，浙７３３和舟９０３等七个品种（系）收获转基因的当代水稻种子并进行大田选育，将外源ＤＮＡ含有ＰＢＩ１２１的转化处理后代种子发芽，在１４天苗龄时分别提取水稻幼苗总ＤＮＡ而后经多种限制性内切酶酶切。

大鼠总基因甲基化状态改变相关危险因素的研究

目的:探讨高胆固醇血症(Hypercholesterolemia,HTC)对wistar大鼠主动脉基因组DNA总甲基化水平及总甲基转移酶活力的影响并比较高同型半胱氨酸血症(Hyperhomocysteinemia,HHCY)和HTC在影响主动脉基因组DNA总甲基化水平、总甲基转移酶活力之间的差异。方法:将wistar大鼠33只,随机分3组:对照组、蛋氨酸组、高胆固醇组,每组11只。对照组给予普通大鼠饲料,其余各组给予相应的配方饲料。持续喂养3个月后心脏取血检测血清同型半胱氨酸(Homocysteine,Hcy)、总胆固醇(Total cholesterol,TC)等相关指标。提取主动脉基因组DNA检测基因组DNA总甲基化水平、提取主动脉核蛋白检测基因组DNA总甲基转移酶活力。结果:经多个样本均数间的多重比较(Dunnett-t检验):高胆固醇组大鼠的血清TC水平明显高于对照组和蛋氨酸组,且差异有统计学意义(P<0.05),血清中甘油三酯(Triglyceride,TG)、低密度脂蛋白胆固醇(Low density lipoprotein-cholesterol,LDL-C)和高密度脂蛋白胆固醇(High density lipoprotein-cholesterol,HDL-C),差异无统计学意义(P>0.05)。蛋氨酸组大鼠血清Hcy水平明显高于对照组及高胆固醇组,且差异有统计学意义(P<0.05)。HHCY和HTC均增加基因组DNA总甲基转移酶活力,可促使基因组DNA去甲基化,与对照组相比,各组均具有统计学意义(P<0.05),但两组之间差异无显著性。结论:HTC降低大鼠主动脉基因组DNA总甲基化水平可能是HTC导致动脉粥样硬化发病重要机制之一,且HHCY和HTC在影响基因组DNA低甲基化之间的差异无显著。

短序十大功劳基因组总DNA提取方法研究

以短序大功劳嫩叶为材料，采用CTAB法、CTAB改良法1、CTAB改良法2、SDS法和试剂盒法五种方法提取短序十大功劳基因组总DNA，用分光光度计和琼脂糖凝胶电泳方法检测所得总DNA的纯度和得率，用ISSR-PCR扩增的方法检测所得总DNA的质量。结果表明，五种方法均能从短序大功劳叶片中提取到基因组DNA，但不同方法提取得的基因组DNA的纯度、浓度和得率存在明显的差异。CTAB改良法2和试剂盒法提取的DNA纯度高，可直接用于下游分子生物学实验，CTAB法、CTAB改良法1和SDS法提取的总DNA质量较差，不利于下游的分子生物学实验；五种方法提取的总DNA的得率在10．836-451．709μg/g之间，呈CTAB法〉SDS法〉CTAB改良法1〉CTAB改良法2〉试剂盒法的现象。此实验获得的结果可以为短序十大功劳分子生物学研究提供基础。

皱边石杉基因组总DNA保存期间的质量检测分析

目的:用CTAB法提取皱边石杉叶片的基因组总DNA,对DNA储存于4℃的TE缓冲溶液与65℃水浴促溶2种实验处理进行为期30d的稳定性对比实验。方法:对比分析保存于4℃的TE缓冲溶液中与65℃进行2～3次水浴促进DNA溶解释放处理的DNA的稳定性及完整性变化。结果:保存于4℃TE缓冲溶液中的DNA样品非常稳定;65℃水浴促进了DNA从包裹的多糖中溶解释放,但2～3次水浴使大片段的DNA降解。结论:DNA保存在TE缓冲溶液中非常稳定;65℃水浴尽管可提高DNA的提取量,但会使DNA发生降解。因此,一般情况下对DNA不要进行水浴促溶。

黄山市几种资源植物基因组总DNA提取方法的初探

为了能在分子生物学基础上进行资源保护和提高综合开发利用价值,对黄山市几种资源植物进行了初步的分子生物学研究。通过3种不同方法(普通CTAB法、改良CTAB法和SDS法)对黄山贡菊、艾草、玉叶金花、柳叶蜡梅和秤锤树这5种黄山市资源植物,进行叶片基因组总DNA提取方法的研究,并用紫外分光光度法和1%琼脂糖凝胶电泳法分别检测所得产物DNA的纯度和完整性。结果表明:对于黄山贡菊、艾草和玉叶金花基因组总DNA的提取采用改良CTAB法所得提取效果最佳;对于柳叶蜡梅以普通CTAB法提取效果最佳;对于秤锤树以SDS法提取效果最佳。琼脂糖凝胶电泳结果显示完整度较好,能够用于进一步的分子生物学研究。

植物耐盐性:演化和盐基因组学

由于社会的发展需要使用盐地产出植物,因此植物耐盐性的研究变得日益重要。植物在长期演化中主要形成嗜盐植物和非嗜盐植物两类,在细胞乃至群体处理盐的机制方面发生了广泛和深度的遗传变异。鉴于发掘植物耐盐、使植物耐盐和改变植物盐环境的各领域都取得了重大进展,结合植物的演化扩展对植物耐盐的认识,从细胞机制、个体机制扩展到群体机制乃至多基因组参与的生态互作,进行以盐基因组学为视角的研究就显得非常重要。本文根据该领域的新近进展,对植物耐盐性进行演化和组学视角的回顾和分析,以期对今后的植物耐盐研究提供参考。

PCR检测牛乳中大肠杆菌基因组DNA的提取方法

为获得一种适于牛乳样品大肠杆菌PCR检测的基因组DNA提取方法,对饱和酚法、CTAB法、试剂盒法及溶剂裂解法等4种提取方法加以比较,通过考察DNA的纯度、定量分析以及PCR分析,确定一套有效、快速、适合牛乳中提取大肠杆菌的基因组DNA方法——溶剂热裂解法。结果显示该方法提取的DNA模板,适于PCR扩增大肠杆菌DNA,可以用于牛乳中的大肠杆菌检测。

正交试验法优化芍药基因组DNA CTAB法提取工艺

[目的]为芍药的杂交育种提供试验依据。[方法]通过正交试验,对利用芍药基因组DNA的CATB法提取工艺进行优化。[结果]CTAB法提取DNA的OD260/OD280为1.8-1.9,不同处理间差异较明显。除泳道c7电泳结果较差外,其他泳道DNA的完整性都相对较好。除c1c、2泳道RNA拖尾现象较严重外,c3、c4、c5c、6泳道脱尾现象均较轻微,而c8和c9电泳条带较为整齐。CTAB法提取芍药基因组DNA的最佳配方为：CTAB 0.75 g、NaCl 3 g、EDTA 2.5 mlT、ris-HCl 5 ml,pH值8.0。[结论]利用该CATB方法,可获得高纯度芍药DNA。

微生物分子生态学技术及其在食品产业中的应用前景

综述了新兴的不依赖于培养的微生物分子生态学技术,包括两类:不基于微生物总基因组DNA的分析方法,主要有单碳源利用图谱法和磷脂脂肪酸图谱法;基于微生物总基因组DNA的分子分析方法,主要有克隆文库分析法、宏基因组文库技术、G+C含量分析法、总DNA复性动力学分析法、群落水平总基因组DNA交互杂交法、荧光原位杂交技术、DNA微阵列芯片技术、变性/温度梯度凝胶电泳、单链构象多态性分析、限制片段长度多态性/扩增核糖体DNA限制性分析、末端标记限制片段长度多态性、核糖体基因间隔区分析、随机扩增多态性DNA等。并提出了这套技术在食品产业中的应用前景:发现新菌种,生产新型酶制剂,优化改造新工艺,确保食品质量与安全等,为食品产业的继续开发提供新的技术支持。

食管癌与微小RNA关系研究进展

食管癌（EC）是世界恶性肿瘤和癌症死亡的常见原因[1]。尽管EC的诊断和治疗已得到快速发展,但其平均5年生存率仅保持在10%~20%[2]。评估EC预后和建立合理的治疗方案显得十分重要。因此,有必要找到食管癌的肿瘤分子标志物。人类基因组计划结束后,人们发现编码蛋白质的基因只占总基因组的2%。

动物宿主遗传背景与肠道微生物关系研究进展

随着新一代高通量测序技术的飞速发展,采用宏基因组测序技术广泛和深入地了解环境微生物的组成、分布和功能等成为可能。截至目前,包括人类在内的多种动物的肠道微生物研究已经贯穿于宿主疾病、营养、代谢、调控、遗传、行为等多个方向。尽管如此,动物肠道微生物的属性,特别是其与宿主和外部环境间的关系还不是十分清楚。文章从基因组、单基因和关联分析层面介绍了动物宿主遗传背景同肠道微生物间关系的研究进展,阐释了肠道微生物的数量性状特性及遗传力估计结果,并提出肠道微生物研究中存在的问题和未来展望,为动物宿主和肠道微生物互作机制研究提供一定的借鉴和参考。

螺旋粉虱成虫体内细菌群落多样性的PCR-DGGE和16S rRNA文库序列分析

为了解螺旋粉虱Aleurodicus dispersus体内细菌多样性和主要优势菌群结构,用PCR-DGGE和16S rRNA文库对采自于海南省番石榴上螺旋粉虱雌、雄成虫体内的细菌群落进行了分析。用PCR扩增体内细菌16S rRNA基因,构建雌、雄虫克隆文库;再用限制性片段长度多态性(restriction fragment length polymorphism,RFLP)方法从文库中筛选不同16S rRNA基因图谱,根据图谱对克隆子进行分型。从螺旋粉虱雌、雄两个样品中共获得10种分类操作单元(operational taxonomic unit,OTUs)。以16S rRNA基因为基础构建系统发育树,系统发育分析表明,螺旋粉虱雌、雄成虫体内优势菌群主要为发酵菌属Zymobacter,杀雄菌属Arsenophonus,泛菌属Pantoea和假单胞菌属Pseudomonas。Candidatus Portiera aleyrodidarum和Arsenophonus sp.可能为其体内共生菌群,在所有样品中均可稳定地检测到。这些微生物可能对螺旋粉虱生长发育、繁殖和性比调控起到重要的协同作用。

DNA甲基化与人肝细胞癌的研究现状

作为转录水平的DNA修饰方式之一,DNA甲基化(DNA methylation)在调节基因表达及维持细胞正常分化中起着重要的作用。正常情况下人的细胞内70％～80％的胞嘧啶-鸟嘌呤碱基对的胞嘧啶(cytosine, C)处于甲基化状态,其是维持细胞稳定性的重要因素。其水平下降或模式改变可导致基因结构和功能的异常,是细胞癌变中重要的一步。

Study on HSP70 Gene Expression in Different Tissue of Cyprinus carpio

[ Objective] The aim of this study was to investigate whether HSP70 can be used as a stress monitoring indicator in Cypnnus carpio breeding. [Method] Based on HSP70 sequence of Cyprinus carpio （AY120894）, one pair of primers was designed and synthesized, while the total RNA of liver tissues in Cyprinus carpio was extracted. Some cDNA fragments of Cyprinus carpio HSP70 were cloned by RT-PCR, and its differential expression in various tissues such as heart, intestine, mucus, gonad, swim bladder, gill and fin in Cyprinus carpio was also studied. [Result] The cDNA sequence of 480 bp was obtained from Cypdnus carpio HSPTO gene by RT-PCR amplification. Homology comparison between the deduced amino acid sequence after sequencing and that of other types of fish showed that the homology among Cyprinus carpio, Danio rerio, Ohcorhynehus mylciss, Paralichthys olivaceus, Xiphophoorus maculates and Carassius auratus was 96%, 98%, 98%, 96%, 98% and 96% respectively. The expression of HSP70 was detected in eight tissues of Cypnnus carpio. The expression was the highest in heart, followed by swim bladder and fin, but there was no significant difference between them （ P 〉 0.05 ）. There was no significant difference among the ex- pression in three tissues of intestine, mucus and fat （ P〉0.05）, but their expression was significantly higher than those in gonad and gill （ P〈 0.05）. [ Conclusion] HSPTO gene expression is a suitable criterion for monitoring the stress degree, stress capacity and healthy conditions in Cyprinus carpio breeding.

Analysis on Interaction between Genotype of Four Main Flavonoids of Barley Grain and Environment

[Objective] This study aimed to analyze the interaction between genotype of flavonoids of barley grain and environment, to increase the flavonoid content of barley grain in cultivation and breeding. [Method] In this study, the content of cate- chin, myricetin, quercetin and kaempferol of barley grain planted in Kunming, Qujing and Baoshan were determined by HPLC, and the genotype, environment, genotype- environment interaction of the flavonoid content of barley grain were analyzed. [Result] According to the experimental results, the genotype variance, environmental variance and G x E interaction variance of catechin and kaempferol contents show the same trend： genotype variation 〉 environmental variation 〉 G × E interaction variation, which all reach a extremely significant level; the genotype variance, envi- ronmental variance and G × E interaction variance of quercetin and total flavonoid contents show the same trend： genetype variation 〉 G × E interaction variation 〉 environmental variation, which all reach a extremely significant level; the genotype variance and environmental variance of myricetin content both reach a extremely sig- nificant level, while the G × E interaction variance reaches a significant level, showing an order of genotype variation 〉 environmental variation 〉 G × E interaction variation; the genotype variance, environmental variance and G x E interaction vari- ance of total flavonoid content show an order of genotype variation 〉 environmental variation 〉 G × E interaction variation. Among different barley varieties, Ziguang- mangluoerling and Kuanyingdamai in Qujing, Kunming and Baoshan have relatively high content of quercetin, while other barley varieties barely contain any quercetin. The grains of Ziguangmangluoerling and Kuanyingdamai are purple, while the grains of other barley varieties are yellow. [Conclusion] Four main flavonoids and the total flavonoids of barley grain are mainly under genetic control and affected by genetic- environment interactions; the purple barley grains contain high content of quercetin.

The Effects of Apolipoprotein E Polymorphism on Serum Lipids, Lipoproteins and Apolipoproteins Variation

Objective: To study the effects of Apolipoprotein E (ApoE) polymorphism onserum levels of lipids, lipoproteins and apolipoproteins. Methods: Fragments of ApoE gene forthex-on containing codon 112 and 158 polymorphic locus were amplified by PCR, and then digested untilCfo I endonuclease. Genotypes and alleles frequencies of 168 healthy persons in Jiangsu area werecalculated. The effects of ApoE genotypes and alleles on serum lipids, lipoproteins andapolipoproteins variation were analyzed. Results: The effects of ApoE alleles on total cholesterol(TC), law density lipoprotein-cholesterol (LDL-C), ApoB was: along a decreasing gradientε_4>ε_3>ε_2. The effect of ε_4 allele was to increase serum levels of TC, LDL-C and ApoB, andthe ε_2 allele had an effect opposite to that of ε_4 allele. Conclusion: ApoE polymorphism is anindependent genetic factor on individual serum levels of lipids and apolipoproteins.

The mitochondrial genomes of Macrocheraia grandis grandis and Myrmoplasta mira(Hemiptera:Heteroptera:Pentatomomorpha) and the unique mitogenome rearrangement in Pyrrhocoroidea

Sequencing technology has developed rapidly in recent years. Complete or nearly complete mitochondrial genomes(mitogenomes) of 155 species from 47 families in Heteroptera have been sequenced. However, the amounts of mitogenomes between those families are unbalanced, which makes it difficult to correctly discern the patterns of mitogenome rearrangement in Heteroptera. Among 21 species from ten families, ten variations in mitogenome rearrangement had been previously reported, among which the translocation between tRNA-Thr and tRNA-Pro was considered as a synapomorphy of Pyrrhocoroidea based on two mitogenomes. As only one mitogenome in each of Largidae and Pyrrhocoridae had been sequenced to conclude the synapomorphy, more mitogenomes of Pyrrhocoroidea need to be explored. In this study, additional two mitogenomes of Pyrrhocoroidea(Macrocheraia grandis grandis(Gray, 1832) and Myrmoplasta mira Gerst-cker, 1892) were sequenced. Both of them also possess the same translocation between tRNA-Thr and tRNA-Pro, which reaffirms that this kind of rearrangement is a molecular synapomorphy of Pyrrhocoroidea. Moreover, we discovered a more complex rearrangement in Myrmoplasta mira, in which six nearly identical duplications of tRNA-Thr were found located downstream of tRNA-Pro. Considering the high biodiversity of Heteroptera, more mitogenomic studies are needed to improve our knowledge about mitogenome rearrangements and the potential synapomorphies.

Study on polymorphism ofArtemisia capillaris by using RAPD for Terengganu population in Malaysia

Artemisia capillaris is a herbaceous aromatic and therapeutic plant. The genetic variability among individuals of Artemisia capillaris from state of Terengganu, Malaysia was examined by using the random amplified polymorphic DNA （RAPD） technique to assess the polymorphism at the species level, The samples from differences regional in Terengganu State. The genomic DNA was extracted from the samples leaves using Sarkosyl method. The results produced by the machine showed clear RAPD banding pattern. Fifty-seven oligonucleotide primers were screened and five primers were selected （OPA 04, OPA 09, OPA 16, OPA 17 and OPA 18） to amplify DNA from five samples of Artemisia capillaris from State of Terengganu, Malaysia. A total of 135 RAPD fragments （RAPDs） with all polymorphic fragments （100%） with size ranging from 250--3000 bp were scored from the population. Genetic distance for samples ranges from 0.0000 to 0.320000. For similarity index samples ranges from 0.0000 to 0.7547.