陆地棉SSR分子标记优化体系的建立

针对陆地棉富含酚类、多糖等次生产物的特点,建立了一种简便、快速的提取高纯度DNA的方法.同时建立了适合陆地棉SSR标记的优化体系和试验方案,并对所构建的QTL定位群体进行了SSRPCR检测,得到了清晰的多态性SSR标记,为SSR分子标记技术在棉花育种中的应用打下了基础.     

鱼糜制品中基因组DNA提取方法的比较

将分别采用普通酚-氯仿抽提法和试剂盒(柱吸附法)提取DNA的两种方法加以比较,以确定最适提取鱼糜制品DNA的方法.将鱼肉和鱼糜制品作为原料,用两种方法提取基因组DNA,利用分光光度计测定其A260与A280的吸光值,计算比率估计核酸的纯度,并利用琼脂糖凝胶电泳观察DNA片段在凝胶中的位置;并用线粒体16S rRNA基因PCR扩增产物鉴定所提取的基因组DNA.     

多子领春木RAPD反应体系的研究

利用RAPD(随机扩增多态性DNA)技术对濒危植物多子领春木进行了RAPD反应体系的研究.以多子领春木叶片为材料,采用改良的CTAB法提取其基因组DNA,进行RAPD反应条件的优化.通过单因子试验,分别研究了模板DNA用量、Mg2 浓度、dNTPs浓度、Taq酶的用量和引物浓度对RAPD反应的影响,建立了适于多子领春木RAPD分析的有效稳定的反应体系,即在25μL总反应体系中,模板DNA的最适用量为10 ng、Mg2 的适宜浓度为2.0 mmol·L-1、dNTPs的适宜浓度2.2 mmol·L-1、Taq酶的用量为1U、随机引物的浓度以2.5 μmol·L-1为佳.     

应用彗星试验研究产毒微囊藻喂食暴露对铜锈环棱螺肝细胞DNA的损伤

把铜锈环棱螺（Bellamya aeruginosa）暴露于3组不同成分的微藻悬浮液中[蓝藻组：只投喂产毒铜绿微囊藻（Microcystis aeruginosa）;混合藻组：50%四尾柵藻（Scenedesmus quadricanda）＋50%产毒铜绿微囊藻;对照组：只投喂四尾柵藻],用酶联免疫检测法（Enzyme-linked immuno sorbent assay,ELISA）检测藻液和螺肝组织中的藻毒素浓度。结果表明,藻液中包括藻相和水相的总微囊藻毒素（MCs）浓度分别为：蓝藻组（36.34±4.12）μg·L-1;混合藻组（18.69±2.12）μg·L-1;对照组未检出。在喂食暴露的前6h内,混合藻组和蓝藻组螺肝组织中微囊藻毒素含量持续增长,而后出现下降趋势;第12h更换藻液并将微藻浓度调整至初始值后,肝组织中MCs含量又迅速回升。同期螺肝细胞DNA损伤指标彗星尾长（TL）、彗星尾距（TM）和彗星尾部DNA百分含量（Tail DNA%）也随螺肝组织中MCs含量发生相应变化,各项DNA损伤指标均在产毒微囊藻喂食暴露6h时达到最大值,之后,各项指标值有所回落,但第12h更换藻液后,DNA损伤再次加剧。整个实验期间（24h）,混合藻组和蓝藻组的DNA损伤指标均显著高于对照组,混合藻组均显著高于蓝藻组。说明铜锈环棱螺经产毒微囊藻喂食暴露后,其肝组织细胞的DNA受到损伤,且螺肝组织MCs积累越多,DNA损伤越严重。     

瑞氏木霉碳源阻遏相关基因Cre1的分子改造

为了提高瑞氏木霉（Trichoderma reesei）纤维素酶的活力,用类似基因改组的方法改造其碳源阻遏相关基因cre1。以瑞氏木霉基因组DNA为模板PCR扩增cre1基因,用DNaseⅠ消化cre1基因后,回收50-100 bp的DNA片段,用T4 DNA连接酶连接,以连接产物作为模板进行无引物PCR,并将PCR产物转入瑞氏木霉原生质体,通过测定滤纸酶活的方法筛选突变菌株,并在NCBI上比对分析突变菌株的cre1基因。结果表明,筛选获得1株纤维素滤纸酶活比出发菌株提高0.7倍的突变菌株cre2-3。cre2-3菌株在液体培养基中呈棉花状,而出发菌株呈小颗粒状,菌株cre2-3发酵液的颜色比出发菌株的更黄亮。推测cre1基因与瑞氏木霉菌株的生长代谢有关。     

胨冻样芽孢杆菌(Bacillus mucilaginosus YNUCC0001)絮凝剂生产、絮凝特性及其系统发育学分析

对BacillusmucilaginosusYNUCC000116SrDNA的1481bp片段与GenBank中最相似的16个分类单位进行了比较。UPGMA,NJ,ME和MP方法构建的系统发育树显示B.mucilaginosusYNUCC0001与B.mucilaginosusHSCC1605T、B.mucilaginosus1480D及Paenibacillussp.NBT形成一个单系群分支。在50L全自动发酵罐中30℃发酵52h后,菌株YNUCC0001产生的胞外生物多聚絮凝剂(EBF)达到最大产率(粘度:3420C.P.)。在pH4.0、用量为0.25mlL-1的条件下,这种EBF对高岭土悬浊液的絮凝活性最大(99.8%);121℃高压灭菌60min后絮凝活性维持在98.6%。Hg2+,Ca2+,Mg2+,K+,Zn2+对其絮凝活性有促进作用,而Fe3+、Al3+、EDTA和Cu2+则有强烈抑制。     

猪瘟DNA疫苗的临床免疫实验

在前期研究工作筛选出2种DNA疫苗质粒的基础上,采用3种免疫策略(DNA疫苗单独免疫法、DNA疫苗初免蛋白免疫加强的prime-boost方法以及沙门氏菌(Salmonella)载体介导法),于某规模化猪场对猪瘟病毒(CSFV)的DNA免疫效果进行了临床应用研究。随机选择未经猪瘟疫苗免疫的断奶仔猪60头,分6组进行免疫接种,检测免疫猪抗体产生情况并对各组免疫猪进行攻毒试验。免疫猪血清抗体ELISA检测结果表明,pcDSW组、pIRE2IL2组、pcDSW E2protein组和pIRE2IL2 E2protein组均产生了较高的抗体水平,而沙门氏菌介导的2种DNA疫苗质粒诱导的抗体水平不显著。攻毒试验结果表明,pcDSW和pIRE2IL2单独免疫组或prime-boost组能够抵抗致死剂量的CSFV石门强毒攻击,保护率87.50%￣90.00%。其中pcDSW效果好于pIRE2IL2。而沙门氏菌介导的DNA疫苗组不能产生有效保护。不同接种途径的免疫效果比较结果表明,颈部肌肉注射免疫后产生的抗体水平较后肢胫前肌注射免疫产生的抗体水平高。     

Measurement of genetic dissimilarity in fieldpea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.) genotypes using RAPD markers

The present investigation was carried out on fifteen germplasm lines of Pisum sativum L. were used for characterization using Randomly Amplified Polymorphic DNA (RAPD) markers. While 12 random primers were taken, out of them 11 primers gave amplification. These primers gave a total of 133 bands out of which 106 were polymorphic. Genetic similarities of the RAPD profiles were estimated by using Jaccard’s coefficient with NTSYSpc 2.0 software. The similarity index values ranged from 0.263 to 0.793 indicating the presence of enormous genetic diversity at molecular level. A dendrogram generated by cluster analysis divided fifteen fieldpea genotypes into two Groups A and B. Major Group A have five genotypes and major Group B have nine genotypes.     

Soil colloids-bound plasmid DNA: Effect on transformation of E. coli and resistance to DNase I degradation

The adsorption and binding of plasmid p34S DNA on four different colloidal fractions from a Brown soil and clay minerals in the presence of various Ca²⁺ concentrations, the ability of bound DNA to transform competent cells of CaCl₂-treated Escherichia coli, and the resistance of bound DNA to degradation by DNase I were studied. DNA adsorption on soil colloids and clay minerals was promoted in the presence of Ca²⁺. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organo-mineral complexes (organic clays) and fine clays (<0.2 μm), DNA was tightly adsorbed by H₂O₂-treated clays (inorganic clays) and coarse clays (0.2-2 μm). The transformation efficiency of bound DNA increased with increasing concentrations of Ca²⁺ at which soil colloid or clay mineral-DNA complexes were formed. DNA bound by kaolinite showed the lowest transformation efficiency, and especially no transformants were observed with kaolinite-DNA complex prepared at 5-100 mM Ca²⁺. Compared to organic clays and fine clays, DNA bound on inorganic clays and coarse clays showed a lower capacity to transform E. coli at different Ca²⁺ concentrations. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. Montmorillonite, organic clays and fine clays showed stronger protective effects for DNA than inorganic clays and coarse clays. The protection mechanisms as well as the differences in transforming efficiency of plasmid DNA molecules bound on various soil colloidal particles are discussed. The information obtained in this study is of fundamental significance for the understanding of the horizontal dissemination of recombinant DNA and the fate of extracellular DNA in soil environments.     

富含多糖的转基因石斛基因组DNA提取方法(英文)

在石斛兰转基因研究中,需通过PCR和Southern杂交等手段检测外源基因是否转入并整合到转化植株基因组.由于转化石斛植株生长缓慢,且含有多糖,因此从转化植株中提取高质量的基因组DNA以尽快对转基因苗进行分子检测存在较大困难.本研究旨在通过改良现有的DNA提取法(CTAB法或SDS法1,克服转化石斛植株基因组DNA提取中碰到的产量低,纯度不高而导致难以进行PCR或酶切等问题.在本研究所用的3种改良法中,方法Ⅱ能从少量的转化石斛苗中提取出高产量和高纯度的基因组DNA.研究结果表明方法Ⅱ提取的基因组DNA完全适用于转基因石斛的外源基因PCR扩增,限制性酶切和Southern杂交分析.