转基因大豆MON89788检测质粒标准分子的构建与应用

[目的]构建适用于转基因大豆MON89788检测的质粒标准分子。[方法]利用定性PCR和连接、转化等分子克隆技术,将大豆内标准基因lectin、MON89788的3′端特异性序列和5′端特异性序列依次克隆到pMD18-T载体上,获得质粒标准分子pMD-LM3M5,并进行适用性验证。[结果]获得了3700bp的质粒标准分子,其中重组DNA片段1029bp。该质粒标准分子的定性PCR检测灵敏度达到10copy。[结论]该研究构建的质粒标准分子pMD-LM3M5能替代MON89788基体标准品,用于MON89788大豆及其产品的定性PCR检测。     

Persistence of an oilseed rape transgene in the environment

Transgenic bromoxynil (BX)-resistant spring oilseed rape (Brassica napus L.) was grown commercially in Canada only for two crop seasons – 2000 and 2001. We investigated the persistence of suspected BX-resistant oilseed rape volunteers in a 64-ha wheat field in Saskatchewan, Canada in 2007, 7 years after the BX-resistant cultivar BX Armor was grown. A small oilseed rape volunteer population, estimated at less than 100 plants, was observed in three main areas or patches in the field. These patches were located in low-lying areas that were too wet to plant or spray with herbicides in 2007. Viable seed was collected from 35 mature volunteers and F₁ progeny screened with BX at 280 g ai/ha in the greenhouse. Progeny of all of the volunteers were visually rated as BX-resistant; the presence of the Oxy 235 transgene in leaf tissue of progeny of all volunteers was confirmed by PCR analysis. This study has documented the longest persistence of oilseed rape volunteers in Canada. Volunteers were not observed in 2008 or 2009, because of drought conditions in spring of both years. Results support the findings from previous studies that persistence of volunteer oilseed rape populations in western Canada is generally ephemeral or transitory in the absence of seed bank immigration. However, this study shows that oilseed rape transgenes can persist in the environment for a number of years even after all cultivars with the conferred trait are removed from the market.     

魔芋叶片中4种总RNA提取方法的比较

本研究分别利用TRIzol法、异硫氰酸胍法、改良的CTAB法及试剂盒这4种方法提取魔芋叶片的总RNA,并用紫外光谱和琼脂糖凝胶电泳对各个提取效果进行鉴定。反复试验的结果表明:TRIzol法难以提取魔芋叶片总RNA,异硫氰酸胍法提取的总RNA浓度低、杂质较多且有部分降解,这两种方法不适宜用于魔芋叶片总RNA提取;试剂盒方法提取的总RNA产率和纯度较高,但稳定性差、成功率低且成本较高;改良的CTAB法利用PVP和β-巯基乙醇去除酚类物质,水饱和酚-氯仿去除蛋白质,醋酸钾去除多糖,该方法提取得到的总RNA的纯度较高,OD260/OD280值为1.893,而且效果稳定,有良好的可重复性。因此,我们认为改良的CTAB法是一种简便、快速且经济的提取魔芋叶片总RNA的方法。     

转基因产品检测方法研究进展

综述了国内外转基因生物（Genetically modified organisms,GMOs）及其产品检测方法的研究进展,并重点介绍了目前广泛应用的一些检测方法。     

杨木材性对酚醛树脂浸渍改性材的影响

采用低分子量酚醛树脂对速生杨木进行浸渍处理、干燥定型,使树脂固化制得改性材。试验表明：低分子量酚醛树脂对杨木具有较好的浸注性,改性材的密度和力学强度均有明显的提高,两组试件改性后浸注性和密度相差不大,但前期经过热水处理的试件改性后密度相对均匀,力学强度略好于直接浸渍的试件。杨木自身材性对改性后物理力学性能的影响各不相同,在实际生产中要根据指标的重要性程度选取最佳方案。     

Cold storage of oil rose (Rosa damascena Mill.) flowers

Oil rose flowers were stored at 0 °C in four different packaging materials [plastic box + stretch film (PB + SF), Xtend^®, Smartbag^® and polyethylene (PE)] for 60 days. During storage, weight loss, O₂ and CO₂ concentrations in the packages, petal color and sensorial attributes were investigated besides essential oil content and composition. Storage duration and packages had significant (p < 0.01) effects on weight loss. At the end of storage, the lowest weight loss was in PE package (1.696%) whereas the highest weight loss was in Xtend^® (10.081%). The essential oil content was significantly (p < 0.01) affected by storage duration and packages. In addition, the essential oil contents obtained from all packages for a storage period of 10 days and the essential oil contents obtained from unstored (control) petals were included in the same group. At the end of storage, the essential oil contents decreased by 91.3, 57.7, 80.0 and 64.3% in PB + SF, Xtend^®, Smartbag^® and PE packages, respectively as compared to control. In addition, storage duration and package types significantly (p < 0.01) affected petal color, O₂ and CO₂ concentrations in the packages and sensorial scores. The concentration of citronellol, a main component of rose oil, increased in all packages during storage of 10 days in comparison to the control group while it varied in other storage durations and package types. However, nerol and geraniol were lower than the control group during storage while concentrations of nonadecane, heneicosane and eicosane were higher. In conclusion, loss of oil yield and quality, due to various reasons and particularly due to fermentation in oil rose from the harvest of petals to their distillation, can be minimized with storage of petals in all package types for up to 10 days.     

玉米秸秆与废弃白菜的混合青贮品质及产沼气能力分析

为实现干玉米秸秆(dried maize straw,DMS)的长时间保质贮存,利用青贮原理将DMS与废弃白菜(cabbage waste,CW)连续混贮90 d,设置DMS单贮组和6个不同质量比例(DMS:CW=29:19、27:21、25:23、23:25、21:27和19:29)混贮组,间隔30 d分析其贮存品质,筛选适宜贮存条件,并考察秸秆贮存前后的微观结构变化和产沼气潜力。结果表明,与单贮组相比,6个混贮组的p H值均显著下降(P0.05),乳酸含量显著升高(P0.05),混贮品质优于单贮。6个混贮组贮存30 d时的p H值均为最低,乳酸含量最高,感官评价均为优级,30 d后p H值有所上升,乳酸含量急剧下降,60d时的木质素含量显著降低(P0.05),综纤维素含量显著升高(P0.05)。随着CW比例增加,混贮组p H值逐渐减小,乳酸含量逐渐增加,MEⅤ组(DMS:CW=21:27)p H值最低,乳酸含量最高,能使DMS连续贮存60 d不变质。扫描电镜结果显示,青贮发酵使干秸秆原先致密复杂的木质纤维结构开始瓦解,青贮秸秆表面有很多裂缝和孔洞,与微生物或酶的可接触面积增加。沼气发酵试验表明,MEⅤ组混贮秸秆的累积产气量略高于未贮存干秸秆,且累积甲烷产量提高了61.67%,是未贮存干秸秆的1.6倍。Modified Gompertz模型显示混贮秸秆的甲烷生成速率增加,产甲烷能力提高。总之,当DMS∶CW质量比为21∶27(含水率为73%)时DMS能连续保质贮存2月,且贮存后秸秆的甲烷产量明显提升。     

转基因抗草甘膦棉花对草甘膦的耐受性研究

为了明确转基因抗草甘膦棉花10-6001对除草剂草甘膦的耐受性,以转基因抗草甘膦棉花材料10-6001和常规棉花品种冀棉958（ CK）为试验材料,在棉苗6片真叶时叶面喷施不同浓度〔0（不喷施除草剂,人工除草, CK）、0.25％和0.50％〕的草甘膦溶液,分别于施药后0（当天下午）、1、7、14和21 d调查棉花的株高、药害级别、受害指数和死苗率,施药后35 d 调查棉花的死苗情况;分析2种棉花对不同浓度草甘膦的反应程度,并对其株高差异进行了显著性方差分析。结果表明：转基因抗草甘膦棉花品系10-6001对草甘膦的耐受性很好,喷施0.25％和0.50％草甘膦溶液的棉花在整个试验期间药害均较轻,表现为植株矮化,生长减慢,叶片略变小,且无死苗,株高方差分析结果显示与人工除草（ CK）相比差异均不显著;随着时间的延长,药害逐渐缓解。而常规品种冀棉958对草甘膦的耐受性很差,喷施0.25％和0.50％草甘膦溶液的棉花药害症状表现为植株矮化,生长缓慢,叶片变小、枯黄、褐化干枯,并且药害程度随着时间的延长而加重,药害级别和受害指数均高于10-6001,株高方差分析结果显示与人工除草（ CK）相比差异均达到了极显著水平,施药后35 d部分棉花出现整株枯死。     

改性生物炭的吸附作用及其对土壤硝态氮和有效磷淋失的影响

在实验室通过生物炭与FeCl3的不同配比确定了生物炭的最佳改性条件,并利用土柱模拟试验,研究改性生物炭对土壤硝态氮和有效磷淋失的影响。结果表明,Fe3+与生物炭质量比为0.7是最佳改性条件,改性作用大大增强了生物炭吸附硝态氮和有效磷的能力。在含N量为50mg.L-1的KNO3和含P量为50mg.L-1的KH2PO4溶液中,最佳改性炭的吸附量分别比改性前提高了12倍和66倍,氮和磷的理论最大吸附量分别为2.47mg.g-1和16.58mg.g-1。土壤中添加最佳改性炭能够延缓并减少硝态氮和有效磷的淋失,添加量为2.5%、5%和10%与不添加任何物质的对照相比,硝态氮的淋失量分别显著降低20%、43%和59%(P<0.05),有效磷的淋失量分别显著降低45%、59%和75%(P<0.05),表明土壤中添加改性生物炭能够有效降低土壤硝态氮和有效磷的淋失风险。     

利用微滴数字PCR分析转基因生物外源基因拷贝数

微滴数字PCR(droplet digital PCR,ddPCR)是一种基于泊松分布原理的核酸分子绝对定量技术,在核酸分子的绝对计数/定量领域具有极大的应用潜力。本研究基于ddPCR平台,以转基因水稻(Oryza sativa)T1c-19和转人乳铁蛋白基因基因山羊(Capra hircus)134为例,建立了转基因生物(genetically modified organisms,GMOs)外源基因拷贝数分析方法,并比较了其与传统的实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)和Southern blot方法的准确性。实验数据表明,T1c-19的杀虫晶体蛋白基因(insecticidal crystal protein,Cry1C*)在qRT-PCR和ddPCR测定结果比较一致,约为2拷贝,但已报道的Southern blot分析结果为1拷贝;ddPCR对bar基因的分析结果高于qRT-PCR,分别为2.09拷贝和1.51拷贝。转人乳铁蛋白基因(human lactoferrin,HLF)山羊134在qRT-PCR和ddPCR的分析结果基本一致,均测得含有1拷贝的HLF基因。研究结果表明,微滴数字PCR方法是一种经济、快速和准确的外源基因拷贝数分析新方法,灵敏度和准确性高,将会在拷贝数分析中广泛应用。