广东省主要红树林植物DNA条形码评价

为评估DNA条形码对鉴定红树林植物的通用性和有效性,在广东红树林分布区域共计采集红树林植物16科22属23种,共144个样品,并进行DNA条形码测序。结果表明:选择的rbcL、matK和trnH-psbA 3个DNA片段的PCR扩增成功率分别为100%、80.29%±8.49%、99.38%±1.25%。测序成果率最高为rbcL 100%,trnH-psbA次之94.57%±5.06%,matK最低75.04%±6.26%。表明rbcL和trnH-psbA片段在红树林群落中都具有较好的通用性。应用BLAST和NJ Tree两种方法计算红树植物的物种识别率。BLAST结果表明,单片段中trnH-psbA的物种识别率最高,为84.48%±12.09%,rbcL次之,matK最低。NJ Tree分析显示单片段中rbcL的物种识别率最高,为66.65%±17.35%;trnH-psbA片段次之,matK片段最低。两张分析方法都显示多个片段组合使用时,rbcL或trnH-psbA是提高物种平均识别率的主要片段。利用单片段rbcL即可获得平均节点支持率最高的红树植物系统发育树,且能准确区分不同树种。trnH-psbA片段可以识别rbcL片段不能识别的物种,可以作为补充片段。综合比较,推荐rbcL、trnH-psbA作为红树林植物DNA条形码片段。     

Comparative analysis of different methods revealing the involvement of proteins during ageing of rice seeds

Seed longevity is a very important characteristic controlling seed quality. However, the mechanism underlying this characteristic is poorly understood. In this study, 'FH7185', a storable rice variety, the germination rate of which was 66.63%, much higher than the control after artificial ageing under relative humidity 88% and 42°C for 21 days. Different methods were applied to reveal the involvement of proteins during ageing of rice seeds. In total, 35 differential proteins were identified from 2D‐PAGE, and 3,719 proteins from iTRAQ analysis. A comparison of these two methods showed that not all protein types could be detected on the 2D‐PAGE gels, and the dynamic range was somewhat limited. So the comprehensive proteome map from the iTRAQ analysis was used to identify the quantitatively regulated proteins that played roles in seed longevity, and found that the most marked change was the increased abundance of many metabolic enzymes, especially the ones involved in α‐linolenic acid metabolism in the embryos during ageing. OsLOX2 and OsLOX3 had a negative effect on seed longevity, which were lower in FH7185 than the control. The dehydrogenase (LOC_Os07g44430) which played a major catalytic role in lipid peroxidation, also changed significantly during ageing. Therefore, OsLOX2, OsLOX3 and Loc_OS 07G44430 may be involved in the regulation of seed longevity with a synergistic effect.     

转基因玉米TC1507质粒DNA标准物质的研制

标准物质具有特定量值、均匀性和稳定性三大典型特征,也是其作为测量标尺的依据。转基因生物标准物质是我国转基因产品标识制度顺利实施的关键技术支撑之一。文章以转基因玉米TC1507为对象,制备了转化体特异性的新型质粒DNA标准物质pTC1507,并对其均匀性、稳定性、量值进行了评价和测定。测试结果表明,制备的质粒DNA标准物质具有良好的瓶间和瓶内均匀性,pTC1507稳定性可靠,可以在-20 ℃稳定放置6个月以上。经过测序和实时荧光定量PCR定值以及不确定度评估,pTC1507标准物质的量值是1.01±0.053。均匀性、稳定性和量值结果表明,研制的转基因玉米TC1507质粒分子标准物质符合标准物质的典型要求,可以代替传统的基体标准物质应用于转基因玉米检测,解决传统标准物质获取困难、制备复杂、成本高等不足。     

DNA条形码技术在中药领域的应用

DNA条形码技术可对物种进行快速自动鉴定,具有鉴定准确、结果稳定、操作简便、适用广泛等特点,目前在中药领域应用较多。从DNA条形码技术在中药材鉴定、种植、流通、市场监管、中成药鉴定和药用植物种质资源调查等中药领域的多个方面进行综述,并探讨DNA条形码技术在中药领域的优势和不足,以期为DNA条形码技术在中药领域的研究提供新的思路。     

Characterization of Genomic Integration and Transgene Organization in Six Transgenic Rapeseed Events

To characterize the DNA rearrangement of both the T-DNA region and the genomic insertion site during T-DNA insertion, the Genomewalker strategy was used to isolate the junctions between the inserted DNA and the plant genomic DNA in six rapeseed events as well as the genomic DNA at the sites before integration. During transformation in each of the six events, portions of both the right border（RB） and left border（LB） regions of the T-DNA were deleted, ranging from a 7 nucleotide deletion of the LB repeats in event RF1 to a 207 bp deletion of the LB region in event RF2. For the six events, T-DNA integration resulted in a deletion at the target site spanning less than 100 bp. Sequence analysis indicated that the T-DNA was integrated into the coding region of various native rapeseed genes in events RF1 and RF2. Duplications of the genomic DNA target site were observed in events RF2, RF3 and Topas 19/2. And multimerization of transgenes was found in event Topas 19/2, in which, the T-DNA was integrated as a head-to-head（RB-to-RB） concatemer into the recipient genome. In event MS1, chromosomal translocation or a large target-site deletion may have occurred during T-DNA integration, which was identified due to a failure to amplify the presumptive insertion site based on the flanking rapeseed DNA sequences. Our results provide comprehensive data concerning transgene organization and the genomic context of the T-DNA in six rapeseed events, which can aid in the developing of insert fingerprinting and the monitoring of long-term genetic stability and potential unintended effects of transgenic events.     

Controlling sprouting in potato tubers using ultraviolet-C irradiance

Legislation limiting the use of chlorpropham (CIPC), the major potato sprout suppressant, has led to a need for new technologies to extend storage life of tubers. Ultra violet C (UV-C) has been used postharvest to reduce disease incidence on many crops, yet its use and efficacy as a sprout suppressant has not been investigated. The aim of this project was to identify the optimum dose and treatment timing of UV-C treatment on potato tubers as an alternative method of sprout suppression to reduce the dependence on chemical sprout suppressants. Up to six potato cultivars over two seasons were treated with varying doses of UV-C ranging from 0 to 30 kJ m⁻² either at harvest or at first indication of dormancy break. The tubers were stored at 9 °C and sprout growth and incidence assessed. Treatment with moderate UV-C doses (5–20 kJ m⁻²) suppressed sprout length and sprout incidence in a range of cultivars. Periderm DNA damage and programmed cell death were not detected in response to any of the UV-C doses. The inactive ABA metabolite, ABA-GE, increased in response to 10 or 20 kJ m⁻² within 72 h of treatment. Multivariate analysis showed a negative relationship between ABA metabolites and sprout growth/incidence during storage. This study found that UV-C reduced sprout growth in potato with no deleterious effects on tuber quality. This suggests potential for further development as an alternative or supplement to conventional sprout suppressant technologies.     

不同发育时期菊芋块茎DNA提取效果比较研究

为筛选菊芋块茎DNA提取的适宜生育时期,以“青芋1号”菊芋品种为试材,用改良CTAB法对不同发育时期菊芋块茎DNA进行提取,经琼脂糖凝胶电泳及全波长分光光度计检测总DNA的纯度、浓度及质量,选用4条ISSR引物进行PCR验证.结果表明:不同发育时期提取菊芋块茎DNA效果较好,DNA浓度呈现单峰曲线变化,峰值出现在第9周,与琼脂糖凝胶电泳检测呈现的亮度结果一致,PCR验证结果显示,提取的DNA能够满足后续相关分子生物学的要求.     

Development of Nucleic Acid Lateral Flow Immunoassay Detection Method for Foot-and-mouth Disease Virus

In this article, through the combination of nucleic acid probes and immune chromatography, a simple, sensitive and specific detection system——nucleic acid lateral flow immunoassay (NALFIA) for amplifing foot-and-mouth disease virus (FMDV) 3D RT-PCR products was established.An ultrasensitive nucleic acid biosensor (NAB) based on streptavidin-labeled gold nanoparticles dual labels and lateral flow strip biosensor (LFSB) were used in this system.The biotinylated goat anti-rabbit IgG was marked to the NC membrane as the alleged strip and the anti-digoxin antibody was labeled to the NC membrane to capture the digoxin probe.After assemblying gold-labeled strip and detecting RT-PCR products, the detection limit of NALFIA was 0.3×10^-3 to 3×10^-3 μg/μL.The NALFIA was compared with agar gel electrophoresis analysis, the results showed that the sensitivity of NALFIA was higher than agar gel electrophoresis.There was an excellent agreement between the two methods.NALFIA was a method with high sensitive, low cost and short time.In conclusion, this method provided a good alternative to detect FMDV.     

利用QuantStudioTM 3D数字PCR分析转基因玉米MON863含量

QuantStudioTM 3D数字PCR (QuantStudioTM 3D digital PCR,3D-dPCR)是一种基于超高密度亲疏水微孔芯片实现数字PCR分液原理的新型核酸绝对定量平台,在转基因生物定量领域具有极大的应用前景.本研究基于3D-dPCR平台,以转基因玉米(Zea mays)MON863混合样品为例,建立基于单重和双重数字PCR体系的转基因生物(genetically modified organisms,GMOs)含量分析方法.与传统qRT-PCR比较发现,在缺乏样品纯度、纯合度信息的情况下,数字PCR能够较好地排除这些因素的影响,测定准确的量值.研究结果表明,QuantStudioTM 3D数字PCR是一种适用于转基因生物含量分析的精确定量方法,还可反映转基因玉米种子的基因型.本研究基于3D-dPCR建立的转基因玉米MON863单重和双重定量方法为转基因检测提供了新的方法和参考.