黑龙江省大豆产区疑似转基因大豆的分子检测

利用表型鉴定和PCR分子检测相结合的方法,对黑龙江省国储库拒收的疑似转基因大豆进行了转基因成分检测.表型鉴定方法为草甘膦叶片涂抹,PCR分子检测对象为CaMV35S启动子、NOS终止子及目的基因CP4-EPSPS.利用大豆自身的凝集素基因作为PCR检测的内标基因,以有效排除DNA质量、实验操作等因素对检测结果的影响.结果表明,所测材料不抗草甘膦除草剂,也不合所检测的外源基因,由此判断该批次材料不是转基因大豆.     

转基因抗虫保铃棉(Bollgard(R))检测方法研究

运用常规PCR方法和实时荧光PCR方法对转基因抗虫保铃棉外源基因CaMV 35S启动子、NOS终止子、标记基因NPTⅡ和目的基因CryIA(c)进行了检测。建立了快速、准确的抗虫保铃棉籽转基因成分的PCR检测方法。     

转基因抗虫保铃棉(Bollgard~ひ)检测方法研究

运用常规 PCR方法和实时荧光 PCR方法对转基因抗虫保铃棉外源基因 Ca MV 35 S启动子、NOS终止子、标记基因NPT II和目的基因 Cry IA(c)进行了检测。建立了快速、准确的抗虫保铃棉籽转基因成分的 PCR检测方法。     

巢式PCR和SYBR Green I实时PCR检测转基因大豆方法的研究

为了建立转基因大豆检测技术,采用巢式PCR和SYBR Green I实时PCR技术,检测转基因大豆 外源基因(CaMV35S、CP4EPSPS).结果表明,利用巢式PCR可检测出1 ng/μL转基因含量1％　的大豆中　的CaMV35S基因,而轮PCR的检测限为100 ng/μL;利用SYBR Green I染料能结合双链DNA的 特点,应用实时PCR技术可检测到CaMV35S、CP4EPSPS基因扩增所产生的信号,通过扩增产物的熔解 曲线能有效地区分特异性产物,CaMV35S基因的检测限为0.1 ng/μL.同时利用该方法对黄豆、炒黄豆、豆干等样品进行检测,样品中未检出CaMV35S基因成分.巢式PCR方法明显提高了PCR的检测限, SYBR Green I实时荧光PCR方法能有效、快速检测CaMV35S转基因成分.     

巢式PCR和SYBR Green Ⅰ实时PCR检测转基因大豆方法的研究

为了建立转基因大豆检测技术,采用巢式PCR和SYBR Green Ⅰ实时PCR技术,检测转基因大豆外源基因(CaMV35S、CP4EPSPS)。结果表明,利用巢式PCR可检测出1 ng/μL转基因含量1%的大豆中的CaMV35S基因,而第一轮PCR的检测限为100 ng/μL;利用SYBR Green Ⅰ染料能结合双链DNA的特点,应用实时PCR技术可检测到CaMV35S、CP4EPSPS基因扩增所产生的信号,通过扩增产物的熔解曲线能有效地区分特异性产物,CaMV35S基因的检测限为0.1 ng/μL。同时利用该方法对黄豆、炒黄豆、豆干等样品进行检测,样品中未检出CaMV35S基因成分。巢式PCR方法明显提高了PCR的检测限,SYBR Green Ⅰ实时荧光PCR方法能有效、快速检测CaMV35S转基因成分。     

转基因大豆种子、豆粕定性PCR检测方法的研究与应用

利用定性PCR检测方法,以大豆凝集素基因(Lectin)为内标基因,检测了转基因大豆种子、豆粕中的四个外源基因:35S-CTP基因、Cp4-epsps基因、nos终止子基因、CaMV35S启动子基因,并通过酶切(XmnI)验证试验验证了定性PCR检测结果的准确性,建立了定性PCR检测转基因大豆种子、豆粕的方法,利用市场上抽检到的大豆种子、豆粕样品,进行了实际检测工作.     

应用复合PCR检测水稻种子的转基因成分

根据转基因水稻中常用的花椰菜花叶病毒（CaMV）35S启动子、胭脂碱合成酶（NoS）终止子、β-葡萄糖苷酸酶基因（GUS）、潮霉素磷酸转移酶基因（Hpt）、新霉素磷酸转移酶基因（Nptn）和Bt毒蛋白基因（Bt Cry 1 Ab）的序列，设计合成6对不同的引物，用简单PCR方法检测了水稻种子中的转基因成分，同时建立应用一班PCR反应同时扩增检测两种或多种外源基因的复合PCR方法。通过对水稻样品进行PCR扩增，分析、比较复合PCR的检测结果与简单PCR的扩增结果。发现两者结果完全一致。表明复合PCR方法不但可以提高检测效率、降低检测成本，还可以有效防止假阳性，是一种值得推广的方法。     

转基因豆粕调控元件在饲料加工中降解变化的初步研究

为了对转基因植物源饲料的安全性提供依据,针对转基因豆粕中的CaMV35S启动子和NOS终止子设计引物,利用PCR技术研究加热、高压蒸汽、膨化以及制粒加工工艺对豆粕中启动子及终止子片段长度的影响.结果表明,加热处理对豆粕中启动子基因和终止子基因的片段长度均没有影响;但高压蒸汽处理对豆粕中对启动子基因和终止子基因的影响明显不同,启动子基因的246,165和101 bp这3个片段在处理后均可被检测到,而终止子基因只有125 bp的片段能被检测到,217 bp片段在处理后检测不到;对以豆粕为原料的饲料产品进行检测也得到相同的结果,即启动子基因较为稳定,终止子基因则较易被破坏.     

转基因水稻品系‘Bt汕优63’微滴式数字PCR定量检测方法的建立

为进一步提升转基因水稻的定量检测效率,推动转基因定量标识制度的实施,对转基因水稻品系‘Bt汕优63’成分进行微滴式数字PCR法定量检测研究。基于转基因水稻品系‘Bt汕优63’的外源插入片段和水稻蔗糖磷酸盐合成酶基因SPS,选择、设计和合成高效特异性的内外源基因引物探针,用于微滴式数字PCR法扩增‘Bt汕优63’的内源基因和品系特异序列。引物探针优化实验表明,SPS60-F/R/P和‘Bt汕优63’品系基因-F/R/P适用于dd PCR双通道法定量检测‘Bt汕优63’成分。准确度验证试验表明,测得值与标准值相近,偏差分别为0.03、0.15、0.15、0.03。测得值的SD介于0.08~0.48之间,RSD介于4.86%~15.10%之间,小于欧盟要求25%,准确度较好。本研究建立的转基因水稻品系‘Bt汕优63’微滴数字PCR定量检测方法简便高效、准确度高,可用于农产品和食品中转基因水稻‘Bt汕优63’成分的定量检测。     

基因及构建特异性PCR方法检测转基因香石竹

摘要：以澳大利亚Florigene公司和日本Suntory公司研发的两种转基因香石竹Moonshade、Moonlite为研究对象,针对内参照基因ANS和外源基因F3’5’H、CHS启动子、D8ter,建立基因特异性定性PCR检测方法。此外,分别在外源MAC启动子和DFR基因上设计PCR引物,开展构建特异性PCR检测,定性PCR方法的检测灵敏度为0.5%。该方法的建立为转基因香石竹的进口检测、国内监管和环境安全评价提供了初步数据。     

利用分子标记快速鉴定甜菜育性的研究

试验旨在优化分子标记鉴定甜菜育性的整个鉴定过程,为分子标记鉴定甜菜育性真正应用于实际育种工作打下基础。以随机抽取的10份糖甜菜和5份甜菜多胚种质资源嫩叶为试材,采用两种甜菜DNA提取方法、双重PCR以及两种电泳方式对甜菜育性进行鉴定。结果表明,裂解液法在提取甜菜DNA上更加高效便捷,1 h就可以提取100份甜菜DNA,效率远远高于CTAB法;采用双重PCR的方式进行甜菜育性鉴定完全可行,使甜菜育性鉴定时间减半。对两种电泳方式进行对比发现,6%聚丙烯酰胺凝胶电泳跑出的结果不能满足实验要求,而1%琼脂糖凝胶电泳跑出的结果完全满足实验要求。通过对用分子标记鉴定甜菜育性的整个鉴定体系进行优化,得出用裂解液法进行甜菜DNA的提取、双重PCR进行甜菜育性的鉴定、电泳方式选用1%琼脂糖凝胶电泳可以达到快速鉴定甜菜育性的目的。     

Weed Control in Sugar Beet using Genetically Modified Herbicide-tolerant Varieties – A Review of the Economics for Cultivation in Europe

Genetically modified sugar beet varieties have been developed to be tolerant to glyphosate and glufosinate. To date, research regarding other active ingredients did not result in additional herbicide‐tolerant varieties and the approval of glufosinate‐tolerant varieties for market access has been withdrawn by the applicant. Therefore, only glyphosate‐tolerant varieties could be introduced for cultivation in the short run. Results concerning efficacy and cost of weed control using these varieties and the complementary herbicides were extensively reported in various contributions, mostly on a national level. Based on these results, the economics of weed control for sugar beet production in Europe were reviewed and aspects of integrated control, risk management, and issues of sustainable development of crop production are discussed. Efficient weed control is possible in almost any field situation with glyphosate at about 2 kg a.i. ha^?1, compared with conventional herbicides at 6 kg ha^?1 or higher, depending on weed infestation level. Cost savings for weed control with glyphosate would amount to an average of €150 ha^?1, without any great deviation across different sites and states. A technology fee of about €40 ha^?1 is assumed. The high selectivity of glyphosate may result in a 1–3 % higher yield performance of the crop. All assumptions being considered, total cost savings of €180 × 10⁶ year^?1 were calculated for the area of 1.7 × 10⁶ ha in the main EU sugar beet‐growing countries. A risk management by implementing a monitoring programme is compulsory, and systems of identity preservation or quality assurance are needed in order to enable the production of conventional and genetically modified sugar beet in coexistence. To date, costs are unknown for these measures. Because of the favourable ecotoxicological behaviour of glyphosate and the possibility of threshold‐based weed control, this new technology could provide an excellent option towards sustainable development of the crop. However, political reasons and the lack of acceptance of genetically modified varieties by the consumer have prevented the market entry of GMHT sugar beet to date, so that conventional herbicides will continuously be used in the future.     

甜菜多重SSR-PCR体系的建立和优化

为了建立甜菜多重SSR-PCR体系,通过利用11对甜菜SSR核心引物,根据SSR扩增产物片段大小的不同,构建甜菜2~5重SSR-PCR反应体系。结果表明,在单一SSR-PCR的基础上,甜菜2~3重SSR-PCR的体系为：每增加一重SSR,仅仅增加相应引物的量以及减少去离子水的量。甜菜4~5重SSR-PCR,要在单一PCR的基础上增加0.5倍DNTPs的含量以及相应引物的量,同时根据个别引物扩增效率的不同,相应减少或者增加0.5倍个别引物的量,并成功的构建了16个4重PCR和9个5重PCR。多重PCR反应能够产生与单一PCR相同的多态性,但是却比单一PCR提高了2~5倍的效率,甜菜多重PCR体系的建立将大大加速甜菜品种纯度和真实性鉴定的速度,也将更快的促进甜菜分子生物学其他领域的发展。     

利用PCR仪快速提取甜菜基因组DNA

为了寻找一种快速提取甜菜基因组DNA的方法,以甜菜干种子、幼苗、种仁以及甜菜叶片干粉为原料,利用PCR仪结合碱裂解法快速提取甜菜基因组DNA,利用微量分光光度计检测取DNA的浓度,并用甜菜SSR引物对提取的DNA进行扩增。结果表明,在4 种材料中均检测到了DNA,干种子、幼苗、种仁以及叶片干粉中提取的DNA平均浓度分别为432、197、158、448 ng/μL,无论是DNA原液还是稀释到20 ng/μL的工作液,均能在SSR-PCR反应中扩增出清晰的条带。该方法提取甜菜基因组DNA简单、快速,仅需要NaOH和HCl两种药品,提取的DNA完全可以用于SSR-PCR反应,为快速鉴定甜菜品种纯度和真实性提供了技术支持。     

Environmental Situation and Yield Performance of the Sugar Beet Crop in Germany: Heading for Sustainable Development

The environmental situation and current yield performance of sugar beet production in Germany are described and compared to those in other European regions. A continuous increase in yield performance and enhanced technical quality have been achieved through progress in breeding and improvements in crop management systems. This rise in yield potential has been brought about not by increased production intensity, but by better use of natural resources and production factors. In Germany, legislation rules many environmental aspects of agricultural plant production, and special laws are in force concerning fertilizer use, soil protection, and pesticide use. In sugar beet, nitrogen fertilizer use has decreased greatly and may be reduced further in some regions. A further reduction of potassium and phosphorus fertilizer use does not seem to be appropriate. Conservation tillage contributes to soil protection and is already performed on > 100 000 ha of land growing sugar beet. Strategies of integrated production aim to reduce pesticide use to the bare minimum. Integrated pest management is effective to control insects, nematodes and leaf spot diseases. Pesticide use in sugar beet is dominated by herbicide application. The most promising strategy to reduce the amount of active ingredient seems to be the growing of genetically modified herbicide‐tolerant varieties. Possible directions for future research are discussed, and the prospects for sustainable development, in terms of economic, ecological and social factors, are considered.     

东北旋耕制度下垄作与平作甜菜产质量差异

为研究东北旋耕制度条件下甜菜平作和垄作对于甜菜产量和质量的影响,2017年以‘H004’为试验材料,采用分区设计的实验方法,在哈尔滨呼兰区多年旋耕地测定了在平作和垄作栽培条件下甜菜的块根产量、绿茎叶产量、含糖率、甜菜地下和地上部位的干物质量比例以及不同耕作条件下不同土层的土壤含水量和容重。研究发现转旋耕条件下平作和垄作甜菜含糖量没有显著差异,但是垄作甜菜块根产量要明显优于平作甜菜,垄作甜菜块根单产达到87.8 t/hm2,而平作甜菜块根单产仅为72.9 t/hm2。此外研究发现平作甜菜地上部分干物质积累较多,如平作甜菜根/地上部干物质比值要显著低于垄作甜菜。同时发现垄作栽培土壤含水量及土壤疏松程度均优于平作,如在20-26 cm土层中垄作土壤的容重和含水量分别为1.38 g/cm3和21.96%,而在20~26 cm平作土壤的容重和含水量仅为1.56 g/cm3和19.35%。本研究表明在东北旋耕制度条件下,垄作栽培更适于甜菜生产,也为下一步研发东北高产高糖甜菜栽培模式鉴定重要基础。     

甜菜幼苗光合生理对干旱胁迫的响应

干旱胁迫是抑制甜菜生长发育和影响产量的重要非生物因素。以耐旱型甜菜种质依安一号（V1）和干旱敏感型种质92011/1-6/1（V2）为试验材料,探讨不同耐旱品种甜菜幼苗光合生理对干旱胁迫的响应。研究了干旱胁迫对甜菜幼苗生长发育、总叶绿素含量和表观光合指标的影响。结果表明,干旱胁迫下2种甜菜幼苗的茎粗、根长、株高、叶鲜重、根鲜重、叶干重和根干重均呈下降趋势,V1下降幅度不明显且各指标降低幅度均小于V2;干旱胁迫降低了2种甜菜幼苗的叶绿素含量,叶绿素含量在第7天降到最低,且V1的含量明显高于V2;干旱胁迫使甜菜幼苗的净光合速率、蒸腾速率、叶片气孔导度和胞间CO₂浓度显著下降,V1受到的影响比V2要小。不同耐旱性甜菜品种对干旱胁迫的响应机制存在一定差异,可以进一步分析其抗旱能力,为甜菜的育种、抗逆栽培和稳产提供理论依据。     

Overwintering of genetically modified sugar beet, Beta vulgaris L. subsp. vulgaris, as a source for dispersal of transgenic pollen

The potential impact of transgenic crops on community ecology will depend on the distribution and establishment of the new transgenic traits, on the sexual transfer of their new genes to the environment (Bartsch &; Pohl-Orf, 1996) and on the potential ecological impact of the transgenic trait. Flowering and pollen dispersal is important for outcrossing of the genetically engineered trait. For a biennial plant, like the cultivars of Beta vulgaris L., overwintering is normally necessary to become generative and to produce pollen and seeds (Abe et al., 1997), which usually does not happen with sugar beet as a field crop harvested in autumn (Longden 1989). The starting point for the project was a transgenic sugar beet, Beta vulgaris L. subsp. vulgaris (Lange et al., 1998), with rhizomania and herbicide ( Basta®, Liberty®) resistance. Cold tolerance is one of the most important factors for survival of sugar beet in Central- and North-Europe. Among other ways, spreading of transgenic traits into weed beet (Boudry et al., 1993) or wild beet can occur if genetically engineered – biennial – plants survive the winter, flower in spring and spread their pollen. Field experiments were performed with transgenic breeding lines and their hybrids, transgenic and non-transgenic hybrids with Swiss chard and three conventional beet cultivars to evaluate winter survival rates at seven different field sites. We could show that survival of sugar beet – transgenic as well as conventional ones – in Germany and at the Dutch border is possible. Survival rates were well correlated with temperature data and were unexpectedly high. Differences between sugar beet hybrids and breeding lines could be detected but not within different breeding lines or hybrids. There were no differences detectable between transgenic and non-transgenic plants. The data are crucial for the risk assessment of the release of transgenic sugar beet and are the basis for further experiments towards outcrossing and establishment.     

Molecular and morpho-physiological characterization of sea,ruderal and cultivated beets

Beta vulgaris genetic resources are essential for broadening genetic base of sugar beet and developing cultivars adapted to adverse environmental conditions. Wild beets (sea beets, B. vulgaris spp. maritima and their naturalized introgressions with cultivated beets known as ruderal beets) harbor substantial genetic diversity that could be useful for beet improvement. Here, we compared molecular and morpho-physiological traits of wild beets collected on the Adriatic coast of Italy with sugar beet using eight primer-pairs amplifying 194 polymorphic fragments and four root traits (glucose and fructose content in the root tip, root elongation rate, number of the of root tips, total root length and its distribution among diameters ranges). Genetic diversity was higher in the sea beet accession, which may be due to the highly variable selection pressures that occur in heterogeneous ecological niches, compared with the ruderal and cultivated beets. Sea and sugar beet accessions showed contrasting root patterns in response to sulfate deprivation: sugar beet showed an increase of reducing sugars in the root tips and higher root elongation rate, and the sea beet accession showed an increase in root tip number, total root length and fine root length (average diameter < 0.5 mm). The ruderal beet showed intermediary responses to sea and sugar beet accessions. AFLP and morpho-physiological cluster analyzes showed sea, ruderal and cultivated beets to be genetically distinct groups. The results of this study indicate variability in response to sulfate deprivation is present in undomesticated beets that could be deployed for sugar beet improvement.     

不同浓度盐胁迫对转基因饲用甜菜种子萌发及幼苗生长的影响

通过不同浓度NaCl溶液处理转基因饲用甜菜种子及其幼苗试验，研究了盐胁迫下种子萌发、幼苗生长及胁迫解除后种子的效应，为盐土农业生产提供理论依据。结果表明，转基因饲用甜菜种子萌发的适宜Nacl浓度为50。150mmol/L，其中100mmoL／LNaCl胁迫下，种子发芽率最高，与对照相比高6．78％。随着盐胁迫浓度的升高，种子萌发延迟，萌发率也呈不同程度的降低。盐胁迫解除后，种子萌芽迅速，萌芽率提高，整齐度增加。转基因饲用甜菜幼苗在不同NaCl浓度胁迫下表现出的反应不同，高盐胁迫对抑制幼根生长影响较大。