一种抗草甘膦转基因油菜的快速鉴定方法

为建立一种快速鉴定抗草甘膦转基因油菜的方法,以抗草甘膦转基因油菜品系及后代分离群体为研究材料,利用不同草甘膦浓度滤纸平板进行种子发芽,观察抗性材料和非抗性材料幼胚抗性反应表型,并通过PCR和苗期草甘膦处理进行抗性验证。结果表明,利用0.5~1 g/L的草甘膦溶液处理的抗性材料胚根根毛生长正常,而非抗性材料胚根生长迟缓且光滑无根毛;利用该浓度的处理BC₁和F₂抗性分离群体,幼胚根毛有无性状分离比符合1:1和3:1,幼胚个体的基因组PCR扩增结果与根毛有无呈共分离。通过观察在该浓度草甘膦发芽处理后的幼胚根毛有无,可有效区分抗草甘膦转基因油菜的抗性和非抗性材料。本研究建立的鉴定方法不仅能够对抗草甘膦油菜材料进行快速、准确鉴定,而且能保证材料成活,对抗草甘膦转基因油菜育种和种子纯度鉴定提供技术参考。     

作物抗草甘膦转基因研究概况

草甘膦是一种非选择性除草剂,其作用机理主要是竞争性抑制莽草酸途径中5-烯醇丙酮莽草酸-3-磷酸合成酶(EPSPS)的活性。该合成酶是真菌、细菌、藻类、高等植物体内芳香族氨基酸生物合成过程中一个关键酶。自从1976年美国孟山都公司的草甘膦类除草剂-农达(Roundup)研制成功并得到广泛应用以来,作物抗草甘膦转基因研究成为抗除草剂基因工程研究的热点。随着抗草甘膦基因克隆的发展,抗草甘膦转基因作物也相继问世并大面积推广应用。对草甘膦作用机理、抗草甘膦基因(EPSPS编码基因)的研究、抗草甘膦作物遗传改良的策略及抗草甘膦转基因作物的推广应用情况进行综述,并简单分析了我国作物抗草甘膦转基因存在的问题及解决途径。     

抗草甘膦转基因大豆生物安全性综述

抗草甘膦转基因大豆目前在转基因作物中占据主导地位,对国内外抗草甘膦转基因大豆的发展现状和存在的安全性问题进行了分析和探讨;介绍了草甘膦和抗草甘膦转基因大豆的作用机制,对抗草甘膦转基因大豆的基因逃逸、产生超级杂草的可能性以及对生物多样性和对土壤微生物的影响等生物安全性方面的问题进行了讨论.同时,还对抗草甘膦转基因大豆能否在我国种植提出了看法和建议.     

抗草甘膦转基因大豆生物测定方法的研究

本研究以非转基因大豆品种黑农37及其3对具有相似遗传背景的大豆抗性品种为材料,分析了室内生物测定法和田间鉴定法的相关性,探讨了光照和黑暗处理对生物测定法鉴定的影响,旨在建立简便快捷的抗性鉴定方法,为培育抗除草剂大豆新品种提供指导。结果表明,光照条件下大豆对草甘膦更为敏感,而且可以用种子发芽过程中下胚轴的抑制率作为评价的标准。利用室内生物测定法分析对草甘膦非常敏感的大豆品种黑农37,其结果可以反映黑农37在田间条件下对草甘膦的抗性。表明本研究建立的快速检测抗草甘膦转基因大豆的室内生物测定方法可以反映大豆对草甘膦的抗性。     

新型转基因抗草甘膦玉米的培育及安全控制技术

在我国转基因作物的种植中,玉米是最早获得审批进行大量种植的农作物之一,同时也是世界上种植面积最大的转基因农作物。在我国的转基因抗除草剂玉米种植中,抗草甘膦玉米是其主要的品种。主要根据我国最新研发的新型转基因抗草甘膦玉米的种植培育情况,探讨其具体的培育技术以及安全控制技术,以消除转基因抗草甘膦玉米潜在的风险。     

抗除草剂转基因玉米快速有效鉴定方法的建立

以含草铵膦乙酰转移酶基因(phosphinothricin acetyl transferase gene,bar)为筛选标记的抗除草剂转基因玉米为研究材料,通过叶片喷雾法和叶片离体平板培养法试验,建立快速有效鉴定转基因玉米的方法,并探索可有效区分转化和非转化植株的筛选剂剂量。研究结果表明:这两种方法都能快速、有效、方便地鉴定抗除草剂转基因玉米;2000mg/L草铵膦叶片喷雾和培养基中含有5mg/L草铵膦叶片离体培养可快速有效区分是否转入bar基因。通过Bar试纸条分子检测法验证了该鉴定方法的准确性。该鉴定方法的建立为快速有效的筛选大量转基因材料节约大量时间和经费。本研究建立的鉴定方法在转基因材料鉴定和转基因安全评估中具有重要意义。     

抗草甘膦转基因技术或将引来草甘膦产业利好

<正>本刊讯近日,一篇名为《中国抗草甘膦转基因技术有望产业化》的文章被广为关注,其提到国内科学家们正在积极地研发抗草甘膦转基因作物,并希望能够在未来三五年内获得品种审定并实现商品化生产,以打破跨国公司在抗除草剂转基因产业上的垄断。在转基因技术出现后,特别是抗草甘膦转基因作物的大面积推广,使得草甘膦的使用量急速增加。据了解,抗草甘膦转基因植物通俗来说,就是通过转基因给作物植入了草甘膦抗体,使得抗草甘膦转基因作物能够耐受更高浓度草甘膦的施用,在草甘膦彻底杀灭杂草的同时,作物生长完全不受影响。这一技术的推广,使得农场除草效率大大增加,节省了很多人工成本,因此较受农场主欢迎。如此省时省力又高效,让抗草甘膦转基因作物成为美国主要的转基因作物。     

转EPSPS基因抗草甘膦棉花的遗传分析

为分析转基因抗草甘膦棉花早代遗传情况,以花粉管通道法获得的26个转5-烯醇式丙酮酰莽草酸-3-磷酸合酶基因(EPSPS)抗草甘膦棉花转化事件为材料,以其背景亲本中棉所49为对照,喷施草甘膦后对转基因棉T1、T2分离比例进行考察。T1田间抗性鉴定结果表明,经卡方检测20个转化事件T1分离符合3∶1的分离规律,即外源基因插入1个位点;6个转化事件不符合1对基因的分离规律,出现了偏分离。T2田间抗性鉴定结果表明,通过花粉通管法共获得152个纯合株系,分别来源于25个转化事件;对T2不纯合株系继续进行分离比例的考察,发现来源于15个转化事件的57个株系符合3∶1的分离规律;此外卡方检测结果表明,每个转化事件都有不符合3∶1分离规律的株系,且其中10个转化事件没有符合3∶1分离规律的株系。表明通过花粉管通道法获得的转基因植株中外源基因的整合和遗传均较复杂。     

孟山都寻求新耐草甘膦转基因油菜在澳大利亚的登记

<正>孟山都已向澳大利亚基因技术管理办公室递交申请,要求批准转基因油菜的商业化推出。此次申请涉及的转基因油菜是MON 88302,该品种含有一个能使作物耐受除草剂草甘膦的基因。孟山都表示,与已经进行商业化种植的     

油菜抗除草剂不育系的抗性效应研究

为探明转育获得的抗除草剂不育系的抗性效应,苗期用不同浓度草甘膦处理抗草甘膦油菜不育系TA6,TA7。结果表明:随着喷药浓度增加,对叶片、茎秆和根的生长表现为不同程度的抑制作用,其中对叶片生长的影响较早且最严重,对茎秆生长的影响较迟,程度相对较轻,对根部生长的抑制作用最小,表现为先抑后扬,但随着时间推移,影响程度逐渐减轻,至喷药60 d后,油菜苗生长基本恢复正常。花期育性调查和套袋自交结果表明,抗性不育系喷施草甘膦除草剂后,育性未受任何影响,TA6,TA7的不育率和不育度均表现正常。     

Inheritance and mapping of a restorer gene for the rapeseed cytoplasmic male sterile line 681A

A novel cytoplasmic male sterility‐fertility restoration system has been developed in rapeseed (Brassica napus). The cytoplasmic male sterile line 681A was derived from a spontaneous male sterile mutant in a newly released double‐low rapeseed cultivar ‘Xiangyou 13′. The restorer line 714R was identified in the interspecific progeny from a B. napus×B. juncea‐cross. Genetic analysis showed that fertility restoration for 681A cytoplasmic male sterility was controlled by a single dominant nuclear gene which might originate from B. juncea. The RAPD marker S1039_‐520 was found to be linked to the restorer gene in F₂ progeny of 681A × 714R with a recombination frequency of 5.45%.     

Identification of AFLP markers linked to the epistatic suppressor gene of a recessive genic male sterility in rapeseed and conversion to SCAR markers

A recessive epistatic genic male sterility (REGMS) two‐type line, 9012AB, has been used for rapeseed hybrid seed production in China. The male sterility of 9012AB is controlled by two recessive duplicate sterile genes (ms1 and ms2) interacting with one recessive epistatic suppressor gene (esp). Homozygosity at the esp locus (espesp) suppresses the expression of the recessive male sterility trait in homozygous ms1ms1ms2 ms2 plants. In this study, we used a combination of bulked segregant analyses and amplified fragment length polymorphism (AFLP) to identify markers linked to the suppressor gene in a BC₁ population. From the survey of 1024 AFLP primer combinations, eight markers tightly linked to the target gene were identified. The two closest markers flanking both sides of Esp, P9M5₃₇₀ and S16M14₇₈₀, had a genetic distance of 1.4 cM and 2.1 cM, respectively. The AFLP fragment from P4M8₁₉₀, which co‐segregated with the target gene was converted into a sequence characterized amplified region marker. The availability of linked molecular markers will facilitate the utilization of REGMS in hybrid breeding in Brassica napus.     

Molecular characterization of novel resynthesized rapeseed (Brassica napus) lines and analysis of their genetic diversity in comparison with spring rapeseed cultivars*

Resynthesized (RS) rapeseed generated from interspecific hybridization between suitable forms of Brassica rapa L. (syn. campestris; genome AA, 2n = 20) and B. oleracea L. (CC, 2n = 18) represents a potentially important resource to expand genetic diversity in the narrow gene pool of oilseed rape (B. napus L., AACC, 2n = 38). In this study 165 RS rapeseed lines originating from crosses between an Indian Yellow Sarson (B. rapa ssp. trilocularis) and five different cauliflower (B. oleracea convar. botrytis) cultivars were studied using amplified fragment length polymorphism (AFLP) markers and their genetic diversity was compared in relationship to an assortment of 40 diverse spring oilseed and fodder rape varieties. Using three AFLP primer combinations, a total of 467 polymorphic bands were scored. Cluster analysis allowed differentiation among the different RS lines, which, as expected, were genetically highly divergent from the cultivars. The genetic diversity of the material is discussed in relation to its morphological variability with a view to the implementation of RS lines in oilseed rape breeding.     

Efficiency of a novel gametocide amidosulfuron on rapeseed (Brassica napus)

The efficiency of a novel gametocide amidosulfuron, 1-(4, 6-dimethoxypyrimidin- 2-yl) -3-mesyl (methyl) sulfamoylurea, was evaluated on rapeseed ( Brassica napus L.). Double application of 0.09–0.12 g/ha amidosulfuron at the uni-nucleate stage of longest buds and 12 days later, made 97.4–100% plants male-sterile in seventeen out of 20 cultivars tested. The shrunken anthers could not release pollen or produced only dysfunctional pollen grains that could not be stained by aceto carmine. The treatment also caused 10–30% reduction on seed setting in comparison to the controls. The purity of hybrid seed from crosses of 'C161 × Huaye' and 'Zhongshuang No.2 × Huaye' based on amidosulfuron was 99.8% and 100.0%, respectively. The results suggested that amidosulfuron is an efficient gametocide for B. napus .     

Development and characterization of SCAR markers associated with a dominant genic male sterility in rapeseed

Rs1046AB is a dominant genic male sterility (DGMS) line in rapeseed, in which the sterility has always been thought to be conditioned by the interaction of a male sterility gene ( Ms ) and its non-allelic restorer gene ( Rf ). This system provides not only a tool for assisting in recurrent selection but also a promising system for hybrid production. Based on previous studies, two amplified fragment length polymorphism markers linked with the Ms gene were converted into a dominant and a co-dominant sequence characterized amplified region (SCAR) marker, respectively. The putative linear order relationship of three dominant SCAR markers with the same genetic distance from the Rf gene, was also determined by an examination of whether the homologues of these markers are present or not in different lines carrying Rf . A bigger fragment generated by the closest marker linked to the Rf gene was observed in all lines carrying the recessive allele rf , suggesting that this marker is a co-dominant marker, which was further confirmed by nucleotide sequence comparison of these fragments. SCAR markers specific for Ms and Rf will be especially valuable in marker-assisted DGMS three-line breeding.     

Breeding a genie and cytoplasmic double-MS line of rapeseed (Brassica napus L.)

All rapeseed lines with Polima male sterility (MS) that are applied in hybrid cultivars have the problem that their sterility varies with temperature. To overcome this problem, two double-MS lines with genie (GMS) as well as cytoplasmic male sterility (CMS) genes were synthesized through seven generations of breeding, based on a systematic study of changes in fertility of the genie and cytoplasmic male sterility lines. The fertility of the new sterility lines was determined by observation of the floral organs and by pollen staining. The results showed that, in the double-MS lines, half the plants maintained the features of the Polima CMS line, while the other half behaved like the GMS line. The GMS genes were correctly expressed in the Pol cytoplasm, but there was little interaction between the GMS and CMS genetic systems.     

Evaluation of and selection criteria for drought resistance in Chinese semiwinter rapeseed varieties at different developmental stages

The impacts of water deficit imposed at either the germination stage or the reproductive stage on biological traits of 37 semiwinter rapeseed varieties were analysed. The osmotic stress induced by 14% polyethylene glycol had a substantial impact on seed germination and caused significant reductions in plant growth and seed vigour. Relative water content was identified as a good indicator for early screening of drought resistance. At the reproductive stage, seed yield, 1000‐seed weight, height of the first branch, plant height and number of pods per plant were all markedly reduced under the exceptionally long and severe drought condition. Two of the top three resistant genotypes at the germination stage were also shown to be resistant based on the drought susceptibility index for seed yield. Phenotypic correlation and path analyses were employed to rank the relative importance of other agronomic traits with regard to seed yield. The important contributors to seed yield under drought conditions, in descending order, were as follows: 1000‐seed weight, days to maturity, number of pods per plant and plant height.     

Development of novel clubroot resistant rapeseed lines (Brassica napus L.) effective against Japanese field isolates by marker assisted selection

Clubroot is an important disease infectible to cruciferous plants and a major threat to rapeseed production in Japan. However, no clubroot resistant rapeseed cultivars have been released. We surveyed pathotype variation of six isolates collected from rapeseed fields and found they were classified as pathotype groups 2 and 4 using Japanese F₁ Chinese cabbage cultivars. We produced the resynthesized clubroot resistant Brassica napus harboring two resistant loci, Crr1 and Crr2, by interspecific crossing and developed resistant rapeseed lines for southern and northern regions by marker-assisted selection and backcrossing. We improved the DNA marker for erucic acid content to remove linkage drag between Crr1 and high erucic acid content and successfully selected lines with clubroot resistance and zero erucic acid for northern regions. A novel line, ‘Tohoku No. 106’, suitable for southern regions showed stable resistance against all six isolates and high performance in infested fields. We conclude that Crr1 and Crr2 are important genes for CR rapeseed breeding and marker-assisted selection is effective in improving clubroot resistance.     

Development of a molecular CAPS marker for the self-incompatibility locus in Brassica napus and identification of different S alleles

Using primers annealing to S locus sequences the cleaved amplified polymorphic sequences (CAPS) method was applied to develop a marker and to characterize different alleles at the self‐incompatibility locus in Brassica napus. A segregating F₂ population from a cross of a self‐incompatible (SI) and a self‐compatible parent, as well as seven SI lines representing four different S alleles were used. Several primers specific to the S locus in B. oleracea and B. campestris, chosen from the literature, allow polymerase chain reaction (PCR) amplification of genomic DNA. However, only one primer pair amplified a single specific and reproducible PCR fragment of the expected length in B. napus. Digestion with restriction endonucleases revealed polymorphisms for two CAPS markers absolutely linked to the S locus. Using the codominant marker efMboI it was possible to discriminate all three F₂ genotypes. With this marker and an additional marker using another primer pair it was possible to distinguish between three of the four different S alleles and five of the seven SI lines, respectively.