油菜的遗传转化及抗溴苯腈转基因油菜的获得

以油菜(Brassica napus L.)的下胚轴和子叶为转化受体,建立了油菜的高效转化系统。在此基础上,将抗除草剂溴苯腈基因(bxn基因)导入油菜,获得了抗溴苯腈转基因油菜。分子检测实验证明,转基因油菜中含有bxn基因。转基因油菜可抗高达10~(-3)mol/L的溴苯腈。     

Induction of Male Sterility in Oilseed Rape by TA29-Barnase Gene

Chimaeric TA29-Barnase gene was introduced into oilseed rape (Brassica napus) of good quality and high yield by Agrobacteriurn tumefaciens transformation. The transgenic plants were obtained and transformed genome was determined by Southern blot analysis. About 90~/40 TA29-Barnase transgenic plants were male sterile. However, about 80% transgenic plants turned to be male fertile at temperature higher than 25 ℃. It suggested that male sterility of these transgenic plants was probably temperature sensitive.     

Chloroplast Transformation in Oilseed Rape

The chloroplast transformation vector pNRAB carries two expression cassettes for the spectinomycin resistance gene aadA and the insect resistance gene cry1Aa10. The two cassettes are sited between the rps7 and ndhB targeting fragments. Biolistic delivery of the vector DNA, followed by spectinomycin selection, yielded chloroplast transformants at a frequency of four in 1000 bombarded cotyledon petioles. PCR analysis and Southern blot of PCR products confirmed the site-specific integration of aadA and cry1Aa10 into the chloroplast genomes of transgenic oilseed rape. When transgenic oilseed rape leaves were fed to second instar Plutella xylostera larvae, 47% mortality was observed against this insect and the surviving larvae had significantly lower weight than the control. This is the first report of chloroplast transformation in oilseed rape and the introduction of novel genes between the rps7 and ndhB genes in the chloroplast genome. This offers an opportunity for improvement of oilseed rape by chloroplast genetic engineering.     

定点整合抗虫基因到油菜叶绿体基因组并获得转基因植株

以基因枪法进行了油菜叶绿体基因组的定点转化,载体pNRAB携带抗壮观霉素的筛选标记基因aadA和抗虫基因cry1Aα10,基因的两侧被添加了可用于同源重组的叶绿体DNA序列,基因枪轰击过的油菜子叶柄经植株再生和壮观霉素筛选,获得了36株抗性植株,PCR检测和Southern杂交显示,其中4株的叶绿体基因组已被转化,外源基因已被定点整合进叶绿体基因组的rps7和ndhB基因之间。用转基因植株的叶片饲喂二龄小菜蛾,1周后幼虫死亡率达33%-47%,存活幼虫的生长明显减慢,转基因油菜的叶片受害较轻。     

Endophytic Bacteria Induce Growth Promotion and Wilt Disease Suppression in Oilseed Rape and Tomato

To determine whether bacteria isolated from within plant tissue can have plant growth-promotion potential and provide biological control against soilborne diseases, seeds and young plants of oilseed rape (Brassica napus L. cv. Casino) and tomato (Lycopersicon lycopersicum L. cv. Dansk export) were inoculated with individual bacterial isolates or mixtures of bacteria that originated from symptomless oilseed rape, wild and cultivated. They were isolated after surface sterilization of living roots and stems. The effects of these isolates on plant growth and soilborne diseases for oilseed rape and tomato were evaluated in greenhouse experiments. We found isolates that not only significantly improved seed germination, seedling length, and plant growth of oilseed rape and tomato but also, when used for seed treatment, significantly reduced disease symptoms caused by their vascular wilt pathogens Verticillium dahliae Kleb and Fusarium oxysporum f. sp. lycopersici (Sacc.), respectively.     

表达核糖核酸酶基因的雄性不育油菜的获得

从细菌Ｂａｃｉｌｌｕｓａｍｙｌｏｌｉｑｕｅｆａｃｉｅｎｓ染色体ＤＮＡ中克隆了ＲＮａｓｅ（ｂａｒｎａｓｅ）基因,构建了ＴＡ－２９基因５＇调控区（－１３００－＋３）与ｂａｒｎａｓｅ基因的嵌合基因,通过农杆菌介导的遗传转化,获得了“双低”甘蓝型油菜“中双８２１”的转基因植株。转化植株与末转化植株在高度、生长速度、花器形态、花色等方面基本相同,但转化植株花丝短小、花药干瘪、没有花粉;自花授粉或以其为父本进行的异花授粉均不能结实,表现为完全的雄性不育。花药的横向解剖结构表明：转基因油菜雄性不育与绒毡层细胞的破坏有关     

Unexpected Diversity of Feral Genetically Modified Oilseed Rape (Brassica napus L.) Despite a Cultivation and Import Ban in Switzerland

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.     

Pea Lectin Expressed Transgenically in Oilseed Rape Reduces Growth Rate of Pollen Beetle Larvae

In several studies plant lectins have shown promise as transgenic resistance factors against various insect pests. We have here shown that pea seed lectin is a potential candidate for use against pollen beetle, a serious pest of Brassica oilseeds. In feeding assays where pollen beetle larvae were fed oilseed rape anthers soaked in a 1% solution of pea lectin there was a reduction in survival of 84% compared to larvae on control treatment and the weight of surviving larvae was reduced by 79%. When a 10% solution of pea lectin was used all larvae were dead after 4 days of testing. To further evaluate the potential use of pea lectin, transgenic plants of oilseed rape (Brassica napus cv. Westar) were produced in which the pea lectin gene under control of the pollen-specific promoter Sta44-4 was introduced. In 11 out of 20 tested plants of the T₀-generation there was a significant reduction in larval weight, which ranged up to 46% compared to the control. A small but significant reduction in larval survival rate was also observed. In the T₂-generation significant weight reductions, with a maximum of 32%, were obtained in 10 out of 33 comparisons between transgenic plants and their controls. Pea lectin concentrations in anthers of transgenic T₂-plants ranged up to 1.5% of total soluble protein. There was a negative correlation between lectin concentration and larval growth. Plants from test groups with significant differences in larval weights had a significantly higher mean pea lectin concentration, 0.64% compared to 0.15% for plants from test groups without effect on larval weight. These results support the conclusion that pea lectin is a promising resistance factor for use in Brassica oilseeds against pollen beetles.     

德国油菜高油酸育种情况(英文)

德国近年来油菜高油酸育种的一些情况,其中包括高油酸育种的意义,育种方法,高油酸突变体的遗传特点,高油酸-低亚麻酸育种的可能性和高油酸油菜的应用前景等,对于我国育种新技术的改进、改良和创新研究有一定的意义.     

Developmental Profile of Diacylglycerol Acyltransferase in Maturing Seeds of Oilseed Rape and Safflower and Microspore-Derived Cultures of Oilseed Rape

Diacylglycerol acyltransferase (EC 2.3.1.20) activity was assayed during the maturation of seeds of oilseed rape (Brassica napus L.) and safflower (Carthamus tinctorius L.). Developmental studies were also conducted with microspore-derived embryos of oilseed rape (B. napus L. cv Topas) and an embryogenic microspore-derived cell-suspension culture of winter oilseed rape (B. napus L. cv Jet Neuf). In the maturing seeds, diacylglycerol acyltransferase activity increased to a maximum during rapid accumulation of lipid and declined, thereafter, with seed maturity. In microspore-derived embryos of oilseed rape (cv Topas), high levels of diacylglycerol acyltransferase activity were found throughout the early torpedo to late cotyledonary developmental stages with maximum enzyme specific activity associated with the mid-cotyledonary developmental stage. The cell-suspension culture of winter oilseed rape (cv Jet Neuf) contained 3 to 4% triacylglycerol on a dry weight basis and represented about half of the total lipid. The fatty acid profile of total lipid and triacylglycerol in the cell-suspension culture was similar in samples taken during a 1-year period. The Jet Neuf culture contained diacylglycerol acyltransferase with specific activity similar to that of Topas microspore-derived embryos. Jet Neuf diacylglycerol acyltransferase also displayed an enhanced specificity for erucoyl-CoA over oleoyl-CoA when assayed with 14 [mu]M acyl-coenzyme A in the reaction mixture. The specific activity of diacylglycerol acyltransferase in homogenates prepared from the Jet Neuf culture ranged from 5 to 15 pmol of triacylglycerol min-1 mg-1 of protein when assayed at intervals during a period of 1 year. Thus, the cell-suspension culture may represent an attractive tissue source for purification and characterization of triacyl-glycerol biosynthetic enzymes.     

不同类型油菜EST-SSR标记的通用性及其应用研究

以10个甘蓝型油菜、11个芥菜型油菜和7个白菜型油菜品种为材料,选用已报道的14对白菜EST-SSR引物和8对油菜EST-SSR引物,探索其在3种类型油菜中的通用性,并利用筛选出的在3类油菜中有通用性和多态性的EST-SSR标记对供试油菜品种进行聚类分析.结果表明:(1)选用的14对白菜EST-SSR引物和8对油菜EST-SSR引物,在供试的3种类型油菜中都有扩增,完全可用.(2)在白菜型、甘蓝型和芥菜型油菜品种中扩增显示出多态性的引物数分别为18、16 和14对,其中有10对引物在3种类型油菜品种间扩增产物具有多态性.(3)利用这10对扩增多态性EST-SSR引物对供试油菜品种聚类分析,结果显示,在遗传相似系数为0.67时,3种类型油菜品种分别独自聚为一大类,表明开发和建立不同类型油菜间可通用的EST-SSR标记是可行且有应用价值的.     

Sulphur Deficiency Causes a Reduction in Antimicrobial Potential and Leads to Increased Disease Susceptibility of Oilseed Rape

The reduction of atmospheric sulphur dioxide pollution is causing increasing problems of sulphur deficiency in sulphur‐demanding crop plants in northern Europe. Elemental sulphur and many sulphur containing compounds such as cysteine‐rich antifungal proteins, glucosinolates (GSL) and phytoalexins play important roles in plant disease resistance. The aim of this work was to analyse the effect of inadequate sulphur supply on disease resistance of oilseed rape (Brassica napus). Compared with fertilized oilseed rape, healthy looking S‐deficient plants showed increased susceptibility to the blackleg fungus Leptosphaeria maculans, to the generalist necrotroph Botrytis cinerea and to the oomycete Phytophthora brassicae. To analyse possible causes of the increased disease susceptibility of S‐deficient plants, protein extracts and methanolic extracts of secondary metabolites of plants grown with and without adequate sulphur supply were tested for antimicrobial activity. None of the protein extracts showed antimicrobial activity. However, extracts containing secondary metabolites from normally grown plants showed a strong antimicrobial activity in in vitro tests with various fungal and bacterial pathogens. This activity was almost totally lost in extracts derived from S‐deficient plants. The antimicrobial activity did not appear to be based on the activity of phytoalexins because it was present in healthy plants and was not increased by a previous inoculation with Botrytis cinerea. The loss of antifungal activity in S‐deficient plants correlated with a strong reduction of various GSL, thus suggesting a reduced level of GSL as a possible cause of the reduced antimicrobial potential. However, limited tests of commercially available GSL or their degradation products did not demonstrate a causal link. Our results show that S‐deficiency of oilseed rape negatively affects disease resistance and suggest that this effect is at least partially caused by a reduction of sulphur‐dependent phytoanticipins.     

特早花甘蓝型油菜的开花特征及特早花候选基因鉴定

开花是植物从营养生长进入生殖生长的重要阶段。油菜早花早熟,可有效解决油菜与其他夏季作物的茬口矛盾。挖掘不同的早花油菜资源,研究油菜早花性状形成的分子机理,可促进利用分子技术快速选育早熟油菜的育种进程。本研究以1个新发现的稳定遗传的特早花油菜迎春一号为研究对象,在覆盖全年9个月的时间中设计了15个不同的播种期,调查其开花特性。结果显示,迎春一号在不同季节播种均可顺利开花,其对低温春化的温度需求更弱于普通春性油菜。将迎春一号与半冬性早花品系FM4和半冬性晚花品系FM5进行比较发现,在10月播种于半冬性生态区种植环境下,迎春一号既能快速进入花期,又表现较好的耐寒性,综合农艺性状表现较好。对迎春一号开展分子生物学试验发现,在高温生长环境下,位于油菜A02、A03、C02和C03染色体上的春化途径关键基因BnaFLC的6个同源拷贝在迎春一号的营养生长阶段均出现了多拷贝基因的同步下调。以半冬性早花品系FM4和半冬性晚花品系FM5为对照,利用50×全基因组重测序技术检测得到与油菜全基因组1064个开花基因相关的16229个SNPs和5842个InDels。其中,迎春一号特异的且会导致开花基因编码蛋白变异的SNPs和InDels分别为69个和34个,包含与BnaFLC基因具有正向调控关系的14个SNPs和4个InDels,最终获得11个潜在候选基因。除了1个BnaFLC基因本身以外,其余基因均处于BnaFLC上游区域,且对BnaFLC基因的表达起正向调控作用。     

Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans

The tomato Cf9 resistance gene induces an Avr9-dependent hypersensitive response (HR) in tomato and transgenic Solanaceae spp. We studied whether the Cf9 gene product responded functionally to the corresponding Avr9 gene product when introduced in a heterologous plant species. We successfully expressed the Cf9 gene under control of its own promoter and the Avr9 or Avr9R8K genes under control of the p35S1 promoter in transgenic oilseed rape. We demonstrated that the transgenic oilseed rape plants produced the Avr9 elicitor with the same specific necrosis-inducing activity as reported for Cladosporium fulvum. An Avr9-dependent HR was induced in Cf9 oilseed rape upon injection of intercellular fluid containing Avr9. We showed Avr9-specific induction of PR1, PR2, and Cxc750 defense genes in oilseed rape expressing CJ9. Cf9 x Avr9 oilseed rape did not result in seedling death of the F1 progeny, independent of the promoters used to express the genes. The F1 (Cf9 x Avr9) plants, however, were quantitatively more resistant to Leptosphaeria maculans. Phytopathological analyses revealed that disease development of L. maculans was delayed when the pathogen was applied on an Avr9-mediated HR site. We demonstrate that the CJ9 and Avr9 gene can be functionally expressed in a heterologous plant species and that the two components confer an increase in disease resistance.     

油菜烯醇酶基因的克隆与分析

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP).我们通过RACE-PCR方法从油菜(Brassica napus L.)中克隆到了编码烯醇酶的全长基因.序列分析表明该基因全长cDNA为1 624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38 kD,等电点为5.78.比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性.Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在.RT-PCR和Northern分析表明烯醇酶基因在100 mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降.该研究表明所克隆基因是植物烯醇酶基因家族的新成员.     

油菜烯醇酶基因的克隆与分析(英文)

烯醇酶(enolase)是糖酵解途径中的一个重要酶类,它能够催化磷酸甘油酸酯(2-PGA)生成磷酸烯醇丙酮酸酯(PEP)。我们通过RACE-PCR方法从油菜(Brassica napus L. )中克隆到了编码烯醇酶的全长基因。序列分析表明该基因全长cDNA为1624bp,拥有一个由444个氨基酸组成的开放读码框,所编码的蛋白质分子量为47.38kD,等电点为5.78。比较发现,油菜烯醇酶与已分离出的其他烯醇酶氨基酸序列有较高的同源性。Southern杂交结果显示烯醇酶以低拷贝形式在油菜基因组中存在。RT-PCR和Northern分析表明烯醇酶基因在100mmol/L盐浓度胁迫条件下表达量上升,而在低温诱导时表达量下降。该研究表明所克隆基因是植物烯醇酶基因家族的新成员。     

Efficient Detection of Leptosphaeria maculans from Infected Seed lots of Oilseed Rape

An efficient DNA extraction protocol and polymerase chain reaction (PCR) assay for detecting Leptosphaeria maculans from infected seed lots of oilseed rape were developed. L. maculans, the causal agent of blackleg, a damaging disease in oilseeds rape/canola worldwide, was listed as a quarantine disease by China in 2009. China imports several millions of tons of oilseeds every year. So there is a high risk that this pathogen will be introduced to China via contaminated seeds. Seed contamination is one of the most significant factors in the global spread of phytopathogens. Detection of L. maculans in infected seed lots by PCR assay is difficult due to the low level of pathogen mycelium/spores on seeds and PCR inhibitors associated with the seeds of oilseed rape. In our study, these two major obstacles were overcome by the development of a two‐step extraction protocol combined with a nested PCR. This extraction protocol (kit extraction after CTAB method) can efficiently extract high‐quality DNA for PCR. Amplification results showed that the detection threshold for conventional PCR and nested PCR was, respectively, 1 ng and 10 fg of DNA per μl in mycelia samples. On contaminated seed lots of oilseed rape, the detection threshold of conventional and nested PCR was 709 fg/μl and 709 ag/μl of DNA, respectively. The DNA extraction protocol and PCR assay developed in this study can be used for rapid and reliable detection of L. maculans from infected seeds of oilseed rape .     

Agronomic Performance of Partly Clubroot-resistant Spring Oilseed Turnip Rape Lines

In field experiment studies that tested the effects of three partly resistant cultivars of spring oilseed turnip rape ( Brassica campestris ), multiplication of clubroot was moderate. When initial soil inoculum levels were 47–72% of infected test plants in the bioassay, only 2.1–10% of the plants were infected after harvest. The yield of partly resistant lines was 5–10% higher compared with that of the susceptible cultivar SW Kulta, which on average yielded 1.73 t/ha. The average disease severity index (DSI) after harvest in these trials was 12.7 for the nonresistant cultivar SW Kulta and ranged between 2.2 and 6.2 for the partly resistant lines. The study demonstrated that in fields where the soil infestation level gives a DSI of less than 10, or when less than 20% of the bait plants are infected in the bioassay, it is possible to avoid the risk of severe yield losses from clubroot infections by integrating a partly resistant cultivar into the crop rotation.     

Determination of a Critical Nitrogen Dilution Curve for Winter Oilseed Rape

Several controlled environmental and field experiments werecarried out to define the critical nitrogen dilution curve forwinter oilseed rape, cultivar Goeland. This curve is describedby the following power equation:N=4.48 W^-0.25,whereNis the totalnitrogen concentration in the shoot biomass andWthe shoot biomass.This curve has been validated over the range of shoot dry matterof 0.88 to 6.3 t ha^-1. For lower shoot biomasses this equationoverestimated the critical nitrogen concentration; we proposea constant value of 4.63 (Nis expressed in reduced N, whichis a more stable N fraction in the shoot at these stages ofdevelopment). These results have been validated in several pedoclimaticconditions in France on a single variety in 1994 and 1995. Thehigher position of this curve relative to the C₃species referencecurve (Greenwoodet al.,Annals of Botany67: 181–190, 1990)can be explained by the experimental conditions obtained byGreenwoodet al. (1990); therefore, all their rape data are ratherclose to the critical curve that we propose. The differencesfound between wheat and winter oilseed rape critical N dilutioncurves correspond to their respective leaf:stem dry matter ratioand the specific leaf loss phenomenon occuring in rape. Winteroilseed rape has a higher capacity of N accumulation in itsshoot than wheat for the same aerial dry matter. The proportionof nitrate in shoots rises with the nitrogen nutrition index(N.N.I.) and is more important for rapeseed than for wheat forthe same N.N.I. This difference is especially high at the beginningof flowering when the shade provided by the canopy of rapeseedflowers decreases nitrate reductase activity.Copyright 1998Annals of Botany Company Winter oilseed rape;Brassica napusL.; plant N concentration; nitrate; reduced N; shoot biomass; critical nitrogen concentration; dilution curve; N productivity.