PDF1.2基因在转草酸氧化酶基因甘蓝型油菜中的表达

采用RT-PCR方法检测茉莉酸（JA）／乙烯（ET）依赖性信号途径中关键基因PDF1。2在转草酸氧化酶基因（OXO）油菜株系与未转化对照中的表达差异。结果表明,在转基因油菜不同株系中PDF1．2都有不同程度的上调表达,预示着转OXO油菜对菌核病的抗性增强可能与激活JA／ET依赖性信号途径有关。     

Expressing a gene encoding wheat oxalate oxidase enhances resistance to Sclerotinia sclerotiorum in oilseed rape (Brassica napus)

Sclerotinia sclerotiorum causes a highly destructive disease in oilseed rape (Brassica napus). Oxalic acid (OA) secreted by the pathogen is a key pathogenicity factor. Oxalate oxidase (OXO) can oxidize OA into CO₂ and H₂O₂. In this study, we show that transgenic oilseed rape (sixth generation lines) constitutively expressing wheat (Triticum aestivum) OXO displays considerably increased OXO activity and enhanced resistance to S. sclerotiorum (with up to 90.2 and 88.4% disease reductions compared with the untransformed parent line and a resistant control, respectively). Upon application of exogenous OA, the pH values in transgenic plants were maintained at levels slightly lower than 5.58 measured prior to OA treatment, whereas the pH values in untransformed plants decreased rapidly and were markedly lower than 5.63 measured prior to OA treatment. Following pathogen inoculation, H₂O₂ levels were higher in transgenic plants than in untransformed plants. These results indicate that the enhanced resistance of the OXO transgenic oilseed rape to Sclerotinia is probably mediated by OA detoxification. We believe that enhancing the OA metabolism of oilseed rape in this way will be an effective strategy for improving resistance to S. sclerotiorum. Xiangbai Dong and Ruiqin Ji contributed equally to this paper.     

水杨酸途径中的防御基因在转草酸氧化酶基因油菜中的表达

采用RT-PCR方法检测对水杨酸依赖的信号途径中PR-1、GLU和CHI3个防御基因在转OXO基因与非转基因的油菜中表达差异的结果表明,在转基因油菜株系中这3个基因都有不同程度的上调表达,暗示转OXO基因的油菜抗菌核病的能力增强可能与对水杨酸依赖的信号途径受诱导有关。     

Identification of prior candidate genes for Sclerotinia local resistance in Brassica napus using Arabidopsis cDNA microarray and Brassica-Arabidopsis comparative mapping

Arabidopsis was the leading model of dicot plant. An accomplished platform had been established for functional genomics studies. The platform had largely facilitated molecular biology research of Arabidopsis itself as well as research on its phylogenetic related plants[1,2]. Brassica napus, as an important cooking oil crop, had a close phylogenetic relationship with Arabidopsis[3―5]. In order to take advantage of avail-able Arabidopsis genetic and molecular tools, Girke et al. had explored …     

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。     

Expression of anti-sclerotinia scFv in transgenic Brassica napus enhances tolerance against stem rot

Canola is an important agricultural crop imparting a significant contribution to global oilseed production. As such, optimizing yield and quality is of paramount importance and canola production can be significantly affected by sclerotinia stem rot. The utility of recombinant antibody technology in plant protection has been explored by many researchers and shows promise for the generation of new lines of agriculturally significant crops with greater resistance to diseases. The objective of the current study was to generate recombinant pathogen specific antibody (scFv)-expressing transgenic Brassica napus plants with increased tolerance to the phytopathogenic fungus, Sclerotinia sclerotiorum. Transgenic canola (B. napus) lines expressing S. sclerotiorum-specific scFv antibody showed a significant level of tolerance towards S. sclerotiorum as compared to their non-transformed counterparts. Both incidence and progression of S. sclerotiorum-induced disease symptoms were reduced in plants expressing the recombinant scFv.     

Oilseed Rape Transformation and the Establishment of a Bromoxynil Resistant Transgenic Oilseed Rape

Using hypocotyls and cotyledons as transformed materials, an efficient transformation system of oilseed rape (Brassica napus L.) was established. Bxn gene (bromoxynil-resistant gene)was introduced into these plants, and bromoxynil-resistant transgenic oilseed rape was obtained. Themolecular monitoring experiments showed that these transgenic plants contained bxn gene. The herbicide experiments showed that these transgenic plants had resistance to 10-3 moL/Lbromoxynil.     

转基因油菜的基因流及生态风险

综合评述了转基因油菜的基因流及其生态风险.油菜作为最早的转基因作物之一目前已在加拿大和澳大利亚大面积商业化应用.(常)异花授粉作物油菜的天然异交率可达30%左右,也易与其它芸苔属作物杂交,因此转基因油菜的生态风险已引起各国科学家的高度重视.转基因油菜主要通过与其野生近缘种的花粉交换和与非转基因油菜的花粉交换两种方式进行花粉的输出.基因可能逃逸到相关野生近缘种,但在大田环境下能够得到杂种的可能性很小;由于基因的漂流在油菜田块间确实存在,因此在种植转基因油菜的过程中必须考虑其间隔距离.     

Overexpression of a gene encoding hydrogen peroxide-generating oxalate oxidase evokes defense responses in sunflower

Oxalate oxidase (OXO) converts oxalic acid (OA) and O(2) to CO(2) and hydrogen peroxide (H(2)O(2)), and acts as a source of H(2)O(2) in certain plant-pathogen interactions. To determine if the H(2)O(2) produced by OXO can function as a messenger for activation of defense genes and if OXO can confer resistance against an OA-producing pathogen, we analyzed transgenic sunflower (Helianthus annuus cv SMF3) plants constitutively expressing a wheat (Triticum aestivum) OXO gene. The transgenic leaf tissues could degrade exogenous OA and generate H(2)O(2). Hypersensitive response-like lesion mimicry was observed in the transgenic leaves expressing a high level of OXO, and lesion development was closely associated with elevated levels of H(2)O(2), salicylic acid, and defense gene expression. Activation of defense genes was also observed in the transgenic leaves that had a low level of OXO expression and had no visible lesions, indicating that defense gene activation may not be dependent on hypersensitive response-like cell death. To further understand the pathways that were associated with defense activation, we used GeneCalling, an RNA-profiling technology, to analyze the alteration of gene expression in the transgenic plants. Among the differentially expressed genes, full-length cDNAs encoding homologs of a PR5, a sunflower carbohydrate oxidase, and a defensin were isolated. RNA-blot analysis confirmed that expression of these three genes was significantly induced in the OXO transgenic sunflower leaves. Furthermore, treatment of untransformed sunflower leaves with jasmonic acid, salicylic acid, or H(2)O(2) increased the steady-state levels of these mRNAs. Notably, the transgenic sunflower plants exhibited enhanced resistance against the OA-generating fungus Sclerotinia sclerotiorum.     

A cysteine-rich antimicrobial peptide from Pinus monticola (PmAMP1) confers resistance to multiple fungal pathogens in canola (Brassica napus)

Canola (Brassica napus), an agriculturally important oilseed crop, can be significantly affected by diseases such as sclerotinia stem rot, blackleg, and alternaria black spot resulting in significant loss of crop productivity and quality. Cysteine-rich antimicrobial peptides isolated from plants have emerged as a potential resource for protection of plants against phytopathogens. Here we report the significance of an antimicrobial peptide, PmAMP1, isolated from western white pine (Pinus monticola), in providing canola with resistance against multiple phytopathogenic fungi. The cDNA encoding PmAMP1 was successfully incorporated into the genome of B. napus, and it's in planta expression conferred greater protection against Alternaria brassicae, Leptosphaeria maculans and Sclerotinia sclerotiorum. In vitro experiments with proteins extracted from transgenic canola expressing Pm-AMP1 demonstrated its inhibitory activity by reducing growth of fungal hyphae. In addition, the in vitro synthesized peptide also inhibited the growth of the fungi. These results demonstrate that generating transgenic crops expressing PmAMP1 may be an effective and versatile method to protect susceptible crops against multiple phytopathogens.     

Detection of Sclerotinia sclerotiorum in Planta by a Real-time PCR Assay

Stem rot caused by Sclerotinia sclerotiorum is a very serious disease on oilseed rape worldwide. In this study, a pair of polymerase chain reaction (PCR) primers was designed based on the nucleotide sequence of a DNA region amplified by a microsatellite primer M13. The primer pair amplified a 252-bp fragment from all S. sclerotiorum isolates collected from oilseed rapes at different locations in different years, but not from any other fungus tested. Using this pair of primers, a real-time PCR assay was developed to rapidly detect early infection of S. sclerotiorum on petals of oilseed rape. The real-time PCR assay developed in this study could help growers make a timely decision on fungicide application.     

Laboratory and field response of the pollen beetle, Meligethes aeneus, to the odour of oilseed rape

Abstract. The behavioural response of adults of the pollen beetle, Meligethes aeneus F. (Coleoptera: Nitidulidae), to the odour of one of their host-plants, oilseed rape ( Brassica napus L.), was observed in the laboratory using a fourarmed airflow olfactometer, and under semi-natural conditions using a markrelease–recapture technique. Oilseed rape leaf and flower odour was attractive to pollen beetles in the olfactometer and they dispersed upwind from the centre of a circular array (radius 20 m) of yellow water traps, baited with extracts of oilseed rape leaves or flowers. The dispersal of pollen beetles in the absence of oilseed rape odour was predominantly downwind and crosswind. The percentage recapture of pollen beetles was significantly greater in traps baited with rape flower extracts than leaf extracts. Pollen beetles can use odour-mediated upwind anemotaxis to locate oilseed rape plants from distances of at least 20 m over open ground.     

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.     

Latent S alleles are widespread in cultivated self-compatible Brassica napus.

The genetic control of self-incompatibility in Brassica napus was investigated using crosses between resynthesized lines of B. napus and cultivars of oilseed rape. These crosses introduced eight C-genome S alleles from Brassica oleracea (S16, S22, S23, S25, S29, S35, S60, and S63) and one A-genome S allele from Brassica rapa (SRM29) into winter oilseed rape. The inheritance of S alleles was monitored using genetic markers and S phenotypes were determined in the F1, F2, first backcross (B1), and testcross (T1) generations. Two different F1 hybrids were used to develop populations of doubled haploid lines that were subjected to genetic mapping and scored for S phenotype. These investigations identified a latent S allele in at least two oilseed rape cultivars and indicated that the S phenotype of these latent alleles was masked by a suppressor system common to oilseed rape. These latent S alleles may be widespread in oilseed rape varieties and are possibly associated with the highly conserved C-genome S locus of these crop types. Segregation for S phenotype in subpopulations uniform for S genotype suggests the existence of suppressor loci that influenced the expression of the S phenotype. These suppressor loci were not linked to the S loci and possessed suppressing alleles in oilseed rape and non-suppressing alleles in the diploid parents of resynthesized B. napus lines.     

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.     

Conserved patterns of chromosome pairing and recombination in Brassica napus crosses.

The patterns of chromosome pairing and recombination in two contrasting Brassica napus F1 hybrids were deduced. One hybrid was from a winter oilseed rape (WOSR) x spring oilseed rape cross, the other from a resynthesized B. napus x WOSR cross. Segregation at 211 equivalent loci assayed in the population derived from each hybrid produced two collinear genetic maps. Alignment of the maps indicated that B. napus chromosomes behaved reproducibly as 19 homologous pairs and that the 19 distinct chromosomes of B. napus each recombined with unique chromosomes from the interspecific hybrid between Brassica rapa and Brassica oleracea. This result indicated that the genomes of the diploid progenitors of amphidiploid B. napus have remained essentially unaltered since the formation of the species and that the progenitor genomes were similar to those of modern-day B. rapa and B. oleracea. The frequency and distribution of crossovers were almost indistinguishable in the two populations, suggesting that the recombination machinery of B. napus could cope easily with different degrees of genetic divergence between homologous chromosomes. Efficient recombination in wide crosses will facilitate the introgression of novel alleles into oilseed rape from B. rapa and B. oleracea (via resynthesized B. napus) and reduce linkage drag.     

转vgb基因油菜的耐涝性

油菜是我国重要的油料作物和蛋白质饲料作物,涝害严重影响了我国油菜产业的发展,提高油菜的耐涝能力对于我国油菜可持续发展具有非常重大的意义。本研究采用PCR、Southern杂交等方法对转vgb基因油菜植株进行鉴定,15 d淹涝实验结果显示,转基因油菜的超氧化物歧化酶、丙二醛和脯氨酸含量在淹涝前后的变幅显著低于对照。对淹水后的农艺性状进行调查,转vgb基因的油菜相比较对照抗涝性明显得到增强,证明vgb基因在油菜中的表达对油菜的抗涝性具有显著作用。     

Homoeologous loci control the accumulation of seed glucosinolates in oilseed rape (Brassica napus).

The genetic control of seed glucosinolate content in oilseed rape was investigated using two intervarietal backcross populations. Four QTLs segregating in the population derived from a Brassica napus L. 'Victor' x Brassica napus L. 'Tapidor' cross, together accounting for 76% of the phenotypic variation, were mapped. Three of these loci also appeared to control the accumulation of seed glucosinolates in a Brassica napus L. 'Bienvenu' x 'Tapidor' cross, and accounted for 86% of the phenotypic variation. The three QTLs common to both populations mapped to homoeologous regions of the B. napus genome, suggesting that seed glucosinolate accumulation is controlled by duplicate genes. It was possible to extend the comparative analysis of QTLs controlling seed glucosinolate accumulation by aligning the published genetic maps generated by several research groups. This comparative mapping demonstrated that high-glucosinolate varieties often carry low-glucosinolate alleles at one or more of the loci controlling seed glucosinolate accumulation.     

Pre-germinated conidia of Coniothyrium minitans enhances the foliar biological control of Sclerotinia sclerotiorum

The relatively slow germination rate of Coniothyrium minitans limits its control efficiency against Sclerotinia sclerotiorum. Pre-germinated conidia of C. minitans enhanced its efficiency significantly: in foliar experiments with oilseed rape, hyphal extension of S. sclerotiorum was inhibited by 68%, while formation of sclerotia was completely inhibited when pre-germinated conidia were applied.