核盘菌液滴凝集素SSL-6基因过表达质粒构建与油菜转化

菌核病是由腐生型真菌核盘菌寄生引起的一种世界范围性严重病害,是我国油菜的主要病害之一,严重影响油菜的品质和产量。目前没有找到针对核盘菌的有效的免疫资源,因此通过不同的途径来开展抗菌核病抗性资源筛选研究具有非常重要的现实意义。本研究本着"以毒攻毒"的思路,将核盘菌自身可能参与致病的分泌型凝集素蛋白SSL-6基因转入甘蓝型油菜中,以期获得具有抗菌核病的油菜种质资源。通过基因克隆、过表达载体构建和借助根癌农杆菌介导的油菜遗传转化,将SSL-6基因转到甘蓝型油菜"中双6号"基因组中;经过潮霉素抗性筛选与PCR鉴定,成功获得了75株转基因油菜;进一步RT-PCR检测,筛选到5株SSL-6基因高表达的转基因植株。这些转基因油菜植株为下一步菌核病的抗性研究提供了重要的基础材料。     

α-甘露糖苷酶SSc4基因过表达质粒构建与油菜转化

菌核病是由核盘菌引起的一种世界性的真菌病害,对我国油菜生产危害尤为严重。但是现在已知的所有植物资源中都找不到对核盘菌完全具有抗性的基因资源。本研究采用"以毒攻毒"的途径,将核盘菌自身参与致病的一个α-甘露糖苷酶SSc4基因转到甘蓝型油菜中,以期获得对能对核盘菌产生抗性的油菜种质新资源。本研究克隆核盘菌的SSc4基因,构建了其过表达质粒,并通过农杆菌介导转化油菜,实验共筛选到159株抗性苗,其中获得了27株转基因油菜,转化率约为1%。对其中的9株转基因苗进一步作RT-PCR检测,显示有6株转基因植株中SSc4基因高表达,实现了设计目标。     

半夏凝集素基因对油菜的遗传转化及REAL-TIMEPCR分析

以甘蓝型油菜陇油2号子叶为转化受体材料,通过农杆菌介导将半夏凝集素抗虫基因(pta)导入陇油2号,经常规PCR检测获得阳性植株3株。以油菜磷酸烯醇式丙酮酸羧化酶基因(pep)作为内参基因、选pta全长序列中高度保守区域中145bp的pta2为目的片段对转基因植株进行REAL-TIMEPCR分析,初步证明外源pta基因已整合到油菜细胞基因组中。     

农杆菌介导抗除草剂基因bar和抗虫基因cry1ab/ac转化甘蓝型油菜的研究

为了获得同时具备抗虫和抗除草剂特性的甘蓝型油菜新种质材料,以湘油15下胚轴为外植体,将人工合成的抗虫融合基因cry1ab/ac与带有草铵膦抗性基因bar的植物表达载体pFGC5941连接,构建了植物表达载体pFGC-Bt。利用农杆菌介导油菜下胚轴遗传转化体系,将载体pFGC-Bt转入到油菜品种湘油15愈伤组织中。经愈伤组织分化和植株再生,获得了7株T0转基因植株。分株收获这7株转基因油菜的种子,种于花盆中,用除草剂basta喷洒,对存活下来的植株用检测引物进行PCR检测。挑选11株2个基因检测结果都呈现阳性的植株,进行Cry1Ab/Ac蛋白双抗体夹心免疫层析技术检测,在这11株油菜中都检测到了Cry1Ab/Ac蛋白。进行转基因油菜抗菜粉蝶幼虫(菜青虫)的离体鉴定,以非转基因湘油15为对照,结果显示,啃食了转基因油菜叶片的菜青虫停止运动和生长,大概7 d后陆续死亡,而非转基因油菜叶片上的菜青虫表现正常,3 d后叶片基本被其啃食干净。饲喂鉴定结果表明,转抗虫融合基因cry1ab/ac油菜对菜青虫具有明显抗性。得到了同时具有菜青虫和草铵膦双抗性的转基因植株,且抗性显著,为油菜的抗性育种提供了新的材料。     

3种筛选NPT-Ⅱ标记转基因油菜方法研究

利用卡那霉素溶液叶片涂抹法、种子浸泡法以及卡那霉素MS培养基筛选法三种方法对含有NPT-Ⅱ标记基因的转基因油菜种子（T0）进行筛选。结果表明,三种筛选方法的最佳卡那霉素选择压为200mg/L;涂抹叶片法4-5d可以明显识别植株对卡那霉素抗性与否,此法筛选到的4株植株PCR检测全部呈阳性,可靠性达100%;浸泡种子法2-3d可以明显识别植株对卡那霉素抗性与否,此法筛选到的5株植株PCR检测全部呈阳性,可靠性100%;MS培养基筛选法一般10d可以明显植株识别对卡那霉素抗性与否,此法筛选到20株植株,移栽后存活5株,PCR检测3株呈阳性,2株是假阳性植株,可靠性为60%。因此可以认为卡那霉素涂抹叶片和浸泡种子两种方法是转基因油菜进行大规模、快速的筛选及后代的遗传分析的理想方法。     

转异质型ACCase复合基因对油菜含油量的影响

乙酰辅酶A羧化酶(ACCase)是脂肪酸生物合成的限速酶,在油料作物脂肪酸代谢和调控基因工程中具有重要的应用价值。本研究利用同尾酶反复酶切的方法,构建了拟南芥Rubisco SSU小亚基转运肽与大肠杆菌异质型ACCase各亚基基因的融合串联种子特异表达载体,在根癌农杆菌(Agrobacterium tumefaciens)介导的下胚轴转化甘蓝型油菜高油品种CY2,研究异质型ACCase转入油菜对含油量的影响。经过抗生素筛选获得11株转化再生植株,PCR、Southern分子检测证明四个目的基因已整合到T0代再生植株油菜基因组中。RT-PCR检测其中4株,说明在转基因株系中目标基因在籽粒中特异表达。对T2代转基因油菜含油量进行测定,研究表明转基因株系的含油量比对照提高了约5.7%。     

基因枪法转Bt/GNA基因超甜玉米植株的获得及抗虫性鉴定

以超甜玉米自交系S1幼胚诱导的愈伤组织为受体材料,利用基因枪法共转化Bt和GNA基因,获得了128株再生植株,移栽成活31株,其中26株可育.经PCR检测外源基因整合进玉米基因组中,含Bt基因11株,其中有5株能扩增出GNA基因.Southern杂交证明,目的基因已经整合在玉米基因组中.对9株转基因T1代植株叶片室内接虫抗性鉴定,结果显示,5株转基因植株叶片饲喂6 d后明显抑制玉米螟幼虫的发育,幼虫体重增加较少,幼虫死亡率高,表现较强的抗性.     

甘蓝型油菜含MATH结构域基因BnMT-1干扰载体的构建及遗传转化

本研究以甘蓝型油菜湘油15种子不同发育时期抑制差减文库为研究对象,设计特异性引物扩增得到一个与油脂代谢相关,含MATH结构域的基因片段,长度为327bp,命名为BnMT-1。根据RNA干扰的基本原理,将目的片段正反向插入到表达载体pFGC5941.nap查尔酮内含子两侧,经限制性内切酶酶切和质粒PCR扩增检测,证明已成功构建了干扰载体PP-MT。采用根癌农杆菌法将PP-MT干扰载体转入甘蓝型油菜中,对转化植株基因组DNA的PCR检测表明,干扰载体已转入到甘蓝型油菜中,共获得了两株转基因植株。这为进一步研究BnMT-1基因在甘蓝型油菜中的功能打下了基础。     

烟草转DFL基因植株的鉴定

LEAFY基因在植物成花过程中具有重要的作用.DFL是甘菊中LFY同源基因.本研究将DFL基因导入烟草中,鉴定该基因的功能作用.对经潮霉素筛选的抗性植株进行了组织化学染色、PCR扩增及Southern杂交检测.结果检测出8株为GUS阳性植株,进一步用PCR方法验证出其中6株为PCR阳性植株:Southern杂交检测最终证实有3株烟草植株的基因组中已经整合进DFL基因.对转基因植株的开花期和植物学性状进行观测,并与非转基因烟草植株做对比,结果发现:转DFL基因烟草植株的花期有所改变,其中3株转基因植株花期提前15～23 d;部分烟草植株发生形态学性状的变异,如叶片褶皱,叶色发黄.本研究可为转基因改良花卉品种开花期提供参考.     

利用花粉管通道法将叶片衰老抑制基因PSAG12-IPT导入普通小麦的研究

利用花粉管通道法将叶片衰老抑制基因PSAG12-IPT导入普通小麦品种西农1376,经PCR﹑GUS组织化学染色﹑点杂交和Southern杂交分析,证明带有特异启动子的目的基因已整合到5个植株的基因组中,且在大部分转基因植株中能够稳定遗传。通过叶片细胞分裂素含量﹑叶绿素含量﹑叶片衰老进程及农艺性状分析,初步证明PSAG12-IPT基因在     

Occasional loss of expression of phosphinothricin tolerance in sexual offspring of transgenic oilseed rape (Brassica napus L.)

The commercial and economic value of genetically modified crops is determined by a predictable, consistent and stable transmission and expression of the transgenes in successive generations. No gene inactivation is expected after selfings or crosses with non-transformed plants of homozygous transgenic oilseed rape plants if the expression of the transgene in homozygous or hemizygous nature in such plants is stable. The segregation ratios of phosphinothricin (PPT) tolerance in successive generations of selfings and mutual crosses of a few independent transgenic PPT-tolerant oilseed rape plants indicated a dominant, monogenic inheritance. In within-variety and between-variety crosses no transgene inactivation was observed. However, after selfings and backcrosses with non-transgenic oilseed rape infrequent loss of the expression of the PPT tolerance transgene was observed independent from its homozygous or hemizygous nature. Molecular analysis of PPT-susceptible plants showed that the loss of expression was due to gene inactivation and not to the absence of the transgene. Methylation and co-suppression are mechanisms that might cause reduced or even loss of expression of the transgene in later generations. The implications of this observation for seed multiplication of varieties and breeding activities with transgenic oilseed rape are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Degradation of oxalic acid by transgenic oilseed rape plants expressing oxalate oxidase

Summary Oxalic acid is thought to have a primary role in the pathogenicity of several plant pathogens, notably Sclerotinia selerotiorum. A gene coding for the enzyme oxalate oxidase was isolated from barley roots and introduced into oilseed rape as a means of degrading oxalic acid in vivo. This report describes the production of several transgenic plants of oilseed rape and the characterisation of these plants by Southern, Western and enzyme activity assays. Plants were shown to contain an active oxalate oxidase enzyme and were tolerant of exogenously supplied oxalic acid.     

农杆菌介导NHX基因转化甘蓝型油菜的研究

以甘蓝型油菜带柄子叶为转化受体，通过根癌农杆菌EHA105（Agrobacterium tumefaciens）介导将盐地碱蓬（Suaeda heteroptera）的Na⁺/H⁺反向运输体基因NHX导入新疆主栽油菜1khp11品系中，获得了抗卡那霉素的再生植株，并对影响遗传转化的一些关键因素进行了研究。结果表明,带柄子叶在含有2,4-D的培养基上经过3 d预培养后于浓度OD₆₀₀为0.3～0.4的菌液中浸染7 min, 卡那抗性绿苗率可达8%～9%，经过对抗性植株的PCR检测、点杂交及PCR-Southern杂交证明外源基因已整合到油菜基因组中，为新疆盐碱土上油菜产量的提高提供了新的途径。     

油菜高效转化系统的研究

以甘蓝型油菜湘油13为试验材料,运用子房注射法将BT毒蛋白基因导入油菜。在授粉后第20小时至第30小时用自制的微玻针从子房中部注射0.5~1.5μg外源DNA,以高达12.8%的频率获得抗卡那霉素植株。分子杂交分析证明BT毒蛋白基因已整合到油菜基因组中,转化频率为1.6%,表明子房注射法是一种有效、实用的油菜遗传转化方法。     

Identification of AFLP markers linked to one recessive genic male sterility gene in oilseed rape, Brassica napus

The commercial utilization of heterosis in seed yield by means of hybrid varieties is of great importance for increasing oilseed rape production in China. This requires a functional system for the production of hybrid seed. The Brassica napus oilseed rape line 9012AB is a recessive epistatic genic male sterility (GMS) two‐type line, in which the sterility is controlled by two pairs of recessive duplicate sterile genes (ms1 and ms2) interacting with one pair of a recessive epistatic inhibitor gene (rf). Homozygosity at the rf locus (rfrf) inhibits the expression of the recessive male sterility trait in homozygous ms1ms1ms2ms2 plants. This study was conducted to identify molecular markers for one of the male fertility/sterility loci in the B. napus male sterility line 9012AB. Sterile bulk (BS) and fertile bulk (BF) DNA samples prepared from male sterile and male fertile plants of the homozygous two‐type line 9012AB were subjected to amplified fragment length polymorphic (AFLP) analysis. A total of 256 primer combinations were used and seven markers tightly linked to one recessive genic male sterile gene (ms) were identified. Among them, six fragments co‐segregated with the target gene in the tested population, and the other one had a genetic distance of 4.3 cM. The markers identified in this study will greatly enhance the utilization of recessive GMS for the production of hybrid seed in B. napus oilseed rape in China.     

Outcrossing frequencies from multiple high erucic acid oilseed rape fields to a central receptor field

Genetically modified oilseed rape is currently grown on about 23 % of the global oilseed rape acreage. In order to separate transgenic and non-transgenic oilseed rape production and to ensure co-existence of different agricultural cultivation schemes, as is specified by the European legislation, confinement measures have to be defined. Pollen-mediated gene flow is the most important means by which transgenes are dispersed between fields. In contrast to the majority of the previous investigations the objective of this study was to assess the extent of gene flow in the case of multiple pollen-donor fields. A high erucic acid rape genotype was used as biochemical marker for the quantification of outcrossing into a low erucic acid oilseed rape variety. Outcrossing data were obtained from two experimental locations. As expected, multiple pollen sources in a fragmented landscape can result in high gene transfer frequencies, thus requiring larger isolation distances than a field design with a single pollen donor source. The results of the study are transferable to homozygous transgenic oilseed rape varieties.     

Predictive study of the advantages of cleistogamy in oilseed rape in limiting unwanted gene flow

Summary The aim of this study was to evaluate the interest of beginning the selection process on a new genetic characteristic, cleistogamy, to manage gene flow in oilseed rape. The first step was to introduce this characteristic in an existing model of gene flow between oilseed rape populations in time and space, GeneSys-Rape. The second step was to evaluate the parameters of the model linked to this characteristic using field experimentations. Cleistogamous oilseed rape was shown to have an autogamy rate as high as 94% and to emit 10 times less pollen than an open-flowered oilseed rape in the same conditions. But the cleistogamous character was also shown to be unstable in the genotypes tested. The third step was to evaluate the interest of cleistogamy using simulations comparing several genotypes with or without cleistogamy in two different cropping systems. These simulations showed that an oilseed rape both dwarf and cleistogamous was interesting to limit gene escape and that a 99%-autogamous oilseed rape was interesting to limit both gene escape from and harvest contamination of the 99%-autogamous oilseed rape.     

Population dynamics of volunteer oilseed rape (Brassica napus L.) affected by tillage

Volunteer plants of oilseed rape (Brassica napus L.) from persistent seeds in soil can affect subsequent crops. Apart from the agricultural disadvantages, the environment and the marketing of the seeds may also be affected, particularly if plants with special ingredients or genetically modified (gm) plants are grown. In order to investigate the influence of soil cultivation and genotype on seed persistence and gene flow via volunteers, a field experiment was set up testing four tillage treatments and two cultivars in a split-plot design. The cultivars tested were near-isogenic to two gm cultivars. To simulate harvesting losses, 10 000 seeds m⁻² were broadcast on a soil in July. The subsequent tillage treatments were combinations of immediate or delayed stubble tillage by a rotary tiller, primary tillage with plough or cultivator, or zero tillage. Over the following year, the fate of the seeds was determined. Immediate stubble tillage with following cultivator or plough resulted in 586 resp. 246 seeds m⁻² in the soil seed bank. After delayed stubble tillage with following plough, 76 seeds m⁻² were found, and no soil seed bank was built up in the zero tillage treatment. Nevertheless, in the zero tillage treatment, several robust volunteer plants survived the herbicide application before the direct drilling in autumn until following spring. In the zero tillage treatment and in the cultivator treatment, 0.19 volunteers m⁻² resp. 0.06 volunteers m⁻² flowered simultaneously to ordinarily sown oilseed rape in the following crop of winter wheat and produced 73 resp. 18 seeds m⁻². Delayed stubble tillage reduced the risk of gene escape via the soil seed bank, while zero tillage resulted in the highest risk of gene escape by pollen and by production of a new generation of seeds. In terms of a labelling threshold for gm food this number of seeds would be below the threshold of 0.9% of transgenic parts in conventially bred food or feed.     

油菜cDNA表达文库构建及核盘菌致病因子互作蛋白的筛选和鉴定

核盘菌 [Sclerotinia sclerotiorum (Lib.) de Bary] 是引起油菜菌核病的病原真菌。核盘菌侵染植物细胞早期,可分泌一些细胞壁降解酶, 如不同类型的多聚半乳糖醛酸酶(polygalacturonase, PG)。这些酶对病菌在植物组织上的定植起关键作用, 同时酶活性的高低也决定病原菌的毒性或致病性。PG可以激活细胞的防卫反应而与其酶活性无关。为了解PG的信号转导途径, 利用RT-PCR方法克隆了核盘菌的PG基因, 将PG的编码区与酵母GAL4的DNA结合功能区融合, 构建到酵母诱饵蛋白表达载体PGBKT7中; 然后在酵母中构建了油菜cDNA表达文库。通过酵母双杂交方法在油菜cDNA表达文库中对与PG互作的蛋白进行了筛选, 分离到一个与PG互作的蛋白, 测序及BLAST分析表明该蛋白含有一个与钙离子结合的结构域, 称为C2结构域(C2-domain), 推测该蛋白是一种钙依赖的膜结合蛋白。该蛋白与拟南芥中一个含C2结构域功能未知蛋白的氨基酸同源性高达80.24%。利用半定量PCR分析了核盘菌诱导后该基因在油菜花、茎、叶中的表达, 结果表明该基因在叶片中表达量最高。克隆到的C2蛋白与其他物种中的C2蛋白比较发现, 与钙离子结合的天冬氨酸残基很保守。