导入双价基因的转基因杂交油菜亲本及其对菌核病抗性的研究

比较了乙酰丁香酮、pH、共培养温度及不同激素配比对根癌农杆菌转化油菜(Brassica napus)的影响,建立了油菜高效转化体系.按该体系,将组成型表达几丁质酶和β-1,3-葡聚糖酶基因转化甘蓝型双低杂交油菜亲本恢复系和保持系,获得了转基因恢复系和保持系植株.对再生植株进行PCR和SouthemBlot检测,结果表明外源基因已整合到油菜基因组中.连续3代的PCR检测及转基因植株抗病性在自交后代中得到保持,证实外源基因已遗传到T3代.对转基因植株T1代离体叶进行菌核病菌丝体接种和T1及T2代大田自然侵染鉴定,结果表明,转基因恢复系棚40-12、棚40-32和保持系96B-2对菌核病的抗性比受体对照大幅度提高,大田鉴定其发病率连续2年均比受体对照减少78%以上,比抗病品种‘中油821'减少75%以上,病情指数比受体对照和‘中油821'减少均达显著水平,其抗病性能在后代稳定遗传,获得了高抗菌核病的转基因育种材料.     

导入β—1,3—葡聚糖酶及几丁质酶基因的转基因可育油菜及其？…

利用农杆菌转化法,将组成型表达β－１,３－葡聚糖酶及必丁质酶基因的双价值植物表达载体ｐＢＬＧＣ转化优质甘蓝型油菜品种Ｈ１６５,并得到了抗卡那毒素（Ｋａｎａｍｙｃｉｎ,Ｋａｎ）的再生植株。我们对所得到的抗Ｋａｎ的再生苗进行了初步分子生物学检测,结果表明,在Ｋ１５（Ｋａｎ １５ｍｇ／Ｌ）培养基上的绿苗中有３０％为ＰＣＲ阳性植株,而在Ｋ２５（Ｋａｎ １０ｍｇ／Ｌ）培养基上的绿苗有５３％的阳性率。对部分Ｐ     

导入β-1,3-葡聚糖酶及几丁质酶基因的转基因可育油菜及其抗菌核病的研究

利用农杆菌转化法,将组成型表达β-1,3葡聚糖酶及几丁质酶基因的双价植物表达载体pBLGC转化优质甘蓝型油菜品种H165,并得到了抗卡那霉素(Kanamycin,Kan)的再生植株。我们对所得到的抗Kan的再生苗进行了初步分子生物学检测,结果表明,在K15(Kan15mg/L)培养基上的绿苗中有30%为PCR阳性植株,而在K25(Kan10mg/L)培养基上的绿苗有53%的阳性率。对部分PCR阳性的转基因植株进行了点杂交分析,结果均表现出较强的杂交信号,这初步说明外源基因已整合到油菜基因组中。转基因植株的活体接种油菜菌核病菌(Sclerotiniasclerotiorium)试验表明,部分转基因植株比对照显示较强抗病性。     

几丁质酶-葡聚糖酶双价基因导入滇型杂交稻恢复系提高稻瘟病抗性的研究

将含有菜豆(Phaseolus limensis)几丁质酶基因和烟草(Nicotiana tabacum)β-1,3-葡聚糖酶基因的pBLGC(16.5 kb)质粒用基因枪法导入滇型杂交稻(Oryza sativa L.ssp.japonica)恢复系"南29"中,总计获得93个转化再生植株,以β-1,3-葡聚糖酶基因制备探针对T1代株系进行Southern杂交分析,17个株系为杂交阳性,证实外源基因完整结构已整合到水稻基因组中;连续多代的PCR追踪检测证实外源基因已遗传至T4代;对所获得的6个PCR检测阳性T4代品系进行了稻瘟病菌(Magnaporthe grisea)生理小种接种鉴定和稻瘟病大田诱发鉴定,结果表明,转基因品系对稻瘟病的抗性较受体对照大幅度提高,获得了稻瘟病新抗源,但不同品系稻瘟病抗性并不相同.     

芥菜型油菜抗虫转基因植株及其后代株系的研究

带有１ ～２ ｍ ｍ 子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加１０ ～２０ ｍｇ／ Ｌ卡那霉素的 Ｍ Ｓ 选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含３０ ～５０ ｍ ｇ／ Ｌ 卡那霉素的选择培养基上继代培养,再转移到含２０ ｍｇ／ Ｌ 卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行 Ｓｏｕｔｈｅｒｎ ｂｌｏｔ 分子杂交,得到阳性结果。 Ｐ Ｃ Ｒ 分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系     

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。     

几丁质酶—葡聚糖酶双介基因导入滇型杂交稻恢复系提高稻瘟病抗性的研究

将含有菜豆（Phaseolus limensis)几丁质酶基因和烟草（Nicotiana tabacum)β-1,3-葡聚糖酶基因的pBLGC(16.5kb)质粒用基因枪法导入滇型杂交稻（Oryza sativa L.ssp.japonica)恢复系“南29”中,总计获得93个转化再生植株,以β-1,3-葡聚糖酶基因制备探讨对T1代株系进行Southern杂交分析,17个株系为杂交阳性,证实外源基因完整结构已整合到水稻基因组中;连续多代的PCR追踪检测证实外源基因已遗传至T4代;对所获得的6个PCR检测阳性T4代品系进行了稻瘟病菌（Magnaporthe grisea)生理小种接种鉴定和稻瘟病大田诱发鉴定,结果表明,转基因品系对稻瘟病的抗性较受体对照大幅度提高,获得了稻瘟病新抗源,但不同品系稻瘟病抗性并不相同。     

芥菜型油菜抗虫转基因植株及其后代株系的研究*

带有1～2mm子叶柄的芥菜型油菜子叶经农杆菌感染后,培养在附加10～20mg/L卡那霉素的MS选择培养基上筛选转化愈伤组织及不定芽。卡那霉素抗性苗相继在含30～50mg/L卡那霉素的选择培养基上继代培养,再转移到含20mg/L卡那霉素的生根培养基上诱导生根。以苏云金杆菌杀虫晶体蛋白基因为探针,进行Southern blot分子杂交,得到阳性结果。PCR分析也证明外源基因整合到油菜基因组并稳定传递到后代。转基因植株的抗虫性和卡那霉素抗性在自交后代中得到保持,筛选得到纯合的转基因植株后代株系。     

用微卫星DNA标记检测中国主要杂交水稻亲本的遗传差异

选用分布于水稻(OryzasativaL.)12条染色体上的20对SSR(Simplesequencerepeats)引物,分析了具有多种质源和较大应用面积的24个水稻胞质雄性不育系、1个光(温)敏核不育系、3个保持系和5个生产中应用较广的恢复系。在以上33份杂交水稻亲本材料间共检测出102个等位基因(alleles),平均每对SSR引物可检测到5.1个等位基因。PIC(polymorphicindexcontent)值的变动范围为0.274～0.773,平均PIC值为0.554。从56对SSR引物中筛选出5对引物,能够有效地区分所有供试的水稻雄性不育系和恢复系。杂交水稻亲本的聚类分析表明:(1)我国水稻雄性不育系遗传变异丰富,但生产中主要应用的水稻雄性不育系遗传背景比较单一。(2)生产中应用面积较大的水稻雄性不育系遗传变异较恢复系差。(3)生产中主要应用的水稻雄性不育系与恢复系分别聚类于不同的类群,且遗传关系较远。     

Cd胁迫对杂交水稻及其亲本叶片丙二醛含量的影响

以15份杂交水稻及其亲本为材料,采用水培试验研究了不同Cd2＋浓度胁迫对水稻叶片丙二醛（MDA）含量的影响。结果表明：（1）在不同Cd处理水平和生育时期,保持系叶片MDA含量存在差异,如D83B叶片MDA含量各处理显著高于对照（p〈0.05）,MDA积累水平较高,受到Cd严重胁迫,而Ⅱ-32B在两个生育时期受低Cd浓度的影响不大,与对照差异不显著,且随着Cd浓度的增大,MDA含量均表现出较低的MDA积累水平,因此保持系Ⅱ-32B是一种较好的保持系材料。（2）Cd胁迫对恢复系各材料叶片MDA的影响不同,其中以R527、R498叶片MDA积累最明显,孕穗期和灌浆期各处理均显著高于对照,其余恢复系材料受不同Cd2＋浓度的影响,MDA积累不稳定。（3）同一保持系（Ⅱ-32B）与不同恢复系（R498、R549、R892）组合配制的3个杂交稻叶片在MDA含量的增加幅度上表现不同,其中Ⅱ优498、Ⅱ优892受Cd影响较小,无论在孕穗期还是灌浆期叶片MDA含量增幅相对较小,而Ⅱ优549受Cd严重胁迫,各处理均显著高于对照,MDA积累增幅最大。（4）同一恢复系（R498）与不同保持系（Ⅱ-32B、D62B）配制的杂交稻叶片MDA含量亦不同,如D优498仅在孕穗期低浓度下MDA较对照有所降低,其余各处理MDA均积累较多,而Ⅱ优498MDA含量随着Cd浓度的增加,MDA增幅相对较小,可见水稻亲本对子代MDA的积累存在差异,可根据优良亲本,寻找优势组合,培育出抗Cd性强的杂交水稻。     

甘蓝型油菜叶表皮蜡质组分及结构与菌核病抗性关系

本试验选用6个抗病性不同的甘蓝型油莱品种,研究其叶表皮蜡质组成及结构与菌核病抗性的关系。结果表明,抗病品种在去除叶表皮蜡质后病情指数显著增加;感病品种无显著变化。不同抗性品种（系）间除酯类组分含量无显著差异外,其余蜡质组分含量差异显著。相关分析表明,蜡质组分中酯类含量与病情指数呈显著负相关关系,醇类、酮类含量与病情指数呈显著正相关,其余组分和蜡质总量与病情指数无显著相关关系。抗性品种叶表皮蜡质中烷类及酯类所占比重较高,而易感品种酮类比重较高。扫描电镜结果显示,抗病品种（系）的蜡质晶体主要为颗粒状、杆状、丝状;而感病品种（系）的蜡质晶体中不规则片状晶体所占比例较大。这些结果说明油菜叶表皮蜡质的组分及结构可能是抗病品种抵抗和延迟病原菌侵入的机制之一。     

Overexpression of Three Glucosinolate Biosynthesis Genes in Brassica napus Identifies Enhanced Resistance to Sclerotinia sclerotiorum and Botrytis cinerea

Sclerotinia sclerotiorum and Botrytis cinerea are notorious plant pathogenic fungi with an extensive host range including Brassica crops. Glucosinolates (GSLs) are an important group of secondary metabolites characteristic of the Brassicales order, whose degradation products are proving to be increasingly important in plant protection. Enhancing the defense effect of GSL and their associated degradation products is an attractive strategy to strengthen the resistance of plants by transgenic approaches. We generated the lines of Brassica napus with three biosynthesis genes involved in GSL metabolic pathway (BnMAM1, BnCYP83A1 and BnUGT74B1), respectively. We then measured the foliar GSLs of each transgenic lines and inoculated them with S. sclerotiorum and B. cinerea. Compared with the wild type control, over-expressing BnUGT74B1 in B. napus increased the aliphatic and indolic GSL levels by 1.7 and 1.5 folds in leaves respectively; while over-expressing BnMAM1 or BnCYP83A1 resulted in an approximate 1.5-fold higher only in the aliphatic GSL level in leaves. The results of plant inoculation demonstrated that BnUGT74B1-overexpressing lines showed less severe disease symptoms and tissue damage compared with the wild type control, but BnMAM1 or BnCYP83A1-overexpressing lines showed no significant difference in comparison to the controls. These results suggest that the resistance to S. sclerotiorum and B. cinerea in B. napus could be enhanced through tailoring the GSL profiles by transgenic approaches or molecular breeding, which provides useful information to assist plant breeders to design improved breeding strategies.     

Transformation of LTP gene into Brassica napus to enhance its resistance to Sclerotinia sclerotiorum

Rapeseed (Brassica napus L.) is one of the most important economic crops worldwide, and Sclerotinia sclerotiorum is the most dangerous disease that affects its yield greatly. Lipid transfer protein (LTP) has broad-spectrum anti-bacterial and fungal activities. In this study, B. napus was transformed using Agrobacterium tumefaciens harboring the plasmid-containing LTP gene to study its possible capability of increasing plant’s resistance. First, we optimized the petiole genetic transformation system by adjusting the days of explants, bacterial concentrations, ratio of hormones, and cultivating condition. Second, we obtained 8 positive plants by PGR analysis of T0 generation. The PGR results of T₁ generation were positive, indicating that the LTP gene had been integrated into B. napus. Third, T₁ transgenic plants inoculated by detached leaves with mycelia of S. sclerotiorum showed better disease resistance than non-transformants. Oxalic acid belongs to secondary metabolites of S. sclerotiorum, and several studies have demonstrated that the resistance of rapeseed to oxalic acid is significantly consistent with its resistance to S. sclerotiorum. The result from the seed germination assay showed that when T₁ seeds were exposed to oxalic acid stress, their germination rate was evidently higher than that of non-transformant seeds. In addition, we measured some physiological changes in T₁ plants and control plants under oxalic acid stress. The results showed that T₁ transgenic plants had lower malondialdehyde (MDA) content, higher super oxide dismutase (SOD), and peroxidase (POD) activities than non-transformants, whereas disease resistance was related to low MDA content and high SOD and POD activities.     

In vitro mutation and selection of doubled-haploid Brassica napus lines with improved resistance to Sclerotinia sclerotiorum

This paper describes a new protocol to develop doubled-haploid (DH) Brassica napus lines with improved resistance to Sclerotinia sclerotiorum. In this protocol, haploid seedlings derived from microspore cultures of B. napus were used to produce haploid calli for in vitro mutation-selection. For routine screening, mutation was induced by EMS (ethylmethane sulfonate) or occurred spontaneously, and screening for resistant mutants occurred on media with added oxalic acid (OA) as a selection agent. In tests with selected lines, the optimal concentration of EMS for mutation was determined to be 0.15%, and the optimal concentration of OA for in vitro screening was 3 mmol/l (half lethal dose was 3.1 mmol/l) for the first cycle of screening. There was an accumulated effect of OA toxicity on calli over two cycles of screening, but the growth and capacity of the surviving calli for regenerating seedlings were not affected by OA. Of the 54 DH lines produced from the in vitro mutation-selection, two DH lines of resistant mutants, named M083 and M004, were selected following seedling and glasshouse tests. The resistance of M083 and M004 to S. sclerotiorum following tests with both mycelial inoculum and OA was greater than that of their donor lines and the resistant control Zhongyou 821. In both glasshouse and field disease nurseries, disease indices on M083 and M004 were less than 50% of those of the control. The time required for M083 and M004 to mature was 14 days and 10 days shorter, respectively, than that of their donor lines. Furthermore, M083 had more pods per inflorescence, a greater 1,000 seed weight and higher yield than its donor line. Random amplified polymorphic DNA characterisation showed that M083 had DNA band patterns that differed from its donor line.     

Quantitative trait loci for resistance to Sclerotinia sclerotiorum and its association with a homeologous non-reciprocal transposition in Brassica napus L.

Sclerotinia stem rot, caused by fungus Sclerotinia sclerotiorum, is one of the most devastating diseases in rapeseed (Brassica napus L.). We report the identification of Quantitative trait loci (QTL) involved in the resistance to S. sclerotiorum in two segregating populations of DH lines: the HUA population, derived from a cross between a partially resistant Chinese winter line (Hua dbl2) and a susceptible European spring line (P1804); and the MS population, derived from a partially resistant French winter cultivar (Major) and a susceptible Canadian spring cultivar (Stellar). A petiole inoculation technique and two scoring methods, days to wilt (DW) and stem lesion length (SLL), were used for the resistance assessment. A total of eight genomic regions affecting resistance were detected in the HUA population, with four of these regions affecting both measures of resistance. Only one region, which affected both measurements, was detected in the MS population. Individual QTL explained 6–22% of the variance. At five of the QTL from both populations, alleles from the resistant parent contributed to the resistance. QTL on N2 from the HUA population had the highest LOD score and R ² value and was detected for SLL in the first evaluation. The N12 resistance allele in Hua dbl2 was detected in a region containing a homeologous non-reciprocal transposition (HNRT) from the resistance-containing portion of N2. This result suggests that QTL in the N12.N2 HNRT enhanced the resistance of Hua dbl2 by increasing the dosage of resistance genes. The relationship of QTL from different genetic backgrounds and their associations with other agronomic traits are discussed.     

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.