Bt基因转化玉米培育抗玉米螟自交系

采用基因枪法将苏云金杆菌(简称Bt)的杀虫毒蛋白基因(CryIA)导入东北春玉米自交系铁7922的幼胚中,诱导愈伤组织.抗虫基因的表达载体是pBI121,包含Bt杀虫毒蛋白基因和新霉素磷酸转移酶(NPTⅡ)基因,筛选标记bar由CaMV 35S启动子驱动.通过对后代植株的除草剂草丁膦(PPT)筛选、分子鉴定和ELISA法检测,证明外源基因已经整合到玉米基因组中并可以稳定遗传.对转基因株系进行田间接玉米螟虫卵,调查不同生长时期的危害指数,最终筛选出一批抗玉米螟自交系.     

兼抗全蚀病和白粉病小麦新种质的创制与鉴定

TaLTP5是从小麦中分离到的一个脂质转移蛋白编码基因。利用基因枪介导法将TaLTP5表达载体pA25-TaLTP5转入抗白粉病的小麦品种扬麦18 (含抗白粉病基因Pm21)中, 旨在选育兼抗全蚀病和白粉病的小麦新种质。对转基因小麦T₀~T₃代植株中引入TaLTP5基因进行分子检测和抗病性鉴定。PCR检测、Southern杂交分析结果表明, 外源TaLTP5基因已转入、整合到3个转基因小麦株系的基因组中, 并能稳定遗传; 荧光定量RT-PCR的分析以及全蚀病菌的接种与鉴定结果表明, 与受体小麦扬麦18相比, 这3个转基因小麦株系中TaLTP5表达量显著提高, 其对全蚀病的抗性也明显增强。对3个转基因株系的Pm21分子标记和白粉病抗性鉴定表明, 外源TaLTP5基因的导入没有影响受体小麦对白粉病抗性, 说明这些转基因株系为兼抗全蚀病和白粉病小麦新种质。     

The introduction into bread wheat of a major gene for resistance to powdery mildew from wild emmer wheat

Summary A new source of resistance to wheat powdery mildew caused by Erysiphe graminis has been transferred to hexaploid bread wheat, Triticum aestivum, from the wild tetraploid wheat, Triticum dicoccoides. The donor was crossed to bread wheat and the pentaploid progeny was then self-pollinated. Plants having a near stable hexaploid chromosome complement were selected in the F₃ progeny and topcrossing and backcrossing of these to a second wheat cultivar to improve the phenotype was undertaken. Monosomic analysis of early backcross lines showed the transferred gene to be located on chromosome 4A. The gene has been designated Pm16.     

Insect-mediated seed-set evaluation of 21 soybean lines segregating for male sterility at 10 different loci

The cowpea trypsin inhibitor gene (CpTI) and neomycin phosphotransferase gene (nptII) were introduced into the embryonic callus cells of immature embryos of wheat elite line Shannong 995604 using Agrobacterium-mediated gene transfer. Independent plantlets were regenerated from kanamycin-resistant calli. PCR and real time PCR analysis, PCR-Southern and Southern blot hybridization indicated that there were three independently-dervied transgenic plants viz. transformed-I, II and III (T-I, T-II and T-III). The segregation of CpTI in the transgenic wheat progenies of T-Iand T-III were consistent with Mendelian inheritance. Resistance to the storage insect pest of wheat viz. the grain moth (Sitotroga cerealella Olivier) was improved significantly in seeds of the three transgenic wheat T₂ lines obtained from T₁ PCR-positive plants. The frequency of moth-eaten seed from T-I, T-IIand T-III was reduced 66.76%, 62.48% and 43.59% respectively. The investigation of agronomic traits of the three transgenic wheat T₁ PCR-positive plants revealed that the three transgenic lines had excellent agronomic traits. They provide good germplasm resource for wheat genetic improvement.     

利用农杆菌介导法将反义Wx基因导入水稻的研究

利用根癌农杆菌将含多基因(hpt选择标记基因、反义Wx基因、GFP和GUS报告基因)的pCAMBIA1304载体导入水稻品种(501R、中花9号和日本晴)的幼胚愈伤组织,分别在含35mg/L、45mg/L和65mg/L潮霉素浓度的筛选培养基上筛选获得抗性愈伤。后经PCR检测,从415株T0代再生植株中选出92株(其中501R、中花9号和日本晴分别为4株、43株和45株)。对这些转基因后代植株进行Southern blotting分析表明：hpt、反义Wx基因已经整合进植物基因组中。绝大多数转基因植株后代的表型正常。成熟种子直链淀粉含量分析表明,部分转基因植株的T1代种子中的直链淀粉含量有不同程度的下降,最低的已下降至6．3％,与对照相比下降了9．8％。     

转SbPIP1基因小麦植株的获得及发芽期耐盐性鉴定

为了培育耐盐小麦新材料,利用基因枪转化法将海蓬子(Salicornia bigelovii Torr.)水通道蛋白(plasma membrane intrinsicprotein,SbPIP1)基因导入到小麦品种宁麦13和扬麦158中。对两个品种的各2500枚幼胚进行轰击,分别获得抗Basta再生植株237株和108株(筛选标记基因为Bar基因),经PCR鉴定阳性转基因植株分别为30株和7株。用130mg/L Basta除草剂对T1代幼苗进行喷施鉴定,发现T1代幼苗的Basta抗性出现了分离。遗传分析发现76.19%的株系表现为1对基因遗传的3:1分离,为单拷贝的基因导入。发芽期耐盐性分析发现81.22%的转基因株系的盐害指数低于受体宁麦13的盐害指数,耐盐性强于受体宁麦13。这些转基因材料将进一步用于小麦的耐盐分子育种研究。     

玉米成熟胚胚性愈伤组织的诱导、高频再生及转化的研究

以玉米自交系CML295、CML304和18-599R的成熟胚为外植体, 结合幼胚离体培养方法, 探讨并优化了成熟胚来源的胚性愈伤组织诱导及继代培养方法。对其愈伤组织的形态和组织切片的研究结果显示, 继代过程可产生良好的Ⅱ型胚性愈伤组织。在分化培养中分别获得68.6%、75.4%和84.8%的高频再生率, 每愈伤组织块成苗数分别为2.45、2.43和2.75。利用基因枪法转化pCAMBIA1301质粒后的GUS瞬时表达效率分别为57.9%、62.5%和73.1%, 转化pCAMBIA1303质粒后检测GFP的瞬时表达效率分别为23.3%、40%和45.5%。以上3种基因型成熟胚来源的愈伤组织转化率与其对应的幼胚来源的胚性愈伤组织转化率相似。这一技术体系为玉米的遗传改良和功能基因组研究提供了重要的技术平台。     

Genetic analysis of in vitro wheat somatic embryogenesis

Summary A spring wheat genotype which produces somatic embryos in vitro, after short and long-term culture, was tested for its ability to sexually transmit this embryogenic trait. Reciprocal crosses were performed between a embryogenic line and a nonembryogenic variety.Immature embryos were cultured on Murashige and Skoog medium plus 2 mg/l 2,4-dichlorophenoxyacetic acid, gelled with 5.5 g/l agarose. Somatic embryogenesis was not expressed in the F₁'s. In contrast, from several hundred immature embryos of the F₂ generation of one cross, 10.7% and 1.6% expressed somatic embryogenesis in short and long-term cultures respectively. These percentages of embryogenic: non-embryogenic fits a model of a few complementary genes. The embryogenic capacity of the F₂ genotypes depends on the presence of recessive alleles at these gene loci. The long-term wheat somatic embryogenesis capacity requires a more complex mechanism than the short-term one.Abbreviations CS Chinese Spring - Aq Aquila - E Embryogenic - NE Nonembryogenic - SC Subculture     

根癌农杆菌介导获得稗草Ecppc转基因小麦的研究

采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1）,对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C₄植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型,并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株,其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示,转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。     

Dosage Response of Rye Genes in a Wheat Background

Wheat (Triticum aestivum L.) breeders often utilize alien sources to supply new genetic variation to their breeding programs. However, the alien gene complexes have not always behaved as desired when placed into a wheat background. The introgressed genes of interest may be linked to undesirable genes, expressed at low levels or not at all. The short arm of rye (Secale cereale L.) chromosome one (1RS) contains many valuable genes for wheat improvement. In order to study rye gene response to varying copy number, wheat lines were constructed which contained zero, two or four doses of 1RS. The meiotic behavior of rye chromosome 1R, and wheat/rye translocation chromosomes, 1AL/1RS and 1BL/1RS was studied in the F₁ hybrids between wheat lines carrying 1R or the translocation chromosomes. The IRS arm was transmitted at a very high frequency; 98 % of the F₂ plants had at least one of the chromosomes with a IRS arm. In addition, 44 % of the F₂ plants received at least one copy of the chromosomes from each parent. Analysis of the meiotic behavior of the IRS arm suggested that few euploid wheat gametes were formed. Therefore, most of the pollen must have contained IRS. It is unknown whether the lack of euploid wheat pollen could account for the high transmission frequency of the rye chromosomes. There may have been differential survival of the embryos receiving the rye chromosome as well.     

BCL、RIP细胞凋亡基因向小麦中的导入和赤霉病抗性鉴定

利用农杆菌介导法将BCL和RIP 2个与细胞凋亡有关的基因分别导入了小麦品种扬麦10号和Bobwhite,转化效率分别为1.60%和1.25%。Southern检测结果表明,细胞凋亡基因已稳定整合到小麦染色体上,多数植株为单拷贝整合。Northern检测结果表明,细胞凋亡基因能以小麦遗传背景高水平转录为RNA。转基因植株T₁代分离比例为2.11～2.33∶1,表明外源基因在后代中能够稳定遗传。BCL转基因植株和RIP转基因植株对赤霉病均表现出一定抗性,其中BCL-20、BCL-21、RIP-8、RIP-18等15个T₁代植株的小穗发病率为5.6%~16.1%,可望进一步培育抗小麦赤霉病新材料。     

Pina~m-Pinb~m-Gsp~m串联三基因表达载体的构建及其在普通小麦中的转化

籽粒硬度是小麦品质改良的重要目标性状之一,主要由5D短臂上的Hardness(Ha)位点的两个主效基因,即Puroindoline a(Pina-D1)和Puroindoline b(Pinb-D1)控制,同时受基因Grain Softness Protein-1(Gsp-1)的影响。本研究以一粒小麦(T.monococcum)DV92作为Pinam、Pinbm和Gspm基因的供体,将三基因各自完整的表达盒串联连接到载体pGEM-TEasy上,构建Pinam-Pinbm-Gspm三基因串联的表达载体。利用基因枪介导的转化方法转化普通小麦科农199幼胚,共轰击5608个小麦幼胚愈伤,经Biolaphos(化学除草剂)筛选,获得933株再生植株,经PCR检测,鉴定出11株阳性植株,有关转基因小麦的籽粒硬度还需进一步实验验证;本实验实现了串联三基因在普通小麦中的遗传转化,为利用基因枪介导的遗传转化方法改善小麦籽粒硬度提供了可行性。     

转TiERF1-RC7双价基因小麦的鉴定及其全蚀病抗性

为探讨TiERF1和RC7基因对小麦抗全蚀病的防御反应,本研究对转TiERF1-RC7双价基因小麦进行了分子检测以及全蚀病抗性的室内和田间鉴定。结果表明,转入的TiERF1和RC7基因在转基因小麦中可以遗传和转录;与受体扬麦18相比,5个转TiERF1-RC7小麦株系在整个生育期抗病性显著提高,苗期的全蚀病严重度在10%以下,成熟期的白穗率在13%以下,而扬麦18的严重度为62.98%,白穗率为26.09%。电子显微镜观察结果表明抗病转基因小麦根表的全蚀病原菌菌丝数量及生长势明显低于感病材料。上述结果说明,转入的TiERF1和RC7基因抑制了全蚀病原菌的侵染及在转基因小麦中繁殖,进而提高了转基因小麦对全蚀病的抗性。     

Inheritance and allelism of resistances to the Russian wheat aphid in seven wheat lines

Summary Studies were conducted to determine the inheritance and allelic relationships of genes controlling resistance to the Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), in seven wheat germplasm lines previously identified as resistant to RWA. The seven resistant lines were crossed to a susceptible wheat cultivar Carson, and three resistant wheats, CORWA1, PI294994 and PI243781, lines carrying the resistance genes Dn4, Dn5 and Dn6, respectively. Seedlings of the parents, F₁ and F₂ were screened for RWA resistance in the greenhouse by artificial infestation. Seedling reactions were evaluated 21 to 28 days after the infestation using a 1 to 9 scale. All the F₁ hybrids had equal or near equal levels of resistance to the resistant parent indicating dominant gene control. Only two distinctive classes were present and no intermediate types were observed in the F₂ segregation suggesting major gene actions. The resistance in PI225262 was controlled by two dominant genes. Resistance in all other lines was controlled by a single dominant gene. KS92WGRC24 appeared to have the same resistance gene as PI243781 and STARS-9302W-sib had a common allele with PI294994. The other lines had genes different from the three known genes.     

用核基质结合区(SAR)序列提高小麦最小表达框转基因表达的稳定性

采用最小表达框技术转化植物可以规避由骨架序列引起的安全风险。核基质结合区序列SAR (scaffold attachment region)可作为边界元件与核基质结合阻挡转基因片段邻近染色质区的作用与影响, 提高外源基因稳定性。本研究在最小表达框序列两端添加SAR序列, 提高小麦最小表达框转基因表达的稳定性, 提高转化基因的表达效率。首先, 以GUS为目的基因构建带有SAR序列的最小表达框, 以科农199为受体进行基因枪转化, 同时以不加SAR序列的最小表达框片段为对照。带有SAR序列的最小表达框片段共轰击857个幼胚, T₀代获得40株再生植株, PCR检测到16株阳性植株, 转化效率为1.87%; 对这16个阳性单株进行GUS染色, 15株显色; 从来自4个T₀阳性植株的18个T₁代植株中随机选取18株进行PCR和GUS染色检测, 有15株表现为阳性。不带SAR序列的对照片段轰击1012个幼胚, 获得31株再生植株, 其中5株PCR阳性, 转化效率0.49%, 这5个阳性植株中仅2株为GUS染色阳性; 来自于5个T₀代PCR阳性株系的10个T₁代单株中没有发现PCR和GUS染色阳性株。表明SAR序列可以提高基因枪转基因效率和目的基因表达稳定性。为了创制抗旱转基因小麦, 以来自大豆的抗旱相关转录因子基因GmDREB3为目的基因, Bar基因为筛选标记基因, 转化受体小麦济麦22, 共轰击6045个幼胚, 获得再生植株130株, PCR检测阳性植株30株, 转化效率为0.50%; 随机选取6株PCR阳性植株进行RT-PCR分析, 其中5株可检测到外源基因的转录。进一步对这5株RT-PCR阳性植株插入片段完整性进行分析, 其中4株插入片段基本完整。通过real-time PCR分析, 发现T₀代6个RT-PCR阳性植株的外源GmDREB3的拷贝数为1~3个。以上结果证明, 在最小表达框两端加上SAR序列后可以提高小麦最小表达框转基因表达的稳定性。     

ω-黑麦碱基因沉默对小麦1B/1R易位系加工品质的影响

ω-黑麦碱是造成小麦1B/1R易位系加工品质差的一个重要因素,为探索解决这一问题,构建了ω-黑麦碱基因沉默表达载体,并通过农杆菌介导转化小麦品种金禾9123,获得3个T_0代转基因植株,再经连续的扩繁和PCR检测,获得纯合转基因T_4代株系。酸性PAGE检测结果表明,这些纯合转基因株系中ω-黑麦碱的总表达量平均下降53%。这些ω-黑麦碱基因沉默的转基因株系的面筋指数、沉降值和稳定时间均显著提高,而其农艺性状,如株高、穗粒数、千粒重和小区产量,均没有受到不良影响,说明沉默ω-黑麦碱基因可以在不影响产量的前提下提高小麦1B/1R易位系的加工品质。     

根癌农杆菌介导获得稗草Ecppc转基因小麦的研究

采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1），对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C₄植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型，并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株，其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示，转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。     

转基因小麦外源品质基因1Dx5表达的遗传研究

转基因小麦B72-8-11b中外源品质基因1Dx5表达量是内源相应基因表达量的6倍。利用小麦转基因品系为父本，常规品种鄂麦12、鄂麦18和日喀则8号分别为母本，配置3个杂交组合。采用SDS—PAGE技术，检测各组合亲本、F1、F2代的HMW-GS组成，研究转基因小麦B72-8-11b中外源品质基因1Dx5表达的遗传。结果表明：外源1Dx5基因有功能拷贝整合在1个位点，如同内源品质基因，遵从孟德尔遗传模式。这对育种选择策略的制订具有指导意义，也表明了基因枪转化法能够使得外源基因有功能拷贝整合在1个位点并能稳定传递。     

猪传染性胃肠炎病毒纤突糖蛋白在转基因玉米中的表达

本研究拟构建携带猪传染性胃肠炎病毒纤突糖蛋白基因(TGEV-S)的转基因玉米植株并检测其表达情况。首先利用PCR方法自携带TGEV-S基因的重组质粒中扩增2.2kb的S基因片段,然后插入植物表达载体pBAC176中,该表达载体以编码除草剂草甘膦抗性的EPSP基因作为选择标记,目的基因以双增强子的CaMV35S(E35S)及其玉米Hsp70第一内含子为驱动。以授粉后10～12d约0.5～1mm大小的玉米幼胚为受体,用基因枪进行转化,经过愈伤组织诱导、草甘膦抗性筛选和分化再生培养,先后获得74株转化再生植株。利用PCR和Southern blot检测表明,T0代转基因苗有9株检测结果为阳性。T1代转基因玉米株系经PCR检测有14个转基因株系为阳性,初步确定了目的基因在这些转基因玉米中的稳定整合。进而通过间接ELISA分析初步确定目的基因在7个T1代转基因玉米株系获得了表达。研究结果为进一步研究表达TGEV-S转基因玉米的生物学活性奠定了基础。     

农杆菌敏感小麦基因型的筛选及其转化

利用C58c1农杆菌菌株（其携带的pPTN290质粒载体上含有nptⅡ 基因和GUS基因）为供体材料和小麦幼胚为受体材料,对我国35个春小麦基因型进行了农杆菌敏感性研究和转化研究。组织化学染色结果表明,PM97034、P187、巴140105、扬麦158、新春9号和克丰6号等基因型农杆菌感染后GUS基因瞬时表达率达到了15.0% ~ 30.0%,显著高于模式基因型Bobwhite。其余幼胚分别在含10-25mg/L G418培养基上诱导愈伤组织和分化植株,抗性再生植株经ELISA检测、PCR检测、叶片离体退绿检测、X-Gluc染色检测和Southerm blot检测,最终从新春9号和PM97034的农秆菌转化效率高于Bobwhite. Southerm blot检测结果同时表明,外源基因在多数转基因植株中表现为单拷贝整合。T₁代植株ELISA检测结果和PCR检测结果表明,外源基因在个别株系中的分离符合孟德尔遗传规律,而在多数株系中的分离偏离了孟德尔遗传规律。阳性植株与阴性植株分离比经X²。适合性检测,表明外源基因在多数株系中的遗传符合3：1分离规律。本文首次对不同小麦基因型进行了农秆菌敏感和转化效率研究,筛选到新春9号和PM97034两个对农杆菌比较敏感且转化效率比较高的基因型,可望广泛用于小麦转基因研究。