大豆凝集素基因lec-s转化烟草>增强对烟草花叶病毒的抗性

 将编码大豆凝集素的lec-s基因插入植物表达载体pBI121中,构建植物重组表达质粒pBI121:: lec-s。由根癌土壤杆菌EHA105介导的叶盘法转化烟草,获得了转基因烟草株系。PCR和RT-PCR检测证明lec-s基因已转入烟草植株中。接种烟草花叶病毒（Tobacco mosaic virus,TMV）进行抗病性试验结果表明,转基因烟草叶片上的病斑数显著减少,说明转基因烟草表现出对TMV的抗性。定量RT-PCR检测发现,接种TMV后,抗病防卫基因（PR-1a、GST1、Pal和hsr515）在转基因烟草叶片中显著上调表达。这些结果表明,大豆凝集素基因lec-s转化烟草可对TMV产生抗性,其作用机制可能在于lec-s基因参与了植物的防卫信号通路,诱导了抗病防卫基因在转基因植株体内的表达,增强了植株对TMV的系统抗性。     

Coat protein-mediated resistance to isolates of barley yellow dwarf in oats and barley

Tissue cultures of GAF30/Park oats were biolistically co-transformed with constructs containing the coat protein (CP) genes of the P-PAV, MAV-PS1 or NY-RPV isolates of barley yellow dwarf virus (BYDV), together with a construct containing the bar gene for herbicide resistance and the uidA reporter gene. Transformed, herbicide-resistant tissue cultures were screened by PCR for the presence of the CP genes. Fertile regenerated plants were recovered from some CP-transformed tissue cultures. T₁ progeny of these plants were screened for resistance to the BYDV isolate corresponding to the introduced gene by inoculation with viruliferous aphids followed by ELISA tests. Variation in ELISA values for GAF30/Park control plants made interpretation of the data difficult, but oat plants resistant to each of the three isolates of BYDV (ELISA values less than 0.3; virus titers equivalent to less than 25% of infected controls) were identified in T₁ generations. Further testing of MAV-PS1 CP-transformed lines to the T₂ generation, NY-RPV CP-transformed lines to the T₃ generation and P-PAV CP-transformed lines to the T₄ generation identified further resistant plants. Similarly, immature embryos and calli of the barley cultivar Golden Promise were biolistically bombarded with constructs containing the CP gene of the P-PAV isolate of BYDV and the bar and uidA reporter genes, lines of self-fertile P-PAV CP-transformed barley plants were developed, and T₁plants were screened for resistance to P-PAV. Eight plants from six lines showed moderate to high levels of resistance to P-PAV that correlated with the presence of the CP gene. Plants giving low ELISA values were also found in other lines, even though the CP gene was not detected in these plants. Some T₂ plants derived from resistant parents that contained the CP gene were themselves highly resistant.     

酵母pac1基因介导对葡萄B病毒的抗性

为明确广谱性抗病毒基因—酵母pac1基因对葡萄B病毒(Grapevine virus B,GVB)的抗性效果,通过农杆菌介导的遗传转化,将pac1基因导入西方烟37B,对转基因植株进行PCR鉴定及Southern blot分析,通过病毒摩擦接种观察症状以及实时荧光定量RT-PCR检测植株体内病毒含量,并对转基因植株抗病性进行初步鉴定。结果表明,目的基因pac1成功导入并整合至西方烟37B基因组,共获得10个转基因株系。不同株系的T1代烟草中阳性植株比例为16.7%~72.7%,表明目的基因可成功遗传到子代。接种病毒后转基因植株普遍延迟发病,但后期症状与非转基因对照相似,其中仅1个转基因株系B6具有不表现典型症状等抗性反应。接种植株病毒含量检测中,所有转基因植株均检测到病毒存在,但表现为抗病的B6株系中病毒含量显著低于非转基因对照,表明该转基因植株虽不能完全抵抗GVB侵染,但对GVB具有耐病性。     

抗青枯病转多肽抗生素基因烟草的选育

分别于温室和田间条件下,对转单价apidaecin、shiva-I及双价apidaecin+shiva-I基因的14个烟草株系材料的T₁和T₂后代群体进行青枯病抗性筛选鉴定及农艺性状评价。温室盆栽试验中,于5~6叶龄期采用伤根法进行接种。接种后15天,转apidaecin、shiva-I和apidaecin+shiva-I基因烟草的病情指数较对照分别下降了16.3%、15.2%和59.7%。田间自然病圃筛选鉴定结果表明:B43-5-4、B43-5-1、B43-5-2、B43-5-3和B43-4-1等5个株系材料病情指数较起始品种K326降低21%~70%,且农艺性状与起始品种K326无显著差异。     

A short‐length single chimeric transgene induces simultaneous silencing of Agrobacterium tumefaciens oncogenes and resistance to crown gall

Transformation with self‐complementary oncogene sequences was used to silence the Agrobacterium tumefaciens oncogenes ipt and iaaM. The silencing response was triggered by using a very short chimeric sequence where conserved fragments from both oncogenes were fused in one unique transgene. Most T₀ transgenic tobacco lines and T₁ seedlings evaluated in vitro had intermediate or very low susceptibility to A. tumefaciens as compared with the wildtype plants. A greenhouse evaluation of whole plants confirmed the lines that were resistant. Low levels of transgene hairpin RNA (hpRNA) coupled with small interfering RNA (siRNA) accumulation correlated with oncogene silencing and, therefore, resistance to crown gall. After infection with the oncogenic strain, much lower levels of the oncogenes’ mRNA were found in resistant lines than in wildtype plants. The frequency of resistant lines, with few or no symptoms, produced with the chimeric construct was similar to the highest reported efficiencies obtained by using sense and antisense whole oncogene sequences.     

含双病原物诱导启动子植物安全表达载体的构建

 本研究用来自烟草的具有高度病原物特异性的两个诱导启动子EAS4和hsr203J,串联驱动GUS基因的表达,同时考虑到转基因植物的安全性,引入双边界序列,构建了含双病原物诱导启动子的植物安全表达载体。将表达载体转化烟草获得转基因植株。分析显示,在正常生长情况下转基因烟草检测不到GUS活性,或活性极低;而受疫霉激发子parasiticein、Phytophthora nicotianea[0]的孢子悬浮液和Ralstonia solanacarum的菌悬液诱导后,转基因烟草叶片中可检测到明显的GUS活性。结果表明,构建的植物表达载体所含双病原物诱导启动子均具有良好的诱导活性,可用于植物遗传转化。     

低温胁迫下转ICE1基因水稻养分变化的研究

采用盆栽试验,选用转ICE1基因水稻T_4-8株系和T_4-9株系及未转基因水稻（Oryza sativa L.）品种垦鉴稻10号为试验材料,研究苗期低温胁迫对转基因水稻氮、磷、钾养分吸收的影响。结果表明,低温胁迫期间,转基因水稻幼苗丙二醛含量维持在较低水平,SOD活性维持在较高水平;地上部分氮、磷、钾含量变化不大,一直维持在较高水平;地下部分氮、磷、钾含量呈缓慢下降趋势,但降幅比非转基因水稻小,在胁迫第10天,T_4-8株系和T_4-9株系氮含量均高出对照13.86%,磷含量分别高出42.31%和50.00%,钾含量均高出85.71%。说明在低温胁迫条件下,ICE1基因过量表达可以增强水稻的抗寒性,减轻低温对水稻的伤害,保持根系较强的吸收养分能力。     

Production of plants of Nicotiana benthamiana and Prunus domestica transgenic for plum pox potyvirus coat protein,and demonstration of PPV resistance in transformed N. benthamiana1

Leaf discs of Nicotiana benthamiana plants were transformed with Agrobacterium tumefaciens and transgenic plants expressing plum pox potyvirus (PPV) coat protein (CP) were generated. Homozygous R₂ progeny from these plants were inoculated with PPV. Plants were scored for the appearance of symptoms and tested for infection by DAS-ELISA. Various levels of resistance were obtained after an initial stage in which PPV was able to multiply in all the transgenic plants. Within 2–3 weeks after inoculation, the transgenic resistant plants fully recovered from virus infection. Conversely, control and susceptible transgenic lines developed severe symptoms and high virus titres. Prunus domestica (plum) was transformed by inoculating hypocotyl slices with A. tumefaciens containing a binary plasmid which included the NPTII, GUS, and PPV CP genes within its T-DNA region. Transgenic shoots were rooted and established in the glasshouse. Analysis of selected transformants by PCR showed that the engineered foreign genes had been integrated, including that for PPV CP. Histological assays on young leaves of these putative transformants gave a positive reaction. This suggests that all genes transferred are expressed in these transformed plums.     

In-plant activation of root-specific expression of a cytotoxic gene disrupts the development of the root-knot nematode,Meloidogyne javanica

Plant hosts can be engineered to disrupt the development of sedentary plant-parasitic nematodes or proper functioning of the feeding sites the nematodes induce. The use of constitutive promoters to express dsRNAs or nematode inhibitor proteins may be unreliable because of possible silencing or yield penalty from continuous expression in a plant host. This ill-effect can be avoided if a root-specific, nematode-responsive promoter (NRP) is used to drive the target nematode-inhibitory message. This study used the In Plant Activation (INPACT) system to express a barstar-controlled barnase in galls of Meloidogyne javanica and assessed how the engineered tobacco lines affected the growth and development of the nematodes. Of the 11 combinations of four NRPs and the CaMV 35S promoter assessed, the AtCel1 and TobRB7 combinations activated specific expression of split β-glucuronidase (GUS) and barnase genes in and around giant cells. The same NRP combination directed expression of the barnase gene in tobacco roots also constitutively expressing the barstar gene (SPBB transgenic lines). On roots of six T₁ SPBB lines, there was up to 94% reduction in the number of galls with significantly smaller adult females compared to those on wild-type plants. Some of the females on lines SPBB4-1 and SPBB-4-2, for example, were not associated with galls. The results indicated the engineered plants disrupted M. javanica development and demonstrate the potential for controlled and localized expression of peptides, such as those that could block specific effectors, to disrupt initiation, formation, establishment, or proper functioning of feeding cells induced by damaging sedentary nematodes.     

Constitutive expression of a phenylalanine ammonia-lyase gene from Stylosanthes humilis in transgenic tobacco leads to enhanced disease resistance but impaired plant growth

Transgenic tobacco plants expressing a phenylalanine ammonia-lyase cDNA (ShPAL), isolated from Stylosanthes humilis, under the control of the 35S promoter of the cauliflower mosaic virus were produced to test the effect of high level PAL expression on disease resistance. The transgenic plants showed up to eight-fold PAL activity and were slowed in growth and flowering relative to non-transgenic controls which have segregated out the transgene. The expression of the ShPAL transgene and elevated PAL levels were correlated and stably inherited. In T₁ and T₂ tobacco plants with increased PAL activity, lesion expansion was significantly reduced by up to 55% on stems inoculated with the Oomycete pathogen Phytophthora parasitica pv. nicotianae. Lesion area was significantly reduced by up to 50% on leaves inoculated with the fungal pathogen Cercospora nicotianae. This study provides further evidence that PAL has a role in plant defence.