A tobacco plastidal transit sequence cannot override the dual targeting capacity of Myxococcus xanthus protoporphyrinogen oxidase in transgenic rice

The effect of a plastidal transit sequence in Myxococcus xanthus protoporphyrinogen oxidase (Protox) on gene targeting ability was investigated by generating transgenic rice that overexpressed M. xanthus Protox with the additional plastidal transit sequence (TTS line). In transgenic lines TTS3 and TTS4, the Protox antibody cross-reacted with the mature M. xanthus Protox protein of 50 kDa. In an in vitro import system using the M. xanthus Protox gene with the plastidal transit sequence, M. xanthus protein was detected in both chloroplasts and mitochondria, confirming that it was targeted into both organelles, as in transgenic rice line, M4, that overexpressed M. xanthus Protox lacking the plastidal transit sequence. A prominent increase in chloroplastic and mitochondrial Protox activity was observed in TTS3 and TTS4 relative to the wild type. However, the increase was lower than that in transgenic line M4. Seeds from all transgenic lines (TTS3, TTS4, and M4) were able to germinate when treated with up to 500 μM of the Protox-inhibiting herbicide, oxyfluorfen, whereas seeds from the wild type failed to germinate even when treated at levels as low as 1 μM. After foliar application of oxyfluorfen, TTS3 and TTS4 exhibited a reduced Protox activity, however, it was much greater than uninhibited Protox activity of wild type. The great increase in conductivity was followed by the great accumulation of photodynamic protoporphyrin IX only in oxyfluorfen-treated wild-type plants, not in oxyfluorfen-treated TTS lines. The presence of the plastidal transit sequence neither excludes the intrinsic ability of subcellular translocation of M. xanthus Protox nor changes herbicide resistance in TTS lines.     

Modifying Myxococcus xanthus protoporphyrinogen oxidase to plant codon usage and high level of oxyfluorfen resistance in transgenic rice

Protoporphyrinogen oxidase (Protox) of Myxococcus xanthus (Mx Protox) is a 49-kDa membrane protein that catalyzes conversion of protoporphyrinogen IX (Protogen IX) into protoporphyrin IX (Proto IX). Upon heterologous expression in transgenic rice plants, Mx Protox is dually targeted into plastids and mitochondria, increasing resistance against the herbicidal Protox inhibitor oxyfluorfen. Here, we describe the chemical synthesis of the Mx Protox gene by assembling several small synthetic DNA fragments derived by ligation-PCR. Codon usage in the resulting 1416-bp gene was modified to correspond to that of the Arabidopsis Protox gene, a change that resulted in a decrease in G+C content from 71 to 49%. The modified Mx Protox gene was used to generate transgenic rice plants via Agrobacterium-mediated transformation. Integration, expression, and inheritance of the transgenes were demonstrated by Southern, Northern, and Western blot analyses. In plants transformed with the modified, low G+C-content Mx Protox gene, levels of Protox expression and enzyme activity were low compared to the levels observed for plants transformed with the native Mx Protox gene. Nonetheless, like the native gene, the modified gene conferred a high level of resistance to the herbicide oxyfluorfen in a seedling growth test.     

Potential gene flow from transgenic rice (Oryza sativa L.) to different weedy rice (Oryza sativa f. spontanea) accessions based on reproductive compatibility

BACKGROUND: The possibility of gene flow from transgenic crops to wild relatives may be affected by reproductive capacity between them. The potential gene flow from two transgenic rice lines containing the bar gene to five accessions of weedy rice (WR1–WR5) was determined through examination of reproductive compatibility under controlled pollination. RESULTS: The pollen grain germination of two transgenic rice lines on the stigma of all weedy rice, rice pollen tube growth down the style and entry into the weedy rice ovary were similar to self‐pollination in weedy rice. However, delayed double fertilisation and embryo abortion in crosses between WR2 and Y0003 were observed. Seed sets between transgenic rice lines and weedy rice varied from 8 to 76%. Although repeated pollination increased seed set significantly, the rank of the seed set between the weedy rice accessions and rice lines was not changed. The germination rates of F₁ hybrids were similar or greater compared with respective females. All F₁ plants expressed glufosinate resistance in the presence of glufosinate selection pressure. CONCLUSIONS: The frequency of gene flow between different weedy rice accessions and transgenic herbicide‐resistant rice may differ owing to different reproductive compatibility. This result suggests that, when wild relatives are selected as experimental materials for assessing the gene flow of transgenic rice, it is necessary to address the compatibility between transgenic rice and wild relatives. Copyright © 2009 Society of Chemical Industry     

Use of Myxococcus xanthus protoporphyrinogen oxidase as a selectable marker for transformation of rice

Protoporphyrinogen oxidase (PPO) is the target enzyme of peroxidizing herbicides. The overexpression of Myxococcus xanthus PPO (Mx PPO) confers a high level of herbicide resistance in rice. Among the peroxidizing herbicides, butafenacil has an efficiency ∼1000-fold that of oxadiazon, as judged by calli susceptibility tests upon herbicide treatment. Butafenacil (0.1 μM) was used to select transgenic rice plants expressing Mx PPO under the control of the constitutive maize ubiquitin promoter. The ectopic expression of the Mx PPO transgene was investigated in the T₀ generation by Northern blot and Western blot analysis. The T₀ transgenic plants expressing the Mx PPO gene were resistant to butafenacil based on in vitro leaf disk and in vivo foliar spray tests.     

Resistance pattern and antioxidant enzyme profiles of protoporphyrinogen oxidase (PROTOX) inhibitor-resistant transgenic rice

We quantified the resistance levels of transgenic rice plants, expressing Myxococcus xanthus protoporphyrinogen oxidase (PROTOX) in chloroplasts and mitochondria, to PROTOX inhibitors, acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon. We also determined whether active oxygen species-scavenging enzymes are involved in the resistance mechanism of transgenic rice. The transgenic rice line M4 was about >200-fold more resistant to oxyfluorfen than the wild-type (WT). M4 was also resistant to acifluorfen, carfentrazone-ethyl, and oxadiazon, but did not show multiple resistance to imazapyr and paraquat, which have different target sites. Acifluorfen, oxyfluorfen, carfentrazone-ethyl, and oxadiazon reduced the chlorophyll content in leaves of WT, but had minimal or no effect on M4. The PROTOX inhibitors also caused significant lipid peroxidation in the treated leaves of WT rice. However, the malondialdehyde production in M4 was not affected by these herbicides. The WT rice had higher activities of superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase than M4 after treatment with PROTOX inhibitors. A similar response was observed in all cases of antioxidant isozyme profiles analyzed. However, the induction in antioxidant activity in WT was not enough to overcome the toxic effects of a PROTOX inhibitor so the plant eventually died.     

Protoporphyrinogen IX-oxidizing activities involved in the mode of action of a new compound N-[4-chloro-2-fluoro-5-{3-(2-fluorophenyl)-5-methyl-4,5-dihydroisoxazol-5-yl-methoxy}-phenyl]-3,4,5,6-tetrahydrophthalimide

N-[4-Chloro-2-fluoro-5-{3-(2-fluorophenyl)-5-methyl-4,5-dihydroisoxazol-5-yl-methoxy}-phenyl]-3,4,5,6-tetrahydrophthalimide (EK-5385) is an experimental substituted bicyclic herbicide. Soil-applied EK-5385 showed good rice selectivity and potent herbicidal activity on barnyardgrass (Echinochloa crus-galli var. oryzicola) at rates of 3.9-250 g a.i./ha. Barnyardgrass was exhibited normal growth under dark condition, however, the growth of shoot and root was severely inhibited under light condition (14/10 h of light/dark, 50 μmol/m²/s of photosynthetically active radiation) when treated with EK-5385, oxadiazon, and oxadiargyl. IC₅₀ of EK-5385 and oxadiargyl to chlorophyll loss in cucumber cotyledons was approximately 0.3 and 0.7 μM, respectively. IC₅₀ of EK-5385 and oxadiargyl to carotenoids loss in cucumber cotyledons was about 0.26 and 0.1 μM, respectively. IC₅₀ concentration of EK-5385 and oxadiargyl on Protox activity was approximately 5.5 and 8 nM, respectively. Cellular leakage occurred without lag period from cucumber leaf squares treated with 1 μM of EK-5385 and oxadiargyl under light exposure.     

Resistance of a soybean cell line to oxyfluorfen by overproduction of mitochondrial protoporphyrinogen oxidase

The diphenyl ether herbicide oxyfluorfen (2-chloro-4-trifluoromethylphenyl 3-ethoxy-4-nitrophenyl ether) inhibits protoporphyrinogen oxidase (Protox) which catalyzes the oxidation of protoporphyrinogen IX (Protogen) to protoporphyrin IX (Proto IX), the last step of the common pathway to chlorophyll and haeme biosynthesis. We have selected an oxyfluorfen-resistant soybean cell line by stepwise selection methods, and the resistance mechanism has been investigated. No growth inhibition was observed in resistant cells at a concentration of 10(-7) M oxyfluorfen, a concentration at which normal cells did not survive. While the degree of inhibition of total extractable Protox by oxyfluorfen was the same in both cell types, the enzyme activity in the mitochondrial fraction from non-treated resistant cells was about nine-fold higher than that from normal cells. Northern analysis of mitochondrial Protox revealed that the concentration of mitochondrial Protox mRNA was much higher in resistant cells than that in normal cells. There were no differences in the absorption and metabolic breakdown of oxyfluorfen. The growth of resistant cells was also insensitive to oxadiazon [5-tert-butyl-3-(2,4-dichloro-5-isopropoxyphenyl)-1,3,4-oxadiazol-2-(3H)- one], the other chemical class of Protox inhibitor. Therefore, the resistance of the selected soybean cell line to oxyfluorfen is probably mainly due to the overproduction of mitochondrial Protox.     

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。     

Host-induced gene silencing for engineering resistance to Fusarium in soybean

Soybean is one of the most economically important crops in the world. Its production is affected by several fungal diseases, such as those caused by Fusarium spp., causing significant losses in yield and seed quality. Management interventions are limited, costly, and associated with environmental problems. Host resistance provides a more convenient and cost-effective approach. Host-induced gene silencing (HIGS) has been demonstrated to be an alternative strategy to engineer fungus resistance in plants. We have generated transgenic soybean lines with an intron-hairpin construction in order to express siRNA corresponding to the CYP51B gene from Fusarium oxysporum. Results showed the presence of siRNA corresponding to the F. oxysporum CYP51B gene in both leaves and roots of the transgenic lines. Plants (T₃ generation) were challenged against F. oxysporum and F. graminearum. Disease severity was evaluated and revealed resistance to F. oxysporum with one line, named 3.22, presenting no symptoms. In addition, transgenic lines presented better plant development (height and root growth) when compared to the nontransgenic line. Moreover, transgenic lines revealed better development when inoculated with F. oxysporum.     

Agronomic performance of F1, F2 and F3 hybrids between weedy rice and transgenic glufosinate-resistant rice

BACKGROUND: Studies of hybrid fitness, of which agronomic performance may be an indicator, can help in evaluating the potential for introgression of a transgene from a transgenic crop to wild relatives. The objective of this study was to assess the agronomic performance of reciprocal hybrids between two transgenic glufosinate‐resistant rice lines, Y0003 and 99‐t, and two weedy rice accessions, WR1 and WR2, in the greenhouse. RESULTS: F₁ hybrids displayed heterosis in height, flag leaf area and number of spikelets per panicle. The agronomic performance of F1 between WR1 and Y0003 was not affected by crossing direction. The tiller and panicle numbers of F1 individuals were higher than their F2 counterparts. However, these traits did not change significantly from the F2 to the F3 generation or in hybrids with weedy rice as maternal or paternal plants. For all hybrids, the in vitro germination rates of fresh pollen were similar and significantly lower than those of their parents, seed sets were similar to or of lower value than those of weedy rice parents and seed shattering characteristics were partially suppressed, but the survival of hybrids over winter in the field was similar to that of weedy rice parents. All F1, F2 and F3 hybrids had similar composite agronomic performance to weedy rice parents. CONCLUSION: There was no significant decrease in the composite agronomic performance of any of the hybrids compared with weedy rice. This implies that gene flow from transgenic cultivated rice to weedy rice could occur under natural conditions. Copyright © 2011 Society of Chemical Industry     

Insect resistance to Nilaparvata lugens and Cnaphalocrocis medinalis in transgenic indica rice and the inheritance of gna+sbti transgenes

Molecular genetic analysis and insect bioassay of transgenic indica rice 'Zhuxian B' plants carrying snowdrop lectin gene (gna) and soybean trypsin inhibitor gene (sbti) were investigated in detail. PCR, 'dot' blot and PCR-Southern blot analysis showed that both transgenes had been incorporated into the rice genome and transmitted up to R3 progeny in most lines tested. Some transgenic lines exhibited Mendelian segregation, but the other showed either 1:1 (positive: negative for the transgenes) or other aberrant segregation patterns. The segregation patterns of gna gene crossed between R2 and R3 progeny. In half of transgenic R3 lines, gna and sbti transgenes co-segregated. Two independent homozygous lines expressing double transgenes were identified in R3 progeny. Southern blot analysis demonstrated that the copy numbers of integrated gna and sbti transgenes varied from one to ten in different lines. Insect bioassay data showed that most transgenic plants had better resistance to both Nilaparvata lugens (Stahl) and Cnaphalocrocis medinalis (Guenee) than wild-type plants. The insect resistance of transgenic lines increased with the increase in transgene positive ratio in most of the transgenic lines. In all, we obtained nine lines of R3 transgenic plants, including one pure line, which had better resistance to both N lugens and C medinalis than wild-type plants.     

水稻抗白叶枯病基因Xa47(t) a的敲除及抗性鉴定

为鉴定水稻Xa47(t)a基因对白叶枯病的抗性,以水稻种质L214为材料,通过构建针对Xa47(t)a基因的Xa47(t)a-Cas9敲除载体来获得敲除突变体,利用生物信息学技术对突变的Xa47(t)a基因进行突变类型分析,同时采用实时荧光定量PCR(quantitative real-time PCR,qPCR)技术分析突变体中Xa47(t)a及病程相关基因的表达情况,并于水稻孕穗期对突变体及其野生型植株接种11株白叶枯病菌Xanthomonas oryzae pv. oryzae菌株进行抗性鉴定。结果表明,在Xa47(t)a基因的第2外显子区域进行基因编辑后成功获得25株T₀代突变体株系;测序分析发现T₀代突变体株系中有13种不同的突变体类型,其中纯合突变体有3种类型,且突变位点均在靶标位点的11位碱基处缺失1～4个A碱基;氨基酸序列分析发现大部分突变体中Xa47a编码的蛋白翻译会提前终止,由原来的803个氨基酸变为144～166个氨基酸;qPCR分析结果表明突变体中Xa47(t)a及大部分病程相关基因的表达水平显著低于野生型株系;抗性鉴...     

含双病原物诱导启动子无选择标记转基因烟草的获得

Transgenic plants are controversial for their edible and environmental security. Marker-free transgenic plants can be produced by the construction and transformation of plant expression vectors carrying twin T-DNAs. The construction of plant expression vectors harboring twin T-DNAs and two pathogen-inducible promoters was previously reported. These vector plasmids were introduced into tobacco plants and the transgenic tobacco plants were obtained. In this paper we report that T₁ transgenic tobacco seedlings were produced through classical genetics approach. Analysis of the seedlings demonstrated that some of them were marker-free lines. The segregation of exogenous genes in T₁ transgenic tobacco seedlings was tested by using two methods. Firstly, the ability of resistance to kanamycin was analyzed in T₁ transgenic tobacco seedlings from 14 transgenic plant lines. It was found that the segregation ratio of NPTII genes met well with Mendel’s law in 13 transgenic tobacco lines. So it was deduced that NPTII gene was as a single copy integrated into one of the homologous chromosomes. Then the NPTII genes and uidA genes of 130 T₁ transgenic seedlings were detected from the above 13 tobacco lines. The results showed that uidA genes were only detected in 20.77% of the seedlings, NPTII genes were solely detected in 22.31% of the seedlings, but both exogenous genes were in 53.85% of the seedlings. The segregation ratio of the two genes was consistent with the law of independent assortment (9∶3∶3∶1). These results suggested that the selective marker gene had no linkage with the reporter gene and they were segregated independently in the T₁ transgenic tobacco plants. This method, as compared with traditional backcross, is confirmed a more easy and rapid way to eliminate the antibiotic resistance gene used as a selective marker in transgenic plants.     

Rice <Emphasis Type="Italic">OsPBL1</Emphasis> (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) enhanced defense of <Emphasis Type="Italic">Arabidopsis</Emphasis> against <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> DC3000

The receptor-like cytoplasmic kinases (RLCK family VII) are required for plant defense against various pathogens. Previously, OsPBL1 (ORYZA SATIVA ARABIDOPSIS PBS1-LIKE 1) was isolated from rice as a potential RSV (rice stripe virus) resistant factor, but its physiological roles in plant defense are yet to be investigated. In this study, we demonstrated that OsPBL1increased defense against P. syringae in transgenic Arabidopsis. To ascertain the role of OsPBL1 gene in plant defense, OsPBL1 tagged with HA (i.e. Hemagglutinin) was overexpressed in Arabidopsis and examined for the resistance against Pseudomonas syringae pv. tomato DC3000 (i.e. Pst DC3000). At 3 dpi of Pst DC3000, transgenic Arabidopsis lines exhibited the reduced chlorotic lesion and propagation of P. syringae, compared to wild type. Elevated pathogen resistance of transgenic lines was correlated with increased H₂O₂ accumulation and callose deposition on the infected leaves. It was also revealed that expression levels of salicylic acid dependent genes such as PR1, PR2, and PR5, were induced higher in transgenic lines than wild type. Taken together, our data suggested that OsPBL1 exerted the role in defense against pathogen attacks in plant via mainly facilitating salicylic acid dependent pathway.     

表达Harpin蛋白质Hpa1功能片段的转基因小麦对赤霉病的抗性

来自水稻黄单胞菌的harpin蛋白质Hpa1有促进植物生长、诱导植物抗病性的功能,Hpa1序列10~42氨基酸片段（Hpa1_10-42）的活性比全长分子高1.3~7.5倍。为研究Hpa1_10-42在转基因小麦体内表达以后对赤霉病的影响和评价转基因小麦抗病水平与应用潜力,对6个小麦转基因系进行了测定。结果显示,Hpa1_10-42在转基因系T₃~T₅代呈现稳定表达,用禾谷镰刀菌一个分离物进行接种以后,转基因系发生赤霉病的程度较非转基因小麦显著降低,且转基因系T₃~T₅代赤霉病降低的趋势一致。另外,转基因系小麦病害减轻的程度,与在非转基因小麦上使用杀菌剂的效果相当,表明Hpa1_10-42转基因表达对小麦赤霉病有防治作用。     

Mining of rice blast resistance gene Pi54 shows effect of single nucleotide polymorphisms on phenotypic expression of the alleles

The rice blast resistance gene Pi54 (formerly Pi-k ^h ) isolated from indica rice line Tetep confers broad spectrum resistance to different strains of Magnaporthe oryzae in India. In this study, we performed PCR based allele mining for blast resistance gene Pi54 from six cultivated rice lines and eight wild rice species to understand its structural variation and its impact on the phenotypes. Sequence analysis indicates presence of more variation between cultivated and wild species (35–90 %) than variation found among cultivated species (1–20 %). Structural analysis of alleles showed presence of variable number of Open Reading Frames (0–2) principally having point mutations in the leucine rich repeats (LRR) regions. The Ka/Ks ratio of LRR region was >1, which shows the effect of selection pressure at this domain. The Pi54 alleles have 142 polymorphic sites with average nucleotide diversity of 0.04522. The K_a/K_s ratio of coding region ranged from 0 to >1 and Tajima’s D test showed negative as well as Darwinian selection within the alleles, which corresponded well with their phenotypic reaction to M. oryzae. The results obtained in this study shows divergent nature of Pi54 allele in wild species and land races of rice. The resistance alleles identified in this study can be used in effective management of rice blast disease through gene pyramiding.     

Bio-potency of serine proteinase inhibitors from Acacia senegal seeds on digestive proteinases, larval growth and development of Helicoverpa armigera (Hübner)

Proteinase inhibitors (AsPIs) with high activity against serine proteinases were purified from seeds of the tree legume, Acacia senegal by ammonium sulfate precipitation followed by DEAE-Sephadex A-25 column and evaluated against Helicoverpa armigera larvae by in vitro and in vivo methods. The molecular weight of AsPIs was found to be approximately 19.58 ± 1.00 and 21.23 ± 1.00 kDa for PI and 18.16 ± 1.00 kDa for PII on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The AsPIs (5 μg/ml) inhibited approximately 70% of midgut trypsin and 61% of elastase-like chymotrypsin. In vitro studies showed that AsPIs have remarkable inhibitory activity towards total gut proteolytic enzymes followed by trypsin and chymotrypsin. The IC₅₀ of AsPIs for midgut trypsin was 0.1 μg/ml and for chymotrypsin was 2.0 μg/ml. The inhibition of gut proteinase enzymes was of the non-competitive type. In larval feeding studies, AsPIs were found to retard growth and development of H. armigera and also affects the fecundity of the pest. The results advocate the use of AsPIs in transgenic technology to develop plant resistance to H. armigera.     

Comparison of the inhibition effect of different anticoagulants on vitamin K epoxide reductase activity from warfarin-susceptible and resistant rat

Anti-vitamin K drugs are widely used as anticoagulant in human thromboembolic diseases. Similar compounds have also been used as rodenticides to control rodent population since 1950s. Massive use of first generation anticoagulants, especially warfarin, has lead to the development of genetic resistances in rodents. Similar resistances have been reported in human. In both cases, polymorphisms in VKORC1 (Vitamin K epoxide reductase subunit 1), the subunit 1 of the VKOR (Vitamin K epoxide reductase) complex, were involved. In rats (Rattus norvegicus), the Y139F mutation confers a high degree of resistance to warfarin. Little is known about the in vitro consequences of Y139F mutation on inhibitory effect of different anticoagulants available. A warfarin-susceptible and a warfarin-resistant Y139F strain of wild rats (Rattus norvegicus) are maintained in enclosures of the Lyon College of Veterinary Medicine (France). Using liver microsomes from susceptible or resistant rats, we studied inhibition parameters by warfarin (K_i = 0.72 ± 0.1 μM; 29 ± 4.1 μM), chlorophacinone (K_i = 0.08 ± 0.01 μM; 1.6 ± 0.1 μM), diphacinone (K_i = 0.07 ± 0.01 μM; 5.0 ± 0.8 μM), coumachlor (K_i = 0.12 ± 0.02 μM; 1.9 ± 0.2 μM), coumatetralyl (K_i = 0.13 ± 0.02 μM; 3.1 ± 0.4 μM), difenacoum (K_i = 0.07 ± 0.01 μM; 0.26 ± 0.02 μM), bromadiolone (K_i = 0.13 ± 0.02 μM; 0.91 ± 0.07 μM), and brodifacoum (K_i = 0.04 ± 0.01 μM; 0.09 ± 0.01 μM) on VKOR activity. Analysis of the results leads us to highlight different anticoagulant structural elements, which influence inhibition parameters in both susceptible and Y139F resistant rats.