Expression of anti-neuroexcitation peptide III of scorpion <Emphasis Type="Italic">Buthus martensii</Emphasis> Karsch BmK ANEP III in plants

Anti-neuroexcitation peptide III of Buthus martensii Karsch (BmK ANEP III) has better anti-epileptic and anticonvulsant effects in the test animal models. The present study is aimed at developing transgenic tomato and tobacco lines overproducing the ANEP III protein. Using the molecular cloning technique, the plant expression vector pBI-ANEP III was constructed successfully. The ANEP III expression cassette included a double CaMV 35S promoter with Ω enhancers, the ANEP III gene with the Kozak sequence, the ER retention signal and the NOS terminator. Recombinant plasmids were transferred into Agrobacterium tumefaciens EHA105 by freeze-thaw transformation methods. By the Agrobacterium-mediated leaf disc transformation method, tobacco (Nicotiana tabacum) and tomato (Lycopersicum esculentum) lines were transformed. Transformants were screened and confirmed by PCR, RT-PCR and western blotting analysis. It was demonstrated that the ANEP III gene was successfully expressed in the genomic DNA of transgenic plants. The ANEP III protein was detected by immunofluorescence analysis, and the results confirmed the high amount of ANEP III protein, being 0.81 and 1.08% of total soluble proteins in transgenic tobacco and tomato. The study of plants with high expression levels of ANEP III has an important theoretical and practical significance and provides valuable information for establishing a new, economical and effective system for industrial protein production.     

豌豆过氧化氢酶在烟草叶绿体中的过量表达提高了植物的抗逆性

通过构建融合番茄RuBP羧化酶小亚基转运肽基因(rbcS-3)和CAT基因编码阅读框(ORF)的双元表达载体,采用农杆菌介导的叶圆盘转化法将融合基因转入烟草,使其能够定向导入叶绿体中发挥作用。在含有50mg/L潮霉素的培养基上筛选获得转CAT烟草30多个株系,并对其进行了分子生物学的验证和生理指标的检测。对获得的抗性植株用PCR、RT-PCR、植株总蛋白Western blot和叶绿体蛋白Western blot分析表明,目的基因已经整合到烟草基因组中,并能正常表达,且在叶绿体rbcS-3转运肽的作用下能定向进入叶绿体中。对转基因植株生理指标的检测发现,在20% PEG₆₀₀₀模拟干旱条件下,野生型烟草的相对电导率提高幅度为43.4%,而转CAT植株的相对电导率仅提高8.8%,表明在干旱胁迫下转CAT烟草的质膜透性小于野生型烟草;经20% PEG₆₀₀₀处理后,野生型和转CAT基因烟草的叶绿素含量都下降,下降幅度分别为68.0%和20.4%;另外,经20% PEG₆₀₀₀处理的野生型烟草叶片的Fv/Fm下降幅度为5.3%,而转CAT基因烟草叶片的Fv/Fm下降幅度0.9%,这些结果表明,在叶绿体中过量表达CAT对干旱胁迫下的细胞质膜、叶绿素和PSⅡ具有一定的保护作用。此外,经150 μmol/L百草枯处理后发现,处理3h后,野生型烟草和转CAT烟草的相对电导率分别比对照提高67.9%和13.5%,而野生型和转CAT烟草的Fv/Fm都下降,降幅分别为23.7%和3.9%,这表明在百草枯氧化胁迫下转CAT烟草的质膜和PSⅡ的损伤程度都小于野生型烟草。总之,豌豆CAT基因在烟草叶绿体中过量表达,提高了转基因烟草的抗旱性和抗氧化性。     

Expression of bioactive human M-CSF soluble receptor in transgenic tobacco plants

The cDNA encoding N-terminal three immunoglobin-like domains of human M-CSFR was linked to His-tag and endoplasmic reticulum retention sequence (KDEL) before being inserted into the genome of tobacco plant, Nicotiana tabacum cv. NC-89, by Agrobacterium tumefaciens-mediated transformation. The insertion and expression of target gene were confirmed by PCR, ELISA, and Western blot. The recombinant M-CSFsR reached a maximum expression level of 1.92% of total soluble protein in transgenic tobacco plant leaf tissues. The recombinant M-CSFsR could be purified through a one-step IMAC process and its bioactivity was confirmed by the inhibition of colony formation of J6-1 cells. The results suggested that we successfully expressed a high level of bioactive human M-CSFsR in tobacco plants.     

Expression of alfalfa mosaic virus coat protein gene confers cross-protection in transgenic tobacco and tomato plants

A chimeric gene encoding the alfalfa mosaic virus (AlMV) coat protein was constructed and introduced into tobacco and tomato plants using Ti plasmid-derived plant transformation vectors. The progeny of the self-fertilized transgenic plants were significantly delayed in symptom development and in some cases completely escaped infection after inoculated with AlMV. The inoculated leaves of the transgenic plants had significantly reduced numbers of lesions and accumulated substantially lower amounts of coat protein due to virus replication than the control plants. These results show that high level expression of the chimeric viral coat protein gene confers protection against AlMV, which differs from other plant viruses in morphology, genome structure, gene expression strategy and early steps in viral replication. Based on our results with AlMV and those reported earlier for tobacco mosaic virus, it appears that genetically engineered cross-protection may be a general method for preventing viral disease in plants.     

过量表达腺苷甲硫氨酸合成酶基因能提高转基因烟草中多聚胺的生物合成

以土壤农杆菌介导的转化方法将盐地碱蓬的腺苷甲硫氨酸合成酶cDNA（SsSAMS2）转化到烟草K326中。PCR分析的结果表明,SsSAMS2整合进K326的基因组内。共筛选到8个转基因纯合品系,分析其中3个品系（ST8．9、ST14—2、ST3．5）基因表达和多聚胺含量的结果表明,SsSAMS2可在转基因烟草中表达,转基因烟草中的多聚胺含量明显高于野生型烟草。这些结果表明,腺苷甲硫氨酸合成酶基因已在转基因烟草中过量表达并导致多聚胺含量提高。     

Overexpression of suadea salsa <Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase gene promotes salt tolerance in transgenic tobacco

The suadea salsa full-length S-adenosylmethionine synthetase (SsSAMS2) was introduced into tobacco (Nicotiana tabacum L.) by Agrobacterium tumefaciens-mediated transformation. The gene transformation and expression in tobacco were confirmed by PCR, RT-PCR and Northern blotting analysis. Several transgenic lines (ST lines) overexpressing SsSAMS2 gene under the control of cauliflower mosaic virus 35S promoter showed more seeds number and weight, and accumulated higher free total polyamines (PAs) than wild-type plants (WT lines) and transformants with blank vector (BT lines). Salt stress-induced damage was attenuated in these transgenic plants, in the symptom of maintaining higher photosynthetic rate and biomass. These results that the transgenic plants overexpressing suadea salsa SAMS2 are more tolerant to salt stress than wild-type plants suggest that PAs may play an important role in contributing salt tolerance to plants.     

铁皮石斛DoSMT2基因的功能分析

为了揭示铁皮石斛(Dendrobium officinale)甾醇C-24甲基转移酶2基因(DoSMT2)在甾醇代谢过程的功能,该研究通过根癌农杆菌介导法将来源于铁皮石斛的DoSMT2基因转化烟草(Nicotiana tabacum),并采用qRT-PCR技术检测DoSMT2基因在转基因烟草叶片中的表达,采用气相色谱质谱法分析菜油甾醇和谷甾醇的含量。结果显示:(1)成功获得DoSMT2基因的开放阅读框(1 119 bp),并成功构建正义植物表达载体质粒pCXSN-DoSMT2,经农杆菌介导的烟草叶盘转化法转化烟草并鉴定,获得4株阳性转基因烟草植株。(2)Southern blot结果表明,4株转基因烟草植株都有1条杂交信号带,而非转基因烟草植株没有,说明外源DoSMT2基因都以单拷贝整合到4株转基因烟草基因组中。(3)qRT-PCR检测显示,非转基因烟草未检测到外源DoSMT2基因的表达,4株转基因烟草都能检测到DoSMT2基因的表达,且表达水平差异极显著,各株系表达量高低依次为P3P1P2(P4)。(4)气相色谱质谱分析显示,转DoSMT2基因烟草叶片的菜油甾醇含量均极显著低于非转基因烟草叶片,而谷甾醇含量均极显著高于非转基因烟草叶片。研究表明,DoSMT2具有催化24-亚甲基胆甾烯醇转化形成24-亚乙基胆甾烯醇活性。     

口蹄疫病毒VP1基因在番茄中的表达及转基因番茄对豚鼠诱导免疫保护

构建克隆有O型口蹄疫病毒China99株VP1基因的植物双元表达载体pBin438/VP1。通过农杆菌介导法转化番茄子叶,经卡那霉素抗性筛选,获得60株抗性植株。对抗性植株分别做PCR、RT-PCR检测目的基因的整合与转录,ELISA筛选约40%的卡那抗性植株阳性,分别提取两株ELISA和Western blot检测阳性的转基因番茄叶片蛋白与弗氏佐剂乳化,于0、15、30d经肌肉途径免疫豚鼠,第三次免疫后28d用100ID50的同源强毒攻击,根据豚鼠抗体水平的消长动态和免疫豚鼠抗强毒攻击的保护率进行转基因植物疫苗免疫原性的评估。结果表明,双元表达载体pBin438/VP1构建正确,PCR、RT-PCR结果证实口蹄疫病毒VP1基因已整合到番茄基因组并在转录水平表达,ELISA和Western blot检测重组蛋白能够与FMDV阳性血清反应。转基因番茄第三次免疫豚鼠后21d血清效价最高可达1∶64,攻毒后两组免疫豚鼠保护率分别达80%和40%,证明转基因番茄表达的VP1蛋白具有良好的免疫原性。     

Improvement of freezing tolerance in transgenic tobacco leaves by expressing the hiC6 gene

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at -1, -3, and -4 degrees C, with the exception that their freezing temperature was -2 degrees C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance tests at -1 - -4 degrees C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at -3 degrees C.     

Improvement of Freezing Tolerance in Transgenic Tobacco Leaves by Expressing the hiC6 Gene

A cryoprotective protein, HIC6, was expressed transgenically in tobacco, a cold-sensitive plant, and the localization of the protein within the cell as well as freezing tolerance of the transgenic tobacco was investigated. For constitutive expression of HIC6 in tobacco, its corresponding gene was subcloned into pBI121. Through the transformation with pBI121/hiC6, fifteen transgenic tobacco lines were acquired, out of which twelve lines expressed the HIC6 protein. None of the transgenic tobacco lines, however, showed significant differences in freezing tolerance from the control plants (wild-type and transformed with pBI121) at ?1, ?3, and ?4°C, with the exception that their freezing temperature was ?2°C. In order to increase the accumulation level of HIC6, pBE2113 with a stronger promoter was used. Eight lines expressed the protein out of thirteen lines transformed with pBE2113/hiC6. The accumulation levels of the protein were clearly higher in the tobacco plants transformed with pBE2113/hiC6 than in those with pBI121/hiC6. The HIC6 protein seemed to be localized in mitochondria of the transgenic tobacco plants. Freezing-tolerance test at ?1 - ?4°C showed that the degree of electrolyte leakage was significantly lower in the plants with pBE2113/hiC6 than in the control plants. A leaf browning observation also showed that high accumulation of HIC6 significantly suppressed injury caused by freezing to the transgenic tobacco at ?3°C.     

Stable transformation of<Emphasis Type="Italic"> Theobroma cacao</Emphasis> L. and influence of matrix attachment regions on GFP expression

We describe a protocol for Agrobacterium-mediated genetic transformation of Theobroma cacao L. using cotyledonary explants from primary somatic embryos (SEs) and A. tumefaciens strain AGL1. Transgenic plants carrying the visible marker, gene green fluorescent protein ( EGFP), the selectable marker gene neomycin phosphotransferase II ( NPTII), the class I chitinase gene from cacao ( Chi), and tobacco nuclear matrix attachment regions (MARs) in different combinations were successfully produced via regeneration of secondary SEs. The presence of the Chi gene or MARs did not influence the number of transgenic plants produced compared to the marker genes alone. However, the inclusion of MARs contributed to increased mean GFP expression in the population of transgenics. Additionally, the presence of MARs reduced the occurrence of gene silencing and stabilized high levels of GFP expression in lines of transgenic plants multiplied via reiterative somatic embryogenesis. Ninety-four transgenic plants were acclimated in a greenhouse and grown to maturity. Detailed growth analysis indicated that there were no differences in various growth parameters between transgenic and non-transgenic SE-derived plants. Seeds produced from two genetic crosses with one of the transgenic lines were analyzed for EGFP expression-a near-perfect 1:1 segregation was observed, indicating that this line resulted from the insertion of a single locus of T-DNA.     

Genetic transformation of Rangpur lime (Citrus limonia osbeck) with thebO (bacterio-opsin) genen and its initial evaluation forPhytophthora nicotianae resistance

Transgenic plants expressing the bacterio-opsin (bO) gene can spontaneously activate programmed cell death (ped) and may enhance broad-spectrum pathogen resistance by activating an intrinsic defense pathway in plant species such as tobacco and potato. In this work, we produced transgenic Rangpur lime plants with thebO gene, viaAgrobacterium tumefaciens-mediated transformation, and evaluated these plants forPhytophthora nicotianae resistance. Two transgenic lines were successfully regenerated and transformation was confirmed by GUS activity assay, PCR analysis, Southern, Northern and Western blot analyses, in addition to detecting the expressed bO protein by an immunological approach. Evaluation forPhytophthora nicotianae resistance was carried out by plant inoculations with the pathogen and quantification of the affected area. One of the two transgenic lines showed greater tolerance to the fungal pathogen as compared to the control, with significantly smaller stem lesions after pathogen challenge. This increase in pathogen tolerance is correlated with a significantly higher level of transgene expression in this line when compared with the other transgenic line. This is the first report of the introduction of a potentially important gene into Rangpur lime to provide novel pathogen tolerance.     

Overexpression of tomato <Emphasis Type="Italic">tAPX</Emphasis> gene in tobacco improves tolerance to high or low temperature stress

In order to investigate the function of chloroplast ascorbate peroxidase under temperature stress, the thylakoid-bound ascorbate peroxidase gene from tomato leaf (TtAPX) was introduced into tobacco. Transformants were selected for their ability to grow on medium containing kanamycin. RNA gel blot analysis confirmed that TtAPX in tomato was induced by chilling or heat stress. Over-expression of TtAPX in tobacco improved seed germination under temperature stress. Two transgenic tobacco lines showed higher ascorbate peroxidase activity, accumulated less hydrogen peroxide and malondialdehyde than wild type plants under stress condition. The photochemical efficiency of photosystem 2 in the transgenic lines was distinctly higher than that of wild type plants under chilling and heat stresses. Results indicated that the over-expression of TtAPX enhanced tolerance to temperature stress in transgenic tobacco plants.     

Assessment of the utility of the tomato fruit-specific E8 promoter for driving vaccine antigen expression

To assess the utility of the tomato fruit-specific E8 gene's promoter for driving vaccine antigen expression in plant, the 2.2 kb and 1.1 kb E8 promoters were isolated and sequenced from Lycopersicon esculentum cv. Jinfeng #1. The 1.1 kb promoter was fused to vaccine antigen HBsAg M gene for the transfer to Nicotiana tabacum, and the CaMV 35S promoter was used for comparison. Cholera toxin B (ctb) gene under the control of the 1.1 kb promoter was transformed into both N. tabacum and L. esculentum. Southern blot hybridization confirmed the stable integration of the target genes into the tomato and tobacco genomes. ELISA assay showed that the expression product of HBsAg M gene under the control of the 1.1 kb E8 promoter could not be detected in transgenic tobacco tissues such as leaves, flowers, and seeds. In contrast, the expression of HBsAg M gene driven by CaMV 35S promoter could be detected in transgenic tobacco. ELISA assay for CTB proved that the 1.1 kb E8 promoter was able to direct the expression of exotic gene in ripe fruits of transgenic tomato, but expression was absent in leaf, flower, and unripe fruit of tomato, and CTB protein was not detected in transgenic tobacco tissues such as leaves, flowers, and seeds when the gene was under the control of the 1.1 kb E8 promoter. The results indicated that the E8 promoter acted not only in an organ-specific, but also in a species-specific fashion in plant transformation.