Fast,simple,efficient Agrobacterium rhizogenes-mediated transformation system to non-heading Chinese cabbage with transgenic roots

Non-heading Chinese cabbage, a variety of Brassica campestris, is an important vegetable crop in the Yangtze River Basin of China. However,the immaturity of its stable transformation system and its low transformation efficiency limit gene function research on non-heading Chinese cabbage. Agrobacterium rhizogenes-mediated(ARM) transgenic technology is a rapid and effective transformation method that has not yet been established for non-heading Chinese cabbage plants. Here, we optimized conventional ARM approaches(one-step and two-step transformation methods) suitable for living non-heading Chinese cabbage plants in nonsterile environments. Transgenic roots in composite non-heading Chinese cabbage plants were identified using phenotypic detection, fluorescence observation, and PCR analysis. The transformation efficiency of a two-step method on four five-day-old non-heading Chinese cabbage seedlings(Suzhouqing, Huangmeigui, Wuyueman, and Sijiu Caixin) was 43.33%-51.09%, whereas using the stout hypocotyl resulted in a transformation efficiency of 54.88% for the 30-day-old Sijiu Caixin.The one-step method outperformed the two-step method;the transformation efficiency of different varieties was above 60%, and both methods can be used to obtain transgenic roots for functional studies within one month. Finally, optimized ARM transformation methods can easily,quickly, and effectively produce composite non-heading Chinese cabbage plants with transgenic roots, providing a reliable foundation for gene function research and non-heading Chinese cabbage genetic improvement breeding.

Application of transgenic mice to the molecular pathogenesis of cataract

One of the most prevalent disorders that cause blindness worldwide is cataract,and its essence is the visual disorder caused by the opacity of the lens.The significant degree of variation in cataracts and the fact that a variety of factors can impact a patient’s lens transparency make it especially crucial to investigate the pathogenesis of cataracts at the molecular level.It has been found that more than 60 genes are linked to the formation of cataracts,and the construction of a transgenic mouse model of cataract similar to the selection of human lens clouding due to a variety of causes has become an important means of studying the pathogenesis of cataract.Therefore,the research on the application of transgenic mice to the molecular pathogenesis of cataracts will be the main topic of this review of the literature.

Barley chitinase genes expression revamp resistance against whitefly (Bemisia Tabaci) in transgenic cotton (Gossypium hirsutum L.)

Background Chitinase is an enzyme that hydrolyzes chitin,a major component of the exoskeleton of insects,including plant pests like whiteflies.The present study aimed to investigate the expression of chemically synthesized barley ch1 and chi2 genes in cotton(Gossypium hirsutum)through Agrobacterium-mediated transformation.Fifty-five putative transgenic cotton plants were obtained,out of which fifteen plants successfully survived and were shifted to the field.Using gene-specific primers,amplification of 447 bp and 401 bp fragments confirmed the presence of the ch1 and chi2 genes in five transgenic cotton plants of the T0 generation.These five plants were further evalu-ated for their mRNA expression levels.The T0 transgenic cotton plants with the highest mRNA expression level and better yield performance in field,were selected to raise their subsequent progenies.Results The T1 cotton plants showed the highest mRNA expression levels of 3.5-fold in P10(2)for the ch1 gene and 3.7-fold in P2(1)for the chi2 gene.Fluorescent in situ hybridization(FISH)confirmed a single copy number of ch1 and chi2(hemizygous)on chromosome no.6.Furthermore,the efficacy of transgenes on whitefly was evaluated through an insect bioassay,where after 96 h of infestation,mortality rates of whitefly were calculated to be 78%–80%in transgenic cotton plants.The number of eggs on transgenic cotton plants were calculated to be 0.1%–0.12 per plant compared with the non-transgenic plants where egg number was calculated to be 0.90–1.00 per plant.Conclusion Based on these findings,it can be concluded that the chemically synthesized barley chitinase genes(ch1 and chi2)have the potential to be effective against insects with chitin exoskeletons,including whiteflies.The transgenic cotton plants expressing these genes showed increased resistance to whiteflies,resulting in reduced egg numbers and higher mortality rates.

Transfer of Lysine-rich Protein Gene into Rice and Production of Fertile Transgenic Plants

Lysine-rich protein gene (lys) was cloned from Psophocarpus tetragonolobus (L.) DC. A plant expression plasmid was constructed and lys gene was under the control of maize ubiquitin promoter which is the highest efficient monocotyledon promoter. The plasmid was introduced into rice embryogenic calli by microprojectile bombardment. The regenerated fertile plants were obtained by effective selection for hygromycin B resistance. Genomic PCR and Southern blotting analyses showed that the lys gene has been integrated into rice genome. Simultaneously, the results of GUS histochemical assay demonstrated that gus report gene is also expressed in leaves, stems and roots of the transgenic rice plants. Data analysis showed that lysine content in most of the 11 transgenic plants is differently improved, and in one of them increased by 16.04%.

Resistance Analysis of the Binary Insect-resistant Transgenic Soybean to Heliothis viriplaca

[Objective] The aim of the research was to analyze the resistance of binary insect-resistant transgenic soybean to Heliothis viriplaca.[Method]In this experiment, resistance analysis of the stabilized binary insect-resistant transgenic soybean to Heliothis viriplaca was conducted in lab and in field conditions.[Result] The results indicated that the leaves of insect-resistant transgenic soybeans T5-150 and T5-195 showed lighter damage than those of non-transgenic soybeans. Meanwhile, the Heliothis viriplaca larvae fed on leaves of these two transgenic soybeans were characterized by less leaf consumption, shortening survival day, slower development and less pupation.[Conclusion]It was concluded that insect-resistance of transgenic soybean to Heliothis viriplaca was increased dramatically and the research provided a reference for selecting binary insect-resistant transgenic soybean to Heliothis viriplaca.

Alterations of Root and Fiber in Transgenic Cotton Plants with Chimeric Ph/P-ipt Gene Expression

The seed_specific phaseolin promoter (Ph/P) was fused to an ipt gene, then was cloned to a plant expression vector containing a gus gene driven by a 35S promoter. Cotton (Gossypium hirsutum L.) plants were transformed through pollen tube pathway methods. After seed germination, histochemical staining of the roots demonstrated that 32 GUS positive plants were obtained and three of which contained the chimeric Ph/P_ ipt transgene as confirmed by PCR analysis. An immunosorbent assay showed that two of the three transgenic cotton lines contained higher levels of zeatin equivalents in seeds than the control. Seedling development of these two transgenic lines differed from the control in a reduction of the shoot growth, showing a stunted phenotype as expected, but a surprisingly developed root system with a 3-4 fold fast_growing lateral roots. In addition, fibers (seed_hairs) of the two transgenic cotton lines were considerably shorter than those of the control. These results indicate that genetic engineering may be used to manipulate the development of cotton plants, particularly cotton fibers.

Cloning of cDNA Encoding Choline Monooxygenase from Suaeda liaotungensis and Salt Tolerance of Transgenic Tobacco

Betaine is a very effective osmoprotectant found in many organisms. In high plants, betaine is synthesized by oxidation of choline in two sequential steps: choline-->betaine aldehyde-->betaine. The first step is catalyzed by choline monooxygenase (CMO). In this study, the full-length CMO cDNA (1 820 bp) was cloned from halophyte Suaeda liaotungensis Kitag by RT-PCR and RACE. It included a 123 bp 5' UTR, a 368 bp 3' UTR and a 1 329 bp open reading frame encoding a 442-amino-acid polypeptide with 77%, 72% and 74% sequence identity compared to CMOs from spinach, sugar beet and Atriplex hortensis, respectively. The CMO open reading frame (ORF) was cloned and the plant expression vector pBI121-CMO was constructed. It was transferred into tobacco ( Nicotiana tabacum L. ev. 89) via Agrobacterium mediation. PCR and Southern blotting analysis showed that the CMO gene was integrated into tobacco genome. Transgenic tobacco plants contained higher amount of betaine than that of control plants and were able to survive on MS medium containing 250 mmol/L NaCl. Relative electronic conductivity demonstrated less membrane damage in transgenic plants as in the wild type.

Photosynthetic Characteristics of Transgenic Rice Plants Overexpressing Maize Phosphoenopyruvate Carboxylase

用转PEPC、PPDK、NADP_ME、PEPC +PPDK 双基因水稻 (OryzasativaL .)及原种为材料 ,以光合酶活性、饱和光合速率及PSⅡ光化学效率 (Fv/Fm)为指标 ,研究了转PEPC基因水稻的光合生理特征。结果如下 :转PEPC基因水稻PEPC活性比原种提高 2 0倍 ;饱和光合速率比原种高 5 5 % ;用高光强或人工光氧化剂甲基紫精 (MV)处理后 ,与原种相比 ,转PEPC基因水稻光化学效率下降较少 ,证明其耐光抑制、耐光氧化能力增强 ,测定其光合日变化看出 :在 1d中不同时间 ,转PEPC基因水稻的光合速率均高于原种 ,且与PEPC活性的日变化有相似的趋势。上述结果为转PEPC基因水稻的生理机制和育种研究提供了依据和途径。

Human Pro-insulin Transgenic Calf Derived from Somatic Cell Nuclear Transfer

The current study was undertaken to evaluate the possibility of producing a human pro-insulin transgenic cow by means of somatic cell nuclear transfer （SCNT）. A double selection system, Neomycin resistance （Neo^r） gene and enhanced green fluorescent protein （EGFP） gene linked through an inner ribosomal entry site （IRES） sequence directed by a Cytomegalovirus （CMV） promoter, was used for enrichment and selection of the transgenic cells and preimplantation embryos. Transgenes were introduced into bovine fetal fibroblast cells （BFF） cultured in vitro through electroporation （900 V/cm, 5 ms）. Transgenic bovine fibroblast cells （TBF） were enriched through addition of G418 in culture medium （800 μg/mL）. Before being used as a nuclear donor, the TBF cells were either cultured in normal conditions （10% FBS） or treated with serum starvation （0.5% FBS for 2-4 days） followed by 10 hours recovery for G1 phase synchronization. Transgenic cloned embryos were produced through GFP-expressing cell selection and SCNT. The results were the percentage of blastocyst development following SCNT was lower using TBF than BFF cells （23.2% VS 35.2%, P 〈 0.05）. No difference in the percentage of cloned blastocysts between the two groups of transgenic nuclear donor of normal and starvation cultures were observed （23.2% VS 18.9%, P 〉 0.05）. Two to four GFP-expressing blastocysts were transferred into the uterus of each synchronised recipient. One pregnancy from of seven recipients （21 embryos） was confirmed by rectum palpation 60 days after embryo transfer and one recipient has given birth to a calf at term. PCR and DNA sequencing analysis confirmed that the calf was produced using human proinsulin transgenic animal.

Aphid-resistant Transgenic Tobacco Plants Expressing Modified gna Gene

A gene sequence coding for the precursor of Galanthus nivalis agglutinin (GNA) was modified by site-directed mutagenesis to change very low usage bias codons to higher usage bias ones for improvement of the gene expression in transgenic tobacco (Nicotiana tabacum L.) plants. Results from Western blot analysis of some of the transgenic tobacco plants showed that the expression level of GNA in plants transformed with the modified gene GNA34m reached 0.25% of total soluble proteins, while that of the GNA34 gene transgenic plants was 0.17%. Since the GNA expression level increased, the aphid resistance of GNA34m transgenic plants were also enhanced significantly as judged by a 71.0% aphid population inhibition in insect bioassay of GNA34m transformed plants and 63.7% for the plants transformed with the natural GNA34 gene.

Novel Halophyte EREBP/AP2-type DNA Binding Protein Improves Salt Tolerance in Transgenic Tobacco

EREBP/AP2-type proteins are members of a large DNA binding protein (DBP) family found in plants. Some members like APETALA2 and AtDREB/CBF can regulate flower development and response to environmental stresses, respectively. To characterize transcription factors involved in plant responses to salt stress, we constructed cDNA library from salt-treated halophyte (Atriplex hortensis) and isolated a novel gene encoding EREBP/AP2-type protein from this library. This cDNA contained an ORF of 723 bp and a long 3'-Untranslated-Region (UTR) of 655 bp. The deduced amino acid sequence showed one conserved DNA binding domain of EREBP/AP2, thus the corresponding gene was named AhDREB1 with a calculated molecular mass of 26.1 kD. AhDREB1 under the control of CaMV 35S promoter was then transformed into tobacco and nine independent transgenic lines were obtained and subjected to long term salt stress. The results suggested that overexpression of AhDREB1 improved the salt tolerance in transgenic tobacco through functioning as a regulatory molecule in response to salt stress. Analysis of Arabidopsis genome in database resulted in dozens of EREBP/AP2-type homologous proteins, of which seven members showed high similarity to AhDREB1. Secondary structure analysis predicted similar arrangement of a-helix in their DNA binding domains.

Insect Resistance of Different Tissues of Transgenic Cotton to Spodoptera exigua(Hbner)

[Objective] The aim was to learn the resistance of different tissues and organs of transgenic cotton to Spodoptera exigua （Hbner）. [Method] Flowers,the 1st,the 3rd,the 6th and the 14th leaves from the top of 33B,GK12 and SGK321 were used to feed S. exigua neonates respectively. Survival larvae and dead ones were counted on the 3rd,the 7th,the 10th,the 16th and the 19th day; meanwhile,the pupae amount was recorded,and the pupae weight was measured at the 24th h after pupation. [Result] The survival curves,pupation rates and pupae weights of S. exigua feeding on different tissues of transgenic cotton were not significantly different from those of S. exigua feeding on the corresponding tissues of conventional cotton; pupation rate of S. exigua feeding on different leaves of the same cotton variety were not significantly different from each other,but all higher than that of S. exigua feeding on the flowers of that cotton; and there were no differences among pupation weights of S. exigua feeding on different leaves or flowers of the same cotton variety. [Conclusion] Transgenic cotton showed weak resistance to S. exigua. Hence,in the transgenic cotton fields,more attention should be paid to occurrence trend of S. exigua and its control.

Increasing Accumulation Level of Foreign Protein in Transgenic Plants Through Protein Targeting

Targeting of the synthesized polypeptide in the cells is an important research field in modern cell biology. Cowpea trypsin inhibitor (cpti) gene has been modified and a fusion protein gene (sck) was produced by fusing a signal peptide sequence at cpti 5' end and an endoplasm reticulum (ER) retention signal peptide at cpti3' end respectively. The signal peptide can direct the newly synthesized polypeptide into ER, while ER retention signal can make the protein retained in the ER and its derivative protein body. ELISA test indicated that the accumulation level of foreign CpTI protein in sck transgenic tobacco (Nicotiana tabacum L.) was two times higher than cpti transgenic tobaccos and some individuals were four times higher. At the same time, sck transgenic tobacco has a high resistance to Lepidoptera pest due to the increased accumulation level of foreign CpTI protein. The strategy of foreign protein targeting can be used to increase the accumulation level of foreign protein in transgenic plants and can be widely applied to other related research field in plant genetic engineering.

Characteristics of CO_2 Exchange and Chlorophyll Fluorescence of Transgenic Rice with C_4 Genes

The responses of photosynthesis of phosphoenopyruvate carboxylase (PEPC), pyrurate dikinase (PPDK), NADP-malic enzyme (NADP-ME) and PPDK+PEPC transgenic rice (Oryza saltiva L.) plant to light, temperature, CO 2 and the characteristics of chlorophyll fluorescence under photoinhibition conditions were studied. The results were as follows: 1. The light-saturated photosynthetic rates of transgenic rice plants were higher than that of wild type, in which the light-saturated point of PEPC and PPDK+PEPC transgenic rice plants was 200 μmol·m -2·s -1 higher than that of untransformed rice and the light-saturated photosynthetic rates were 51.6% and 58.5% respectively. The carboxylation efficiency of PEPC transgenic rice plant increased by 49.3% and the CO 2 compensation point decreased by 26.2% than that of untransformed rice. Under high temperature (35 ℃), the photosynthetic rate of PEPC transgenic rice plant was higher over 17.5% than that of untransformed rice. 2. On the 8th day after photoinhibition treatment, the PSⅡ photochemical efficiency (F v/F m) and photochemical quenching (qP) of PEPC and PPDK+PEPC transgenic rice plants decreased by about 20%-30% while the non-photochemical quenching (qN) increased by approximately 30%. But F v/F m and qP of untransformed rice decreased by over 50% while qN increased by less than 10%. The result suggested that transgenic rice plants were more tolerant to photoinhibition.

Effects of two kinds of transgenic poplar on protective enzymes system in the midgut of larvae of American white moth

The leaves of Bt (Bacillus thuringiensis) transgenic poplar (Populus nigra L.) and CpTI (Cowpea trypsin inhibitor) transgenic poplar ((P. tomentosa×P. bolleana)×P. Tomentosa) were taken to feed the 4th-5th-instar larvae of American white moth (Hyphantria cunea (Drury)) for determination of the activities of the protective enzyme system inside larvae’s body. The physiological and biochemical effects of the transgenic poplars on the larvae were studied. The results showed that the two kinds of transgenic poplars had similar effects on the protective enzyme system in the midgut of larvae. The activities of superoxide dismutase, catalase, and peroxidase in midgut of the larvae increased gradually, reached the highest value at a certain time, and then decreased suddenly. For the larvae that were fed with the leaves of Bt transgenic poplar, the peak value of superoxide dismutase and catalase presented at the time of 24-h feeding, while the peak of peroxidase took place at the time of 12-h feeding. The activities of these protective enzymes for the larvae that were fed with leaves of CpTI transgenic poplar peaked 12 h later than that of those fed with leaves of Bt transgenic poplar. The comparison of activities of the protective enzymes was also carried out between the larvae with different levels of intoxication. It was found that the activities of protective enzyme of the seriously intoxicant larvae were higher than that of the lightly intoxicant larvae. This difference was more obvious in the group treated with CpTI transgenic poplar.

Effect of Transgenic Bt plus CpTI Cotton on Carboxylesterase and Acetylcholinesterase of Cotton Aphid Aphis gossypii

[Objective] The research aimed to assess the effect of transgenic Bt plus CpTI cotton variety SGK321 on carboxylesterase and acetylcholinesterase of cotton aphid Aphis gossypii and provide theoretical basis for studying the biosafety of transgenic cotton.[Method] Cotton aphids were fed with SGK321 and Shiyuan321（normal parental varieties） for over 40 generations.Enzyme activities were compared between cotton aphids feeding on SGK321 for 1,2,3,41,42 and 43 generations with those on Shiyuan321.[Result] The carboxylesterase activity of cotton aphids feeding on SGK321 for 1 generation was significantly higher than those feeding on Shiyuan321.Acetylcholinesterase activity of cotton aphids feeding on SGK321 for 1,2 and 3 generations were significantly higher than those feeding on Shiyuan321 in the same generation.But there was no significant difference of enzyme activity between cotton aphids feeding on SGK321 for a long term and those feeding on parental cotton.[Conclusion] The cotton aphid that feeding on transgenic Bt plus CpTI cotton SGK321 for a long time has adaptivity to SGK321 by regulating the detoxifying enzyme.

Isolation of a Genomic DNA for Gastrodia Antifungal Protein and Analyses of Its Promoter in Transgenic Tobacco

A genomic DNA containing 5'-upstream region and complete open reading frame of a Gastrodia antifungal protein was isolated by screening of a genomic library from Gastrodia elata B1. To investigate the promoter activity, the 5'-flanking region - 1 157 lip upstream from the putative transcription start site was fused to the coding sequence of beta-glucuronidase (GUS) gene and transformed into Nicotiana tabacum. The strongest GUS activity was detected in the roots of transgenic tobacco, followed by stems. The leaves only showed a low GUS activity. Furthermore, the promoter established inducible expression pattern in transgenic tobacco upon fungus Trichoderma viride inoculation and jasmonic acid and salicylic acid treatments.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Obtaining High Pest_resistant Transgenic Upland Cotton Cultivars Carrying cry1Ac3 Gene Driven by Chimeric OM Promoter

Hypocotyl segments from aseptic seedlings of two important cultivars of upland cotton ( Gossypium hirsutum L.) in Northwest China, 'Xinluzao_1', 'Jinmian_7', 'Jinmian_12' and 'Jihe_321' were transformed respectively by two efficient plant expression plasmids pBinMoBc and pBinoBc via Agrobacterium tumefaciens . In pBinMoBc, cry 1Ac3 gene, which encodes the Bt toxin, is under the control of chimeric OM promoter. In pBinoBc, it is under control of CaMV 35S promoter. After co_cultivation with Agrobacterium tumefimpfaciens LBA4404 (containing pBinMoBc or pBinoBc), kanamycin_resistant selection, somatic embryos were induced and regenerated plants were obtained. Then the regenerated plantlets were grafted to untransformed stocks in greenhouse to produce descendants. The integration of cry 1Ac3 gene and its expression in T 2 generation of transgenic cotton plants were confirmed by Southern hybridization and Western blotting. The analyses of insect bioassay indicated that the transgenic plants of both constructions have significant resistance to the larvae of cotton bollworm ( Heliothis armigera ) and that cry 1Ac3 gene driven by chimeric OM promoter could endue T 2 generation cotton with high pest_resistant ability, implicating that it has a profound application in genetic engineering to breed new pest_resistant cotton varieties.

Expression of Bt Protein in Transgenic Pest-resistant Rice

[Objective] The aim of this study was to study on expression of Bt protein in transgenic pest-resistant rice. [Method] Enzyme-linked immunosorbent assay （ELISA） was used to measure Bt protein expression in different tissues of transgenic pest-resistant rice at same growth stage. [Result] Absolute content of Bt protein from high to low was as follows： leaves 〉 immature seeds and glumes 〉 roots 〉 stems in different tissues of transgenic rice in grain-filling stage; Bt protein content of trans- genic rice changed a little in different growth stages （including tillering stage, booting stage, and grain-filling stage）; in general, its level declined a little in later growth stage, but the resistibility would not be influenced significantly. [Conclusion] The ex- periment is significant for pest prevention and transgenic rice breeding.