枝状枝孢霉MD2的候选基因A00981的克隆及转基因菌株的构建

目的:为揭示真菌紫杉醇合成分子机制.方法:克隆了产紫杉醇内生真菌枝状枝孢霉MD2的候选基因A00981,该基因DNA和cDNA序列分别为969 bp和894 bp;其编码蛋白含有298个氨基酸、无跨膜结构和信号肽,与酰基辅酶A脱氢酶(ACAD)具有73%的一致性.进一步构建该基因的超表达载体pTFC-A00981,并转化枝状枝孢霉MD2,共获得9株转基因菌株.结果:Southern blot分析证明其中5株转基因菌株的外源基因是以单拷贝方式插入.结论:该结果为深入分析超表达候选基因A00981对枝状枝孢霉MD2紫杉烷类合成的影响奠定了基础.

紫杉醇合成相关候选基因A02725的克隆及转基因菌株的构建

真菌紫杉醇合成相关基因的功能研究是揭示真菌紫杉醇合成机制的关键。为了揭示产紫杉醇内生真菌枝状枝孢霉MD2的候选基因A02725对宿主紫杉烷类合成的作用,本研究克隆了候选基因A02725的DNA全长序列(1 524 bp);生物信息学预测结果表明该基因编码蛋白为亲水蛋白,含508个氨基酸,无信号肽和跨膜结构,与牻牛儿基牻牛儿基焦磷酸合成酶(geranylgeranyl pyrophosphate synthase,GGPPS)具有79%的一致性;进一步构建该基因的超表达载体并转化枝状枝孢霉MD2,通过抗性筛选以及Southern blot检测,共获得27株转基因菌株,其中5株转基因菌株的外源基因是以单拷贝方式整合。该结果为深入分析超表达候选基因A02725对宿主紫杉烷类合成的影响奠定了基础。

转基因生防菌308R(pCPP430)对番茄根围菌群的影响

研究目的在于了解转基因生防菌308R(pCPP430)对番茄根围菌群代谢能力和群落结构的影响。实验中使用了两种互为补充的方法,即单一碳源利用测试(SCSU)和ERIC-PCR,对分别以308R(pCPP430)悬液、308R悬液和无菌水蘸根处理的番茄植株根围菌群进行比较。SCSU菌落计数的聚类分析表明,308R(pCPP430)和308R处理的根围菌重复之间相似性好,水处理的相似性差。主成分分析也得到了相同的结果。ERIC-PCR聚类结果表明,10种碳源,其中8种水处理和308R处理聚为一类。实验为生防菌与植物的互作提供一些依据,为根围菌群结构研究提供一些新的思路。

转基因溶菌酶羊hLZ-J1品系特异性数字PCR方法及样本中痕量转基因成分检测

人溶菌酶是一种天然抗生素,具有抗细菌、真菌,抗炎症,预防感染等功能。转基因溶菌酶山羊可以在乳腺中特异性表达,从而可以从羊乳中规模化制备溶菌酶。本研究旨在建立一种适用于痕量样本中转基因溶菌酶羊成分定量检测的品系特异性数字PCR方法,并利用该方法对山羊的血液、羊奶、粪便和环境土壤样本等进行检测,用于对转基因溶菌酶山羊成分的溯源。建立的数字PCR方法具有高特异性、灵敏度、准确性,检测限和定量限低至15个拷贝每个反应,可以对羊血、羊乳等制品进行准确的定量检测。研究结果还表明该方法可以准确测定粪便和环境土壤样本中的痕量转基因溶菌酶羊成分,可进一步用于转基因动物外源基因的水平转移检测及环境风险评估。

诺卡氏菌株C-14-1基因工程菌ZM1对试验动物的安全性评估

为了对诺卡氏菌株C-14-1以及转基因工程菌ZM1的安全性进行合理评价,并为该产品的生产和应用过程中安全防护措施的制定提供依据,根据国家标准《农药登记毒理学试验方法》(GB15670-1995),就C-14-1菌株以及ZM1菌株的粉剂对试验动物进行了相关的毒理学试验。试验结果表明:两株菌株对昆明小白鼠的急性经口LD50>5000mg/kg,急性经皮毒性LD50>2000mg/kg,均属于微毒或无毒类;对大耳白兔皮肤刺激平均指数72h后为0,皮肤刺激强度为无刺激性;眼刺激平均指数48h后为0,眼刺激强度为无刺激性。

真养产碱杆菌聚羟基烷酸合成酶基因在欧文氏菌中的表达

将含有真养产碱杆菌（Ａｌｃａｌｉｇｅｎｅｓｅｕｔｒｏｐｈｕｓ）合成聚羟基烷酸（ＰＨＡ）基因（ｐｈａＣＡＢ）的质粒ｐＴＺ１８ＵＰＨＢ改造成为具有卡那霉素抗性的质粒ｐＪＭＣ１，并以电击法将ｐＪＭＣ１引入利用碳源广泛的胡罗卜软腐欧文氏菌（Ｅｒｗｉｎｉａｃａｒｏｔｏｖｏｒａ）非致病菌系（Ｅｃｃ１３Ｂ）中，ｐｈａＣＡＢ可获得高效表达，膨大的转基因菌［Ｅｃｃ１３Ｂ（ｐＪＭＣ１）］细胞内几乎充满ＰＨＡ颗粒。以蔗糖为碳源，初步在５Ｌ发酵罐中对转基因菌进行分批补料培养３５ｈ，菌体干重达２８ｇ／Ｌ，ＰＨＡ占菌体干重的６８％，具有生产潜力。将该菌合成的ＰＨＡ提取纯化（纯度达９９％）后，进行核磁共振分析。

Agrobacterium _mediated Transformation of Rice with Help of Bombardment

Oryza sativa L. ssp. japonica cv. Zhonghua 8, which is recalcitrant to infection of Agrobacterium tumefaciens (Smith et Townsend) Conn strain EHA105 with ordinary binary vector pCambia 1301, was transformed through Agrobacterium-mediated transformation with help of bombardment. The transformation efficiency can be raised greatly. Single copy of gene insertion in the genome of transgenic rice plants was proved by Southern analysis and the expression of GUS gene was observed. GUS gene and hygromycin-resistant gene show 3:1 segregation in progenies of the transgenic rice plants.

Expression of Exogenous Antimicrobial Peptide and Resistance to Stem Rot Disease of Houttuynia cordata Thunb.

[Objective] This study was to identify the expression of exogenous antimicrobial peptide in transgenic Houttuynia cordata Thunb. plants,and analyze their resistance to stem rot disease. [Methods] SDS-PAGE and Western blot analysis were employed to detect expression of exogenous antimicrobial peptide in transgenic H. cordata plants. Both wild type and transgenic H. cordata plants were inoculated with different concentrations of Rhizoctonia solani spores for detecting their resistance. [Results] The exogenous antimicrobial peptide was detected at translation level. The optimal parameters for detecting the resistance of transgenic H. cordata plants to R. solani was inoculation of spores at a concentration of 3×105 ind./ml and cultured for three days. The results showed that resistance of transgenic H. cordata plants to R. solani was enhanced in comparison with CKs. [Conclusion] Expression of exogenous antimicrobial peptide can enhance the resistance of transgenic H. cordata plants to stem rot disease.

Pathogen_resistant Transgenic Plant of Brassica pekinensis by Transfering Antibacterial Peptide Gene and Its Genetic Stability

The soft rot infected by pathogenic bacterium Erwinia aroideae Holland is one of the three serious diseases of Chinese cabbage ( Brassica pekinensis Rupr.). By constructing vector system of high frequency transformation mediated by Agrobacterium tunefaciens EHA105, anti-bacterial peptide gene with strong bactericidal action to pathogenic bacteria was introduced into Chinese cabbage AB-81 self-bred line and the transgenic plants were obtained. PCR and Southern blotting detection showed that target gene was integrated into plant genome of Chinese cabbage. The tests of bacteriostasis action of the extract from transgenic plants in vitro, and the assay of disease-resistant of transgenic plantlets in the tube and the pot by perfusing inoculation with pathogenic bacteria showed obvious resistance to soft rot. This resistance can be a stable heredity by genetic analysis of generations of transgenic plants self-bred, separation ratio of its R, was 3:1. The resistance to Km and disease of soft rot was still kept in the R-5. These results indicated the possibility of breeding new varieties of anti-soft rot Chinese cabbage by transgenic plants as parents.

Improvement of the Hydroponic Growth and Waterlogging Tolerance of Petunias by the Introduction of vhb Gene

The coding sequence of Vitreoscilla hemoglobin (vhb) was cloned with PCR technique from Vitreoscilla stercoraria Pringsheim. The plant expression vector with vhb gene under the control of CaMV 35S promoter was constructed and used in the transformation of Petunia hybrida Vilm by the Agrobacterium mediated procedure. The results of PCR amplification and Southern hybridization indicated that the vhb gene had been integrated into the petunia genome and the vhb gene expression had been detected by RT-PCR amplification. In hydroponic culture the transgenic petunias grew much better than non-transgenic controls. For further analysis of hypoxia tolerance of transgenic petunia, the petunia plants with vhb gene were submerged into liquid MS medium. The transgenic plants survived in hypoxic condition and grew out of the liquid surface in a few weeks, while non-transgenic plants were still submerged and suffocated in culture solution without ability to grow out of liquid medium in submersed culture for four to five weeks. The vhb gene transformed petunia plants had been planted and tested in a simulated flooding condition, and showed obvious tolerance to water-logging. It seen is that hemoglobin gene from Vitreoscilla might have the potential use in molecular breeding for the improvement of plant resistance to hypoxia and flooding.

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.

转溶菌酶基因山羊乳腺上皮细胞的原代培养及特性分析

采用组织块法、胰酶差速消化法分离了人溶菌酶(hLYZ)转基因山羊的乳腺上皮细胞,建立了转基因羊乳腺上皮细胞(TGMEC)的体外培养体系,并对其生长与体外分泌特性进行了分析。研究显示其体外生长曲线符合典型的S型,传代期间二倍体染色体数正常;通过免疫荧光染色、RT-PCR发现该细胞能表达上皮细胞所特有的角蛋白18,并能表达内源性乳蛋白(酪蛋白与乳球蛋白);进一步通过酶联免疫试验发现该乳腺上皮细胞在体外培养中可持续稳定地分泌重组人溶菌酶。由此获得的人溶菌酶转基因羊乳腺上皮细胞系为乳腺生物反应器研究提供了一个重要细胞膜型。

Effects of Transgenic DREB Soybean Dongnong 50 on the Diversity of Soil Ammonia-oxidizing Bacteria

ObjectiveThe aim was to understand the effects of transgenic DREB soybean on the ammonia-oxidizing bacteria. MethodThe diversity of the cto gene in pot-planted transgenic soybean and near-isogenic non-transgenic soybean under normal water condition and drought stress was analyzed by PCR-DGGE and sequence analysis. ResultRhizosphere community diversity of ammonia-oxidizing bacteria showed no difference between the treatments of transgenic soybean and its non-transgenic isolines, moreover transgenic soybean under normal water condition and drought stress improved the diversity of the ammonia-oxidizing bacteria in the harvest time. The phylogenetic analysis revealed that all the sequences of excised DGGE bands were closely related to members of the genus Nitrosovibrio and Nitrosospira of the β-subclass Proteobacteria. ConclusionTransgenic DREB soybean has no adverse impact on soil ammonia-oxidizing bacteria.

Study on Agrobacterium tumefaciens-mediated Transformation of Brassica campestris L. with Fusion Gene Ycoil-bFGF

[ Objective] The study is to generate pharmaceutical protein via plant transgenic technique. [Methed] Using the cotyledons with petiole as transformation receptor, the fusion gene of rapeseed oil-body gene and bFGF was introduced into the rapeseed （ Brassica campestris L. ） by Agrobacterium tumefaciens-mediated transformation; meanwhile regeneration conditions of rapeseed were also optimized, and the regenerated resistant plantlets were detected by PCR and Southern blot. [ Result] This fusion gene had been integrated into rapeseed genome successfully, and the optimized conditions of transformation and regeneration were as follows： explants pre-culture for 2 d, co-culture for 3 d, bacteria solution OD600 for 0.3 and infection time for 5 min. [ Conclusion] The results laid a solid foundation for extraction, isolation and purification of protein in transgenic plant seeds.

Establishment of the Agrobacterium-mediated Genetic Transformation System of Ginkgo biloba and the Construction of the Expression Vector of Gb-DXR

[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.

Antibacterial Effects of Nanometallic Materials in Genetic Transformation of Anthurium by Agrobacterium-mediated Method

[Objective] This study was conducted to investigate the application of nanometallic materials in inhibiting Agrobacteriurn contamination in genetic transformation of Anthurium. [Method] Different nanometallic materials were added into Agrobacterium medium and Anthurium callus medium, to investigate the effects of their effects on Agrobacterium growth, callus growth and differentiation, and Agrobacterium contamination. [Result] Among the 4 nanometallic materials, NanoAg-2 showed a significant inhibitory effect on the growth of Agrobacterium, with a minimal inhibitory concentration of 25 mg/L. Even for the Anthurium calli or transgenic material contaminated by the Agrobacterium, a good antibacterial effect could be achieved after treating with 25 mg/L NanoAg-2 for 1 d with oscillation, the antibacterial rate reached 100%, and the Anthurium calli could grow and differentiate normally. [Conclusioa] NanoAg-2 could effectively inhibit Agrobacterium contamination, and its an- tibacterial effect is significantly better than cephalosporin and carbenicillin.

Enhancement of germ cell apoptosis induced by ethanol in transgenic mice overexpressing Fas Ligand

It was suggested that chronic ethanol exposure could result in testicular germ cell apoptosis, but the mechanism is still unclear. In the present study, we use a model of transgenic mice ubiquitously overexpressing human FasL to investigate whether Fas ligand plays a role in ethanol-induced testicular germ cell apoptosis. Both wild-type (WT)mice and transgenic (TG) mice were treated with acute ethanol (20% v/v) by introperitoneal injection for five times.After ethanol injection, WT mice displayed up-regulation of Fas ligand in the testes, which was shown by FITCconjugated flow cytometry and western blotting. Moreover, TG mice exhibited significantly more apoptotic germ cells than WT mice did after ethanol injection, which was demonstrated by DNA fragmentation, PI staining flow cytometry and TUNEL staining. In addition, histopathological examination revealed that degenerative changes of epithelial component of the tubules occurred in FasL overexpressing transgenic mice while testicular morphology was normal in wild-type mice after acute ethanol exposure, suggesting FasL expression determines the sensitivity of testes to ethanol in mice. In summary, we provide the direct evidences that Fas ligand mediates the apoptosis of testicular germ cells induced by acute ethanol using FasL transgenic mice.

Agrobacterium-mediated transformation and assessment of factors influ-encing transgene expression in loblolly pine (Pinus taeda L.)

This investigation reports a protocol for transfer and expression of foreign chimeric genes in loblolly pine (Pinus taeda L.). Transformation was achieved by co-cultivation of mature zygotic embryos with Agrobacterium tumefaciens strain LBA4404 which harbored a binary vector (pBI121) including genes for β-glucuronidase (GUS) and neomycin phosphotransferase (NPTII). Factors influencing transgene expression including seed sources of loblolly pine, concentration of bacteria, and the wounding procedures of target explants were investigated. The expression of foreign gene was confirmed by the ability of mature zygotic embryos to produce calli in the presence of kanamycin, by histochemical assays of GUS activity, by PCR analysis, and by Southern blot. The successful expression of the GUS gene in different families of loblolly pine suggests that this transformation system is probably useful for the production of the genetically modified conifers.

Establishment of genetic transformation system via Agrobacterium in tall fescue cultivar

Tall fescue （Festuca arundinacea Schreb.） is a cool-season turfgrass used on fairways in golf courses. The object of this study was to develop a more efficient, reliable, and repeatable approach in transforming the grass using Agrobacterium （EHA105）, where β-glucuronidase gene （uidA） was used as a reporter and hygromycin phosphotransferase gene （hyg） as a selectable marker. An effective expression of transgene was observed in transforming 2-month-old calli derived from mature seeds （cv. Bingo） cultured on MS medium supplemented with 9 mg·L^-1 2, 4-D. A two-step solid medium selection with increasing hygromycin concentration （from 30 to 50 mg· L^-1） was used to obtain resistant calli. Transgenic plants have been produced from many independent transformed calli. The presence of functional β-glucuronidase gene （uidA） was detected in hygromycin-resistant calli. Transgenic plants were regenerated and PCR and Southern blot confirmed transgene integration in the tall fescue genome.

Comparison of Biolistic and Agrobacterium-mediated Transformation Methods on Transgene Copy Number and Rearrangement Frequency in Rice

Transferring foreign DNA into plant cells by biolistic and Agrobacterium _mediated methods may result in random integration of different copy numbers of the transgene, and different proportions of intact vs. rearranged copies of the transgene. This may, in turn, affect transgene expression levels. To test the above hypothesis, we first introduced the same plasmid, pAc1PG_CAM, into rice (Oryza sativa L.) calli separately by the biolistic method and by the Agrobacterium _mediated method. To show whether different plasmids may affect the results, we also introduced pTOK233 by the Agrobacterium _mediated method and pJPM44 by the biolistic method. Transgene expression of R0 plants was monitored by histochemical analysis of GUS activity. Transgene copy number was determined by Southern blot analysis after digesting genomic DNA with an enzyme that has a unique cutting site within the input plasmid. The total genomic DNA was also digested by a two_cut enzyme (the cuts are located at two sides of a given transgene expression cassette), followed by Southern blotting analysis, for determining the number of intact transgene expression cassettes. Our data showed that Agrobacterium _mediated transformation resulted in lower transgene copy number (average between 2.1 and 2.3) in transgenic rice plants, compared with those plants obtained by the biolistic method (average between 4.2 and 5.6). The frequency of DNA rearrangement in expression cassettes is lower in transgenic rice plants obtained by the Agrobacterium _ mediated method than those obtained by the biolistic method. The average rearrangement frequency is 0.07 to 0.106 for the Agrobacterium _mediated method, and 0.57 to 0.66 for the biolistic method. Our results suggest that it is better to compare the number of intact expression cassettes instead of the total copy number of the transgene in demonstrating their influence on the level of transgene expression. This is the first report on the frequency of expression cassette rearrangement in transgenic plants transformed with the same plasmid by two different transformation methods.