Agrobacterium tumefaciens-mediated genetic transformation of the phytopathogenic fungus Penicillium digitatum

Agrobacterium tumefaciens-mediated transformation (ATMT) system was assessed for conducting insertional mutagenesis in Penicillium digitatum, a major fungal pathogen infecting post-harvest citrus fruits. A transformation efficiency of up to 60 transformants per 106 conidia was achieved by this system. The integration of the hph gene into the fungal genome was verified by polymerase chain reaction (PCR) amplification and sequencing. These transformants tested were also shown to be mitotically stable. Southern blot analysis of 14 randomly selected transformants showed that the hph gene was randomly integrated as single copy into the fungal genome of P. digitatum. Thus, we conclude that ATMT of P. digitatum could be used as an alter-natively practical genetic tool for conducting insertional mutagenesis in P. digitatum to study functional genomics.

Optimization of Agrobacterium tumefaciens-Mediated Immature Embryo Transformation System and Transformation of Glyphosate-Resistant Gene 2mG2-EPSPS in Maize(Zea mays L.)

Since maize is one of the most important cereal crops in the world,establishment of an efficient genetic transformation system is critical for its improvement.In the current study,several elite corn lines were tested for suitability of Agrobacterium tumefaciens-mediated transformation by using immature embryos as explants.Infection ability and efficiency of transformation of A.tumefaciens sp.strains EHA105 and LBA4404,different heat treatment times of immature embryos before infection,influence of L-cysteine addition in co-cultivation medium after transformation,and how different ways of selection and cultivation influence the efficiency of transformation were compared.Glyphosate-resistant gene 2mG2-EPSPS was transformed into several typical maize genotypes including 78599,Zong 31 and BA,under the optimum conditions.Results showed that the hypervirulent Agrobacterium tumefaciens sp.strain EHA105 was more infectious than LBA4404.Inclusion of L-cysteine（100 mg L-1） in co-cultivation medium,and heating of the immature embryos for 3 min prior to infection led to a significant increase in the transformation efficiency.Growth in resting medium for 4-10 d and delaying selection was beneficial to the survival of resistant calli.During induction of germination,adding a high concentration of 6-BA（5 mg L-1） and a low concentration of 2,4-D（0.2 mg L-1） to regeneration medium significantly enhanced germination percentage.Using the optimized transformation procedure,more than 800 transgenic plants were obtained from 78599,Zong 31 and BA.By spraying herbicide glyphosate on leaves of transgenic lines,we identified 66 primary glyphosate-resistant plants.The transformation efficiency was 8.2%.PCR and Southern-blot analyses confirmed the integration of the transgenes in the maize genome.

Establishment of Agrobacterium tumefaciens-Mediated Transformation System for Rice Sheath Blight Pathogen Rhizoctonia solani AG-1 IA

To construct the T-DNA insertional mutagenesis transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1 IA,the virulent isolate GD118 of this pathogen was selected as an initial isolate for transformation.The conditions for transformation of isolate GD118 were optimized in five aspects,i.e.pre-induction time,co-culture time,acetosyringone(AS) concentration at the co-culture phase,co-culture temperature and pH value of induction solid medium(ISM) at the co-culture phase.Finally,a system of Agrobacterium tumefaciens-mediated transformation(ATMT) for R.solani AG-1 IA was established successfully.The optimal conditions for this ATMT system were as follows:the concentration of hygromycin B at 30 μg/mL for transformant screening,8 h of pre-induction,20 h of co-culture,200 μmol/L of AS in ISM,co-culture at 25 ℃ and pH 5.6 to 5.8 of ISM at the co-culture phase.The transformants still displayed high resistance to hygromycin B after subculture for five generations.A total of 10 randomly selected transformants were used for PCR verification using the specific primers designed for the hph gene,and the results revealed that an expected band of 500 bp was amplified from all of the 10 transformants.Moreover,PCR amplification for these 10 transformants was carried out using specific primers designed for the Vir gene of A.tumefaciens,with four strains of A.tumefaciens as positive controls for eliminating the false-positive caused by the contamination of A.tumefaciens.An expected band of 730 bp was amplified from the four strains of A.tumefaciens,whereas no corresponding DNA band could be amplified from the 10 transformants.The results of the two PCR amplifications clearly showed that T-DNA was indeed inserted into the genome of target isolate GD118.

Scarabaeid Larvae- and Herbicide-Resistant Transgenic Perennial Ryegrass (Lolium perenne L.) Obtained by Agrobacterium tumefaciens-Mediated Transformation of cry8Ca2, cry8Ga and bar Genes

Insect pest and weeds are two major problems for forage and turf grasses. In this study, scarab larvae- and herbicide-resistant transgenic perennial ryegrass （Lolium perenne L.） was obtained by transforming it with cry and bar genes simultaneously via the Agrobacterium-mediated method. To optimize the callus induction and plant regeneration conditions, various concentrations of 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine were assayed. The transformation efficiencies of different Agrobacterium suspension media, used during Agrobacterium-mediated transformation, were compared. Then, plasmids of pCAMBIA3301 containing cry gene （cry8Ca2 or cry8Ga） and bar gene, driven by ubiquitin promoter, were transformed into perennial ryegrass. The transformants were generated and confirmed by both Southern hybridization analysis and Western hybridization analysis. Further, the resistance of transgenic perennial ryegrass plants to scarab larvae and herbicide were analyzed. After 30 d of co-cultivation with scarab larvae, the damage to the root system of transgenic plants was less than that of non-transgenic control plants. Additionally, the leaves of transgenic plants were resistant to Basta, while leaves of the wild plants wilted after Basta spraying. These results show that cry gene and bar gene were successfully transferred into perennial ryegrass by the Agrobactgerium-mediated method, and convey resistance to scarab larvae and herbicide in transgenic perennial ryegrass plants.

Establishment of Agrobacterium tumefaciens-mediated Genetic Transformation System for Aspergillus awamori

[ Objective ] This study aimed to establish Agrobacterium tumefaciens-mediated genetic transformation system for Aspergillus awamori and investigate the feasibility of expressing heterologous proteins in A. awamori. [ Method] Appropriate A. awamori host strains were determined according to the secretory protein profile. Selectable marker was selected for genetic transformation by drug sensitivity analysis. The established A. awamori genetic transformation system was used for transformation and expression analysis of Rhizomucor miehei lipase （RML）. The feasibility of using A. awamori to express heterologous proteins was investigated by identification of transformants and property analysis. [ Result~ Based on the analysis of secretory protein profile, A. awamori strains CBS115.52 and CICC2257 were determined as the host strains for heterologous protein expression ; drug sensitivity analysis shows that hygromycin B resistance gene （ HygBr ） is an effective ge- netic seleetable marker; by using Agrobacterium tumefaciens-mediatcd transformation （ATMT） method, the plasmid pHGW-amdS containing HygBr was successfully transformed into A. awamori strain CBS115.52 to establish the genetic transformation system ofA. awamorl with HygBr as selectable marker. RML was transformed into A. awamori and its expression was validated by substrate hydrolysis test, SDS-PAGE and Western blot. [ Conclusion] This study demonstrates that the genetic transformation system of A. awamori mediated by Agrobacterium tumefaciens has potential feasibility for expression of heterologous proteins.

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.

番茄愈伤组织的诱导与遗传转化体系的建立

番茄是植物研究领域常用的模式植物之一,番茄高效稳定的遗传转化体系是研究番茄基因生物学功能的基础,但常规番茄遗传转化耗时过长。以番茄愈伤组织为侵染对象,最终获得转基因番茄愈伤组织,可极大地缩短获得番茄转基因材料的时间。本文以番茄Micro-Tom(Solanum lycopersicum cv. Micro-Tom)为实验材料,探索番茄愈伤诱导条件,建立标准的番茄愈伤组织遗传转化体系。结果表明,胚根为外植体、激素配比2,4-二氯苯氧乙酸(2,4-Dichlorophenoxyacetic acid, 2,4-D)1 mg/L+6-苄氨基腺嘌呤(6-Benzylaminopurine, 6-BA)1 mg/L、蔗糖浓度30 g/L、光照强度3 000 lx为最佳诱导条件。标准番茄愈伤遗传转化体系包括愈伤组织培养、悬浮细胞建立、农杆菌侵染、共培养、抗性愈伤筛选、核酸鉴定等。该实验结果能够为以番茄为研究对象的分子机理研究及其所涉及的免疫沉淀、高通量测序、亚细胞定位等实验提供科研实验材料和方法参考。

Development of Alfalfa (Medicago sativa L.) Regeneration System and Agrobacterium-Mediated Genetic Transformation

The regeneration ability of four alfalfa （Medicago sativa L.） cultivars, Xinjiang Daye, Longdong, Gannong 1 and Gannong 3, was studied, and the effects of various cultivars, explant sources and medium recipes on regeneration were compared. The better callus forming frequency obtained from hypocotyls of Xinjiang Daye is 88.5% and regeneration frequency is 9.8% in our initial experiments. To further optimize regeneration system for genetic transformation, we therefore changed concentrations of plant growth regulators and supplemented with glutamine into callus-induction and shoot-regeneration media. Callus forming frequency and shoot differentiation frequency were increased to 100%. The time taken to generate transgenic plants （16 weeks） was shorter than that for previouse procedure （25 weeks） and regeneration frequency was promoted to 15.1%. The results show that addition of glutamine is particularly important for shortening period of regeneration and promoting regeneration frequency. For study of genetic transformation of alfalfa, Agrobacterium tumefaciens-mediated transformation of Xinjiang Daye was developed based on this optimized regeneration system. The plant expression vector carrying two glutamine synthetases （GS 1 and GS2） and △1-pyrroline-5-carboxylate synthetase （P5CS） gene was used for alfalfa in vitro transformation. Six transgenic alfalfa plantlets with resistance to PPT were obtained. The introduction of foreign genes into plants was assessed in the transformants by PCR analysis and Southern hybridizations.

褐角苔遗传转化体系的建立

本研究旨在筛选适用于农杆菌(Agrobacterium)介导的角苔植物遗传转化体系,为今后研究早期陆生植物的进化提供理论基础。实验使用携带p CAMBIA1305.2质粒的农杆菌AGL1菌株对褐角苔(Folioceros fuciformis)外植体进行侵染,将潮霉素作为筛选标记,通过比较外植体预培养时间、菌液OD值、菌液添加量、乙酰丁香酮(AS)浓度和共培养时间来构建该物种的遗传转化体系。结果表明,褐角苔抗性植株筛选所用潮霉素的最适浓度为15 mg/L,预培养4 d的外植体侵染后状态最好。最佳侵染条件为共培养基中添加80μL OD600为0.8的农杆菌菌液,AS浓度为100μmol/L,共培养时间为3 d。通过潮霉素筛选、PCR、实时荧光定量PCR(RT-q PCR)和β-葡萄糖苷酸酶(GUS)染色鉴定获得转基因植株褐角苔。研究结果可为后续角苔植物基因功能的研究提供技术支持。

重组质粒导入根癌农杆菌冻融法的研究

对重组质粒导入农杆菌冻融法的几个技术环节进行改进与优化。结果表明:该方案可简便、快速、有效地将重组质粒导入农杆菌,转化效率高、重复性好。应用该改进和优化的标准冻融法将重组质粒pBISP1,pBIP1,pBIAP1和pBISgp120分别导入根癌农杆菌EHA105和LBA4404,构建了植物双元表达载体,为用农杆菌介导法进行植物遗传转化提供了良好的载体系统。

农杆菌介导慈竹4CL基因遗传转化梁山慈竹

以梁山慈竹2种类型成熟胚的愈伤组织为材料,采用农杆菌遗传介导的方法,将已构建好的具有降低木质素含量的PBI121-4CL-RNAi表达载体导入愈伤组织,探讨愈伤组织预培养时间、菌液浓度、侵染时间、共培养时间和温度对遗传转化的影响。研究结果表明,淡黄色、颗粒状、疏松易碎的胚性愈伤组织是较好的遗传转化材料。以在愈伤组织培养基上预培养8天的淡黄色、颗粒状、疏松易碎的胚性愈伤组织为转化受体,在菌液浓度为OD600=0.05的EHA105中侵染20min后,在25℃、黑暗条件下共培养2天(共培养基表面加一层无菌滤纸),在含有卡那霉素为55mg.L-1的抗性筛选培养基上筛选30天,抗性愈伤组织率为90%,经PCR检测,慈竹4CL基因已导入梁山慈竹愈伤组织中。抗性愈伤组织在芽诱导培养基上诱导30天,可获得丛生芽,待丛生芽长至3～5cm后,在生根培养基上经过20～30天的诱导,可产生1～8条根,获得再生植株。经PCR检测,慈竹4CL基因已导入梁山慈竹再生植株中,获得了转基因植株,转化效率为9%。RT-PCR检测结果表明,转4CL基因的梁山慈竹愈伤组织和植株的内源4CL基因的表达受到抑制,且表达量比对照明显降低。

西瓜NPR1抗病基因的遗传转化

比较了不同预培养时间、侵染时间、共培养时间等因素对农杆菌转化西瓜的影响。西瓜遗传转化的适宜条件；3d苗龄的子叶，预培养1d，农杆菌侵染10～15min，共培养3d；不定芽与根的潮霉素（Hyg）筛选浓度分别为20mg·L^-1和6mg·L^-1。对20株西瓜转化苗进行DNA提取，PCR检测，其中5株呈阳性，初步证明目的基因已经整合到西瓜基因组中。

农杆菌介导的苏云金杆菌抗虫基因cryIA(b)和cryIA(c)在水稻中的遗传转化及蛋白表达

通过农杆菌介导法用含有抗潮霉素和 Ｇ Ｕ Ｓ 基因的双元载体将杀虫结晶蛋白基因ｃｒｙ Ｉ Ａ（ ｂ） 和ｃｒｙ Ｉ Ａ（ｃ） 导入到籼、粳稻幼穗愈伤组织中，然后经过在含有不同浓度潮霉素的培养基上进行数次筛选，获得一批 Ｂｔ 转基因株。经 Ｐ Ｃ Ｒ、 Ｓｏｕｔｈｅｒｎ 杂交及 Ｗｅｓｔｅｒｎ 印迹分析证实此二基因已整合进水稻中，饲虫试验结果表明，转基因株具有１００ ％ 杀虫率。

转Cry30Fa1基因抗褐飞虱水稻的获得及鉴定

将编码高效杀褐飞虱蛋白的苏云金芽胞杆菌基因Cry30Fa1密码子改造后,通过农杆菌介导法转入蜀恢818(R818),并最终获得46个转基因植株。通过定量PCR及Western Blot鉴定了Cry30Fa1在转录水平和蛋白水平的表达,并通过分子检测固定稳定表达的抗性基因,并结合传统育种系谱法选择具有优良农艺性状株系。对选育的株系在室内和大田环境下进行了抗虫性鉴定,选育的R818-Cry30Fal株系抗性明显优于亲本材料并达到抗的水平;在单株抗虫试验中,观察到了转基因株系对褐飞虱具有致死作用。说明转入Cry30Fa1基因使水稻产生了对褐飞虱抗性。培育出了具有抗褐飞虱蛋白的新型恢复系R818-Cry30Fal,为三系杂交育种提供了新的抗性材料并丰富了抗褐飞虱水稻种质资源。

农杆菌培养方式和预培养基激素配比对甘蓝型油菜下胚轴转化效应分析

利用根癌农杆菌共培养法转化甘蓝型油菜"华双4号"下胚轴,通过组织化学染色法检测报告基因gus的表达情况,研究了农杆菌培养方式和预培养基激素配比对转化效果的影响.结果表明,用LB培养基活化后的农杆菌再用添加AS的MS培养液继续培养6～30h对转化有明显的促进作用;不同的农杆菌培养方式对转化的影响有明显的差异,在试验的6种农杆菌培养方式中s2-5的转化效果最好,即收集活化后的农杆菌用MS+AS+抗生素重悬培养24h,再用MS+AS培养6h;2,4-D与6BA之间的相互作用对转化有重要影响,在试验的预培养基激素组合中,2,4-D2.00mg/L6BA0.25mg/L的组合转化效率最高;抗性愈伤率与转化效率之间不一定具有相关关系.

cry2Aa9m抗虫基因植物表达载体构建及对大豆的遗传转化

cry2Aa9m基因编码的杀虫晶体蛋白(ICPs)对鳞翅目(Lepidoptera)和双翅目(Dipter)昆虫具有显著的毒杀作用,可防治大豆食心虫等害虫。试验构建了由豆荚特异性启动子Pmsg调控的cry2Aa9m基因的植物表达载体pCMB2A,以抗除草剂bar基因为筛选标记,通过农杆菌介导法对绥农28大豆子叶节进行遗传转化,获得抗性株系8株。经PCR和RT-PCR检测结果证明,cry2Aa9m基因已经整合到大豆基因组中并得以表达。研究为抗大豆食心虫转基因育种产业化奠定基础。

Bt-CryV基因对马铃薯的遗传转化

文章以马铃薯品种东农303的脱毒微型薯为外植体,利用农杆菌介导法将Bt-CryV基因转入马铃薯中,同时优化影响遗传转化的因素,包括激素种类与浓度、休眠期、脱菌抗生素种类与浓度、共培养时间、筛选方式等。获得21株Kan抗性植株进行PCR和PCR-Southern检测,其中10株为阳性植株,证明外源基因已经整合到植物的基因组上。

棉花黄萎病菌致病的分子机理

阐述了大丽轮枝菌(Verticillium dahliae Kleb)产生的细胞壁降解酶、蛋白酶和毒素在致病中的作用及微菌核形成机制,全面分析了基因组学途径、RNA干涉和农杆菌介导的转化等真菌功能基因组学技术在大丽轮枝菌致病分子机理研究上的进展。

抗冻蛋白(afp)基因表达载体的构建及对香蕉胚性细胞悬浮系的转化

通过PCR从‘京都七寸人参'胡萝卜基因组DNA中扩增抗冻蛋白基因,测序结果表明该基因的核苷酸序列与从宁夏‘吴忠'胡萝卜中克隆的完全一致。先后将获得的胡萝卜afp基因克隆和亚克隆至pMD18-T和pBI121,构建植物表达载体pBI121-afp。通过冻融法将pBI121-afp导入根癌农杆菌EHA105中。以香蕉栽培品种‘北大矮蕉'的胚性细胞悬浮系为受体,采用农杆菌介导法将胡萝卜afp基因导入其中,然后在Kanamycin的选择压力下通过体细胞胚发生途径进行植株再生。共获得抗性再生植株9株,其中两株经PCR检测呈阳性,可初步确定目的基因已经整合到这两株转基因香蕉植株的基因组中。

农杆菌介导玉米幼胚遗传转化体系的建立

以玉米（Zea mays L.）HiII的幼胚为外植体，β-葡萄糖苷酸酶基因（GUS）为报告基因，通过农杆菌介导转化法对影响遗传转化体系的外植体大小、农杆菌浓度、热预处理温度、侵染时间、共培养及恢复培养时间、抗生素、筛选剂等因素进行优化，以建立农杆菌介导玉米幼胚遗传转化体系。结果表明，幼胚大小为1.0~1.5 mm，农杆菌菌液的OD600 nm为0.5，40℃热处理3 min，侵染8 min，共培养3 d和恢复培养4 d，100 mg/L羧苄青霉素作为抑菌剂，双丙氨膦作为筛选剂时为最佳遗传转化条件。通过该优化体系已获得多种转基因玉米材料，表明该体系具有较高的可重复性和可靠性。