平菇疏水蛋白Po.hyd基因RNAi载体构建及转化

为研究平菇疏水蛋白基因Po.hyd的功能,首次构建了Po.hyd的RNAi载体,并通过根癌农杆菌介导获得了平菇转化子。通过聚合酶链式反应（polymerase chain reaction,PCR）扩增Po.hyd序列,利用Golden Gate克隆法构建了干扰载体p1302-GG-ihyd,将其导入根癌农杆菌GV3101中,并在乙酰丁香酮的诱导下完成根癌农杆菌对平菇幼嫩菌丝的介导转化。转化子经PCR检测、荧光显微镜观察、实时定量荧光PCR技术（real-time PCR）以及Southern杂交等技术进行验证。结果显示,本研究最终获得1 株可稳定遗传的平菇转化子,该转化子具有潮霉素抗性,且在荧光显微镜下可检测到绿色荧光信号,Po.hyd基因的表达量为野生型的43%,干扰载体T-DNA片段以单拷贝的形式整合在转化子基因组内。     

根癌农杆菌介导的尖孢镰刀菌遗传转化体系构建

  背景和目的  尖孢镰刀菌（Fusarium oxysporum）可引起烟草镰刀菌根腐病（Fusarium root rot of tobacco）,为明确病原菌致病相关基因。  方法  以烟草强致病性尖孢镰刀菌菌株B-9-1的分生孢子为受体,利用携带潮霉素B（hygromycin B,hyg）质粒的根癌农杆菌（Agrobacterium tumefaciens）介导转化,获得能够稳定表达绿色荧光蛋白（green fluorescent protein, GFP）的尖孢镰刀菌转化菌株,构建烟草尖孢镰刀菌遗传转化体系。  结果  （1）尖孢镰刀菌的最优转化体系为:潮霉素B最适质量浓度50 μg/mL,在25℃条件下,以1 cm宽的条形硝酸纤维膜为载体,将携带有pCAM-GFP-hyg质粒的根癌农杆菌摇菌培养至OD₆₀₀=0.7,与震荡培养24 h获得的浓度为106个/mL的尖孢镰刀菌分生孢子悬浮液在含有200 μmol/L乙酰丁香酮的共培养基上共培养48 h。（2）转化子继代培养、PCR检测和荧光显微观察结果表明外源GFP基因成功整合到烟草尖孢镰刀菌基因组中并可以进行稳定遗传表达。（3）Sourthern blot分析结果进一步证明T-DNA成功整合到尖孢镰刀菌基因组中,且60%以上为单拷贝插入。  结论  本研究成功构建了根癌农杆菌介导的烟草尖孢镰刀菌遗传转化体系,为烟草尖孢镰刀菌基因功能及病原菌的致病机制研究提供基础。      

影响根癌农杆菌的电击转化条件

采用电击转化方法将双元载体质粒pCAM3300导入根癌农杆菌LBA4404,研究了不同实验条件对转化率的影响.结果表明:当电场强度为11 kV/cm时,转化率最高,每微克DNA得到9.8×103CFU;在细胞OD600为1.0时进行电转化,转化率最高,每微克DNA得到9.54×103CFU;在一定的细胞浓度范围内,电击转化率与细菌浓度密切相关,转化率随着细菌浓度的上升而上升;质粒大小与电击转化率之间没有明确的关系.将含pCAMBIA3300的根癌农杆菌与粗糙脉胞菌共培养转化,在含有膦化麦黄桐(PPT)(405 mg/L)的平板上筛选到转化子.PCR扩增转化子的染色体,初步证明Bar基因整合进粗糙脉胞菌的染色体中.     

根癌农杆菌介导的亚麻遗传转化研究进展

随着亚麻组织培养技术的逐渐成熟,有关应用根癌农杆菌介导进行的亚麻遗传转化方面的研究工作开展得越来越多,根癌农杆菌介导法已成为亚麻遗传转化的主要方法。概述了农杆菌介导法的转化机理、转化亚麻具体方法的优缺点以及基因型、农杆菌菌株、侵染时间和菌液浓度等对亚麻遗传转化的影响,并从亚麻抗病、抗除草剂等方面对近年来亚麻的转基因研究进展进行了总结。     

根癌农杆菌介导水稻香味基因的遗传转化

以日本腈为材料,研究根癌农杆菌介导水稻香味基因的遗传转化。研究结果表明,通过农杆菌介导的方法将水稻香味基因badh2导入了水稻粳稻品种日本腈中,并获得转基因植株。通过PCR检测,初步证明外源基因badh2已整合到日本腈基因组中。     

根癌农杆菌介导酸性蛋白酶在泡盛曲霉中表达的研究

利用Gateway克隆技术快速构建丝状真菌表达载体,以米曲霉α-淀粉酶A启动子(amyB)、α-糖苷酶终止子(agdA)及黑曲霉酸性蛋白酶基因(pepA)分别构建入门载体,以带有潮霉素抗性标记(hygB)的农杆菌双元载体pHGW为目的载体,通过LR连接反应,构建根癌农杆菌介导丝状真菌重组表达载体pHGW-apA。转化泡盛曲霉宿主菌,筛选的转化子经传代培养确定其遗传稳定性,PCR及测序分析表明,hygB和目的基因pepA整合到了泡盛曲霉基因组。转录水平上的RealTime-PCR定量分析、表达水平上的Western Blotting分析以及培养液酶活分析表明酸性蛋白酶基因pepA在泡盛曲霉中得到正确表达。为丝状真菌表达载体快速构建与外源基因在丝状真菌中快速表达提供了可行的方法。     

一种根癌农杆菌介导转化平菇菌丝体的新方法

设计了一种简单高效的根癌农杆菌介导转化平菇菌丝体的方法。利用嵌套平板的方法,在内层平板培养平菇菌丝,待菌丝生长至平板边缘,将经诱导的根癌农杆菌与共培养培养基(CCM)混合,倒入内层平板与外层平板的间隙中,与正在生长中的菌丝共培养,实现转化,利用潮霉素抗性筛选获得转化子,平均转化效率33%左右。此方法操作简单,周期短,极易扩大实验规模,为平菇的分子育种以及其他丝状真菌的遗传转化提供参考。     

三孢布拉霉遗传转化体系的构建及应用

为解决三孢布拉霉转化过程中原生质体转化率低、孢子细胞壁厚难以导入外源载体等问题,本研究中构建了根癌农杆菌侵染原生质体的转化体系,同时克隆了与非同源末端连接修复途径相关的ku80基因,并将该转化体系应用于ku80的敲除中。将ku80基因敲除框插入双元载体pDHt-sk上构建了重组敲除载体pDH-85H3,通过化学转化法将敲除载体pDH-85H3导入根癌农杆菌LBA4404,并基于农杆菌介导法转化三孢布拉霉原生质体。结果表明,经潮霉素筛选和PCR鉴定,得到的20株转化子中,有18株的基因组插入了敲除框,转化率达90%。经鉴定有2株转化子发生了同源重组,敲除率为10%。该结论表明了根癌农杆菌介导三孢布拉霉原生质体转化技术的可行性。     

转基因番茄表达口服乙肝疫苗

将构建好的植物表达载体pCAMBIA1301/HB转化到根癌农杆菌LBA4404中,并通过农杆菌介导转化番茄。所得再生植株经PCR和Southern杂交鉴定确认成功整合了HBsAg(hepatitis B surface antigen)基因,SDS-PAGE显示蛋白表达与非转化植株相比有明显差异,ELISA检测和Western blotting证实HBsAg已在转化植株中表达,分子量约为24kD。     

农杆菌介导小球藻快速转化方法的建立与条件优化

基于根瘤农杆菌（Agrobacterium tumefaciens）介导的方法实现异养蛋白核小球藻快速遗传转化,并优化转化条件。结果表明,小球藻异养生长条件的碳氮组合配比为葡萄糖20 g/L和酵母粉2 g/L时,生长速率为自养的3～5倍;根瘤农杆菌GV3101和LBA4404均可成功实现对异养小球藻的遗传转化,且转化效率无显著性差异（P＞0.05）。最优转化条件为根瘤农杆菌GV3101初始OD600 nm值= 0.8,小球藻（Chlorella pyrenoidosa）浓度为107个/mL,各取100 μL混合涂布在pH=5.4、乙酰丁香酮浓度为200 μmol/L的CM平板上,24 ℃避光培养40 h后转到筛选平板,仅2 d转化子便清晰可见,数目达88个/106微藻。转化过程整体耗时仅5 d,普遍比自养微藻转化时间缩短3倍以上,为小球藻代谢工程改造提供技术手段,同时为其他可异养培养微藻的短时高效遗传转化提供科学依据。     

Expression of the Drosophila 70,000 Dalton heat shock protein is translationally controlled in yeast

Plasmid pPW229, containing the 2.25 kilobase transcribed sequence for the 70,000 Dalton heat shock protein of Drosophila, was integrated into plasmid CV13 and used to transform Saccharomyces cerevisiae. Upon a heat shock, at 41 degrees C for 20 min, a new 70,000 Dalton protein appeared in the transformants. This protein was not detected in transformants grown at 23 degrees C, nor in transformants carrying the hybrid plasmid from which the structural gene for the 70,000 Dalton protein had been deleted. RNA was isolated from transformants grown at 23 degrees C and from transformants heat shocked at 41 degrees C. RNA complementary to the Drosophila heat shock gene was present in the transformants, grown either at 23 degrees C or heat shocked. No complementary RNA was detected in yeast cells transformed with the hybrid plasmid from which the structural gene had been deleted. The Drosophila heat shock gene in yeast appears to be transcribed constitutively but translated only under heat shock conditions.     

黑曲霉转化系统的优化及重组菌株高效筛选

黑曲霉(Aspergillus niger)是食品工业中重要的生产菌株,广泛应用于酶和有机酸生产.为提高黑曲霉遗传操作效率,对黑曲霉转化及重组菌株筛选策略进行优化.基于已报道的最佳转化条件对黑曲霉AG11进行电转化、农杆菌介导和聚乙二醇(polyethylene glycol,PEG)介导转化(Agrobacteriu...     

DNA transformations of Candida tropicalis with replicating and integrative vectors.

The alkane-assimilating yeast Candida tropicalis was used as a host for DNA transformations. A stable ade2 mutant (Ha900) obtained by UV-mutagenesis was used as a recipient for different vectors carrying selectable markers. A first vector, pMK16, that was developed for the transformation of C. albicans and carries an ADE2 gene marker and a Candida autonomously replicating sequence (CARS) element promoting autonomous replication, was compatible for transforming Ha900. Two transformant types were observed: (i) pink transformants which easily lose pMK16 under non-selective growth conditions; (ii) white transformants, in which the same plasmid exhibited a higher mitotic stability. In both cases pMK16 could be rescued from these cells in Escherichia coli. A second vector, pADE2, containing the isolated C. tropicalis ADE2, gene, was used to transform Ha900. This vector integrated in the yeast genome at homologous sites of the ade2 locus. Different integration types were observed at one or both ade2 alleles in single or in tandem repeats.     

Genetic transformation of auxotrophic mutants of the filamentous yeast Trichosporon cutaneum using homologous and heterologous marker genes

A transformation system for the filamentous yeast Trichosporon cutaneum based on auxotrophic markers is presented and techniques for the induction, isolation and characterization of mutants are described. A number of auxotrophic mutants were isolated and characterized by using biosynthetic precursors and/or inhibitors. A mutant unable to grow in the presence of ornithine could be complemented successfully by spheroplast transformation experiments using the cloned Aspergillus nidulans ornithine transcarbamoylase gene (argB gene) as selection marker with an efficiency of 5-100 transformants per microgram of DNA. In these transformants the heterologous argB gene was present in multiple tandem copies and the transforming DNA was found to remain stable after more than 50 generations in non-selective media. The same mutant could be complemented by a T. cutaneum cosmid gene library and a complementary cosmid was subsequently isolated from this library by a sib-selection strategy. This cosmid transformed T. cutaneum spheroblasts with an efficiency of 50-200 colonies per microgram of DNA. Southern blot analyses were consistent with the view that the transforming sequences became stably integrated into the host genome at the homologous site.     

Transformation of oil-producing fungus, Mortierella alpina 1S-4, using Zeocin, and application to arachidonic acid production

The arachidonic acid-producing fungus Mortierella alpina 1S-4, an industrial strain, was endowed with Zeocin resistance by integration of the Zeocin-resistance gene at the rDNA locus of genomic DNA. Plasmid DNA was introduced into spores by microprojectile bombardment. Twenty mg/ml Zeocin completely inhibited the germination of M. alpina 1S-4 spores, and decreased the growth rate of fungal filaments to some extent. It was suggested that preincubation period and temperature had a great influence on transformation efficiency. Four out of 26 isolated transformants were selected. Molecular analysis of these stable transformants showed that the plasmid DNA was integrated into the rDNA locus of the genomic DNA. We expect that this system will be applied for useful oil production by gene manipulation of M. alpina 1S-4 and its derivative mutants. On the basis of the fundamental transformation system, we also tried to overexpress a homologous polyunsaturated fatty acid elongase gene, which has been reported to be included in the rate-limiting step for arachidonic acid production, thereby leading to increased arachidonic acid production.     

低双乙酰啤酒酵母工程菌的构建

利用PCR技术以啤酒酵母QY的染色体为模板扩增出含有乙酰羟酸合成酶（AHAS）基因ILV2的片段，将ILV2基因的内部EcoRI片段连接到整合载体YIp5上，并在该载体的Bam HI-SalI位点插入铜抗性基因CUP1－MT1，构建了YIpCE质粒，将其转化啤酒酵母QY，所得到的转化子AHAS酶的活力比受体菌QY降低75％左右， 在发酵测试中，转化了产生双乙酰的量比原始菌株降低30％。     

融合疏水蛋白在银耳中的异源表达

为改善疏水蛋白的性质,充分利用疏水蛋白和银耳的可食性优势,将平菇疏水蛋白基因hyd与去除终止密码子的hyd拼接,形成融合疏水蛋白H-H,并构建其表达载体pEGH-H-H。通过电击法将pEGH-H-H导入到根癌农杆菌EHA105中,并转化到银耳芽孢。转化子经潮霉素抗性筛选,PCR检测后,随机挑取10个转化子进行Southern blot,筛选获得高拷贝且能稳定遗传的转化子。实时荧光定量PCR分析显示5号转化子的疏水蛋白相对表达量是野生型的11.4倍。最后提取5号转化子中的融合疏水蛋白,经SDS-PAGE检测,证明融合疏水蛋白在银耳中成功异源表达。起泡性和乳化性实验表明,转化子融合疏水蛋白较平菇疏水蛋白有一定的提升。     

Transformation of Candida albicans by electroporation

In contrast to a variety of other yeasts, Candida albicans has proved difficult to transform with high efficiency. Lithium acetate transformation is fast and simple but provides a very low efficiency of DNA transfer (50-100 transformants/microg DNA), while spheroplast transformation, although more efficient ( approximately 300 transformants/microg integrative DNA and 10(3)-10(4) transformants/microg replicative DNA), is complicated and time-consuming. In this study we applied various yeast transformation techniques to C. albicans and selected an electroporation procedure for further optimization. Transformation efficiencies of up to 300 transformants/microg were obtained for an integrative plasmid and up to 4500 transformants/microg for a CARS-carrying plasmid. This reasonably high transformation efficiency, combined with the ease and speed of electroporation in comparison to alternative techniques, make it the preferred method for transformation of C. albicans.