根癌农杆菌介导的木霉插入转化及其应用

根癌农杆菌介导的遗传转化法(ATMT)已广泛地应用于真菌的转化研究,该方法具有操作简单、转化效率高和转化子遗传稳定等特点。木霉作为土传植物病原菌的重要生防真菌,对其生防分子作用机制方面的研究也越来越受到关注,目前所面临的问题是如何基因突变和基因替换。ATMT法为木霉(Trichodermaspp.)的分子生物学研究提供了一个强有力的工具,在木霉的插入突变、遗传转化以及基因标记和克隆方面起着重要的作用。本文综述了根癌农杆菌介导木霉遗传转化的原理、特点、转化方法和应用,并且对有关发展趋势进行了展望。     

Efficient transformation and expression of gfp gene in the edible mushroom Pleurotus nebrodensis

An efficient transformation method mediated by PEG-protoplasts was developed for the newly commercial edible mushroom Pleurotus nebrodensis. Two plasmids were used to co-transform protoplasts of P. nebrodensis. One plasmid is pAN7-1 containing a positive selectable marker gene hph conferring hygromycin B resistance. Another plasmid is pBlue-GFP containing a reporter gene gfp conferring green fluorescent protein. PCR and Southern blot analysis showed that hph gene or/and gfp gene were integrated into the genome of P. nebrodensis transformants. The transformation efficiency of the positive selectable marker gene hph was 3 transformants per microgram of plasmid pAN7-1 DNA, which was about 30 times higher than that previously reported in thoroughly studied Pleurotus species such as Pleurotus ostreatus. The transformation efficiency of the reporter gene gfp was 9 transformants per microgram of plasmid pBlu-GFP DNA. The co-transformation efficiency was 23.68%. This is the first report that a ＂reporter＂ gene, green fluorescent protein gene can be successfully stably exoressed in this Pleurotus species.     

T-DNA transfer and integration as a tool for insertional mutagenesis in the taxol-producing fungus

Agrobacterium tumefaciens-mediated DNA transformation method was applied to transform Nodulisporium sylviforme fusant HDF-68, a taxol-producing fungus. We constructed a binary vector pBI121-43 carrying a hygromycin-resistant gene cassette between the right and left borders of T-DNA. Optimal co-cultivation of N.sylviforme with A.tumefaciens containing pBI121-43 led to 110～130 hygromycin-resistant transformants per million conidia. Putative transformants were found to be mitotically stable. The molecular analysis of transformants demonstrated the random integration of single copy of the T-DNA into the host genome. This transformation system serves as a basic tool for insertional mutagenesis in N.sylviforme fusant HDF-68, and the development of such system lays a solid foundation for constructing high-yied gene engineering strain and clarifying taxol biosynthesis pathway in this fungus.     

利用根癌农杆菌介导的转化方法改良木霉菌

根癌农杆菌介导的转化系统在丝状真菌的研究中具有重要的意义。通过农杆菌介导,成功实现了丝状真菌绿色木霉菌(Trichoderma viride)遗传转化,转化率约为30～80个转化子／10^5个孢子。PCR检测和几丁质酶分析表明含有编码几丁质酶外源基因(Cli 113)的T-DNA已整合进木霉菌基因组中,而且转化子都能够稳定遗传。农杆菌介导的遗传转化方法具有转化率高、操作简便、遗传稳定等优点,在丝状真菌的遗传转化中具有重要的意义。     

根癌农杆菌介导的丝状真菌简青霉遗传转化的研究

利用根癌农杆菌LBA4404介导,建立了丝状真菌简青霉(Penicillium simpli-cissimum)H5的遗传转化系统.潮霉素抗性筛选、PCR和Southern blot分子鉴定等结果表明:筛选获得的转化子能够稳定遗传,外源的T-DNA以单拷贝随机整合到简青霉的基因组中.实验初步研究了农杆菌浓度、乙酰丁香酮浓度和共培养时间等因素对转化效率的影响,经过优化后转化体系的效率可达50个转化子/105个孢子.农杆菌介导的遗传转化方法在简青霉上的运用,将为研究该菌的基因工程改造提供强有力的工具.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Functional genomics in the rice blast fungus to unravel the fungal pathogenicity

A rapidly growing number of successful genome sequencing projects in plant pathogenic fungi greatly increase the demands for tools and methodologies to study fungal pathogenicity at genomic scale. Magnaporthe oryzae is an economically important plant pathogenic fungus whose genome is fully sequenced. Recently we have reported the development and application of functional genomics platform technologies in M. oryzae. This model approach would have many practical ramifications in design and implementation of upcoming functional genomics studies of filamentous fungi aimed at understanding fungal pathogenicity.     

Nonspecific integration of the HTLV provirus genome into adult T-cell leukaemia cells

Human T-cell leukaemia virus (HTLV), previously also reported as ATLV, is a recently identified retrovirus which is closely associated with adult T-cell leukaemia (ATL) endemic in southwestern Japan and the Caribbean. Determination of the total nucleotide sequence of the HTLV genome has revealed no typical onc gene acquired from the cellular sequence. Screening of the HTLV provirus genome in tumour cells has shown that in all cases of ATL examined, the primary tumour cells contained the provirus genome and were monoclonal with respect to the integration site of the provirus. These findings suggest that ATL leukaemogenesis may be due to insertional mutagenesis in which the provirus genome is integrated into a specific locus on the chromosomal DNA and then activates an adjacent cellular onc gene, a mechanism already demonstrated in avian lymphoma and erythroblastosis induced by avian leukosis viruses. A common site of HTLV provirus integration in leukaemic cells among some ATL patients was reported by Hahn et al. but subsequently retracted. However, this retraction does not imply the random integration of the proviruses. Independently, we have been testing this insertional mutagenesis model in ATL and report here that the provirus did not have a common locus of integration in 35 ATL patients and did not integrate on the same chromosome in 2 ATL patients.     

Construction of Streptomyces lydicus A01 transformant with the chit33 gene from Trichoderma harzianum CECT2413 and its biocontrol effect on Fusaria

Streptomyces lydicus A01 resists many plant pathogens (including Fusarium spp.) by producing the antifungal agent natamycin, which binds to the ergosterol of fungal cell membranes and inhibits the growth of pathogens. Trichoderma harzianum CECT2413 is a widely-distributed soil fungus that antagonizes several plant fungal pathogens (including Fusarium spp.) by producing chi-tinase and degrading chitin, a major component of the fungal cell wall. This study attempted to enhance the biocontrol effect of S. lydicus A01 on Fusarium spp. by transforming the chitinase gene of Trichoderma. Chitinase and natamycin could act synergisti-cally on both the cell walls and cell membranes of pathogens. The 33-kD chitinase-encoding gene (chit33) was cloned and conju-gal-transformed from T. harzianum CECT2413 to S. lydicus A01, and then confirmed via polymerase chain reaction (PCR) assays. Subsequent analyses using the 3,5-dinitrosalicylic acid (DNS) method and ultraviolet spectrophotometry showed that compared with its wild type strain (WT), the S. lydicus A01 conjugal transformant (CT) with chit33 gene exhibited substantially higher chi-tinase activity and natamycin production. The resistance of S. lydicus A01-chit33 CT and WT to four Fusaria in crops and vegetables was tested via the cup-plate method. Compared with the WT, the conjugal transformant of S. lydicus A01 with chit33 gene from T. harzianum CECT2413 showed greatly increased biocontrol effect on fusarium disease. This study would be beneficial to the development of high-quality antifungal bio-agents for agricultural applications via the synergy between the previously non-existent and pre-existing functions achieved through heterogeneous gene transformation.     

Salt tolerance conferred by over-expression ofOsNHX1 gene in Poplar 84K

OsNHX1 gene (Na⁺/H⁺ antiporter gene ofOryza sativa L.) was introduced into Poplar 84K withAgrobacterium tumefaciens- mediated transformation. PCR, Southern and Northern blot analysis showed thatOsNHX1 gene was incorporated successfully into the genome of Poplar 84K and expressed in these transgenic plants. Salt tolerance test showed that three lines of transgenic plants grew normally in the presence of 200 mmol/L NaCl, while the Na⁺ content in the leaves of the transgenic plants grown at 200 mmol/L NaCl was significantly higher than that in plants grown at 0 mmol/L NaCl. The osmotic potential in the transgenic plants with high salinity treatment was lower than that of control plants. Our results demonstrate the potential use of these transgenic plants for agricultural use in saline soils.     

Stable expression of Arabidopsis vacuolar Na+/H+ antiporter gene AtNHX1, and salt tolerance in transgenic soybean for over six generations

The Arabidopsis vacuolar Na+/H+ antiporter gene,AtNHX1,was introduced into soybean by Agrobacterium-mediated transformation.Four independent kanamycin resistant lines were obtained.The result of PCR,Southern blotting and Northern blotting analyses demonstrated that the AtNHX1 gene was successfully inserted into the soybean genome and stably expressed in these kanamycin resistant lines.The stability of AtNHX1 expression and salt resistance were evaluated in the soybean transformants for over 6 generations.Tw...     

低温菌穿梭质粒的构建及转化方法研究

 由于低温微生物在细胞结构上的特殊性,使得对它们进行遗传操作受到很大的限制.以分离自冻土的低温菌Acinetobacter sp.DWC6为宿主菌,构建了一套外源DNA导入系统.通过在质粒pBR322和pUC118中插入一段Acinetobacter属特异性的Ori片段,成功构建了一系列穿梭质粒,并建立了稳定的转化方法,所有重组质粒均可在Escherichia coli和Acinetobacter sp.DWC6中正常复制.通过优化转化方法,使质粒在低温菌Acinetobacter sp.DWC6中的转化率达3×10⁶转化子/μg DNA.     

莱茵衣藻外源基因整合特征分析及蛋白表达

将衣藻表达载体pSP108转化莱茵衣藻细胞壁缺陷型藻株CC-400,通过接头PCR获得并分析阳性转化子中ble基因的侧翼序列发现:外源基因的插入位点呈现随机性,并且有些转化子中衣藻基因组和质粒序列发生断裂和重排.通过间接ELISA分析外源蛋白表达量,并结合外源基因整合状态时发现:在ble基因启动子核心区域缺失的阳性转化子中,ble可以利用自身基因启动子进行蛋白表达,但表达量相比使用质粒载体PSP108上RBCS2启动子的表达量要低;在部分启动子完整的阳性转化子中蛋白表达量低下,可能与衣藻基因组大片段的缺失相关.     

A Gγ subunit promoter T-DNA insertion mutant――A1-412 of Magnaporthe grisea is defective in appressorium formation, penetration and pathogenicity

Heterotrimeric G-proteins consisting of α, β and γ-subunits are essential for the transduction of ex- tracellular signals to various downstream intracellular effectors in eukaryotes. Previous studies showed that Gα and Gβ were involved in regulating     

Expression of the mouse metallothionein mutant ββ-cDNA in the lettuces (Lactuca sativa L.)

Mouse metallothionein (MT) domain mutant ββ-cDNA gene has been inserted into the plant expression vector pGPTVd35S which has herbicide (PPT)-resistance gene bar as a selective marker. The chimeric gene was introduced into the lettuce (Lactuca sativa L. cv Salinas 88) by Agrobacterium-mediated transformation. PCR and Southern blot analysis of some putative transformants indicated that the introduced ββ-cDNA was integrated into lettuce genome and inheritted by sexual reproduction. The expression of ββ gene in lettuce plants was demonstrated by Northern and Western blot analysis. Meanwhile, the zinc content of the transformed lettuce plants is up to 400 μg/g dry weight, remarkably higher than the control plants.     

Studies of biouptake and transformation of mercury by a typical unicellular diatom Phaeodactylum tricornutum

Mercury(Hg) is a toxic heavy metal with its biogeochemical cycling in the ocean depending on the type and behavior of the oceanic microalgae.The present work aimed to evaluate bioaccumulation and transformation of Hg by Phaeodactylum tricornutum,a typical unicellular diatom,when exposed to the extremely high level of Hg in order to understand the possible mechanisms of acute stress response.P.tricornutum can accumulate Hg(its bioaccumulation factor is at 104 level),and the 96 h EC 50 was estimated to be 145μg L-1.The amounts of surface-bound Hg being about 1.2 to 4.8 times higher than those of intracellular Hg under exposure to HgCl 2(from 20 to 120μg L-1 concentrations) suggested that the cell wall of P.tricornutum is an important "fence" towards Hg.After entering the P.tricornutum cell,Hg underwent transformation in its chemical form via interactions with high molecular weight sulfur-containing proteins(accounting for 68% of the intracellular Hg),and glutathione as well as the induced phytochelatins(PCs)(24% Hg) which alleviated the toxicity of HgCl2.In addition,the existence of organic ligands greatly influenced the uptake and transformation behavior of P.tricornutum towards HgCl2,especially in the case of cysteine(Cys),which increased the uptake of Hg,but alleviated the toxicity of Hg towards P.tricornutum due to the fact that Cys is an important precursor for the synthesis of PCs inside the cell.The uptake process of Hg by P.tricornutum was in agreement with the Freundlich isotherm,suggesting a typical heterogeneous sorption process.More importantly,we observed the conversion of HgCl2 into methylmercury inside the P.tricornutum cells and its release into the culture solution using HPLC/CVG-AFS and GC-MS,although the mechanism needs to be further investigated.     

Fungal diversity on fallen leaves of Ficus in northern Thailand

Fallen leaves of Ficus altissima, F. virens, F. benjamina, F. fistulosa and F. semicordata, were collected in Chiang Mai Province in northern Thailand and examined for fungi. Eighty taxa were identified, comprising 56 anamorphic taxa, 23 ascomycetes and l basidiomycete. Common fungal species occurring on five host species with high frequency of occurrence were Beltraniella nilgirica, Lasiodiplodia theobromae, Ophioceras leptosporum, Periconia byssoides and Septonema harknessi. Colletotrichum and Stachybotrys were also common genera. The leaves of different Ficus species supported diverse fungal taxa, and the fungal assemblages on the different hosts showed varying overlap. The fungal diversity of saprobes at the host species level is discussed.     

Improving DNA damage repair ability ofE. coli to UV-radiosensitivity by DNA-mediated gene transfer of low energy ion beam

Using calcium chloride method of transfer gene as control, a new technique of transferring gene by low energy ion beam has been applied to the study of improving DNA damage repair ability ofE. coli to UV-radiosensitivity. The genome DNA pieces ofDeinococcus radiodurans, as “foreign” genetic materials, were introduced into the UV-radiosensitive strains ofE. coli by implantation of 20 keV Ar⁺ at doses ranging from 1 × 10¹⁵ to 2 × 10¹⁵ ions/cm². Results show that the transfected strains present higher UV-radioresistance than that of un-transfected ones and start ones. The survival rate of transfected strains and their unscheduled DNA synthesis (UDS) ability is increased, indicating that the transfer gene is a success.     

转座子诱变甘蓝黑腐病菌所获胞外多糖突变体的类型

据对转转子Ｔｎ５ｇｕｓＡ５诱变甘蓝黑腐病民获得３９株胞外多糖突变休的３种胞外酶活性和在含２％葡萄ＮＹＧＡ培养基上的菌落形态的检测结果，将突变株分成５种类型。Ｓｏｕｔｈｅｒｎ杂交结果表明它们的突变位于基因组中的８个不同的位置，其中１个位于已鉴定的ｒｐｆ调控基因簇。