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利用根癌农杆菌介导的转化方法改良木霉菌(英文)


第 18 卷   3 期 第 2005 年 8 月
Artical ID :100224026 (2005) 0320030206
1
5

山 东 科 学 SHANDONG SCIENCE

Vol. 18   3 No. Aug. 2005

fungi. We successfully

transformed the Trichoderma viride LTR22 by A . tumef aciens mediated method with an efficiency of 30 - 80

subtilis was integrated into the genome , and the foreign gene chi113 remained stably in transformants. The transformation system mediated by Agrobacterium tumef aciens may prove to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high

of protoplast with polyethylene glycol or the electroporation . However , all these techniques to fungi are very time2consuming with low efficacy colonization
[11 ] [3 ,4 ,5 ]

wide range of soil2borne plant2pathogenic fungi
Received Date :2005202228 3 Corresponding Author.

transformants per 10 conidia. PCR analysis showed that only the T DNA containing foreign gene chi113 encoding chitinase from Bacillus 2 transformation frequency , simplicity of T DNA integration , and genetic stability of transformants. 2 CLC number :   Q784     Document code :   A Key words : Agrobacterium tumef aciens ; Trichoderma viride ;transformation ; Hygromycin resistance ;biocontrol

the fungi transformation in 1973

transforms plants by integrating its T DNA into the cellular genome with the express of the vir genes. Besides the plant 2 hosts , A . tumef aciens has also been demonstrated to have the ability to transform yeast
[6 ]

method has proved to be a relatively simple , efficient and reproducible tool than protoplast fusion or REMI for the

transformation of filamentous fungi .

natural soils may be limited by soil fungistasis

before with the molecular biology. But there are so many disadvantages as above in filamentous fungi tradtional

transformation , in this research we report the modification of Trichoderma viride by Agrobacterium tumef aciens2mediated transformation with foreign gene chi 113 and with hygromycin B resistance as the selective marker.
? 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.

Abstract : Agrobacterium tumef aciens2mediated transformation (ATMT) system is a significant technical method in the study of many filamentous

Introduction

The study on transformation of filamentous fungi has progressed substantially in recent years since Mishrah reported
[1 ]

Filamentous fungi , in particular Trichoderma spp . , have been used as potential biological control agents against a
[8 ,9 ]

Modification of Trichoderma viride by Agrobacterium tumef aciens2mediated transformation
HUANG Yu2jie , YANG He2tong
[9 ]

(1. Shandong Provincial Key Lab for Applied Microbiology ,

Biotechnology Research Center ,Shandong Academy of Sciences , Jinan 250014 ,P. R. China ; 2. Life Science College , Shandong University of Technology ,Zibo 255049 ,P. R. China)

. The traditional procedure for transforming filamentous fungi involves the preparation
[2 ]

, limiting the utility of the system. Agrobacterium tumef aciens genetically
[7 ]

. However , the efficacy of Trichoderma spp . as biocontrol agents in , competition by other soil microorganisms
[10 ]

, or unfavorable environmental conditions. Recently , the utility of the biocontrol agents is more better than

1 ,2 3

, CHEN Kai , ZHOU Hong2zi
1

1

, filamentous fungi

. This

, poor plant root

第3期

1  Materials and Methods
1. 1  Fungal isolates

glycerol . The incubation period to form conidia was about 3 days at 28 ℃ Conidia were collected from the plates with a .
4 5 sterile scalpel , resuspended in sterile water ( about 10 - 10 per ml ) , and filtered through two layers of sterile

cheesecloth to remove large particles and mycelial debris. 1. 2  Plasmid and A . tumefaciens strain

over two layers of sterile cheesecloth and dry in oven about 24h at 65 ℃ and ground to fine powder with grinder. The

μ ml min with kanamycin ( 100 gΠ ) for 1 days at 28 ℃on a rotary shaker at 200rΠ , then
ml acetosyringone (AS) and were allowed to grow for an additional 4h at 28 ℃ with was used as control . 1. 4  Transformation 1. 5   NA isolation D 1. 6  Polymerase chain reaction conidial suspension of T . viride LTR22 , and the mixture was allowed to stand at emergency of putative transformants. RNA present in the DNA preparations was destroyed by treatment with RNase.
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μ with two volumes of absolute ethanol , washed in 70 % ethanol , air2dried , and dissolved in 200 l ddH2 O. Contaminating
Putative transformants were screened for the presence of chi 113 encoding chitinase gene from B . subitilis by PCR annealing at 53 ℃ and 2 min of extension at 72 ℃, then 20 min extension at 72 ℃ DNA from untransformed wild2type . using primers : chi 1132F ( 5 CCACATACGGTTTG AAG AGGCG 3 ) and chi 1132R ( 5 CG ACTCTGCGG AAATGTTGTGG 3 ) . The PCR program used was 10 min denaturation at 94 ℃, and 30 cycles of 1 min of denaturation at 94 ℃, 1 min of

laboratory ( Huang et al , unpublished) and contained chitinase2encoding gene chi 113 from Bacillus subtilis used for the the selective marker. ( Fig. 1) 1. 3   Cultivation of A . tumefaciens The plasmid pCA1300CHI113 was transfer to A . tumef aciens EHA105 by freeze thawing. A . tumef aciens strain EHA1052C1 was grown in LB medium supplemented The strain A . tumef aciens EHA1052C1 was mixed with an equal volume of

transformation. The plasmid pCA1300CHI113 ( about 11kb) contains the hygromycin B phosphotransferase gene ( hph) as

μ ml μ the cells were inoculated in LB medium containing 100 gΠ kanamycin and 200 gΠ
shaking at about 200rpm. And strain EHA1052C1 which grown in medium without AS

μ 28 ℃for 6h with shaking at 200rpm with AS. From the co2cultivated mixture , aliquots of 200 l were plated on potato

mycelia were resuspended in an extraction buffer ( 100 mM EDTA , 50mM Tris2HCl , pH 8. 0 ) and mixed with 10 % SDS , 5M NaCl and CTABΠ NaCl , then extracted twice with chloroform : isoamylalcohol ( 24 :1 ) . DNA was precipitated

μ ml dextrose agar ( PDA ) containing Hygromycin B ( 100 gΠ ) as a selection agent for transformation , and Ampicillin
(100 gΠ ) to kill the A . tumef aciens cells. The plates were incubated at 28 ℃ and frequently checked for the μ ml For DNA isolation , conidia from monocinidial culture were grown in 100ml of potato dextrose broth ( PDB) containing

μ ml Hygromycin B ( 100 gΠ ) in 2502ml flasks at 28 ℃on a rotary shaker at 200rpm for up to 4 days. Mycelia were collected

A . tumef aciens strain EHA1052C1 carrying the binary plasmid pCA1300CHI113 which was constructed in this
Fig. 1 Organization of binary vector pCA1300CH1113 ,which consists of pCAMBIA1300 containing the kanamycin resistance ( R) gene and the hygromycin B resistance ( hph) gene and chitinase from Bacillus subtilis . (Unpublished)

Trichoderma viride LTR22 was used in the study

HUANG Yu2jie ,et al :Modification of Trichoderma viride by Agrobacterium tumef aciens2mediated transformation
[12 ]

31

. This isolate was stored as conidial culture at - 80 ℃ in 30 %

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山                           东 科 学 2005 年

was also carried out using primers of hph and Kan : hph2F ( 5 CGGTTTCCACTATCGGCG 3 ) and hph2R ( 5 AAAGCCT A PCR programs for the amplification of Hygromycin B resistant gene and the kanamycin gene were as for chi 113 except annealing temperatures of 60 ℃ and 56 ℃, respectively. 1. 7   Chitianse assays and genetic stability was hydrolyzed by chitinase. The chitin medium contained : (NH4 ) 2 PO4 , 3g ; KH2 PO4 , 4g ; K2 HPO4 , 3g ; MgSO4 , 0. 2g ; yeast extract , 0. 2g ; boric acid , 5. 6mg ; CuSO4 , 0. 4mg ; ZnSO4 , 0. 5mg ; sodium molybdate , 1. 5mg ; ferric citrate , 1mg ; CaCl2 , 10mg ; chitin , 5mg ; agar , 13g ; water , 1000ml . pH7. 2 - 7. 4. T . viride LTR22 and the days at 28 ℃ .
5

medium was opaque for chitin granules were suspended in the medium , but became translucent in the area in which chitin

transformants were inoculated onto chitin medium plates and incubated at 28 ℃ about 5 days , then the hydrolyzed zones

were investigated.

2  Results

2. 1  Transformation of T . viride

AS produced so many colonies. In contrast , there are no colonies to appear in the plates lacking AS used for the transformation. Individual transformed colonies were picked at random and grown on PDA medium with Hygromycin B transformation efficiency was calculated to be 30 - 80 transformants per 10 conidia co2cultivated with A . tumef aciens . glycerol at - 80 ℃ until further analysis. 2. 2  PCR the only DNA transferred into the host genome. 2. 3  Genetic stability and chitinase production of transformed Trichoderma isolates
? 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.

transformants contained chi 113 , but T . viride didn ’ ( Fig. 2a ) . This result showed that chi 113 which encoding t chitinase had been transformed into T . viride . T identify whether genome DNA of transformants contain T DNA , PCR o 2 respectively. The expected products size , about 1kb ( hph ) ,were amplified from the plasmid ( pCA1300CHI113 ) and

transformed DNA templates. No product was amplified from the untransformed wild2type isolate of T . viride LTR22 ( Fig.

2b) . And no amplified product for Kan from the analyzed transformants has been obtained. All that suggested T DNA was 2

2C28 , LTR22C34 , LTR22C102 and LTR22C147 expressed stronger activity than the wild strain LTR22. The transformed

μ ml and subcultured for ten generations , then incubated on the selective medium with hygromycin B ( 100 gΠ ) about 3 - 5
PCR screening using genome DNA as template from transformants and T . viride LTR22 indicated that all the

μ ml Conidia from these monoconidial cultures of Hygromycin B ( 100 gΠ ) resistant transformants were stored in 30 %
Trichoderma isolates were plated on fresh PDA medium and subcultured for ten generations , then incubated on the

using primers specific to Hygromycin B resistance gene and primers to kanamycin resistance gene had been done ,

strains grow better than the wild ones on the chitin medium , but on the PDA medium they do not . The transformed

G AACTCACCGC 3 ) ; Kan2F ( 5 TTTCTCCCAATCAGGCTT 3 ) and Kan2R ( 5 G AATATCACCGG AG AATTG 3 ) . The A

(LTR22) served as negative control DNA template. T check the presence of vector sequences in the transformants , PCR o Chitinase activity was assessed using colloid chitin , prepared by dissolution of chitin in concentrated HCl . Chitin T test the genetic stability of the transformed Trichoderma isolates , the strains were plated on fresh PDA medium o (100 gΠ ) and purified for five times. Almost all transformants continued to grow on the selection plates. And the μ ml All the T . viride isolates tested showed a moderate chitinase activity in vitro and the transformants LTR22C2 , LTR2 It ’ about 5 - 7days that co2culture of T . viride and A. tumefaciens on PDA medium containing Hygromycin B and s

第3期

3  Discussion

poorly developed , and to our knowledge that there are so few transformation system which has been developed. This limits progress in carrying out gene manipulations as well as molecular genetic studies in this fungus. Most filamentous fungi have been transformed by protoplast technology in the presence of polyethylene glycol and calcium chloride. But applying

protoplast and low transformation efficiency often resulting in abortive or transient transformation.
[13 ]

previously shown to integrate randomly in a variety of different plants and molds heterologous integration but also with homologous
[14 ]

AS during co2cultivation. There are no positive strains appeared in the medium without AS , which showed it is indispensable that vir genes induce the transfer of T DNA during the transformation of Trichoderma . PCR ( hph gene and 2
? 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.

the molecular genetics of the fungi including the molecular basis of bio2control mechanism. Trichoderma spp . are well integration in filamentous fungi . comparable to the frequency reported by Gao et al
[14 ]

this technique to transform T . viride resulted in limited success. The major problems encountered were low yield of

transformation by protoplast method with PEG CaCl2 mediated. In this research the foreign gene ( chi 113 ) was 2 medium. ATMT of T . viride , as in other filamentous fungi
[15 ]

transformed into the genome DNA of T . viride , but the transformants grew slower than the wild strains did on PDA ,is enhanced by induction of the A . tumef aciens cells with

grew not only on selective medium but also on no2selective medium. Therefore the genetic characteristic of the recombinant T . viride is stable.
Fig. 2 Polymerase chain reaction of the chi113 gene ( Fig. 2a) and hph gene ( Fig. 2b) from randomly selected
5 generating recombinant fungal strains. The transformation efficiency ( 30 - 80 putative transformants per 10 conidia ) is

known as biocontrol agents against the plant soil2borne pathogen. However , the genetic characteristics of the fungus are kanamycin gene ) showed that only the T DNA , including the target gene , is inserted into the chromosome DNA of 2
A . tumef aciens2 imediated transformation method can alter those disadvantages in essential . TheT DNA , which was 2

μ ml selective medium with hygromycin B ( 100 gΠ ) about 3 - 5 days at 28 ℃ The results showed that the transformants .
Our results demonstrate that A . tumef aciens2mediated transformation of fungi can be used as an efficient tool for , the transformation efficiency improved about 20 folds compared to
λ primers hph F and R for amplification. 2EcoT14 I digest DNA molecular size marker(M) ,DNA from the wild2type
strain (Fig. 2a ,lane6 ;Fig. 2b ,lanes1 ,2) ,DNA from plasmid (Fig. 2a ,lane5 ;Fig. 2b ,lanes 3) and DNA from transformants(Fig. 2a ,lane124 ;Fig124 ;Fig. 2b ,lanes 4 7) 2 transformants of T . viride ,the plasmid pCA1300CHI113 and the wild type strain T . viride LTR22 using 2

The development of an efficient transformation system is an important tool for genetic manipulation and for studying

HUANG Yu2jie ,et al :Modification of Trichoderma viride by Agrobacterium tumef aciens2mediated transformation

33

However ,

, can also be used not only with

Trichoderma viride LTR22. And this characteristic is significant for the transgene safety. Because if we use auxotroph as

34

山                           东 科 学 2005 年

plant or fungi except T DNA fragments. 2 References :
Genet Res ,1977 ,29 : 9 - 19. - 153. [J ] . Mycol Res ,2002 ,106 :132 - 137. 23 - 54. 2004 ,5 :32 - 35.

protoplast2requiring transformation protocols. Fortunately , it should be possible to adapt the approaches used here to many additional fungi . The hygromycin B resistance cassette in pCAMBIA1300 is specific and its function is so clear that application of this relatively simple and useful methodology to a variety of other filamentous fungal species.
[1 ]   Mishra N C. Characterization of the new osmotic mutant (os) which orginated during genetic transformation in Neurospora crassa [J ] . [2 ]  Hynes M J Genetic transformation of filamentous fungi[J ] . J Genet , 1996 , 79 : 297 - 311
grisea to hygromycin2B resistance[J ] . Current Geneics ,1990 ,17 :409 - 411.

can be used in a range of species. The broad utility and availability of the reagents used in our study should allow the

selective marker , the other fragments in Ti2vector , especially bacterium antibiotic would not integrate into genome DNA of
[3 ]   Leung H. Lehtinen U. Karjalainen R. Skinner D. T ooley P. Leong , S Ellingboe A. Transformation of the rice blast fungus Magnaporthe [5 ]  Marmeisse R. Gay G. Debaud J . C , Casselton L A. Genetic transformation of the symbiotic basidiomycete fungus Hebeloma
cylindrosporum . Current Genetics ,1992 ,22 :41 - 45.

[4]   Durand N. Reymond P ,fevre M. Transformation of Penicillium roqueforti to Phleomycin2B2Resistance[J ] . Current Genetics ,1991 ,19 :149 [6 ]  Bundock P , Den Dulk2Ras A. Beijersbergen A , Hooykass PJJ Trans kingdom T DNA transfer from Agrobacterium tumef aciens to 2 2
Saccharomyces cervisiae [J ] . EMBO J , 1995 , 14 :3206 - 3214

[7 ]   Pardo AG, Hanif M , Raudaskoski M , G orfer M Genetic transformation of ectomycorrhizal fungi mediated by Agrobacterium tumef aciens

[8 ]   Chet I. Trichoderma2application , mode of action , and potential as biocontrol agent of soilborne plant pathogenic fungi [ A ] . Innovative approaches to plant disease control[ C] . John Wiley & Sons , Inc. , New Y , N. Y. 1987 ,137 - 160 ork [9 ]   Papavizas G C. Trichoderma and Gliocladium : biology , ecology , and potential for biocontrol[J ] . Annu. Rev. Phytopathol. 1985 ,23 : [10 ]  Hubbard J P , G E Harman , Y Hadar. Effect of soilborne Pseudomonas spp . on the biological control agent , Trichoderma hamatum , on pea seeds[J ] . Phytopathology ,1983 , 73 :655 - 659. [11 ]   Ahmad J S ,R Baker. Rhizosphere competence of Trichoderma harzianum [J ] . Phytopathology ,1987 , 77 : 182 - 189. agents of Trichoderma spp [J ] . Journal of Shandong University of Technology ( Sci & Tech) ,2004 ,Vol. 18 (6) :15 - 23. [ 13 ]  Groot M J A ,de , Bundock P , Hooykaas P J J ,Beijersbergen A G M. Agrobacierium tumef aciens2mediated transformation of filamentous fungi[J ] . Nat. Biotechnol. 1998 16 , 839 - 842. [15 ]  Yeshitila Degefu , Mubashir Hanif , Agrobacterium2tumef aciens2mediated transformation of Helminthosporium turcicum , the maize leaf2 blight fungus[J ] . Arch Microbiol 2003 ,180 :2792284. [14 ]  Gao Xingxi ,et al. Agrobacerium tumef aciens2mediated Transformation of Trichoderma harzianum [J ] . HIGH TECHNOLOGY LETTERS

[ 12 ]  Yang Hetong ,Tang Wenhua ,Maarten Ryder ,etal. Lectins of fungal pathogens as potential tools in selecting promising biological control

In our experience , Agrobacterium tumef aciens2mediated transformation is simpler and less labor2intensive than
? 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.

第3期

利用根癌农杆菌介导的转化方法改良木霉菌
黄玉杰 ,杨合同
1 1 ,2

(1. 山东省科学院中日友好生物技术研究中心 ,济南 250014 ;2. 山东理工大学生命学院 ,淄博 255049)

摘要 : 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Π 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 10

基因 ( chi113) 的 T2DNA 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 。 关键词 : 根癌农杆菌 ; 绿色木霉菌 ; 转化 ; 潮霉素抗性 ; 生物防治 中图分类号   Q784        文献标识码 :A ? 1995-2005 Tsinghua Tongfang Optical Disc Co., Ltd. All rights reserved.

HUANG Yu2jie ,et al :Modification of Trichoderma viride by Agrobacterium tumef aciens2mediated transformation

35

,陈凯 ,周红姿

1

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