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元旭
YUAN Xu
文献标识码: 1671-9964(2016)01-0085-06
文章编号: 1671-9964(2016)01-0085-06
收稿日期: 2014-11-13
网络出版日期: 2016-01-20
版权声明: 2016 上海交通大学期刊中心 版权所有
基金资助:
作者简介:
作者简介:元旭(1989-), 男, 硕士生, 研究方向:分子植物病理学;
通讯作者: 陈捷(1959-), 男, 博士, 教授, 博士生导师, 研究方向:植物病虫害综合防治, 环境微生物与生物农药基因工程, E-mail:jiechen59@sjtu.edu.cn
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摘要
本研究对木霉菌T30野生株和限制性内切酶介导的DNA整合技术(REMI)构建的突变株进行降解敌敌畏能力和最佳条件的研究。结果表明, 野生株和几个REMI突变株均能降解有机磷农药敌敌畏, 但以突变株TK-3降解活性最高, 其降解效率达98%。TK-3突变株降解有机磷农药效率取决于葡萄糖剂量、敌敌畏初始浓度、初始pH等。突变株降解敌敌畏的葡萄糖最佳浓度为1 000 μg/mL, 最适pH为7.0, 敌敌畏浓度500 μg/mL。敌敌畏浓度超过1 000 μg/mL则会抑制木霉菌降解能力。
关键词：
Abstract
The dichlorvos degradation abilities of Trichoderma koningii mutants obtained by REMI (restriction enzyme mediated integration) and the optimal conditions for effective degradation were studied.Results showed both the wild type (T30) and mutants (TK-1, TK-2, TK-3, TK-5, TK-7, TK-8, TK-21, TK-38, TK-53, TK-30, TK-42)were able to degrade dichlorvos.TK-3 could reach 98% dichlorvos decomposition, which is the most efficient among mutants.Dichlorvos degradation rate was closely associated with glucose, dichlorvos initial concentration and pH of the medium.It was found that glucose at 1 000 μg/mL, pH 7.0 and dichlorvos at 500 μg/mL were the optimal conditions for effectively degrading dichlorvos by the mutant TK-3.The degradation efficiency of TK-3 would decline if dichlorvos concentration exceeded 1 000 μg/mL.
Keywords：
Trichoderma, as a bio-control microbe, has been widely applied in the control of soil borne diseases[1-2], which is a resource-rich antagonistic organism as well as a useful soil remediation microorganism. As people pay more attention to agricultural environment pollution control, the Trichoderma strain is required not only with antimicrobial activity but also with potential to reduce pollutants[3-6].Therefore, in the future, multi-function Trichoderma agents would be highly valuable for comprehensive application in agriculture[7].
In the past decades, numerous microorganisms have been genetically modified for improving degradation activity of organophosphorus pesticide. Lorz et al.[8]successfully used protoplast transformation to construct a multi-functional genetically engineered strain. Woloshuk et al.[9]transferred insect detoxifying gene into microbial, creating a new pesticide degrading strain. Sun et al.[10]took advantage of gene recombination in different microbe and finally obtained a strain with better dichlorvos degradation performance.
Nowadays, studies on the degradation of organophosphorus pesticides by Trichoderma are increasingly emphasized because of extensive concerns over environment pollution [11].However, as most wild type strains from Trichoderma seemed not effective enough to reduce organophosphorus pesticide residues in environment, genetic modification should be a rational choice to improve their degradation ability. REMI(Restriction Enzyme Mediated Integration)is a relatively new approach to create diversified molecular mutations by random insertion of DNA fragment into chromosome, elite functional strain may be obtained from mutated population. Liu et al.[12]used REMI to obtain Trichoderma viride T21, which works better than Ttrm31, Ttrm34 and Ttrm55 in bio-control tomato grey mold caused by Botrytis cinerea. The three REMI mutants produce higher chitinase and β-1, 3 glucanase activity than wild type, suggesting the bio-control activity is significantly improved. Zhou[13] had studied cyanides degradation efficiency of different Trichoderma REMI mutants.It is found that the mutants T30, TKB6 and TaK1 show higher rhodanese activity than wild type strain and other mutants. Therefore, REMI is a promising method to effectively create genetic variation to improve theagro-chemicals residue degradation ability of Trichoderma. Huang[14]also used REMI to get plenty of transformants, it is proved that there are significant improvement of Trichoderma in the inhibition against barnyard grass and tall fescue grass growth, which means that selected mutants of Trichoderma are useful to inhibit the growth of some herbs. Taken together, Trichoderma REMI mutants can be used in many fields.
To date, however, reports on the role of REMI Trichoderma mutants in the degradation of dichlorvos are pretty limited. Previous studies demonstrated microorganisms that can degrade dichlorvos are mainly focused on bacteria. In this study, we intend to evaluate the activity of Trichoderma koningii REMI mutants in dichlorvos degradation, and find out the optimal conditions for REMI transformants to degrade DDV efficiently, eventually open a new way to treat harmful pollutants present in agricultural environment.
T.koningii wild type T21, T23, T30, and REMI mutants TK-1, TK-2, TK-3, TK-5, TK-7, TK-8, TK-21, TK-38, TK-53, TK-30, TK-42, were stored in School of Agriculture & Biology, Shanghai Jiaotong University.
Potato dextrose agar(PDA), Potato dextrose(PD), Burk media(g/L:KH2PO4, 0.8; MgSO4·7H2O, 0.2; CaSO4·2H2O, 0.1, Na2MoO4·2H2O, 0.0033; FeSO4·7H2O, 0.005; (NH4)2SO4, 1.0; glucose, 1.0)(pH 7.0).
80% dichlorvos, 100 μg/mL dichlorvos acetone solution(purchased from Beijing Zhongwei Food Hygienic Technology Company), 10%(V/V)ethanol-water solution, 1%(m/V)resorcinol aqueous solution, 0.5%(m/V) NaOH solution, etc.
The Trichoderma koningii strains stored with silicone beads were cultured on PDA for 3-4 days until the spores were produced. With hole punchers, the colony were inoculated into Burk solid media containing dichlorvos(100 mg/mL), incubated at 28 ℃ for 5 days. Tolerance of dichlorvos for different T.koningii mutants was compared.
The spores were washed by sterile water, diluted into 1×107conidia/mL, and then inoculated in PD(100 mL).Flasks were shaked at 28 ℃ for 2 days. After filtration with Buchner funnel and washing with sterile water, thallus were weighed and transferred to Burk media containing dichlorvos, cultured at 30 ℃ with shaking(180 r/min)for 60 h, the concentration of dichlorvos was determined after filtration.
Resorcinol fluorescence technique was used to determine the contents of dichlorvos. The sample was diluted with culture medium containing dichlorovs to 10 mL by adding 4 mL 10%(V/V)ethanol solution, 0.96 mL 0.5%(m/V)NaOH solution and 0.24 mL 1%(m/V)resorcinol aqueous solution. After blending, samples were placed in boiling water bath for 3 min, when it’s cool to room temperature by water, the fluorescence intensity at excitation wave length λex 491.6 nm and fluorescent wave length λem 521.1 nm was determined. Meanwhile, media without dichlorvos was used as control. If the initial concentration of dichlorvos was C1, concentration after treatment was C2, the dichlorvos degradation rate could be represented as(C1-C2)/C1×100%.
Three factors(pH, glucose concentration and dichlorvos concentration)were studied to evaluate their influence on dichlorvos degradation by REMI transformant TK-3.Detailed experimental procedures were as follows:
Burk media containing 300 μg/mL dichlorvos was adjusted to different pH value(pH 3-10), inoculated with the REMI mutants of T.koningii, and then it was cultured shakily(120 r/min)at 28 ℃ for 60 h, the residual concentration of dichlorvos was determined to calculate the decomposition rate.
The mutants of T.koningii was inoculated into Burk media containing 300 μg/mL dichlorvos, and gluocose was added to different concentration(500, 1 000, 5 000, 10 000 μg/mL), cultured with shaking(120 r/min)at 28 ℃ for 60 h, dichlorvos degradation rate was determined as described.
Then, 500 μg/mL gluocose was added to Burk media, containing different concentration of dichlorvos(50, 100, 300, 500, 1 000 μg/mL), then the mutants of T.koningii were inoculated and cultured at the above mentioned conditions.Then, dichlorvos degradation rate was determined as described.
It showed all transformants of Trichoderma koningii could grow on Burk solid media containing dichlorvos, indicating REMI mutants of T.koningii had tolerance for dichlorvos.Clones with relatively fast growth were subjected to re-screen.
After further analysis, it was found there were great differences between different transformants(Fig.1).Among the transformants from T30, TK-3 had the highest degradation rate(up to 96.9%).Spore suspension, fermentation broth and the mycelium broken by ultrasonication of TK-3 were studied separately when exposed to dichlorvos to identify the degradation process.It indicated the mycelium of the mutants of Trichoderma koningii played an important role in degrading dichlorovs.
TK-3 could grow well at different pH(6-9), when pH was 7, the degradation rate of TK-3 reached maximum(Fig.2).As pH rose, the degradation rate gradually decreased.
Fig.3 showed that at different glucose concentration, the degradation rate was different. When it was 1 000 μg/mL, the degradation rate could reach up to 95.9%.However, if the glucose concentration was reduced, the degradation rate was dropped down as well. But if glucose concentration was over 1 000 μg/mL, the mutants of T.koningii were induced to use glucose in priority and the degradation rate decreased as a result.
Fig.4 indicated when dichlorvos concentration was 500 μg/mL, the degradation rate was up to the highest.The degradation rate increased linearly with initial dichlorvos concentration differed from 50 μg/mL to 500 μg/mL.When increased to 1 000 μg/mL, the degradation rate declined.It declared that the tolerance dichlorvos concentration for transformant of T.koningii was 500 μg/mL, if the concentration was too high, it may poison the transformant and make the degradation rate decreased.
Fig.4 Degradation efficiency of TK-3 under different initial dichlorvos concentrations
To date little work has been reported on degradation of dichlorvos by Trichoderma.The major target pesticides for being investigated on microbial degradation includes parathion, monocron and dimethoate and the majority of investigated microbe are bacteria rather than fungi.Even so those bacteria are not effective enough in dichlorvos degradation.Tang et al.[15] reported that Trichoderma had a relatively high degradation rate for dichlorvos.Similarly, in this study, we researched the degradation of dichlorvos by T.koningii and its REMI transformants.The results indicated that both the wild type and mutants had ability to degrade dichlorvos.In general, there was a closely connection between dichlorvos degradation and experimental conditions including amount of glucose, dichlorvos initial concentration and pH of the medium.
As to organophosphate pesticide degradation mechanism, some authors have reported the significance of co-metabolism.
Sun et al.[16]has studied T.atroviride on degrading pesticides and found that degradation was closely related to its growth, indicating co-metabolism degradation.When glucose concentration was 5 g/L, the mycelium grew quickly while the degradation rate of methamidophos decreased.However, when glucose concentration was increased to 10 g/L, degradation of methamidophos was fully inhibited, suggesting that substrate may play an important role in pesticide degradation by microbes which means the excess of nutrients may inhibit the degrading activity of enzymes.Therefore, researches on the type and proportion of carbon, nitrogen and phosphorus were needed to clarify dichlorvos degradation mechanism by Trichoderma.In this study, it was demonstrated that the degradation rate of REMI mutants of T.koningii was lower when glucose concentration was 500 μg/mL and 5 000 μg/mL than 1 000 μg/mL, which implied presence of co-metabolism between REMI mutants of T.koningii and dichlorvos, and depended on the concentration of carbon source.In addition, based on our previous experiment, we hypothesis that higher concentration of glucose over 1 000 μg/mL may influence the function of some transporters that pump dichlorvos out of cells.
Dichlorvos concentration is also a major factor impacting biodegradation of dichlorvos.When the concentration of dichlorvos is too high, that would be toxic to the microbe, leading to microbial population dropped significantly.To the contrary, if the concentration of dichlorvos is too low, degradation rate is also declined as lacking of carbon and nitrogen source to support microbe growth.Zhao et al.[5]confirmed when the concentration of dimethoate was 0.2%, the degradation by microorganism was the best, while lower in degradation at 0.1% and 0.5%. Here, in this article, we’ve got the similar results.When dichlorvos concentration was 500 μg/mL, the degradation rate was the highest, but declined at 100 μg/mL and 1 000 μg/mL.Thus, it is not a linear relation between pesticide concentration and degradation level by microbe.In other words, the biodegradation relied on specific concentration could provide a scientific basis to face up to serious pollution incited by over release of pesticide into environment.Meanwhile, studies on degradation of low concentration pesticides could provide theoretical basis and reference to bioremediation on environmental pollution caused by pesticide residues.
4 Acknowledgements
We would like to acknowledge the financial assistance of the China Agriculture Research System Project(CARS-02), the National Natural Science Foundation of China(31270155)and the key basic research project of Shanghai Municipal Science and Technology Commission(12JC1404600).We are also deeply indebted to all the teachers in our lab for their help, special thanks should go to Sun Jianan and Zhang Tailong who support us so much and encourage us to finish the article.
The authors have declared that no competing interests exist.
| [1] |
Trichoderma-plant root colonization:escaping early plant defense responses and activation of the antioxidant machinery for saline stress tolerance [J].DOI:10.1371/journal.ppat.1003221 URL PMID: 23653566 [本文引用: 1] 摘要
Author Summary Trichoderma fungi have been developed as biocontrol agents and are applied to protect and improve crop yields. Colonization of plant roots by Trichoderma can protect plants against diseases and environmental stresses such as salinity and drought, and an improve plant growth and development. To better understand the mechanism underlining the plant- Trichoderma interaction we followed changes in global gene expression in colonized Arabidopsis roots. We associate the known gene biological function to the processes of root colonization and abiotic stress tolerance mediated by Trichoderma . Using Arabidopsis mutant lines we show the function of a subset of those genes in root colonization. We show that wrky18 and wrky40 transcription factors activate and suppress the expression of different genes in order to allow successful root colonization. We also combine the gene expression data together with the measurement of ascorbic acid level to demonstrate that salt stress tolerance offered by Trichoderma is dependent on activation of the plant antioxidant defense machinery. Using Trichoderma lines mutated in the ACC deaminase gene, we demonstrate that reduction of ethylene levels is also essential in achieving salt tolerance. This study represents an important step forward in understanding the nature of the non-pathogenic plant Trichoderma interaction, and may contribute to the efforts to improve Trichoderma biocontrol abilities.
|
| [2] |
Understanding Trichoderma:between biotechnology and microbial ecology [J].DOI:10.1007/s101230100001 URL PMID: 11770814 [本文引用: 1] 摘要
Author information: (1)emv@gugu.usal.es
|
| [3] |
Growth inhibition of the cyanobacterium Microcystis aeruginosa and degradation of its microcystin toxins by the fungus Trichoderma citrinoviride [J].DOI:10.1016/j.toxicon.2014.05.008 URL PMID: 24874888 [本文引用: 1] 摘要
Harmful cyanobacterial blooms are recognized as a rapidly expanding global problem that threatens and ecosystem health. Many bacterial strains have been reported as possible agents for inhibiting and controlling these blooms. However, such algicidal activity is largely unexplored for . In this study, a fungal strain kkuf-0955, isolated from decayed cyanobacterial bloom was tested for its capability to inhibit phytoplankton species in batch cultures. The strain was identified as Based on its morphological characteristics and DNA sequence. co-cultivated with living fungal mycelia rapidly decreased after one day of incubation, and all completely died and lysed after 2 days. The fungal filtrate of 5-day culture also exhibited an inhibitory effect on , and this inhibition increased with the amount of filtrate and incubation time. Conversely, and have not been influenced by either living fungal mycelia or culture filtrate. Interestingly, the fungus was not only able to inhibit but also degraded produced by this . The toxins were completely degraded within 5 days of incubation with living fungal mycelia, but not significantly changed with fungal filtrate. This fungus could be a potential bioagent to selectively control blooms and degrade toxins.
|
| [4] |
Tmac1, a transcription factor which regulated high affinity copper transport in Trichoderma reesei [J].DOI:10.1016/j.micres.2012.02.002 URL PMID: 22397974 摘要
The Mac1 protein was a transcriptional activator that sensed very low concentration of copper and regulated the copper transport in. Here, we cloned a gene fromnamed, whose deduced amino acid sequence showed 29% identical to Mac1p. Furthermore, two Cys-His repeats metal binding motifs of Tmac1p, one in the 354–369聽C terminus and one in the 475–490聽C terminus were also present in Mac1p. A deletion mutant ofwas hypersensitive to the copper starvation and showed poor growth. Subsequently, the function was recovered by the gene complementation experiment. Furthermore, thegene fully complemented growth defects of yeast 螖mutant. The expression of Tmac1p was activated at low concentration of copper and depressed when the concentration of copper excess 1聽mM. Furthermore, the fluorescence intensity enhanced at copper starvation and decreased under copper excess by fusion the eGFP to the Tmac1p. It proved that the expression of Mac1p was exactly regulated by copper concentration, because eGFP and Mac1p were expressed under the control of the same one promoter. We also cloned a gene namedwith bioinformatics. With a series of experiments, we proved it was the target gene of. To sum up,may encode a transcriptional activator regulated high-affinity copper transport in.
|
| [5] |
A brief study on the degradation kinetics of seven organophosphorus pesticides in skimmed milk cultured with Lactobacillus spp. at 42° C [J].
<h4 id="secGabs_1">Highlights</h4><div id="sp005"><p>► The degradation kinetics of seven organophosphorus pesticides in skimmed milk was investigated. ► The kinetics of the pesticides was impacted by the inoculated <em>Lactobacillus</em> spp. ► <em>L. bulgaricus</em> and <em>L. plantarum</em> exhibited stronger impact than <em>L. paracasei.</em> ► Dimethoate and methyl parathion were most stable while malathion was most labile.</p>
|
| [6] |
Hidden fungi, emergent properties:endophytes and microbiomes [J]. |
| [7] |
Sánchez-Arreguín J A, Herrera-Estrella A H.Trichoderma:sensing the environment for survival and dispersal [J].DOI:10.1099/mic.0.052688-0 URL PMID: 21964734 [本文引用: 1] 摘要
Species belonging to the genus Trichoderma are free-living fungi common in soil and root ecosystems, and have a broad range of uses in industry and agricultural biotechnology. Some species of the genus are widely used biocontrol agents, and their success is in part due to mycoparasitism, a lifestyle in which one fungus is parasitic on another. In addition Trichoderma species have been found to elicit plant defence responses and to stimulate plant growth. In order to survive and spread, Trichoderma switches from vegetative to reproductive development, and has evolved with several sophisticated molecular mechanisms to this end. Asexual development (conidiation) is induced by light and mechanical injury, although the effects of these inducers are influenced by environmental conditions, such as nutrient status and pH. A current appreciation of the links between the molecular participants is presented in this review. The photoreceptor complex BLR-1/BLR-2, ENVOY, VELVET, and NADPH oxidases have been suggested as key participants in this process. In concert with these elements, conserved signalling pathways, such as those involving heterotrimeric G proteins, mitogen-activated protein kinases (MAPKs) and cAMP-dependent protein kinase A (cAMP-PKA) are involved in this molecular orchestration. Finally, recent comparative and functional genomics analyses allow a comparison of the machinery involved in conidiophore development in model systems with that present in Trichoderma and a model to be proposed for the key factors involved in the development of these structures.
|
| [8] |
Gene transfer to cereal cells mediated by protoplast transformation [J].DOI:10.1007/BF00330256 URL [本文引用: 1] 摘要
Direct gene transfer to cereal cells was achieved by transformation of protoplasts with naked DNA. Protoplasts isolated from cultured cells of Triticum monococcum were incubated in the presence of pol
|
| [9] |
Genetic transformation system for the aflatoxin-producing fungus Aspergillus flavus [J].DOI:10.1016/S0065-2164(08)70320-4 URL PMID: 2495764 [本文引用: 1] 摘要
ABSTRACT A heterologous transformation system was developed for Aspergillus flavus with efficiencies greater than 20 stable transformants per micrograms of DNA. Protoplasts of uracil-requiring strains of the fungus were transformed with plasmid and cosmid vectors containing the pyr-4 gene of Neurospora crassa. Transformants were selected for their ability to grow and sporulate on medium lacking uracil. Vector DNA appeared to integrate randomly into the genome of A. flavus with a tendency for multiple, tandem insertion. Transformants with single or multiple insertions were stable after five consecutive transfers on medium containing uracil. Uracil-requiring recipient strains were obtained either by UV-irradiating conidia and selecting colonies resistant to 5-fluoroorotic acid or by transferring the mutated pyr locus to strains by parasexual recombination. This is the first report of a transformation system for an aflatoxin-producing fungus. The transformation system and the availability of aflatoxin-negative mutants provide a new approach to studying the biosynthesis and regulation of aflatoxin.
|
| [10] |
Conidia immobilization of T-DNA inserted Trichoderma atroviride mutant AMT-28 with dichlorvos degradation ability and exploration of biodegradation mechanism [J]. |
| [11] |
Accumulation of copper in Trichoderma reesei transformants, constructed with the modified Agrobacterium tumefaciens-mediated transformation technique [J].DOI:10.1007/s10529-010-0365-y Magsci [本文引用: 1] 摘要
A modified <i>Agrobacterium tumefaciens</i>-mediated transformation method was established for the construction of mutants with improved copper tolerance and accumulation capability in <i>Trichoderma reesei</i>. One transformant, AT01, exhibited the highest copper accumulation capability. With copper at 0.7 mM, AT01 removed 13 mg copper/g biomass (removal rate of 96%), whereas the wild-type strain removed only 6 mg copper/g biomass (removal rate of 50%). Optimal conditions were a pH of 5.0 at 28°C. The pigment change of <i>Trichoderma</i> mycelia was a potential indicator of copper accumulation. Electron microscopy revealed that copper was mainly accumulated in cell vacuoles.
|
| [12] |
Isolation and transformation of Trichoderma viride protoplasts [J]. |
| [13] |
Degradation of cyanide by Trichoderma mutants constructed by restriction enzyme mediated integration(REMI) [J].DOI:10.1016/j.biortech.2006.09.047 URL PMID: 17112721 [本文引用: 1] 摘要
REMI technique was used to construct mutants with improved cyanide-degradation ability from biocontrol fungus Trichoderma koningii strain T30. The plasmid pV2 transformation was confirmed by PCR and Southern blot analysis. Out of 21 transformants, 15 single-copied transformants (71.4%) were found. To compare enzyme activities of rhodanese and cyanide hydratase, T. atroviride T23, T. harzianum T21 and their transformants constructed by REMI previously were also included. Transformants TkB6 (0.173 micromols thiocyanate formed min(-1)mg protein(-1)) from T30 and TaK1 (0.174 micromols thiocyanate formed min(-1)mg protein(-1)) from T23 showed higher rhodanese activity than other transformants and their wild strains. TkA9 (5.53 micromols formamide formed h(-1)mg protein(-1)) from T30 and Th64 (5.35 micromols formamide formed h(-1)mg protein(-1)) from T21 had higher cyanide hydratase activity than other transformants and their wild strains.
|
| [14] |
Production and Regeneration of Trichoderma Strain T23 Protoplast [J].
The best conditions for protoplast preparation and regeneration were studied on Trichoderma T23 strain. The course of releasing and regeneration of protoplast was observed. The optimal conditions for protoplast preparation of Trichoderma T23 strain were as follows:24 h mycelium growth time;the content of enzyme is 15 mg/mL;3~4 h reaction at 30~35 ℃. Trichoderma T23 strain releases protoplast through mycelium rupture. Two modes forming irregular outshoot were found to regulate regeneration of T23 protoplast.
|
| [15] |
Disruption of hex1 in Trichoderma atroviride leads to loss of Woronin body and decreased tolerance to dichlorvos [J].DOI:10.1007/s10529-013-1409-x URL PMID: 24243234 [本文引用: 1] 摘要
The tolerance of species to is necessary for their application in the bioremediation of -polluted environments. In some cases, such a requirement is also key to the synergistic use of these with chemical pesticides, aiming to broaden the scope of control targets to include both plant pathogens and insect pests. However, the mechanism of tolerance of remains unclear. To address this, we have analyzed the function of the putative -tolerance gene by knocking it out. The -deleted mutant showed loss of Woronin bodies and decreased tolerance to the organophosphate, . Moreover, localizes at the septal plugs in mycelium which may be involved in controlling movement of . thus is involved the tolerance to and the formation of Woronin bodies in .
|
| [16] |
根癌农杆菌介导的深绿木霉菌T23遗传转化研究 [J].DOI:10.3969/j.issn.1671-9964.2009.05.011 URL [本文引用: 1] 摘要
采用根癌农杆菌介导的方法,成功地建立了丝状生防真菌深绿木霉 (Trichoderma atroviride)菌株T23的遗传转化体系.并且,通过提高筛选培养基中潮霉素B的浓度和调整农杆菌的培养时间,对转化体系作了进一步的优化.转化 效率约为50个突变体/107个分生孢子.所有转化子经继代培养5代,潮霉素B抗性筛选后,共得到118个遗传稳定的转化子.随机抽取部分转化子,进行 PCR和Southern blot分子鉴定,结果证实外源的T-DNA已经随机整合到T23的基因组中.通过形态学观察,筛选出3个在产孢量和菌丝体生长速度方面显著不同于野生型 菌株T23的转化子.农杆菌介导的遗传转化方法在深绿木霉菌株T23上的成功运用,将为研究该菌的功能基因组提供强有力的工具.
Agrobacterium tumefaciens-mediated Transformation(ATMT)of Trichoderma atroviride T23 [J]. |
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