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Progress in Natural Science:Materials International 2007年第6期17卷 703-707页

作者： Chen Zhi, Lin Wen, Yu Ping and Song Yuan(Department of Microbiology, College of Biological Sciences, China Agricultural University, Beijing 100094, China) Department of Microbiology College of Biological Sciences China Agricultural University Beijing 100094 China

Intergenric protoplast fusion between Aspergillus terreus CA99 and Monascus anka M-3, the high and low producers of monacolin K respectively,was performed for enhancement of monacolin K production. The 24-hour-old mycelia of A. terreus CA99 and M. anka M-3 were treated with 0.5% lywallzyme, 0.3% snailase and 0.3% cellulase at 34℃ for 5 h and at 30℃ for 3.5 h, and their protoplasts formation reached 1.76×107/mL and 1.68×107/mL respectively. Parental protoplasts were irradiated with a 30 W UV light away from 30 cm for 3 min and then mixed. The mixture was incubated with 30% PEG 6000 for 15 min. The reviving fusants were isolated on the regeneration plates. Of the 363 fusants isolated, over 100 showed enhanced monacolin K production compared with the parental strain M. anka M-3. Ten of them produced monacolin K about 1.6-fold of that *** M-3 does and the monacolin K titer of two fusants (F49 and F104) increased by about 1-fold. The monacolin K yields of F49 and F104 were 460 μg/mL and 457 μg/mL respectively. In optimized fermentation medium, the monacolin K titer of F49 reached 1216 μg/mL.

关键词： monacolin K Monascus anka Aspergillus terreus protoplast fusion fermentation.

Regeneration of intergeneric somatic hybrids by protoplast fusion between Onobrychis viciaefolia and Medicago sativa

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Science China(Life Sciences) 1997年第4期40卷 363-370页

作者：徐子勤贾敬芬 Cell Biology Laboratory of Lanzhou University Lanzhou 730000 China

protoplast fusion was induced between sainfoin and alfalfa by an improved polyethyleneglycol (PEG) method. The intergeneric somatic calluses were selected based on complementation of hydroxyproline-resistance of sainfoin and hormone autonomy growth of alfalfa transformation cell line. 17 somatic hybrid plantlets were regenerat-ed. PEG could induce the tight agglutination of protoplasts. During diluting and washing process, cyclization of the linked membrane and formation of vesicle-like structures were observed, resulting in protoplast fusion. 5%-10% glycerol supplemented in the fusion inducing solution markedly increased the frequency of heterogeneous fusion. Better fusion results were obtained when mixed protoplast suspension was dripped in petri dishes in which PEG solution was previously placed. Chromosome number of regenerated hybrid buds varied from 30 to 60. The genome of hybrids in-cluded the small chromosome from sainfoin and two chromosomes with two clear constrictions from alfalfa. The hybrid-ity of obtained hybrid calluses was confirmed by their isozyme banding patterns and their nopaline synthetase activity.

关键词： hydroxyproline-resistant cell line of sainfoin (Hyp^r) Agrobaeterium tumefaciens 702 transformed cell line of alfalfa (M_(7)) PEG glycerol vesicle protoplast fusion somatic hybrid plant let.

protoplast fusion of Cosmos bipinnatus and Cosmos sulphureus

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Agricultural Biotechnology 2013年第1期2卷 24-26,41页

作者： Xiaoxia LIU Huoxu WANG College of Life Science Liaoning Normal University Key Lab of Plant Biotechnology of Liaoning Province Liaoning Normal University

[ Objective] The aim was to study appropriate fusion conditions of protoplasts from 2 species Cosmos bipinnatus and Cosmos sulphureus. [ Method ] The protoplasts were separated from the blossom petals of Cosmos bipinnatus and Cosmos sulphureus availed with the function of pectinase and cellulase, and the fusion of two pretoplasts was performed with PEG. [ Results] A great deal of free pretoplasts were obtained from Cosmos bipinnatus by using 6% mannitol ＋ 1.3% cellu- lase ＋ 1.2% pectinase, and from Cosmos sulphureus by using 10% mannitol ＋ 1.3% cellulase ＋ 1.2% pectinase. Five hours after of enzymolysis treatment, large amount of dissociated protoplasts were observed in the solution. Pretoplast fusion was be obtained by using 30% PEG fusion liquid and 0. lmol/1 CaC12 solution （pH value = 9.5）. [ Conclusion] Our study established an optimal separation and fusion system of protoplasts from different coreopsis species, laying foundation for the breeding of novel coreopsis varieties.

关键词： Cosmos bipinnatus Cosmos sulphureus protoplast fusion

Research Progress of Algae protoplast fusion Technique

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Asian Agricultural Research 2022年第2期14卷 39-40页

作者： Xiaoying XU Wenkai SHI Lei LI Ya'nan CAO Xiaoguang LU Peng LIU Yantai Marine Economic Research Institute Yantai 264006 China

This paper discussed the technique of algal protoplast fusion,including the method of protoplast-induced fusion,the screening and identification of hybrid cells and so *** this paper,the research progress of protoplast-induced fusion of microalgae was reviewed,and the problems existing in the process of protoplast-induced fusion were discussed in order to provide a theoretical basis for the development of algae genetics and breeding.

关键词： Algae protoplast fusion Research progress

protoplast culture and fusion of citrus

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Progress in Natural Science:Materials International 1995年第3期 53-61页

作者：邓秀新章文才 China Huazhong Agricultural University State Key Laboratory of Crop Genetic Improvement Wuhan 430070

1 Introduction Citrus is the most important fruit crop in the southern provinces of China. Citrus production increased by 10 times in the past 15 years, reaching 6.6 million metric *** development in great degree reli... 详细信息

关键词： citrus protoplast fusion somatic hybrid cultivar improvement.

The protoplast two-way fusions and fusant characteristics of Antrodia cinnamomea and Cordyceps militaris

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Food Science and Human Wellness 2022年第5期11卷 1240-1251页

作者： Wei-Kuang Lai Yang-Chang Wu Tai-Sheng Yeh Chun-Ren Hsieh Yi-Hong Tsai Chien-Kei Wei Chi-Ying Li Ying-Chen Lu Fang-Rong Chang Graduate Institute of Natural Products Kaohsiung Medical UniversityKaohsiung 807TaiwanChina Graduate Institute of Integrated Medicine China Medical UniversityTaichung 404TaiwanChina Chinese Medicine Research and Development Center China Medical University HospitalTaichung 404TaiwanChina Department of Food Science and Nutrition Meiho UniversityPingtung 912TaiwanChina Department of Food Science National Chiayi UniversityChiayi City 600TaiwanChina Drug Development and Value Creation Research Center Kaohsiung Medical UniversityKaohsiung 807TaiwanChina Department of Medical Research Kaohsiung Medical University HospitalKaohsiung Medical UniversityKaohsiung 807TaiwanChina Department of Marine Biotechnology and Resources National Sun Yat-sen UniversityKaohsiung 804TaiwanChina

This study generated two fused protoplasts of Antrodia cinnamomea and Cordyceps militaris in two *** protoplasts of *** were inactivated by heat to inactivate biochemical processes and enzymatic activities in the cytoplasm,and the protoplasts of *** were inactivated by UV radiation to invalidate their genome function,then they were fused under optimal conditions to get a fusion rate as(7.42±0.8)×10^(-6) fusants/mL;the new fusants were abbreviated as *** the other hand,when *** and *** were treated with heat and UV oppositely using similar experiments,the fusion rate was(9.70±0.68)×10^(-5) fusants/mL,and the new fusants were abbreviated as *** selected each of two best-growing fused colonies Ac-Cm-1,Ac-Cm-2,Cm-Ac-1,and Cm-Ac-2,together with parental *** and ***,and studied their morphology,growth antagonism tests,and genetic relationships by 18 S rRNA *** comparison with the initial cultures of 4 fusants,the yields of adenosine,biomass,cordycepic acid,cordycepin,total polysaccharide,and total triterpenoids were increased up 1.305-50.1563 times in the optimal medium *** gene stability tests,those of the four fusants and their outputs were stabilized within 10 generations.

关键词： Medicinal fungi protoplast fusion Antrodia cinnamomea Cordyceps militaris Secondary metabolite

Analysis of parental strain DNA fragments existing in GEMs-Fhhh

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Journal of Environmental Sciences 2003年第5期15卷 590-594页

作者： HAOChun-bo YANJun QUMeng-meng WANGDong CHENGShu-pei GUJi-dong QIUWan-fei WANGYin-yin TheNationalKeyLaboratoryofPollutionControlandResourceReuse SchoolofEnvironmentalScienceNanjingUniversityNanjing210093China DepartmentofEcology&Biodiversity TheUniversityofHongKongPokfulamRoadHongKongChina

There were 6 target DNA fragments of the three parental strains existing in the cell of GEMs(genetically engineered microorganism strain) Fhhh measured in this research by PCR(polymerase chain reaction). The determination showed that GEMs Fhhh contained all the 6 target DNA fragments, mnp 1, mnp 2、 lip 1、 lip 2, FLO 1 and 16S rDNA, and had the molecular genetic stability. Meanwhile the PCR production of each parental strain could only had its target DNA fragments and was different from each other. It may illustrate that the technique of the inter kingdom protoplast fusion for the construction of GEMs Fhhh through the process of intercellular gene recombination could be used as a reliable bioengineering technique to create the specific functional stain for the pollution control.

关键词： GEMs protoplast fusion target gene DNA fragment PCR pollution control

Construction of hybrid cell with Phanerochaete chrysosporium to degrade terephthalic acid wastewater——(I) phenotype evidence

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Journal of Environmental Sciences 2000年第4期12卷 394-397页

作者： CHENG Shu pei 1 , ZHONG Zhi hui 1 , LU Peng 1 , CHEN Jun 2 , ZHU Jian zhong 2 , JUE Zi long 2 (1. The National Key Laboratory of Pollution Control and Resource Reuse, Department of Environmental Sciences and Engineering, Na The National Key Laboratory of Pollution Control and Resource Reuse Department of Environmental Sciences and EngineeringNanjing University Nanjing 210093 China Water Plant Yangzi Petrochemical Corporation Nanjing 210048 China

The fungi Phanerochaete chrysosporium (PC) and Saccharomyces cerevisiae Y99 and the native bacterium YZ1 were the three parental strains for construction of hybrid cells through protoplast fusion to degrade terephthalic acid (TPA) wastewater. The results showed that the native bacterium YZ1 protoplasm could integrate with that of PC to form the hybrid cell Fhh and the fungus Y99 protoplasm also could integrate with that of Fhh to form the hybrid cell Fhhh. The protoplasts of YZ1 and Y99 could change the morphology of PC spore and mycelium for two times. The hybrid cell Fhhh got the best growth and degradation abilities in the wastewater. It suggested that the hybrid strains obtained from the inter\|kingdom protoplast fusion of the three parental strains could create potential for the purification of TPA wastewater.

关键词： Phanerochaete chrysosporium YZ1 bacterium Saccharomyces cerevisiae protoplast fusion terephthalic acid wastewater

Accelerating the menaquinone‑7 production in Bacillus amyloliquefaciens by optimization of the biosynthetic pathway and medium components

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Systems Microbiology and Biomanufacturing 2023年第4期3卷 776-791页

作者： Chang‑Long Li Meng Li Wei‑Guo Zhang Jian‑Zhong Xu The Key Laboratory of Industrial Biotechnology Ministry of EducationSchool of BiotechnologyJiangnan University1800#Lihu RoadWuXi 214122People’s Republic of China

Menaquinone-7(MK-7)has an important role in preventing diseases such as cardiovascular disease and *** this study,a combination strategy of strain improvement and medium optimization is investigated to increase MK-7 production in Bacillus *** breeding method was first used to modify the biosynthetic pathway to construct a MK-7 high-producing strain by atmospheric and room temperature plasma mutagenesis and protoplast *** resulted strain Ba-4 with resistance to sulfaguanidine,1-hydroxy-2-naphthoic acid,menadione,2-deoxy-d-glucose and rifampicin as well as sensitive toβ-fluoropyruvate produced 73.57±1.61 mg/L of MK-7,which was 1.36 times more than that of the parent strain H.β.D.R.-5(i.e.,31.12±1.40 mg/L).Subsequently,single-factor optimization and response surface method-ology(RSM)were used to optimize the medium components for increasing MK-7 production by strain *** Ba-4 produced 90.43±1.32 mg/L of MK-7 under the single-factor optimized ***,the results of response surface methodology indicated that glycerol,soy peptone and Tween-80 had significant effects on MK-7 production,and the highest MK-7 production(i.e.,95.03±1.01 mg/L)was obtained under the optimized medium,which was 0.29 times higher than that of the initial *** results confirmed that the conventional breeding methods and fermenter control system are effective strategies in improving MK-7 production by ***.

关键词： Bacillus amyloliquefaciens Menaquinone-7 ARTP mutagenesis system protoplast fusion Medium optimization

Effect of Riboflavin Operon Dosage on Riboflavin Productivity in Bacillus Subtilis

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Transactions of Tianjin University 2005年第1期11卷 1-5页

作者：陈涛陈洵王靖宇赵学明 School of Chemical Engineering and Technology Tianjin University Tianjin 300072 China

After deregulating the purine and riboflavin synthesis in the Gram-positive bacterium Bacillus subtilis,it is critical to amplify riboflavin operon with appropriate dosage in the host strain for remarkable increase of riboflavin *** subtilis RH13, a riboflavin-producing strain, was selected as host strain in the construction of engineering strains by protoplast fusion. The integrative plasmid pRB63 and autonomous plasmid pRB49, pRB62 containing riboflavin operon of *** 24 were constructed and transformed into the host strain respectively. Increasing one operon copy in *** RH13 results in about 0.4 g/L improvement in riboflavin yield and the appropriate number of operon copies was about 7—8. Amplifying more riboflavin operons is of no use for further improvement of yield of riboflavin. Furthermore, excessive operon dosage results in metabolic unbalance and is fatal to the host cells producing riboflavin.

关键词： Bacillus subtilis riboflavin operon protoplast fusion gene amplification