利用原生質(zhì)體融合技術選育高產(chǎn)菌株

1、利用原生質(zhì)體融合技術選育高產(chǎn)菌株李小俊,孫海虹,趙寶華,邊艷青(河北師范大學生命科學學院,河北石家莊050016摘要:以龜裂鏈霉菌(S terpomyces rimosus 98#和76#為出發(fā)菌株,分別用溶菌酶制得2個親本的原生質(zhì)體,其中76#采用15W 紫外燈下30cm 照射25min ,100%滅活,用PEG 誘導進行了原生質(zhì)體的融合.通過比較菌落外觀、顏色挑選融合子328株,結(jié)合前期代謝情況進一步篩選高單位的融合菌株8株,經(jīng)反復傳代考察融合子的穩(wěn)定性后得到1株新菌株102#,其發(fā)酵單位比親本提高了13.0%.關鍵詞:原生質(zhì)體融合;滅活;篩選中圖分類號:Q 939.97文獻標識碼:A 文

2、章編號:100025854(20070120112204土霉素(terramycin 是屬于四環(huán)類的一種廣譜抗生素,以其抗菌譜廣、抑菌效果好、毒副作用小、飼料利用率高等優(yōu)點,在醫(yī)療、畜牧業(yè)和農(nóng)業(yè)上有廣泛的用途和廣闊的市場前景.近年來,隨著菌種選育及工藝優(yōu)斜面培養(yǎng)基:麩皮,瓊脂.母瓶培養(yǎng)基:淀粉30g ,黃豆餅粉3g ,CaCO 35g ,(N H 42SO 44g ,NaCl 5g ,玉米漿4g ,KH 2PO 40.15g ,水1L.發(fā)酵培養(yǎng)基:淀粉150g ,黃豆餅粉20g ,CaCO 314g ,(N H 42SO 414g ,NaCl 45g ,玉米漿4g ,KH 2PO 40.1g

3、,淀粉酶0.15g ,消沫劑0.1g ,水1L.將76#原生質(zhì)體懸浮液5mL 置于直徑9cm 的雙碟中,震蕩照射5min ,紫外燈為15W ,垂直照射距離30cm 4.收稿日期:20060911;修回日期:20061115作者簡介:李小俊(1981,女,河北清苑人,碩士研究生,研究方向為應用微生物.第31卷/第1期/2007年1月河北師范大學學報/自然科學版/總糖和還原糖測定:斐林試劑法.氨基氮測定:甲醛法.生物量測定:取發(fā)酵液稱重,1500r/min 離心10min ,傾去上清液稱重,計算濕菌體的百分比.發(fā)酵單位測定:參照文獻5的方法.2結(jié)果與討論2.1G ly 質(zhì)量濃度對原生質(zhì)體形成和再生

4、的影響圖1G ly 質(zhì)量濃度對原生質(zhì)體形成和再生的影響G ly 在細胞生長過程中能取代細胞壁肽聚糖中的D Ala 殘基,干擾交聯(lián),引起細胞壁結(jié)構(gòu)的不完整,有利于去除細胞壁并釋放原生質(zhì)體.文獻6表明,放線菌G ly 以240g/L 為宜.筆者實驗采用3個G ly 質(zhì)量濃度進行對比.結(jié)果表明,G ly 為10g/L 時的原生質(zhì)體形成量最大;G ly 為5g/L 時的原生質(zhì)體的再生率最大,因此實驗采用5g/L G ly (見圖1.2.2滅活效果將紫外滅活的76#原生質(zhì)體涂布在再生培養(yǎng)基上培養(yǎng),37培養(yǎng)7d ,菌落數(shù)為0,滅活效果為100%.2.3原生質(zhì)體融合效果對上述菌株進行穩(wěn)定性考察,102#菌株

5、的傳代結(jié)果見表2.表14天殘氮和7天效價結(jié)果菌株4天殘氮/%7天效價/%02-25#99.010602-51#90.211502-83#95.010802-102#90.211302-156#95.010602-208#98.011802-289#95.010702-296#96.010876#130.010098#100.086表2102#菌株傳代穩(wěn)定性傳代效價/%94.33112.5代謝特性考察對102#菌株進行生理代謝考察,結(jié)果見圖 2.圖2102#菌株的代謝特性1以76#菌株的孢子顏色為篩選條件篩選得到328株菌株,以4天殘氮水平、7天發(fā)酵單位為篩選條件初篩得到8株菌株.篩選得到的8株

6、菌株4天殘氮明顯低于76#,略低于98#菌株;7天的發(fā)酵單位明顯高于98#菌株,而且高于76#菌株,表現(xiàn)出氨氮代謝和發(fā)酵水平均優(yōu)于雙親本的特點.因此,所選8株菌株為融合子,融合率為2.43%,其中102#菌株氨氮代謝和發(fā)酵單位明顯優(yōu)于兩親本.2102#菌株生理代謝特點為糖代謝、氨氮代謝明顯快于雙親本,因此生物量明顯高于雙親本.發(fā)酵前期102#菌株的發(fā)酵單位和雙親本沒有太大的差異,是因為前期的快速生長抑制了抗生素的產(chǎn)生;在發(fā)酵中后期,102#菌株的發(fā)酵單位明顯高于雙親本.3102#菌株經(jīng)傳代6次,菌落顏色仍然保持灰色,4天的殘氮水平仍然明顯低于雙親本,由此證明102#菌株的融合基因發(fā)生了重組并能

7、穩(wěn)定遺傳.隨著傳代次數(shù)的增加,7天發(fā)酵單位有所降低,但通過自然選育,菌種可以恢復原有發(fā)酵水平.這是部分孢子發(fā)生突變退化的原因,經(jīng)過純化,仍能保持原有的優(yōu)良性狀.4與常規(guī)育種方法比較,原生質(zhì)體融合使兩親本的遺傳物質(zhì)更加充分融合,得到的重組子遺傳特性更穩(wěn)定、突變幅度更大,在工業(yè)化生產(chǎn)中有較強的實用價值.參考文獻:1邊艷青,李保榮,趙寶華,等.耐磷酸鹽土霉素菌株選育和發(fā)酵研究J .中國抗生素雜志,2003,28(8:4562458.2賀筱蓉,黃小倩.鏈霉菌原生質(zhì)體的制備J .生物學通報,1998,33(1:40241.4113姚曙光,劉瑞華,劉金榮.應用原生質(zhì)體融合法選育林可霉素高產(chǎn)菌株J .安徽醫(yī)

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9、ua ,B IAN Yan 2qing(College of Life Science ,Hebei Normal Universit y ,Hebei Shijiazhuang 050016,China Abstract :The protoplasts of strains 98#and 76#of S terpom yces ri mosus were obtained by dissolvent en 2zyme respectively.The protoplasts of strains 76#were treated with UV light for 15W ,30cm and

10、 25min ,the death rate reached 100%.The protoplasts fusion was induced by PEG.328strains were obtained by selecting the fusion 2stains with the character and color of colony ,then 8strains were obtained by further selecting with metabolism ,the stability of more progeny was tested and at last one st

11、rains 102#was obtained ,the ferment level was improved 13.0%than parent strains.K ey w ords :protoplast fusion ;kill ;select(責任編輯柴鍵(上接第111頁4G OODRID GE L ,CHEN J ,GRIFFITHS M.The Use of a Fluorescent Bacteriophage Assay for Detection of Escherichia coliO157:H7in Inoculated Ground Beef and Raw Milk J

12、 .Int J Food Microbiol ,1999,47(122:43250.5楊水云,崔亞麗,趙文明,等.一種制備高效價噬菌合格懸液的有效方法J .西北大學學報,1999,29(1:15217.6WEBER 2DABROWSK A B ,ZIMECKI M ,MULCZYK M.E ffective Phage Therapy is Associated with Normalization of Cy 2tokine Production by Blood Cell Cultures J .Arch Immunol Ther Exp (Warsz ,2000,48(1:31237.7

13、魯會軍,韓文瑜.雷連成治療性噬菌體制劑研究進展J .中國獸藥雜志,2002,36(6:39241.Phage Isolates and Lysis of Chicken Escherichia coliSUN Hai 2hong 1,MA Tong 2suo 2,YU Shan 1,ZHAO Bao 2hua 1(1.College of Life Science ,Hebei Normal Universit y ,Hebei Shijiazhuang 050016,China ;2.College of Biology Science and Biotechnology ,Hebei Un

14、iversity of Economics and Trade ,Hebei Shijiazhuang 050061,China Abstract :The antimicrobial 2resistant strains of Escherichia coli separated from clinic is very wide.Experi 2ment shows that average resistant ratio of E.coli to eighteen kinds of antibacterial agents is nearly 80percent.Common antibacterial agents therapy effects are usually far from