高雄山虫草无性型细脚拟青霉的核型分析  

Molecular karyotype analysis of Paecilomyces tenuipes,the anamorph of Cordyceps takaomontana

作　　者：荣跃文[1] 徐莉[1] 汪小艳[1] 李春如[1] 樊美珍[1] 李增智[1] 

机构地区：[1]安徽农业大学微生物防治省重点实验室,合肥230036

出　　处：《菌物学报》2010年第1期31-36,共6页Mycosystema

基　　金：国家高技术研究发展计划资助项目(863计划)(No.2007AA021506);国家自然科学基金(No.30570004);安徽省自然科学基金(No.070411011)

摘　　要：对高雄山虫草无性型细脚拟青霉的酶解条件、原生质体制备、电泳条件和核型进行了研究。以10mg/mL相同浓度的溶壁酶、纤维素酶和蜗牛酶配成混合酶解液,在30℃、120r/min振荡处理18h菌龄的菌丝2.5h,获得约107个/mL的原生质体悬浮液。浓缩至约108个/mL原生质体悬浮液与1.4%的低熔点琼脂糖等体积混合制胶,50℃蛋白酶K酶解48h,ET液洗3次(每次1h),浸没,4℃保存。以温汉逊氏酵母Hansenula wingei YB-4662-VIA和粟酒裂殖酵母Schizosaccharomyces pombe 972h-染色体DNA作为标准,利用钳位均匀电场脉冲电泳系统电泳,细脚拟青霉染色体组被分离成8条带,利用AlphaEase Fc软件分析,估计其大小在0.43-5.78Mb之间,大小分别为:0.43、2.45、2.55、2.88、3.28、3.94、4.70、5.78Mb,推测细脚拟青霉核型大小约为26.01Mb。The enzyme composition and concentration, protoplast preparation, electrophoresis conditions and molecular karyotype of Paecilomvces tenuipes （Teleomorph： Cordvceps takaomontana） were investigated. The higher production （1.0×10^7/mL） of the protoplasts were obtained under the following conditions： the mycelia cultured for 18h, digested by the mixed enzyme solution consisting of 1% lywallzyme, 1% cellulase and 1% snailase with shaking of 120r/min for 2.5h at 30℃, then the suspension concentration of protoplasts was adjusted approximately to 1.0×10^8/mL and mixed with 1.4% agarose （L.M.P）. The agarose plugs with 2mg/mL proteinase K and proteolysis was performed at 50℃ for 48h. Finally, the agarose plugs were washed 3 times of lh each with washing ET buffer and kept in storage solution at 4℃. The electrophoresis experiment was carried out by using CHEF system with Hansenula wingei YB-4662-VIA and Schizosaccharomyces pombe 972h-chromosome DNA as molecular weight markers, and CHEF patterns obtained were analyzed by AlphaEase Fc software. The results showed P. tenuipes had eight chromosomes varied from 0.43 to 5.78 mega base pairs （Mb）, ranked as 0.43, 2.45, 2.55, 2.88, 3.28, 3.94, 4.70 and 5.78Mb. The total genome sizes were estimated to be 26.01Mb.

关 键 词：虫生真菌 原生质体 钳位均匀电场电泳 染色体组 

分 类 号：S567.35[农业科学—中草药栽培]