【作者】 郑重谊；

【导师】 谢达平；

【作者基本信息】 湖南农业大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 为了选育对多种白菜病害具有拮抗作用的菌株用于生物防治，本试验以革兰氏阳性菌芽孢杆菌KR和革兰氏阴性菌假单胞菌B13为亲本进行原生质体融合，主要结果如下：在以0.6 mol／L蔗糖为稳定剂，pH 8.0，溶菌酶8 mg／mL，37℃，酶解80 min的条件下，KR原生质体得率可达31.6％；在以.0.6 mol／LNaCl为稳定剂，pH 7.0，溶菌酶6 mg／mL，37℃，酶解45 min，B13原生质体可达95.2％。为了提高再生率，KR酶解20 min，采用夹层培养，再生培养基中以0.6 mol／L蔗糖做稳定剂，Ca²⁺ 0.03mol／L，Mg²⁺ 0.02 mol／L，琥珀酸钠0.3 mol／L，L-色氨酸0.2 g／L，培养基在37℃下放置72 h原生质体再生率可达42.7％；B13酶解15 min，采用夹层培养，培养基中以0.6 mol／L NaCl做稳定剂，Ca²⁺ 0.02 mol／L，Mg²⁺ 0.01 mol／L，琥珀酸钠0.3 mol／L，L-色氨酸0.1 g／L，培养基在37℃下放置48 h原生质体再生率可达15.3％。2.0单位／mL的硫酸妥布霉素和5×10^-3 g／mL的氯霉素可分别抑制B13和KR，培养基中添加这两种抗生素可选出融合子。本试验共得到182个融合子，连续传10代后，得到一株能稳定遗传的融合子KB1。KB1长2.99381μm，宽0.45195μm，杆状，周生鞭毛，有芽孢，革兰氏染色阴性。KB1在A培养基中，经35℃，pH 6.5，60 h发酵的滤液，用平板打孔法，置于平板的孔内，其对软腐菌产生了1.70 cm的抑菌圈。在PDA中添加经35℃，pH 8，60 h发酵的KB1的A培养基滤液10％，黑斑病菌饼生长8 d后大小仅为1.61 cm，明显小于对照。更多还原

【Abstract】 This experiment used Bacillus KR strains and Pseudomonas B13 strains tooperate as arch_strains to conduct protoplast fusion,established fusants.the mainresults were presented as follows:The highest formation rate of protoplast of KR was 31.6% under the condition ofthe concentration of sucrose 0.6 mol/L, pH 8.0,the concentration of lysozyme 8mg/mL, 37℃,the time of lysozyme treatmen 80 min; the highest formation rate ofprotoplast of B13 was 95.2% under the condition of the concentration of NaCl 0.6mol/L, pH 7.0,the concentration of lysozyme 6 mg/mL, 37℃,the time of lysozymetreatment 45 min.In order to improve the regeneration rate, KR regeneration rate under thecondition of the time of lysozyme treatment 20 min, interlayer, the concentration ofsucrose 0.6 mol/L, Ca²⁺ 0.03mol/L, Mg²⁺ 0.02 mol/L,the concentration of Sodiumsuccinate acid 0.3 tool/L, the concentration of L-tryptophan 0.2 g/L,culture mediumplaced 72 h under 37℃; B13 regeneration rate under the condition of the time oflysozyme treatment 15 min,interlayer, the concentration of NaCl 0.6 mol/L, Ca²⁺0.02mol/L, Mg²⁺ 0.01 mol/L, the concentration of Sodium succinate acid 0.3 mol/L,the concentration of L-tryptophan 0.1 g/L, culture medium placed 48 h under 37℃.Using drug resistance as the genetic markers of protoplast fusion between KRand B13. In this protoplast fusion experiment between two bacteria, the applyingdensity in selective culture medium of Tobramycin and Chloramphenicol are 2.0U/mL and 5×10^-3g/mL.Using PEG, we got 182 fusants,generating continuously 10 times,derived asteady fnsant KB1. Judging of characters of fusants on morphological andconfiguration、physics and chemical: The KB1 are rhabditiform,their averagemacro-axis are 2.99381μm, average brachy-axis are 0.45195μm, Gram negative,periichous flagellum, sporangium. The studies on biology characters of KB1: theantimicrobial zone on Soft Rot Disease are 1.70 cm under condition of A culturemedium ferment,35℃, pH 6.5, 60 h; Black Spot Disease are 1.61 cm after 8 d onPDA which contain 10% A culture medium ferment under 35℃, pH 8, 60,distinctness smaller than CK.更多还原