灭蚊真菌贵阳腐霉原生质体诱变育种实验研究

灭蚊真菌贵阳腐霉原生质体诱变育种实

验研究

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【摘要】目的：建立采用紫外线和氯化锂诱导灭蚊真菌发生突变的方法，为培育理想菌株开辟新的途径。方法：对出发菌株贵阳腐霉的原生质体进行诱变处理，通过正突变率与紫外、化学诱变剂量的相互关系，确定最佳诱变条件，经过酪蛋白平板透明圈和菌粉得率粗筛突变株，连续8代传代培养及摇瓶实验检测突变株的遗传稳定性。结果：当用0.6%氯化锂处理90 min时，突变株的致死率和正突变率分别为86.9%和26.9% ;用20 W紫外灯30 cm照射180 s，突变株致死率为81.3%、正突变率为78.5% ;筛选了7株突变株，其中U22经传代实验证明其遗传性状稳定。结论：本研究所建立的对贵阳腐霉原生质体诱变系统基本成功，为该菌的细胞工程育种创造了条件。

【关键词】贵阳腐霉；原生质体；紫外线；氯化锂；诱变；育种

[Abstract ] Objective： To establish a mutation induction system for mutati on breed ing of Pythium guiya ngense Su, a fungal pathogen of mosquitoes. Methods: Protoplasts of original strain were treated with UV or LiCl for mutatio n in ducti on. Case in plate tran spare

nt loop method and mycelial weight determ ine were used to scree n muta nt col on ies. Pert inent in ducti on con diti on comb in ati on were summarized based on the an alysis of the relationship between the positive mutation rates and treatment dosages and periods of UV or LiCl. One of the mutational colonies was continu ously cultivated for 8 gen erati ons on KPYG2 plates for the observation of genetic stability. Results: A lethal rate of 86.9% and a positive mutation rate of 26.9% were achieved when the protoplasts were treated by 0.6% of LiCl for

90mins, and a lethal rate of 81.3% and a positive mutati on rate of

78.5% were obta ined when the protoplasts were irradiated by a UV lamp of 20W in a dista nee of 30cm for 180s. Seve n muta nts were obta in ed, and one of them, U22, was proved genetically stable. Conclusions: The mutation induction system of P. guiyangense by UV and LiCl is successful, and can provide basis for the mutati on breedi ng of P.

guiya ngense in the future.

[Key words ] Pythium guiyangense Su; protoplasts; ultraviolet rays; lithium chloride; mutation breeding

丝状真菌贵阳腐霉(Pythium guiyangense Su )具有灭蚊

能力强，繁殖速度快，易于人工培养以及对其它非靶生物相对安全等优点［1］，在蚊虫生物防治中具有潜在的应用前景。但是，与大多数

微生物一样，贵阳腐霉在人工培养基经过多次传代后其毒力和产孢量会有所下降；同时，与大多数丝状真菌一样，其有效贮藏期不够长。

为将贵阳腐霉真正用于灭蚊，提高其灭蚊能力和延长有效贮藏期，对贵阳腐霉进行了改良。微生物改良育种方法较多，原生质体诱变技术是一种行之有效的方法［2，3］。杜海英［4］等通过原生质体诱变选育乳糖酶高产菌株。目前有关利用原生质体紫外诱变和氯化锂诱变提高丝状真菌遗传稳定性方法报道较少。贵阳腐霉是发现的新种，其诱变研

究尚未见报道。2008年1月〜8月对贵阳腐霉原生质体进行紫外诱变和（或）氯化锂诱变，为该真菌的细胞工程改良奠定基础。

1材料与方法

1.1实验材料

1.1.1菌种来源于贵阳医学院蚊虫生物防治实验室。菌丝在固体培养基上每7天转种1次，培养温度为（25 ±1 ）C。

1.1.2培养基

菌丝培养液（KPYG2 ）:葡萄糖1.5 g/L，酵母1.0 g/L，蛋白胨1.0 g/L，氯化钙0.075 g/L，胆固醇0.02 g/L，玉米油10 g/L。菌丝固体培养基：KPYG2加入琼脂粉10 g/L。酪蛋白水解培养基：NaHPO4

7H2O 1.07g/L，KH2PO4 0.36 g/L，酪蛋白4 g/L，琼脂粉20 g/L。再生培养基：用0.6 mol/L进口蔗糖渗透压稳定剂溶解KPYG2 各成分。氯化锂的诱变培养基为LiCI（0.2%〜1%）加再生培养基。

1.1.3酶液配制

配制浓度为0.6 mol/L的进口蔗糖作为渗透压。取1 g酶用渗透压稳定剂溶解成质量分数为1%的酶液，经0.22呵微孔滤膜过滤除菌后使用。

1.2诱变方法

1.2.1贵阳腐霉原生质体的制备与再生

参考文献［5］制备足够量（6.48 X106个/ml）的原生质体，经系