草菇基因枪法遗传转化体系的建立

The Transformation System for Volvariella volvacea by Particle Bombardment

草菇菌株V1308对潮霉素、卡那霉素、遗传霉素、膦丝菌素的抗性测验结果表明,该菌株对潮霉素敏感,而对其它三者不敏感。进一步测定结果显示,潮霉素的最低筛选浓度在PDSA固体培养基上为75μg·mL-1,在PDSB液体培养基中为50μg·mL-1。以含有35S启动子、gus报告基因、潮霉素抗性基因的质粒pCAMBIA1301为表达载体,采用基因枪法对草菇菌丝体进行遗传转化。结果表明,基因枪的最佳轰击参数为:氦气压力1100Psi,真空压力87·88kPa,靶距离6cm,轰击次数1次。Southern杂交结果显示,gus基因已整合进V1308基因组中。GUS组织化学检测结果证明,gus基因在V1308菌丝体中获得了表达。草菇出菇试验结果显示,转基因草菇和对照都能正常形成子实体,子实体组织分离长出的菌丝体经GUS组织化学检测,证明了外源基因gus能够在转基因草菇的菌丝体中稳定表达。

The resistant test of Volvariella volvacea strain V1308 to hygromycin, kanamycin, G418 and PPT showed that the strain was sensitive to the former but resistant to the latter 3 reagents. The lowest selection concentrations of hygromycin for V1308 were 75 μg· mL^-1 on PDSA solid medium and 50μg· mL^-1 in PDSB liquid medium. The expression vector pCAMBIA1301, carrying 35S promoter, hygromycin selectable gene and gus report gene, was transferred into intact mycelia of V1308 by particle bombardment. The most effective bombardment parameters were 1 100 Psi of helium pressure, 87. 88 kPa of vacuum pressure and 6 cm of target-shelf distance. The result of southern blotting indicated that the gus gene was integrated into the V1308 genome. GUS histochemical assay confirmed that gus had expressed in V1308 mycelia. Cultivation of V1308 showed that both the transformant and the control were able to form normal fruit-bodies. GUS histochemical assay indicated that the exogenous gus gene expressed stably in the mycelia derived from the fruitbodies of transformant.