抗冷冻蛋白基因遗传转化草菇的研究

采用RT PCR技术从瑞典的北极云杉卷叶蛾幼虫中扩增出抗冷冻蛋白基因 ,利用基因枪法遗传转化草菇。PCR检测和Southern杂交结果证明 ,抗冷冻蛋白基因已整合进草菇基因组。低温胁迫试验结果表明 ,转基因草菇具有较强的耐低温能力。转基因草菇生物学特性观测结果显示 ,大多数草菇转化子的生长速率明显地慢于对照的宿主菌株 ,多数转化子的菌丝也明显地比宿主菌丝细弱。转化子筛选结果表明 ,采用三轮的转化子筛选程序 ,即第一轮在固体培养基上筛选、第二轮和第三轮在液体培养基中筛选 ,有利于获得真实转化子和淘汰假转化子。转基因草菇一代低温胁迫结果证明 ,转基因草菇后代仍然具有较强的低温耐受能力 ,这说明转基因草菇的耐低温性能在世代之间是稳定的

Transformation of Volvariella volvacea with a thermal hysteresis protein gene by particle bombardment

A cDNA encoding a thermal hysteresis protein was isolated from the Swedish Arctic insect spruce budworm by RT-PCR amplification. Volvariella volvacea strain V34 was transformed with this cDNA through particle bombardment. PCR detection and Southern blotting analysis show that the thermal hysteresis protein gene is integrated into Volvariella volvacea genome. Cold stress assay reveals that transgenic Volvariella volvacea lines exhibit stronger cold tolerance than host strain. The morphological observation of transgenic Volvariella volvacea lines shows that growth rates of most Volvariella volvacea tansformants are significantly slower than that of negative control strain. And hypha of most Volvariella volvacea tansformants is thinner than host strain's hypha. Transformant screening result indicates that three-round of selection procedure with first selection on PDSA solid selective medium followed by second and third selection in PDSB liquid selective medium is favorable to get genuine transformants and to eliminate false transformants. Cold tolerance assay of transgenic Volvariella volvacea F1 generation demonstrates that the progeny of transgenic Volvariella volvacea still possesses stronger cold tolerance than non-transformed host strain. This suggests that the cold tolerant characteristic of transgenic Volvariella volvacea is meiotically stable between generations.