莱茵衣藻外源基因整合特征分析及蛋白表达

The characteristic of transgene insertion and exogenous protein expression in Chlamydomonas reinhardtii

摘要:

将衣藻表达载体pSP108转化莱茵衣藻细胞壁缺陷型藻株CC-400，通过接头PCR获得并分析阳性转化子中ble的侧翼序列发现：外源基因的插入位点呈现随机性，并且有些转化子中衣藻基因组和质粒序列发生断裂和重排。通过间接ELISA分析外源蛋白表达量，并结合外源基因整合状态时发现：在ble启动子核心区域缺失的阳性转化子中，ble可以利用自身基因启动子进行蛋白表达，但表达量相比使用质粒载体上RBCS2启动子的表达量要低；在部分启动子完整的阳性转化子中蛋白表达量低下，可能与衣藻基因组大片段的缺失相关。

Abstract:

Transformation of the cell wall deficient C.reinhardtii CC-400 with the plasmid pSP108 was carried out. Adapter PCR analysis on flanking sequences of ble in positive transformants showed that: the foreign gene ble inserted randomly, plasmid sequences rearranged with nuclear genome sequences and fracture phenomenon occurred. ELISA was used to analyze the expression of foreign gene, combined with the gene insertion which indicated that: In the positive transformants of which promote is partially truncated, the promoter in the unknown gene can initiate the expression, but the level is lower than the RBCS2 promoter initiation; In the other transfomants of which the promoter is complete, the protein expression level is low, may be due to the lacking of partial C.reinhardtii genome.