植物多基因干扰载体体系构建与效用分析

Construction of Multi-gene Interference System for Plant and Analysis of Its Application Efficiency

多数重要的功能基因属于多基因家族,这些家族成员间存在功能冗余,高效的多基因干扰体系对研究多基因家族成员的生物学功能及其分子调控机制具有重要意义。对pCAMBIA1301载体改造,构建了适用于植物的多基因干扰体系pCAMBIA1301m和pCAMBIA1301s。使用该多基因干扰体系构建了四基因的干扰载体pCAMBIA1301m:35S∷SlPP2C1-2-3-4,4个目标基因为来源于番茄PP2C家族A组的PP2C1、PP2C2、PP2C3和PP2C4,并通过遗传转化导入番茄,用GUS染色和PCR检测转基因阳性植株,再利用RT-qPCR技术检测T_(1)和T_(2)代转基因植株中目标基因的干扰效率,用T_(2)代种子分析转基因番茄对ABA敏感性。结果表明,应用该干扰体系成功获得了四基因干扰的转基因植株35S∷SlPP2C1-2-3-4。在转基因番茄中4个目标基因的表达量显著低于野生型,其干扰效率均高于70%,转基因番茄种子萌发具有强烈的ABA不敏感性。多基因干扰体系能高效地同时沉默多个目标基因。

Most important functional genes belong to polygenic families,and abundant functional redundancy is enriched among those family members.An efficient multi-gene interference system is of great significance for investigating the biological function and molecular regulation mechanism of polygenic family members.The pCAMBIA1301 vector was modified to construct multi-gene interference systems pCAMBIA1301m and pCAMBIA1301s for plants.The four genes interference vector,pCAMBIA1301m:35S∷SlPP2C1-2-3-4,containing PP2C1,PP2C2,PP2C3 and PP2C4 from tomato PP2C family A group,was constructed using this system and then introduced into tomato by genetic transformation.The positive plants were detected by GUS staining and PCR,the interference efficiency in T_(1)and T_(2)transgenic plants was tested by RT-qPCR,and the sensitivity of T_(2)transgenic seeds to ABA was analyzed.The results showed that the transgenic plant 35S∷SlPP2C1-2-3-4 with four interfered genes was successfully obtained by using this multi-gene interference system.In the transgenic plants,the expressions of the four target genes were significantly lower than that in wild plants,and the interference efficiency was above 70%.The achieved transgenic seeds demonstrated strong insensitivity to exogenous ABA.The multi-gene interference system may effectively and synchronously silence multiple target genes.