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突变基因的编码序列是研究基因功能的一个有效途径。三亲杂交是利用细菌结合的特性使外源DNA进入受体细胞，通过外源DNA与基因组目标序列发生同源重组，进而实现突变目标基因的有效方法。本研究通过分析同源臂的长度和混合菌比例，明确其对三亲杂交效率的影响。以根瘤菌HH103的Ⅲ型效应因子编码基因nopB、nopC、nopL为例，对混合菌(根瘤菌，帮助菌株，供体菌株)的比例和同源臂的长度设置了不同的梯度组合，探讨根瘤菌Ⅲ型效应因子突变的高效方法。结果显示，左右同源臂的长度为521 bp、861 bp时和三种菌混合的比例为2：1：1的因素组合，是突变目标基因最高效的组合方法。本研究所改进方法可为根瘤菌及其它革兰氏阴性菌的基因高效突变研究提供参考。“,”Mutate gene coding sequence is a key approach to detect the function of special gene. Triparental mating is an efficient method to mutate specific gene, which allows the foreign DNA enter into the receptor cell using the bacterial conjugation character. Then the target gene could be replaced by foreign DNA via homology crossing. Some factors could influence the efficiency of triparental mating, such as the length of the homologous arms, and the ratio of mixture bacteria (donor stain, helper strain and recipient strain). In this study, we want to promote the efficiency of triparental mating procedure by test the effect of homologous arms and ratio of mixture bacteria. The coding sequence of typeⅢeffector nopB, nopC, nopL in Sinorhizobium fredii HH103 were mutated to detect the effect of homologous arm's length and the ratio of mixture bacteria. We demonstrate that homologous arm's length of about 521 bp and 861 bp were the most efficiency length, and the ratio of 2:1:1 (rhizobium:helper:donor) was the highest efficiency ratio. Our results supply a modified and high efficiency ratio. Our results supply a modified and high efficiency protocol for the gene mutate in gram-negative bacteria.