无选择标记转中国对虾抗菌肽基因水稻的获得及其遗传分析、抗病检测

Generation of Marker-free Transgenic Rice with Antimicrobial Peptide Genes from Penaeus chinensis and Its Genetic Analysis and Disease Resistance

为了获得不含筛选标记、抗病性显著增强的转抗菌肽基因水稻,利用双T-DNA载体系统将中国对虾抗菌肽基因dp3和选择标记基因Hpt导入到水稻恢复系明恢86中。结果表明,T1代110个株系中88.2%的株系出现抗感分离,其余11.8%的株系全为阴性。PCR检测7个分离比例符合3∶1的株系显示,dp3和Hpt基因在3个株系中为自由组合,在4个株系中出现连锁。在468个检测单株中筛选出31个无选择标记的转基因植株,占总样本的6.6%。RT-PCR检测T1代转基因植株显示,抗菌肽基因在RNA水平上得到表达。用白叶枯病小种PXO86、PXO99和纹枯病小种GD-118、C-30对上述3个转基因株系进行接种,转基因阳性植株明显提高了对白叶枯病和纹枯病的抵抗力。

To obtain marker-free transgenic rice with significantly enhanced disease resistance,the antimicrobial peptide gene dp3 and selectable marker gene Hpt were integrated into Oryza sativa L. restoring line Minghui 86 by a double T-DNA binary vector system. Hygromycin resistance analysis revealed that88. 2% of the lines showed obvious resistant and sensitive separation,while the others 11. 8% were sensitive in 110 T1 lines. PCR analysis was carried out with seven lines that matched Medel＇s ratio of 3∶ 1;the results showed that dp3 and Hpt genes were assorted independently in three lines,while the other four lines were detected with linkage of these two genes. Thirty-one marker-free transgenic rice plants were screened out from the generated 468 seedlings,accounting for about 6. 6% of the total samples. RT-PCR analysis showed that the antimicrobial peptide gene was expressed in T1 generation. T1 generation were inoculated with the strains PXO86 and PXO99 of Xanthomonas campestris pv. Oryzae（ Xoo）,GD-118 and C-30 of Rhizoctonia solani. The results indicated that the transgenic lines had a significantly enhanced resistance to bacterial blight and sheath blight caused by the four Xoo and Rhizoctonia solani strains.