利用正义RNAi技术提高玉米直链淀粉含量效果的研究

Increasing of Maize Amylose Content by Sense RNA Interference

为了研究利用正义RNAi技术提高玉米直链淀粉含量效果,用基因枪法将构建的sbeⅡb正义RNAi表达载体pBAC506和pBAC508(筛选标记基因分别为35S启动子及Adh1-intron1增强驱动的epsps和bar)导入玉米(Zeamays)自交系幼胚愈伤组织,经过筛选、分化和再生获得了44株转基因当代植株,经PCR扩增、PCR-Southern检测有30株为阳性。选择9株健壮的阳性植株进行基因组Southern-blotting分析,结果表明7株T0植株整合了目的基因,其中4株整合了1个转基因拷贝,2株整合了2个转基因拷贝,1株整合了3个转基因拷贝。这7株中有2株结实,直链淀粉含量分别为23.22%和24.60%,有一定的小幅提高。下一步要进行较大规模的基因枪转化,得到比较大的转基因群体,以对更多转基因后代进行鉴定,期望筛选出外源基因多拷贝插入和直链淀粉含量大幅提高的株系。

In order to explore the effect of sense RNAi （RNA interference） on increase the amylose content of maize （Zea mays） ,two sense-RNAi vectors pBAC506 and pBAC508 with the sense sbeⅡb and selection gene epsps or bar were constructed and transformed to maize callus induced from immature embryos of the elite inbred lines by biolistic PDS1000 /He. Forty-four T0 transformed plantlets were regenerated,and thirty plants were positive by analysis of PCR and PCR-Southern hybridization. Southern-blotting results of 9 strong PCR-positive plants showed that the RNAi constructs in 7 plants were successful integrated into their genomes,4 with one copy of transgene,2 with two copies and 1 with three copies. The amylose content of 2 T2transgenic lines were 23. 22% and 24. 60% ,respectively. The results suggested that a big transgenic population was necessary for screening the transgenic plants with multi-copy transgene and high amylose content.