利用ihpRNA介导的基因沉默培育抗冷糖化马铃薯

Regeneration of Potato Plants Resistant to Cold Induced Sweetening via ihpRNA Technique

为培育抗冷糖化的转基因马铃薯材料,克隆了368 bp的马铃薯酸性转化酶(AcInv)基因3′端非翻译区(3′UTR)和281 bp的马铃薯VP1-ABI3-like protein基因的第一内含子,构建了内含子连接的马铃薯AcInv基因3′UTR的发夹型RNA(ihpRNA)载体pBIV;采用微束激光穿刺技术转化马铃薯品种,得到了149个转基因株系,其中139个转基因株系在低温贮藏35 d时块茎还原糖含量低于亲本材料。RT-PCR分析表明还原糖含量降低的转基因植株中检测不到AcInv基因mRNA的积累。本研究结果表明以3′UTR作为RNAi载体的干涉片段可以有效地抑制靶标基因mRNA的积累,通过对AcInv沉默可获得抗低温糖化的马铃薯育种材料。

The present work aimed to produce transgenic potato plants resistant to cold induced sweetening. RT-PCR was employed to clone the 368 bp 3＇ untranslated region （3＇UTR） of potato Aclnv gene and PCR was used to clone the first intron of potato VP1-ABI3 like protein gene. The intron-spliced hairpin RNA （ihpRNA） vector pBIV was constructed by inserting the 3＇UTR in both sense and anti-sense orientation into pBI121 and spliced with the first intron of VP1-ABI3-1ike protein gene. Laser micro-beam puncture technique was employed to transform potato varieties and 149 transgenic lines were attained. Reduced sugar content indicated that 139 out of 149 transgenic lines were more resistant to cold induced sweetening （CIS） than their parental lines. Semi-quantitative RT-PCR analysis showed that the accumulation of mRNAs derived from Aclnv was undetectable in the CIS resistant transgenic lines. This research suggested that the ihpRNA of 3＇UTR can be used efficiently to inhibit the accumulation of target mRNAs and CIS resistant potato lines can be attained by silencing the expression of Aclnv gene.