采用一种新型RNAi载体培育转基因高直链淀粉马铃薯

Development of Transgenic High-Amylose Potato Using a Novel RNAi Vector

采用PCR技术分别克隆nos终止子、烟草axi1内含子和烟草ubi.u4启动子,亚克隆部分与马铃薯Sbe1同源、部分与Sbe2同源的融合基因SIII,构建具有“ubi.u4启动子—反向SIII—反向nos终止子—axi1内含子—正向nos终止子”结构的异源3′端UTR反向重复序列型RNAi载体pCUSNI,采用农杆菌介导法转化马铃薯品种陇薯3号、甘农薯2号和大西洋,获得了16个转基因植株,其中14个的块茎直链淀粉含量大幅度增加,表观直链淀粉含量介于53.80%~85.33%;但随着直链淀粉含量的升高,转基因马铃薯淀粉含量下降。半定量RT-PCR分析表明,在直链淀粉含量超过80%的转基因株系中检测不到Sbe1和Sbe2基因mRNA的积累。结果表明,异源3′端UTR反向重复序列型RNAi载体pCUSNI能够高频、高效地同时抑制马铃薯Sbe1和Sbe2基因的表达,是一个优良的RNAi载体。以载体pCUSNI为基础,再以植物的其他基因为靶,只需在pCUSNI的BamHI和XbaI位点之间插入干涉片段替换SIII即可完成载体构建,而不必构建靶标基因的反向重复序列,使载体制备迅速便捷。

Amylose from potato starch is of great advantage for applications in many fields because of its higher degree of polymerization and lower gelling temperature compared with cereal starch. However, there is no natural mutant of high-amylose potato. RNAi technique is efficient and specific for plant gene silence but traditional RNAi vector is laborious to prepare. To design an easily-prepared RNAi vector and to develop transgenic high-amylose potato, PCR technique was employed to amplify the nos terminator, the tobacco axil and the tobacco ubi.u4 promoter, and to sub-clone a fused fragment Sill which is partially homologous to potato Sbel and to Sbe2. Then, a newly designed vector pCUSNI containing ＂ubi.u4 promotor-antisense Slll-antisense nos terminator-aM/gene intron-sense nos terminator＂ was generated and transformed into potato varieties Longshu 3, Gannongshu 2, and Atlantic by Agrobacterium-mediated transformation. Sixteen transgenic potato plants were obtained, and the amylose content in 14 of them increased significantly, which ranged from 53.80% to 85.33% of total starch. But with the increase of amylose content, starch content decreased in transgenic potato plants. Results of semi-quantitative RT-PCR indicated that the accumulation of mRNAs for Sbel and Sbe2 was not detectable in transgenic plants with amylose content higher than 80%, indicating that the novel RNAi vector pCUSNI was highly efficient for the simultaneous silence of Sbel and Sbe2 in potato. The generation of the RNAi vector pCUSNI makes it much easier to prepare RNAi vectors targeting to other plant genes. The only thing is to insert a fragment of the target gene or the target genes in restriction sites between BamH I and Xba I in vector pCUSNI to replace Sill and there is no need to construct an inverted repeat of target gene.