马铃薯块茎颗粒结合型淀粉合酶基因的克隆及其RNAi载体的构建

Cloning of Granule-Bound Starch Synthase Gene and Construction of Its RNAi Vector in Potato Tuber

GBSSI是马铃薯块茎中控制直链淀粉合成的关键酶,为培育高支链淀粉含量或纯支链淀粉含量的转基因马铃薯材料,根据GenBank登录号X58453的序列设计特异引物,采用RT-PCR技术获得马铃薯块茎GBSSI相似基因,并利用生物信息学相关软件分析,预测GBSSI相似基因cDNA序列编码的蛋白质结构和功能。结果表明,克隆的GBSSI相似基因与报道的GBSSI基因序列相似性达到99.78%,其开放阅读框长1824bp,编码607个氨基酸,具有许多重要功能位点。三级结构预测结果表明,该蛋白具有淀粉合成功能,基因序列已注册到GenBank,序列登录号为EU403426。以此基因CDS内542bp的靶标序列作为干扰区段,扩增GBSSI的正反向基因片段,并引入237bp的内含子序列,构建由Patatin启动子驱动的具有"正义基因片段gbssA-内含子VP1-ABI3-like protein-反义基因片段gbssB"的植物干扰表达载体pBI121g-PgABI。本研究结果将为淀粉合成的进一步研究和高支链淀粉含量或纯支链淀粉含量的马铃薯品种的培育奠定基础。

There is about 17% starch in potato （Solanum tuberosum L.） tubers. Potato starch granules are composed of two poly- saccharides, unbranched amylose （approximately 20% to 25%） and branched amylopectin （approximately 75% to 80%）. To de- velop transgenic potato with high-amylopectin in tubers, we got a cDNA sequence of the potato GBSSI from the total RNA of potato tubers by RT-PCR using specific primers of conserved domain of GenBank Accession Number X58453 sequence. The GBSSI cDNA sequence shared 99.78% similarity with the GBSSI gene in GenBank （accession No. X58453）. The full-length of cDNA was 1 824 bp, which contained 607 amino acids, three conserved domains and many important functional sites. The 3D structure of GBSSI was highly similar to that of the glycogen synthase, indicating that GBSS1 has a function of starch synthesis. GBSSI similar gene obtained here was granule-bound starch synthase, and its sequence was submitted to GenBank, with the ac- cession number of EU403426. On the basis of a 542 bp RNAi target region from the CDS of GBSSI, the sense and antisense fragments were amplified and separated by a 237 bp intron to construct the RNA interference expression vector pBI121g-PgABI containing ＂sense gbssA-VP1-ABl3-1ike protein intron-antisense gbss B＂ regulated by Patatin promoter, which will lay a solid foundation for the study on synthesis of starch and breeding of transgenic potato with high amylopectin content or pure amy- lopectin.