小麦SBEⅡa、SSⅡa基因片段的克隆及VIGS重组载体的构建

Cloning of Wheat SBEⅡa and SSⅡa Genes Partial Sequence and Construction of Their VIGS Recombinant Vectors

【目的】克隆小麦SBEⅡa、SSⅡa基因部分序列并构建二者的VIGS重组载体,为在小麦上利用VIGS技术研究SBEⅡa、SSⅡa基因与小麦淀粉合成的关系奠定基础。【方法】根据GenBank中公布的小麦SBEⅡa(AF286319.1)、SSⅡa(AF155217.2)基因序列分别设计特异引物,利用RT-PCR技术克隆SBEⅡa、SSⅡa基因的特异片段。然后以BSMV:γ-PDS为原载体,通过酶切连接用SBEⅡa、SSⅡa基因片段分别将原载体中的PDS基因进行替换,从而构建BSMV:γ-SBEⅡa和BSMV:γ-SSⅡa重组载体。【结果】克隆的SBEⅡa和SSⅡa基因片段长分别是178 bp和171 bp。序列分析表明:SBEⅡa基因片段与小麦SBEⅡa(AF286319.1)序列同源性为100%;、SSⅡa基因片段与SSⅡa(AF155217.2)序列同源性也为100%;酶切鉴定结果表明成功构建BSMV:γ-SBEⅡa和BSMV:γ-SSⅡa重组载体。【结论】构建的BSMV:γ-SBEⅡa和BSMV:γ-SSⅡa重组载体将为下一步利用VIGS技术在小麦上研究SBEⅡa、SSⅡa基因与小麦淀粉合成的关系奠定基础。

[ Objective] In order to lay the foundation for researching the relationship between SBEⅡ a, SS Ⅱ a genes and wheat starch synthesis by using VIGS technology, wheat SBE Ⅱ a and SSⅡ a genes partial se- quence were cloned and their VIGS recombinant vectors were built. [ Method ] According to wheat SBE Ⅱa （AF286319.1）, SS Ⅱ a（AF155217.2） genes sequence published in the GenBank, speeial primers were de- signed and wheat SBE Ⅱ a and SS Ⅱ a genes partial sequence were cloned though RT - PCR. BSMV ： ~/- PDS vector was used as the original vector. The PDS gene of BSMV ： γ- PDS vector was replaced by wheat SBE Ⅱ a and SS Ⅱ a genes to build BSMV ：γ- SBE Ⅱ a and BSMV ：γ-SS Ⅱ a recombinant vectors. [ Result] The se- quencing results showed that SBE Ⅱ a and SS Ⅱ a genes partial sequence were 178 bp and 171 bp and the se- quence analysis displayed that 100% sequence homology was between SBE Ⅱ a genes partial sequence and SBE Ⅱ a（ AF286319.1 ） ,SS Ⅱa genes partial sequence and SS Ⅱ a（AF155217.2）. The enzyme digesting re-sult showed that BSMV ： ~/- SBE 11 a and BSMV ： ~/- SS Ⅱa recombinant vectors were successfully constructed. [ Conclusion ] BSMV：γ-SBE Ⅱ a and BSMV：γ-SS Ⅱ a recombinant vectors will lay the foundation for re- searching the relationship between SBE Ⅱ a, SS Ⅱ a genes and wheat starch synthesis by using VIGS technology in wheat.