CAJ | 학술논문

为探讨以PDS基因为指示基因利用VIGS技术研究小麦SBEⅡb基因的功能，本研究拟构建小麦SBEⅡb和PDS的双基因串联VIGS重组载体。根据GeneBank中公布的小麦SBEⅡb（AY740401.1）和PDS（FJ517553.1）基因序列分别设计搭桥引物，利用RT-PCR技术克隆SBEⅡb和PDS基因的特异片段，利用SOE-PCR技术获得双基因融合片段SBEⅡb：PDS。然后以BSMV：γ-PDS为原载体，通过酶切连接，用SBEⅡb：PDS基因片段将原载体中的PDS基因片段进行替换，从而构建BSMV：γ-SBEⅡb：PDS重组载体。结果显示克隆的SBEⅡb和PDS基因片段长分别是181和190 bp，SBEⅡb：PDS片段长是370 bp。序列分析表明：SBEⅡb基因片段与小麦SBEⅡb（AY740401.1）序列同源性为97%；小麦PDS基因片段与PDS（AF155217.2）序列同源性也为97%；酶切鉴定结果表明成功构建BSMV：γ-SBEⅡb：PDS重组载体。本研究构建的BSMV：γ-SBEⅡb：PDS重组载体将在PDS基因的指示下为下一步利用VIGS技术在小麦上研究SBEⅡb基因与小麦淀粉合成的关系奠定了基础。
위탐토이PDS기인위지시기인이용VIGS기술연구소맥SBEⅡb기인적공능，본연구의구건소맥SBEⅡb화PDS적쌍기인천련VIGS중조재체。근거GeneBank중공포적소맥SBEⅡb（AY740401.1）화PDS（FJ517553.1）기인서렬분별설계탑교인물，이용RT-PCR기술극륭SBEⅡb화PDS기인적특이편단，이용SOE-PCR기술획득쌍기인융합편단SBEⅡb：PDS。연후이BSMV：γ-PDS위원재체，통과매절련접，용SBEⅡb：PDS기인편단장원재체중적PDS기인편단진행체환，종이구건BSMV：γ-SBEⅡb：PDS중조재체。결과현시극륭적SBEⅡb화PDS기인편단장분별시181화190 bp，SBEⅡb：PDS편단장시370 bp。서렬분석표명：SBEⅡb기인편단여소맥SBEⅡb（AY740401.1）서렬동원성위97%；소맥PDS기인편단여PDS（AF155217.2）서렬동원성야위97%；매절감정결과표명성공구건BSMV：γ-SBEⅡb：PDS중조재체。본연구구건적BSMV：γ-SBEⅡb：PDS중조재체장재PDS기인적지시하위하일보이용VIGS기술재소맥상연구SBEⅡb기인여소맥정분합성적관계전정료기출。
To explore the foundation for pro bing the silencing effect of SBEⅡb with the indicator gene PDS on wheat,we constructed the VIGS vector carrying SBEⅡb and PDS fragments.According to the wheat SBEⅡb (GenBank accession number:AY740401.1) and PDS (GenBank accession number:FJ517553.1) genes sequences published in the GenBank,specific primers were designed and wheat SBEⅡb and PDS partial sequences were cloned through RT-PCR.With SOE-PCR,SBEⅡb:PDS was cloned in one step by using SOE-PCR.BSMV:γ-PDS vector was used as the original vector.The PDS gene of BSMV:γ-PDS vector was replaced by SBEⅡb:PDS fusion gene to construct BSMV:γ-SBEⅡb:PDS recombinant vector. Sequencing results showed that SBEⅡb and PDS genes partial sequences were 181 bp and 190 bp, respectively.The sequence analysis displayed that SBEⅡb genes partial sequence has a 97% homology with SBEⅡb,and the same with PDS gene's partial sequence compare to PDS.The enzymes digesting result confirmed that BSMV:γ-SBEⅡb:PDS recombinant vector was successfully constructed. The recombinant vector will lay the foundation for researching the relationship between SBEⅡb gene and wheat starch synthesis using VIGS technology with the PDS gene as a indicator in wheat.