摘要
【目的】克隆大豆豆荚特异性启动子,并对其进行功能分析,为研究抗病虫基因在大豆荚中的特异性表达奠定基础。【方法】利用PCR技术克隆得到豆荚特异性启动子(Pod Specific Promoter,PSP)的核心序列,用PSP序列取代质粒pBI121中的CaMV 35S启动子,构建与GUS基因融合的豆荚特异性报告载体pPSP-GUS,通过农杆菌介导法转化烟草"NC89",对转基因植株进行分子生物学检测,将鉴定为阳性的植株经GUS组织化学染色,验证PSP片段的功能。【结果】所获得的豆荚特异性启动子PSP大小为1 270bp,与已报道序列的同源性为98%,具有多种典型的启动子表达调控元件,如A/T-rich core、CAATBOX、TATABOX、GATABOX等。将pPSP-GUS质粒转化烟草后,经PCR和Southern blot检测结果显示,成功获得了转基因阳性烟草植株。GUS活性检测结果显示,在转pPSPGUS质粒的烟草根和叶片中均未检测到GUS活性,在萼片中可检测到低水平的GUS活性;而在花荚中可检测到高水平的GUS活性,且明显高于转pBI121质粒的对照植株。【结论】克隆得到的豆荚特异性启动子PSP片段具有启动基因在豆荚中特异性表达的功能。
【Objective】The aim of this study was to clone soybean pod specific promoter and analyze its functions.【Method】The core sequence of pod specific promoter(PSP)was cloned by PCR method.The cloned PSP core sequence was fused with the GUS reporter gene to construct plant reporter vector pPSPGUS by replacing the 35 Spromoter sequence in the pBI121 vector.Then the plant reporter vector was introduced into Nicotiana tabacum NC89 via Agrobacterium-mediated transformation.Molecular biological detection of the transgenic plants was conducted and functions of PSP in positive plants were validated by GUS histochemical staining.【Result】The obtained PSP was 1 270 bp,having 98%homology with the reported sequences.It contained many typical regulatory elements for promoter expression such as A/T-rich core,CAATBOX,TATABOX,and GATABOX.PCR and Southern blot results showed that the transgenic plants were obtained successfully.GUS activity assays indicated that the expression of GUS was highly active in pods rather than in roots and leaves.The GUS activity was stronger than that of control plants.【Conclusion】The obtained PSP fragment could express specifically in soybean pod.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2014年第10期63-69,80,共8页
Journal of Northwest A&F University(Natural Science Edition)
基金
国家自然科学基金项目(30971805)
转基因生物新品种培育重大专项(2011ZX08004-004)
