CAJ | 학술논문

启动子位于转录起始位点上游并能特异性地结合 RNA 聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得 Gerb 和 Bntt 两个种皮特异性启动子,并对其进行生物信息学分析,构建了 Gerb ：GUS和 Bntt ：GUS 植物表达载体并转化拟南芥,通过组织化学染色观察了 GUS 的表达情况。结果表明：两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件；转基因拟南芥幼苗期,大麦Gerb 种皮特异启动子驱动GUS 全株表达且子叶和下胚轴较真叶和根中表达量高；油菜 Bntt 种皮特异启动子表达较弱；成株期,Gerb 在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性；Bntt 仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb 表达模式未发生显著变化,而 Bntt 仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子 Gerb 和油菜种皮特异性启动子 Bntt 在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。
계동자위우전록기시위점상유병능특이성지결합 RNA 취합매,기작위조공서렬구동외원기인재이원식물중표체,종이실현전기인적고효성,구유시공표체특이성적계동자대획득유효전기인식물급산물구유중요의의。위료해충피특이계동자적표체모식,해연구기우전기보도적서렬,통과동원극륭적방법분별종대맥화유채중극륭획득 Gerb 화 Bntt 량개충피특이성계동자,병대기진행생물신식학분석,구건료 Gerb ：GUS화 Bntt ：GUS 식물표체재체병전화의남개,통과조직화학염색관찰료 GUS 적표체정황。결과표명：량충계동자서렬중도함유다고패충피특이표체계동자원건이급다충협박유도향응원건；전기인의남개유묘기,대맥Gerb 충피특이계동자구동GUS 전주표체차자협화하배축교진협화근중표체량고；유채 Bntt 충피특이계동자표체교약；성주기,Gerb 재불동조직(협편、경、화서화각과)중균유표체,미현시조직특이성；Bntt 부재협편급각과유관속중유미약표체。재각충비생물협박하,Gerb 표체모식미발생현저변화,이 Bntt 부재염협박하현시흔강적각과화충자특이성표체,기타협박미견명현표체。이상결과현시,대맥충피특이성계동자 Gerb 화유채충피특이성계동자 Bntt 재시간화공간표체모식상존재차이,저대금후선택충피특이계동자구유삼고작용,단기구체궤제잉수진일보연구험증。
Promoter is a segment of DNA molecule which locates in the upstream of transcription start site and specifical-ly binds to RNA polymerase.Efficient genetic modification of transgenic plant requires promoter as the regulatory se-quence to drive the expression of foreign gene.Specific promoters with spatial and temporal control of ectopic gene ex-pression are important for acquirement of the transgenic plant and its product with high quality.To understand the ex-pression pattern of seed-coat specific promoters,based on the reported sequences,two seed-coat specific promoters-Gerb and Bntt were isolated from Brassica napus and Hordeum vulgare by homologous-based cloning method,analysis of the elements in both promoters,then Gerb ::GUS and Bntt ::GUS were constructed and introduced into Arabidopsis , and the expression patterns of Gerb and Bntt were observed by GUS histochemical staining.The results of bioinformatics showed that multi-copies of seed specific expression cis-acting elements and various stress response elements were identi-fied in both promoters.Histochemical GUS staining of the transgenic plant indicated that in seedling stage,Gerb pro-moter showed strong blue GUS staining with the cotyledon and hypocotyl while relatively weaker of GUS activity with the true leaves and roots;Bntt promoter showed weak GUS staining in whole seedling;for adult plant,various tissues (leaf,stem,inflorescence,silique,etc .)with promoter Gerb presented visible GUS staining while with promoter Bntt a very weak staining was only observed on vascular bundle of silique and the leaf.Under various stresses,the expression pattern of transgenic line with Gerb ::GUS showed no significant change,however,for Bntt ::GUS ,NaCl stress could cause strong GUS staining with silique and seeds,while no significant staining was detected in other tissues or under oth-er stresses.These results indicated that Gerb and Bntt promoters displayed different temporal and spatial expression pat-terns,which may be helpful for screening seed-coat specific promoter in practice,however,the details of regulation mechanism needs further study to validate.