CAJ | 학술논문

水稻Pib基因的表达受茉莉酸、乙烯等激素诱导,为了确定该基因启动子响应茉莉酸和乙烯诱导的必需区域,进一步阐明茉莉酸和乙烯响应分子元件,文章用PCR制备了Pib全长启动子-3 572～2 bp及3个5'端有不同长度缺失的Pib启动子片段-2 692～2 bp,-1 335～2 bp、-761～2 bp.4个不同长度Pib启动子分别置换掉双元质粒中gus基因上游的35S构建为重组质粒.经农杆菌介导转入水稻获得转基因植株.转基因水稻中gus活性的蛋白质水平和mRNA水平的定性和定量分析结果表明,全长Pib启动子(-3 572～2 bp,pNAR901)启动活性最强,茉莉酸或乙烯诱导6 h后,其驱动gus基因在转基因植株各部组织中的表达量明显上升.而-3 572～-2 692 bp区段序列缺失后不但Pib启动子启动活性显著降低而且也丧失了对茉莉酸和乙烯的诱导活性.pNAR902(-2 692～2 bp),pNAR903(-1 335～2 bp)和pNAR904(-761～2 bp)中的Pib启动子序列的缺失长度相差达2倍和3倍以上,但其对茉莉酸和乙烯的诱导响应没有区别.这些结果显示3个Pib启动子缺失体构建中,其共同缺失序列即-3 572～-2 692 bp区域是Pib启动子茉莉酸和乙烯诱导响应的必需区域.软件检索证实,Pib启动子序列中只在上述共同缺失区段之内的-2 722 bp处有一个GCCGCC基序.文章报道的转基因实验表明GCCGCC基序可能是Pib基因中有关茉莉酸和乙烯诱导响应的顺式分子元件.
수도Pib기인적표체수말리산、을희등격소유도,위료학정해기인계동자향응말리산화을희유도적필수구역,진일보천명말리산화을희향응분자원건,문장용PCR제비료Pib전장계동자-3 572～2 bp급3개5'단유불동장도결실적Pib계동자편단-2 692～2 bp,-1 335～2 bp、-761～2 bp.4개불동장도Pib계동자분별치환도쌍원질립중gus기인상유적35S구건위중조질립.경농간균개도전입수도획득전기인식주.전기인수도중gus활성적단백질수평화mRNA수평적정성화정량분석결과표명,전장Pib계동자(-3 572～2 bp,pNAR901)계동활성최강,말리산혹을희유도6 h후,기구동gus기인재전기인식주각부조직중적표체량명현상승.이-3 572～-2 692 bp구단서렬결실후불단Pib계동자계동활성현저강저이차야상실료대말리산화을희적유도활성.pNAR902(-2 692～2 bp),pNAR903(-1 335～2 bp)화pNAR904(-761～2 bp)중적Pib계동자서렬적결실장도상차체2배화3배이상,단기대말리산화을희적유도향응몰유구별.저사결과현시3개Pib계동자결실체구건중,기공동결실서렬즉-3 572～-2 692 bp구역시Pib계동자말리산화을희유도향응적필수구역.연건검색증실,Pib계동자서렬중지재상술공동결실구단지내적-2 722 bp처유일개GCCGCC기서.문장보도적전기인실험표명GCCGCC기서가능시Pib기인중유관말리산화을희유도향응적순식분자원건.
The expression of Pib gene in rice was induced by hormone, such as jasmonic acid and ethylene. In order to determine the necessary regions of sequence or motifs for response to jasmonic acid and ethylene in Pib promoter, the full length promoter of Pib (-3 572～2 bp) and three different 5' deletion fragments of Pib promoter (-2 692～2 bp,-1 335～2 bp, -761～2 bp) were synthesized by PCR and then were substituted for 35S upstream gus in a binary plasmid to construct re-combined plasmids of Pib promoter-gus fusions. Transgenic rice plants of the four recombined plasmids were produced by Agrobacterium-mediated transformation. Quality and quantum analysis of gus activities in transgenic plants at both protein and mRNA levels were conducted. The promotion activity of the full length promoter of Pib (-3 572～2 bp, pNAR901) was the highest in the four recombinants and the gus activities in its transgenic plant organs were enhanced obviously at 6 h after treatment with jasmonic acid or ethylene. The promotion activity of the deleted Pib promoters was significantly decreased and the response to jasmonic acid or ethylene treatment was not present when the-3 572～-2 692 bp sequence was knocked out from the Pib promoter. Although the disparity in the lengths of the deleted Pib promoter of pNAR902 (-2 692～2 bp), pNAR903 (-1 335～2 bp), and pNAR904 (-761～2 bp) was more than 2 or 3 times, the response to jasmonic acid or ethylene treatment was not different among their transgenic plants. All these results indicated that the common deleted sequences (-3 572～-2 692 bp) in the three deleted Pib promoter constructs were the essential region to the response to jasmonic acid and ethylene treatment. The result of pib promoter sequence searching indicated that there was only one GCCGCC motif at -2 722 bp of this common deleted segment in the Pib promoter sequence. Our rice transgenic results showed that the GCCGCC may be a cis-motif for Pib gene conferring response to jasmonic acid and ethylene for Pib gene.