CAJ | 학술논문

1,6-二磷酸果糖酶(EC3.13.11)催化1,6-二磷酸果糖分解为6-磷酸葡萄糖和无机磷酸.在高等植物的光合作用细胞中,存在两种1,6-二磷酸果糖酶:即叶绿体型1,6-二磷酸果糖酶和细胞质型1,6-二磷酸果糖酶.由于细胞质型1,6-二磷酸果糖酶在植物碳水化合物代谢中起重要作用,且具有表达特异性,本试验通过Genome Walking分离了水稻细胞质型1,6-二磷酸果糖酶基因的上游序列,并将其与β-葡糖醛酸酶(GUS)报告基因构建成嵌合表达载体.采用基因枪法转化水稻,在转基因水稻中分析了GUS的表达活性和特异性.组织化学检测表明,在转基因水稻的成熟叶片中,GUS基因只在叶肉细胞中表达,在表皮细胞、泡状细胞、维管组织中均无表达;在叶鞘中的表达与叶片中相似,仅仅在叶肉细胞中表达;在根、茎所有细胞中均没有蓝色反应.为进一步研究1,6-二磷酸果糖酶基因启动子在水稻中的表达量,对12株独立来源的转基因水稻的GUS 活性进行了荧光定量分析.结果显示,水稻成熟叶片中的GUS活性平均值为7 031.5 pmol 4-MU-1*min-1*mg蛋白.在不同器官及组织中表达活性有差异,在转基因水稻的叶片、叶鞘中GUS均有较强的表达,在根、茎中未检测到GUS活性.实验结果表明,ATG上游1 195 bp调控区足以导致GUS基因在水稻中的特异性表达,因此该片段包含有使报告基因在叶肉细胞中特异性表达的所有顺式调控元件.
1,6-이린산과당매(EC3.13.11)최화1,6-이린산과당분해위6-린산포도당화무궤린산.재고등식물적광합작용세포중,존재량충1,6-이린산과당매:즉협록체형1,6-이린산과당매화세포질형1,6-이린산과당매.유우세포질형1,6-이린산과당매재식물탄수화합물대사중기중요작용,차구유표체특이성,본시험통과Genome Walking분리료수도세포질형1,6-이린산과당매기인적상유서렬,병장기여β-포당철산매(GUS)보고기인구건성감합표체재체.채용기인창법전화수도,재전기인수도중분석료GUS적표체활성화특이성.조직화학검측표명,재전기인수도적성숙협편중,GUS기인지재협육세포중표체,재표피세포、포상세포、유관조직중균무표체;재협초중적표체여협편중상사,부부재협육세포중표체;재근、경소유세포중균몰유람색반응.위진일보연구1,6-이린산과당매기인계동자재수도중적표체량,대12주독립래원적전기인수도적GUS 활성진행료형광정량분석.결과현시,수도성숙협편중적GUS활성평균치위7 031.5 pmol 4-MU-1*min-1*mg단백.재불동기관급조직중표체활성유차이,재전기인수도적협편、협초중GUS균유교강적표체,재근、경중미검측도GUS활성.실험결과표명,ATG상유1 195 bp조공구족이도치GUS기인재수도중적특이성표체,인차해편단포함유사보고기인재협육세포중특이성표체적소유순식조공원건.
A genomic DNA fragment containing the 5′-upstream sequence and part of the open reading frame corresponding to the cytosolic fructose-1,6-bisphosphatase (cyFBPase) cDNA was isolated by Genome Walking. The 1 195 bp 5′-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding β-glucuronidase (GUS) and stably transferred to rice via particle bombardment. Strong GUS activity was detected in leaves and leaf sheaths of transgenic rice, but not in culms and roots. Histochemical localization revealed that GUS expression was exclusively restricted to mesophyll cells in transgenic rice. Our results indicate that the 1 195 bp fragment contains all the cis-elements required for directing mesophyll-specific expression pattern in rice.