Isolation of a 1 195 bp 5′-Flanking Region of Rice Cytosolic Fructose-1,6-bisphosphatase and Analysis of Its Expression in Transgenic Rice

水稻细胞质1 ,6-二磷酸果糖酶基因1195 bp的上游调控序列的克隆及其在转基因水稻中的表达研究(英文)

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 lip 5'-flanking region which started from the translation initiation ATG codon was fused to reporter gene encoding beta-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.

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GenomeWalking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 70 31.5pmol4_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