Klein R R, Crafts-Brandner S J, Salvucci M E
USDA-ARS, University of Kentucky, Lexington 40546-0076.
Planta. 1993;190(4):498-10. doi: 10.1007/BF00224789.
A 561-base-pair (bp) polymerase-chain-reaction (PCR) product of sucrose-phosphate synthase (SPS) was amplified using degenerate oligonucleotide primers corresponding to tryptic peptides of SPS (EC 2.4.1.14) from spinach (Spinacia oleracea L). Crucial to the primer specificity and the synthesis of the 561-bp product was the use of primer pools in which the number of degenerate primer species was limited. A full-length cDNA was subsequently obtained by screening a cDNA bacteriophage library with the 561-bp product of SPS and 5' PCR-RACE (Rapid Amplification of cDNA Ends). The 3530-bp cDNA of SPS encoded for a 1056-amino-acid polypeptide of predicted molecular mass of 117 kDa. The deduced amino-acid sequence of spinach SPS showed regions of strong homology with SPS from maize (A.C. Worrell et al., 1991, Plant Cell 3, 1121-1130); amino-acid identity was 54% over the entire protein. Western and Northern analyses of root, petiole and spinach leaf tissue showed that SPS was expressed in an organ-specific manner, being predominantly localized in the leaf. The accumulation of SPS protein and mRNA during leaf development coincided with the early rapid phase of leaf expansion and the apparent transition of the leaf from sink to source status. Levels of SPS mRNA and protein were reduced during the acclimation of leaves to low-irradiance conditions. Transfer of low-irradiance-adapted leaves to higher-irradiance conditions resulted in a gradual increase in SPS protein and mRNA. Diurnal changes in irradiance did not alter SPS protein or transcript levels, indicating that short-term regulation of SPS primarily involves a modulation of enzyme activity.
使用与菠菜（Spinacia oleracea L）中蔗糖磷酸合酶（SPS，EC 2.4.1.14）胰蛋白酶肽段对应的简并寡核苷酸引物，扩增出一段561个碱基对（bp）的蔗糖磷酸合酶聚合酶链反应（PCR）产物。使用简并引物种类数量有限的引物池对于引物特异性和561 bp产物的合成至关重要。随后，通过用SPS的561 bp产物和5' PCR-RACE（cDNA末端快速扩增）筛选cDNA噬菌体文库，获得了全长cDNA。SPS的3530 bp cDNA编码一个预测分子量为117 kDa的1056个氨基酸的多肽。菠菜SPS推导的氨基酸序列显示出与玉米SPS有高度同源区域（A.C. Worrell等人，1991年，《植物细胞》3，1121 - 1130）；整个蛋白质的氨基酸同一性为54%。对根、叶柄和菠菜叶组织的蛋白质免疫印迹和Northern分析表明，SPS以器官特异性方式表达，主要定位于叶片中。叶片发育过程中SPS蛋白和mRNA的积累与叶片早期快速扩展阶段以及叶片从库到源状态的明显转变相一致。叶片适应低光照条件期间，SPS mRNA和蛋白质水平降低。将适应低光照的叶片转移到高光照条件下，导致SPS蛋白和mRNA逐渐增加。光照的昼夜变化不会改变SPS蛋白或转录本水平，表明SPS的短期调节主要涉及酶活性的调节。
