Purification and molecular genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from maize
Purification and molecular genetic characterization of ZPU1, a pullulanase-type starch-debranching enzyme from maize
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DOI：
10.1104/pp.119.1.255
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发表时间：
1999-01-01
期刊：
影响因子：
7.4
通讯作者：
James, MG
中科院分区：
文献类型：
--
作者：
Beatty, MK;Rahman, A;James, MG
本研究鉴定并纯化了发育中的玉米（Zea mays L.）胚乳中特异的异淀粉酶和普鲁兰酶型淀粉脱支酶（DBEs）。利用与水稻（Oryza sativa L.）编码普鲁兰酶型DBE的cDNA同源性，分离到cDNA克隆Zpu1。将蛋白产物ZPU1与其他18个DBEs进行比较，发现了异淀粉酶和普鲁兰酶型酶共有的基序，以及类特异性序列块。Zpu1与基因组DNA杂交确定了位于2号染色体上的单拷贝基因Zpu1。在淀粉生物合成过程中，胚乳中富含Zpu1 mRNA，但在叶片和根中均未检测到。抗ZPU1抗血清在发育中的胚乳中特异性识别约100-kD的ZPU1蛋白，而在叶片中不识别。从发育中的玉米籽粒提取物中纯化出普鲁兰酶型和同淀粉酶型DBEs。普鲁兰酶型活性鉴定为ZPU1，同淀粉酶型活性鉴定为SU1。已知糖1 （su1）基因的突变会导致su1异淀粉酶和普鲁兰酶型DBE的缺乏。在su1突变体中，ZPU1活性、蛋白水平和电泳迁移率发生改变，表明它是受影响的普鲁兰酶型DBE。Zpu1的转录水平在非突变体和土壤突变体籽粒中是相同的，这表明Zpu1和SU1的协调调控发生在转录后。
This study identified and purified specific isoamylase- and pullulanase-type starch-debranching enzymes (DBEs) present in developing maize (Zea mays L.) endosperm. The cDNA clone Zpu1 was isolated based on its homology with a rice (Oryza sativa L.) cDNA coding for a pullulanase-type DBE. Comparison of the protein product, ZPU1, with 18 other DBEs identified motifs common to both isoamylase- and pullulanase-type enzymes, as well as class-specific sequence blocks. Hybridization of Zpu1 to genomic DNA defined a single-copy gene, zpu1, located on chromosome 2. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in the leaf or the root. Anti-ZPU1 antiserum specifically recognized the approximately 100-kD ZPU1 protein in developing endosperm, but not in leaves. Pullulanase- and isoamylase-type DBEs were purified from extracts of developing maize kernels. The pullulanase-type activity was identified as ZPU1 and the isoamylase-type activity as SU1. Mutations of the sugary1 (su1) gene are known to cause deficiencies of SU1 isoamylase and a pullulanase-type DBE. ZPU1 activity, protein level, and electrophoretic mobility were altered in su1-mutant kernels, indicating that it is the affected pullulanase-type DBE. The Zpu1 transcript levels were equivalent in nonmutant and sol-mutant kernels, suggesting that coordinated regulation of ZPU1 and SU1 occurs posttranscriptionally.