BT1, A STRUCTURAL GENE FOR THE MAJOR 39-44 KDA AMYLOPLAST MEMBRANE POLYPEPTIDES
BT1, A STRUCTURAL GENE FOR THE MAJOR 39-44 KDA AMYLOPLAST MEMBRANE POLYPEPTIDES
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DOI：
10.1111/j.1399-3054.1995.tb00825.x
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发表时间：
1995-10-01
影响因子：
6.4
通讯作者：
SHANNON, JC
中科院分区：
文献类型：
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作者：
CAO, HP;SULLIVAN, TD;SHANNON, JC
本工作的目的是研究Bt 1基因（Bt 1）与玉米（Zea mays L.）脆粒（Bt 1）淀粉体膜上缺失的主要39-44 kDa淀粉体膜多肽之间的关系。本文描述了一种从未成熟籽粒中分离造粉体的快速而温和的方法。这些淀粉体是相对免费的污染，由其他细胞成分，免疫学研究表明，它们含有的多肽与抗体反应，玉米淀粉漂白酶和ADP-Glc焦磷酸化酶。从淀粉体分离的纯化膜含有一种多肽，该多肽与菠菜叶绿体中P-转运蛋白的抗体反应。然而，一簇39-44 kDa的多肽占W 64 A籽粒淀粉体膜蛋白总量的约40%。这些多肽被针对由BT 1蛋白羧基端的56个氨基酸和谷胱甘肽S-转移酶组成的融合蛋白产生的抗体特异性识别，BT 1抗体还与杂种籽粒造粉体膜中丰富的多肽反应（Doebler 66 XP和Pioneer 3780），以及shrunken 1和shrunken 2突变基因型，但在来自bt 1突变籽粒的造淀粉体膜中不存在BT 1反应多肽，我们无法检测BT 1的免疫印迹程序中的微粒体膜从胚胎和果皮组织从内核，从幼苗根和芽，或在膜从线粒体和叶绿体。从发育中的胚乳和纯化的造淀粉体膜的微粒体膜中提取的蛋白质获得了相同的BT 1免疫印迹模式。Bt 1等位基因的拷贝数和胚乳微粒体膜中的BT 1水平之间的线性关系被证明在一个基因剂量系列。BT 1不提取从淀粉体膜氯仿/甲醇或碱性缓冲液在pH 11.5，但部分提取0.1 M NaOH。这些证据支持Bt 1是主要的39-44 kDa造粉体膜多肽的结构基因的结论，这些多肽是胚乳造粉体膜特异性的整合蛋白。
The aim of the present work was to investigate the relationship between the Bt1 gene (Bt1) and the major 39-44 kDa amyloplast membrane polypeptides which were deficient in amyloplast membranes of brittlel (bt1) kernels of maize (Zea mays L,). A rapid yet gentle procedure for the isolation of amyloplasts from immature kernels is described. These amyloplasts were relatively free of contamination by other cellular components, and immunological studies showed that they contained polypeptides which reacted with antibodies to maize starch blanching enzyme and ADP-Glc pyrophosphorylase. Purified membranes isolated from the amyloplast contained a polypeptide which reacted with antibodies to the P-translocator from spinach chloroplasts. However, a cluster of 39-44 kDa polypeptides accounted for about 40% of the total amyloplast membrane protein from W64A kernels, These polypeptides were specifically recognized by antibodies raised against a fusion protein consisting of 56 amino acids of the carboxyl terminus of the BT1 protein and glutathione S-transferase, The BT1 antibodies also reacted with the abundant polypeptides in amyloplast membranes from hybrid kernels (Doebler 66XP and Pioneer 3780), and the shrunken1 and shrunken2 mutant genotypes, but no BT1 reacting polypeptides were present in amyloplast membranes from bt1 mutant kernels, We were unable to detect BT1 by the immunoblot procedure in microsomal membranes from embryo and pericarp tissues from the kernel, from seedling roots and shoots, or in membranes from mitochondria and chloroplasts. The same BT1 immunoblot pattern was obtained for proteins extracted from microsomal membranes from developing endosperm and from purified amyloplast membranes. A linear relationship between the number of copies of Bt1 alleles and the levels of BT1 in endosperm microsomal membranes was demonstrated in a gene dosage series. BT1 was not extracted from amyloplast membranes by chloroform/methanol or by alkaline buffer at pH 11.5, but was partially extracted by 0.1 M NaOH. These lines of evidence support the conclusion that Bt1 is the structural gene for the major 39-44 kDa amyloplast membrane polypeptides and that these polypeptides are integral proteins specific to amyloplast membranes from the endosperm.