cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons
cDNA cloning and expression of a gene for 3-ketoacyl-CoA thiolase in pumpkin cotyledons
复制标题
DOI：
10.1007/bf00019471
复制
发表时间：
1996-07-01
影响因子：
5.1
通讯作者：
Nishimura, M
中科院分区：
文献类型：
--
作者：
Kato, A;Hayashi, M;Nishimura, M
3-酮脂酰-CoA硫解酶的cDNA克隆（EC 2.3.1.16）从黄化南瓜子叶的poly（A）（+）RNA构建的λ gt 11 cDNA文库中分离。该cDNA插入片段全长1682个核苷酸，编码461个氨基酸残基。体外表达的cDNA的研究和蛋白质的氨基末端序列的分析表明，南瓜硫解酶是作为前体合成的，其具有33个氨基酸的可切割的氨基末端前序列。氨基端前序列与典型的氨基端et蛋白和微体高度同源。免疫印迹分析表明，硫解酶的量显着增加，但在萌发过程中显着下降，光诱导的过渡期的微体从乙二醛到叶过氧化物酶体。相比之下，mRNA的量在萌发的早期阶段暂时增加。在衰老的子叶中，硫解酶的mRNA和蛋白质的水平再次增加，从叶过氧化物酶体的微体到乙醛酸酶体的反向过渡，但蛋白质的积累模式略有不同，从苹果酸合成酶。这些结果表明，在种子萌发和萌发后生长过程中，硫解酶的表达以类似于其他乙醛酸酶体酶（如苹果酸合酶和柠檬酸合酶）的方式进行调节。相比之下，在衰老过程中，硫解酶的表达以与其他乙醛酸酶不同的方式调节。
A cDNA clone for 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was isolated from a lambda gt11 cDNA library constructed from the poly(A)(+) RNA of etiolated pumpkin cotyledons. The cDNA insert contained 1682 nucleotides and encoded 461 amino acid residues. A study of the expression in vitro of the cDNA and analysis of the amino-terminal sequence of the protein indicated that pumpkin thiolase is synthesized as a precursor which has a cleavable amino-terminal presequence of 33 amino acids. The amino-terminal presequence was highly homologous to typical amino-terminal et proteins to microbodies. Immunoblot analysis showed that the amount of thiolase increased markedly during germination but decreased dramatically during the light-inducible transition of microbodies from glyoxysomes to leaf peroxisomes. By contrast, the amount of mRNA increased temporarily during the early stage of germination. In senescing cotyledons, the levels of the thiolase mRNA and protein increased again with the reverse transition of microbodies from leaf peroxisomes to glyoxysomes, but the pattern of accumulation of the protein was slightly different from that of malate synthase. These results indicate that expression of the thiolase is regulated in a similar manner to that of other glyoxysomal enzymes, such as malate synthase and citrate synthase, during seed germination and post-germination growth. By contrast, during senescence, expression of the thiolase is regulated in a different manner from that of other glyoxysomal enzymes.