SEQUENCE OF A CDNA AND EXPRESSION OF THE GENE ENCODING EPIDERMAL AND GUT CHITINASES OF MANDUCA-SEXTA
SEQUENCE OF A CDNA AND EXPRESSION OF THE GENE ENCODING EPIDERMAL AND GUT CHITINASES OF MANDUCA-SEXTA
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DOI：
10.1016/0965-1748(93)90043-r
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发表时间：
1993-09-01
影响因子：
3.8
通讯作者：
MUTHUKRISHNAN, S
中科院分区：
文献类型：
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作者：
KRAMER, KJ;CORPUZ, L;MUTHUKRISHNAN, S
昆虫在蜕皮过程中使用几丁质分解酶来消化外骨骼中的几丁质。我们从烟草天蛾Manduca sexta中分离并测序了一个几丁质酶编码cDNA，将其序列与其他来源的几丁质酶编码基因进行了比较，并研究了几丁质酶基因在幼虫-蛹转化过程中的表达和激素调控。该克隆中的插入DNA为2452个核苷酸长，具有1662个核苷酸的开放阅读框，其编码分子量为62 kDa的554个氨基酸的蛋白质。该蛋白质中氨基酸序列的几个区域与酵母、黄瓜和细菌内-β-N-乙酰氨基葡糖苷酶中的序列相似。杂交选择的mRNA和在体外翻译产生了一个免疫反应性蛋白的表观分子量为75 kDa，这是类似于存在于pharate蛹蜕皮液中的几丁质酶的大小。Southern杂交分析表明，与该cDNA克隆相关的一个或两个基因编码Manduca基因组中的几丁质酶。几丁质酶基因主要在表皮和肠中表达，在大肠杆菌中表达量最高。5-7日龄幼虫。注射20-hydroxyecdysone到结扎的五龄幼虫引起的表皮和肠道的mRNA水平增加约10倍，局部应用保幼激素模拟物，苯氧威，抑制蜕皮激素诱导的几丁质酶RNA的积累。
Insects use chitinolytic enzymes to digest chitin in the exoskeleton during the molting process. We have isolated and sequenced a chitinase-encoding cDNA from the tobacco hornworm, Manduca sexta, compared its sequence with genes encoding chitinolytic enzymes from other sources, and studied chitinase gene expression and hormonal regulation during the larval-pupal transformation. The insert DNA in this clone is 2452 nucleotides long with an open reading frame of 1662 nucleotides that encodes a protein of 554 amino acids with a molecular weight of 62 kDa. Several regions of the amino acid sequence in this protein are similar to sequences in yeast, cucumber and bacterial endo-beta-N-acetylglucosaminidases. Hybrid-selection of mRNA and in vitro translation yielded an immunoreactive protein with an apparent molecular mass of 75 kDa, which is similar to the size of a chitinase present in pharate pupal molting fluid. Southern blot analysis indicated that one or two genes related to the cDNA clone are encoding chitinases in the Manduca genome. The major tissues expressing chitinase genes were the epidermis and gut with mRNA levels highest on c. days 5-7 during the fifth larval instar. Injection of 20-hydroxyecdysone into ligated fifth instar abdomens caused about a 10-fold increase in mRNA levels in both epidermis and gut, and topical application of the juvenile hormone mimic, fenoxycarb, suppressed the ecdysteroid-induced accumulation of chitinase RNA.