Characterization of cold-responsive extracellular chitinase in bromegrass cell cultures and its relationship to antifreeze activity
Characterization of cold-responsive extracellular chitinase in bromegrass cell cultures and its relationship to antifreeze activity
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DOI：
10.1104/pp.106.081497
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发表时间：
2008-05-01
期刊：
影响因子：
7.4
通讯作者：
Oda, Aska
中科院分区：
文献类型：
--
作者：
Nakamura, Toshihide;Ishikawa, Masaya;Oda, Aska
从无芒雀麦‘Manchar’悬浮细胞中分离到一个冷响应几丁质酶基因BiCHT1。在未受胁迫的无芒雀麦细胞中检测到低水平的BiCHT1信使RNA，而Northern-Blot和Western-Blot分析表明，在10℃和4℃的培养条件下，BiCHT1信使RNA的积累被诱导。BiCHT1与黑麦CHT9高度同源，已知编码一种抗冻蛋白。利用遗传转化方法在大肠杆菌和无芒雀麦细胞中高效表达了BiCHT1。在两种体系中表达的BiCHT1产物都具有几丁质酶活性，但表达的蛋白在所测试的任何条件下都不影响冰晶的生长。除了冷胁迫，BiCHT1基因的表达在35摄氏度的环境中上调，但不受盐或渗透胁迫、脱落酸或乙烯利的影响。BiCHT1信使RNA在茉莉酸甲酯和水杨酸的作用下不积累，但在25℃下长时间培养后略有增加，仅被甲壳素短暂增加。在培养基中检测到的抗冻活性在4℃时被诱导，但在10℃时仅有轻微的诱导，它也被乙烯利处理，但不被脱落酸、几丁质或在25℃下的长时间孵育所诱导。转基因和表达分析结果表明，BiCHT1产物是在经冷处理的细胞的培养基中分泌的具有几丁质酶活性的主要蛋白质，不太可能与在培养基中检测到的抗冻活性有关。
A cold-responsive chitinase gene, BiCHT1, was isolated from bromegrass (Bromus inermis) 'Manchar' suspension cells. BiCHT1 messenger RNA was detected at low levels in nonstressed bromegrass cells, whereas its accumulation was induced by incubation at 10 degrees C and 4 degrees C as detected by northern- and western-blot analyses. BiCHT1 was highly homologous to rye CHT9, known to encode an antifreeze protein. BiCHT1 was overexpressed in Escherichia coli and bromegrass cells using genetic transformation procedures. BiCHT1 products expressed in both systems had chitinase activity, but the expressed proteins did not affect the growth of ice crystals in any conditions tested. Besides cold stress, the expression of the BiCHT1 gene was upregulated by exposure to 35 degrees C, but not by salt or osmotic stress, abscisic acid, or ethephon. BiCHT1 messenger RNA did not accumulate in response to methyl jasmonate and salicylic acid, but was slightly increased by prolonged culture at 25 degrees C and only transiently by chitin. Antifreeze activity detected in the culture medium was induced at 4 degrees C but only slightly at 10 degrees C. It was also induced by ethephon treatment, but not by abscisic acid, chitin, or prolonged incubation at 25 degrees C. The results of transgenics and expression analyses suggest that the BiCHT1 product is a major protein with chitinase activity secreted in the medium of cold-treated cells and is unlikely to be responsible for the antifreeze activity detected in the culture medium.