Kragh K M, Hendriks T, de Jong A J, Lo Schiavo F, Bucherna N, Højrup P, Mikkelsen J D, de Vries S C
Danisco Biotechnology, Copenhagen, Denmark.
Plant Mol Biol. 1996 Jun;31(3):631-45. doi: 10.1007/BF00042235.
To characterize the acidic endochitinase EP3, able to rescue somatic embryos of the carrot cell line ts11, the enzyme was purified from the medium of wild-type suspension cultures. Peptide sequences, deduced amino acid sequences of corresponding PCR-generated cDNA clones, serological relation and biochemical properties showed that there were at least five closely related chitinases, four of which could be identified as class IV EP3 chitinases with an apparent size of 30 kDa. Two other proteins were identified as a serologically related class I acidic chitinase (DcChitI) of 34 kDa, and a serologically unrelated 29 kDa class II acidic chitinase (DcChitII), respectively. Additional cDNA sequences, Western and Southern analysis showed the presence of a least two, but possibly more, highly homologous class IV EP3 genes in the carrot genome. Two class IV EP3 chitinases were tested and found to be able to increase the number of ts11 globular embryos formed under non-permissive conditions. One of the class IV EP3 chitinases as well as the class I chitinase DcChitI promoted the transition from globular to heart-stage ts11 embryos. The class II endochitinase and a heterologous class IV chitinase from sugar-beet were not active on ts11. This suggests that there are differences in the specificity of chitinases in terms of their effect on plant somatic embryos.
为了鉴定能够拯救胡萝卜细胞系ts11体细胞胚的酸性内切几丁质酶EP3，从野生型悬浮培养物的培养基中纯化了该酶。肽序列、相应PCR产生的cDNA克隆的推导氨基酸序列、血清学关系和生化特性表明，至少有五种密切相关的几丁质酶，其中四种可鉴定为表观大小为30 kDa的IV类EP3几丁质酶。另外两种蛋白质分别被鉴定为血清学相关的34 kDa I类酸性几丁质酶（DcChitI）和血清学不相关的29 kDa II类酸性几丁质酶（DcChitII）。额外的cDNA序列、蛋白质免疫印迹和Southern分析表明，胡萝卜基因组中至少存在两个，但可能更多的高度同源的IV类EP3基因。对两种IV类EP3几丁质酶进行了测试，发现它们能够增加在非允许条件下形成的ts11球形胚的数量。其中一种IV类EP3几丁质酶以及I类几丁质酶DcChitI促进了ts11胚从球形向心形阶段的转变。II类内切几丁质酶和来自甜菜的异源IV类几丁质酶对ts11没有活性。这表明几丁质酶在对植物体细胞胚的作用方面存在特异性差异。
