Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea)
Differential expression of ribosome-inactivating protein genes during somatic embryogenesis in spinach (Spinacia oleracea)
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DOI：
10.1111/j.1399-3054.2008.01129.x
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发表时间：
2008-10-01
影响因子：
6.4
通讯作者：
Masuda, Kiyoshi
中科院分区：
文献类型：
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作者：
Kawade, Kensuke;Ishizaki, Takuma;Masuda, Kiyoshi
菠菜 (Spinacia oleracea L. cv. Jiromaru) 幼苗的根段形成胚胎发生愈伤组织 (EC)，该愈伤组织通过积累与胚胎发生潜能的表达相关的 31-kDa 糖蛋白 [BP31 或 S. oleracea 核糖体失活蛋白 (EC 3.2.2.22) (SoRIP1)] 对外源 GA(3) 作出反应。该蛋白质的微测序显示与 1 型 RIP 显着相似。我们鉴定了 SoRIP1 和甘蓝 RIP2 (SoRIP2) 的 cDNA，这是一种具有共有志贺/蓖麻毒素毒性结构域的新型 RIP，并对 SoRIP 在体细胞胚胎发生过程中的表达进行了比较分析。蛋白质印迹和定量聚合酶链反应分析表明，愈伤组织中 SoRIP1 的表达显着增加，与胚胎发生潜力的获得相关，尽管体细胞胚胎中的表达随着发育而适度下降。然而，SoRIP2在愈伤组织中的表达仍然较低且恒定，但随着体细胞胚胎的发育而显着增加。用 GA(3) 和/或 ABA 处理愈伤组织 24 小时，或用 ABA 处理较长时间，都不能刺激任一基因的表达。免疫组织化学显示，SoRIP1优先在发育早期体细胞胚胎的原胚和外周分生组织中积累。在愈伤组织中没有检测到 SoRIP2 的明显表达，但在体细胞胚胎的表皮中发现了强烈的表达。这些结果表明，菠菜 RIP 基因的表达在菠菜体细胞胚胎发生过程中以发育依赖性方式受到差异性调节。
Root segments from spinach (Spinacia oleracea L. cv. Jiromaru) seedlings form embryogenic callus (EC) that responded to exogenous GA(3) by accumulating a 31-kDa glycoprotein [BP31 or S. oleracea ribosome-inactivating protein (EC 3.2.2.22) (SoRIP1)] in association with the expression of embryogenic potential. Microsequencing of this protein revealed significant similarity with type 1 RIPs. We identified cDNAs for SoRIP1 and S. oleracea RIP2 (SoRIP2), a novel RIP having a consensus shiga/ricin toxic domain and performed a comparative analysis of the expression of SoRIPs during somatic embryogenesis. Western blotting and quantitative polymerase chain reaction analyses revealed that the expression of SoRIP1 in calli increased remarkably in association with the acquisition of embryogenic potential, although the expression in somatic embryos decreased moderately with their development. However, the expression of SoRIP2 in calli remained low and constant but increased markedly with the development of somatic embryos. Treatment of callus with GA(3) and/or ABA for 24 h, or with ABA for a longer period, failed to stimulate the expression of either gene. Immunohistochemistry showed that SoRIP1 preferentially accumulated in the proembryos and peripheral meristem of somatic embryos early in development. Appreciable expression of SoRIP2 was not detected in the callus, but intense expression was found in the epidermis of somatic embryos. These results suggest that the expression of spinach RIP genes is differentially regulated in a development-dependent fashion during somatic embryogenesis in spinach.