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作 者 ：张秀娟，杨冠宇，石 蕊，韩晓敏，王瑞刚，李国婧*

期 刊 ：西北植物学报 2012年 9期 页码：1719~1725

关键词：拟南芥；CRK45；差异表达；钙离子；CICBP；

Keywords:Arabidopsis thaliana, CRK45, differential expression, Ca2+, CICBP,

摘 要 ：以拟南芥野生型和类受体蛋白激酶基因CRK45的T-DNA插入突变体crk45为材料，采用差异基因表达筛选技术检测ABA处理后野生型和crk45中基因表达的差异。结果显示：（1）crk45突变体中有1个基因的表达比野生型高约4倍。（2）NCBI数据库检索表明，该基因编码的蛋白具有EF手型结构，蛋白序列全长为130个氨基酸，是典型的Ca²⁺结合蛋白，故命名为CRK45抑制的钙离子结合蛋白（CICBP）。（3）Northern blotting分析结果显示，ABA处理后crk45突变体中CICBP的表达明显升高，证明CICBP基因的确受ABA诱导，且其表达受CRK45的抑制。（4） 外源75 mmol/L的Ca²⁺处理后，crk45突变体的萌发率（30.8%）显著高于野生型（17.16%），说明在Ca²⁺介导下CRK45的功能是抑制种子萌发。（5）qRT-PCR检测显示，野生型中CRK45的表达受Ca²⁺诱导明显升高，而crk45突变体中的表达一直保持很低，说明crk45突变体是一个基因敲除突变体；Ca²⁺处理后crk45突变体中CICBP基因表达上调，而野生型中CICBP的表达反而降低，说明Ca²⁺处理下CRK45抑制CICBP基因的表达。研究表明，ABA或Ca²⁺处理后，CRK45通过负调控CICBP基因的表达，从而抑制拟南芥种子萌发。

Abstract:In this study,differential display technique was used to compare the gene expression difference between Arabidopsis wild type and the crk45 mutant with the T-DNA inserted in the CRK45 gene under ABA treatment.The results showed that:(1)One gene showed 4 fold higher expression in the mutant than that in the wild type.(2)Searches in the NCBI database revealed that this gene encoding a Ca²⁺ binding protein composed by 130 amino acid and with the typical EF hand,so it was dubbed as CICBP (CRK45 inhibited Ca²⁺ binding protein).(3)Northern blotting analysis further confirmed that CICBP was indeed significantly induced by ABA treatment in the crk45 mutant,and its expression was inhibited by CRK45.(4)After treated by 75 mmol/L exogenerous Ca²⁺,the germination rate of the crk45 mutant is 30.8%,which is obviously higher than that of the wild type (17.16%),indicating that CRK45 plays a role in seed germination inhibition under Ca²⁺ treatment.(5) qRT-PCR analysis showed that the expression of CRK45 was induced highly by Ca²⁺ in wild type,but keeping very low level in the mutant,indicated that the gene was completely knocked out.In addition,the transcript of CICBP gene was also upregulated in the crk45 mutant after Ca²⁺ treatment compared with wild type.These results indicated that CRK45 inhibited germination of Arabidopsis seeds by down regulating CICBP after ABA and Ca²⁺ treatment.

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