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75mmol/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。
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.
出处
《西北植物学报》
CAS
CSCD
北大核心
2012年第9期1719-1725,共7页
Acta Botanica Boreali-Occidentalia Sinica
基金
国家自然科学基金(30860030)
教育部新世纪优秀人才支持计划(NCET-08-0871)
内蒙古农业大学科技创新团队培育计划(NDPYTD2010-3)
