[Objective]Gene(BoRACK1)sequence of tyrosine kinase receptorC1(RACK1)from Brassica oleracea was cloned;subcellular localization and expression analysis were also conducted,in order to provide theoretical basis for research on the regulation mechanism of gene RACK1 in the process of plant growth and development.[Method]Gene BoRACK1 was amplified by PCR and expression vector of subcellular localization was constructed.Onion epidermal cells were transformed by the particle bombardment method.The distribution of green fluorescent protein(GEP)was observed under a laser scanning confocal microscope.The fluorescent quantitative PCR(qRT-PCR)method was used to detect the expression of gene BoRACK1 in different tissues and seedlings under abiotic stress(200 mmol/L NaCl,100 μmol/L ABA and 1 mmol/L H2O2solution).[Result]The open reading frame(ORF)sequence of gene BoRACK1 was acquired by PCR amplification.cDNA was 980 bp in length,encoding 326 amino acids.Amino acid sequence hadhigh homology with the RACK1 of other plants,which was over 65%.In particular,the homology with Arabidopsis thaliana reached 93%.Gene BoRACK1 expressed in roots,stems,leaves,flowers and seeds of B.oleracea.The expressions in roots,leaves and seeds were high while those in flowers,seedlings and stems were low.The overall expression of gene BoRACK1 was down-re-gulated under abiotic stress of ABA,H2O2and NaCl. The expression level reached the lowest point at 6 h under NaCl stress,and the expression level reached the lowest point at 4 h under other stresses.BoRACK1 was localized in the cyto-plasm,cell nuclear and plasma membrane.[Conclusion]The expression of gene BoRACK1 has no tissue specificity.The gene has different degrees of correlations with development of flowers,fruits and vegetative organof B.oleracea;and the gene may also be involved in the regulation process of plant stress resistance.%[目的]克隆羽衣甘蓝蛋白激酶C1受体(RACK1)基因(BoRACK1)序列,并对其进行亚细胞定位及表达分析,为研究RACK1基因在植物生长发育过程中的调控机制提供理论参考.[方法]PCR扩增BoRACK1基因,构建其亚细胞定位表达载体,通过基因枪法转化洋葱表皮细胞后在激光共聚焦显微镜下观察绿色荧光蛋白(GEP)的分布情况.利用荧光定量PCR(qRT-PCR)检测BoRACK1基因在不同组织中及在非生物胁迫(200 mmol/L NaCl、100 μmol/L ABA和1 mmol/L H2O2溶液)下幼苗中的表达情况.[结果]PCR扩增获得BoRACK1基因的开放阅读框(ORF)序列,其cDNA全长980 bp,编码326个氨基酸,氨基酸序列与其他植物的RACK1具有较高同源性,在65%以上,尤其与拟南芥的同源性高达93%.BoRACK1基因在羽衣甘蓝的根、茎、叶、花和种子中均有表达,其中在根、叶和种子中的表达量较高,而在花、幼芽和茎的表达量较少.BoRACK1基因在ABA、H2O2和NaCl胁迫下的表达量整体上呈下调趋势,其中NaCl胁迫下其表达量在6 h时降至最低,而其他胁迫下其表达量均在4 h时降至最低.BoRACK1定位于细胞质、细胞核和细胞质膜.[结论]BoRACK1基因表达无组织特异性,除了与羽衣甘蓝花、果实及营养器官的发育存在关联外,还可能参与了植物的抗逆性调控过程.
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