龙眼体胚CDC48基因的克隆、原核表达及其在体胚发生过程中的表达分析

Cloning and Prokaryotic Expression of CDC48,and Its Expression during Somatic Embryogenesis in Dimocarpus longan Lour.

为探讨龙眼(Dimocarpus longan)体胚CDC48基因的表达方式,采用RT-PCR和RACE方法,从龙眼胚性愈伤组织中克隆得到1条长度为2620 bp、含有完整开放阅读框的DlCDC48基因cDNA序列(GenBank登录号:EU606206)和长度为2418 bp的DNA序列(GenBank登录号:FJ590953)。DlCDC48编码1个含有805个氨基酸的蛋白质。DlCDC48基因不含内含子。生物信息学分析表明:DlCDC48蛋白为不具跨膜结构域的亲水性胞质蛋白,不具有信号肽,定位在细胞核;与其他植物的CDC48有较高的同源性。将DlCDC48基因构建成原核表达载体,经IPTG诱导表达了1个分子量约为89 kD的蛋白。利用实时荧光定量PCR(qPCR)技术,DlCDC48在龙眼体胚发育过程中的各个阶段均有表达,其中球形胚时的表达量最低,胚性紧实球形结构阶段的表达量最高。这为进一步研究CDC48基因在植物体胚发生中的作用奠定基础。

In order to understand the expression of CDC48 from embryo of Dimocarpus longan, a 2620 bp cDNA sequence （GenBank accession No.： EU606206） with complete ORF and a 2418 bp DNA sequence （GenBank accession No.：FJ590953） was cloned from calli of Dimocarpus longan, named DlCDC48 gene. The DlCDC48 gene is intronless, encoding 805 amino acids. Bioinformatic analysis indicated that DlCDC48 was a hydrophilic cytoplasmic protein without transmembrane domain and signal peptide, located in nucleus. The amino acid sequence had high similarity between DlCDC48 and CDC48 from other plants. Prokaryotic expression vector fused with DlCDC48 was constructed, and a protein about 89 kD was expressed after induced by IPTG. DlCDC48 expressed at all somaitc embyogenesis stages of longan by using qPCR method. The expression of DlCDC48 was the lowest at globular embryo stage, while the highest was at compact globular embryo stage. These lay a foundation for future study the CDC48 gene function during somatic embryogenesis in plants.