水稻MYB cDNA的克隆和表达分析

Cloning and Expression Analysis of MYB cDNA in Rice

根据植物MYB类转录因子DNA结合功能域的保守区设计一对简并引物 ,以水稻根、小苗和未成熟种子中的RNA为材料 ,用RT PCR方法扩增出约 180bp的片段。序列分析表明 ,它们与MYB基因的保守区有很好的同源性。以未成熟种子中获得的这一 180bp片段作探针 ,从水稻未成熟种子cDNA文库中分离到 5个新的MYB基因家族成员 ,它们是OsMYB12、13、14、15和5 1。在酵母系统中证实OsMYB13、OsMYB15和Os MYB5 1蛋白具有转录激活功能。Northern印迹分析表明 ,OsMYB5 1主要在未成熟种子中表达 ,在根和小苗中表达水平较低。RT PCR分析表明 ,OsMYB15在根、茎、小穗。

A pair of degenerate primers was designed according to the conserved regions which encode the MYB DNA binding domains in plant MYB genes. Using the method of RT-PCR, some 180 bp fragments were amplified from various rice tissues, including root, leaf and immature seed (Fig.1). The 180 bp fragments were then cloned into pUC18 vector. One clone from the rice immature seed, named pMYB23, was sequenced and confirmed to contain the conserved region of the plant MYB genes (Fig.2). Sixteen positive clones were obtained by screening a rice immature seed cDNA library with 32P labeled fragment of pMYB23. The results of sequencing showed that there were five new MYB cDNA in sixteen clones. Among them, OsMYB13, OsMYB15, OsMYB51 genes contain complete ORF (open reading frame) respectively. The N-termini of the deduced protein sequences are very similar with the known plant R2R3-MYB proteins while the C-termini are characterized with the transcriptional activation domains (Fig.3). The transcriptional activation abilities of OsMYB15 and OsMYB51 proteins were confirmed by the yeast one-hybrid system (Table 1). Northern blotting showed that the OsMYB51 gene was clearly expressed in the immature seed and weakly expressed in rice root and leaf (Fig.5), which suggested that OsMYB51 gene may be involved in the development of rice seed. RT-PCR showed that OsMYB15 gene was expressed at a very low level in root, stem, leaf, panicle and immature seed (Fig.5). The relationships among OsMYB12, OsMYB13, OsMYB15, OsMYB51 and other plant MYB proteins were discussed (Fig.6).