【目的】为探索薄壳山核桃嫁接愈合的分子机理,对CiMYB46基因进行克隆,并分析其表达模式及启动子区的诱导元件。【方法】提取薄壳山核桃芽接愈合部位不同发育时期的RNA,根据转录组学分析结果设置引物,通过RT-PCR进行基因克隆。以基因组DNA为模板,使用PCR方法克隆目标基因的启动子区域。【结果】克隆得到1条序列,该序列的开放阅读框(ORF)从起始密码子ATG开始,到终止密码子TAA结束共969 bp,编码322个氨基酸,所推导的氨基酸含有MYB结构域,与拟南芥中的AtMYB46聚为一类,将其命名为CiMYB46。实时定量PCR分析显示,CiMYB46在嫁接体发育的维管组织形成期具有高表达,并与部分次生壁合成相关功能基因具有共表达趋势。克隆得到CiMYB46起始密码子上游1 070 bp的启动子区域,经PlantCARE分析,结果显示CiMYB46启动子区域具有CAAT-box及TATA-box的基本顺式作用元件和多个胁迫诱导元件,同时还具有响应包括脱落酸、茉莉酸甲酯、赤霉素、水杨酸在内的激素调控元件。【结论】CiMYB46可能与薄壳山核桃嫁接愈合过程中维管组织的形成有关,并受赤霉素诱导。

【Objective】 To explore the molecular mechanism of graft healing in pecan, we cloned the CiMYB46 gene and analyzed its expression pattern and cis-elements. 【Method】 We extracted RNA from graft unions of pecan plants at different developmental stages and designed primers, based on our previous transcriptome results, to clone the desired gene by RT-PCR. In addition, genomic DNA was used as a template for PCR amplification and cloning of the promoter area of the desired gene.【Result】 The amplified fragment obtained by RT-PCR was cloned and sequenced. It contained an open reading frame of 969 bp that started with the initiation codon, ATG and ended with the termination codon, TAA, encoding a polypeptide of 322 amino acids. The deduced amino acid sequence of the cloned gene contained a MYB domain and was closely clustered withArabidopsis AtMYB46. Thus, we designated our sequence as CiMYB46. Real-time PCR analyses demonstrated that CiMYB46 was highly expressed during the vascular formation stage of graft union development in pecan and had a similar expression profile to that of some functional genes involved in secondary cell wall synthesis. A 1 070 bp promoter region upstream of the initiation codon of CiMYB46 was obtained through PCR and cloning, using genomic DNA as a template. PlantCARE analysis revealed that the promoter region of CiMYB46 contained two basic cis-acting elements, a CAAT box and a TATA-box, drought responsive elements, and hormone responsive elements, including ABA, Me-JA, GA, and SA. 【Conclusion】 CiMYB46 might be associated with vascular bundle formation during the graft healing process of pecan. It may be induced by GA.