MO Zhenghai,LI Fengda,et al.Cloning and expression analysis of CiMYB46 in the graft healing process of Carya illinoinensis[J].Journal of Nanjing Forestry University(Natural Science Edition),2019,43(05):156-162.[doi:10.3969/j.issn.1000-2006.201811041]





Cloning and expression analysis of CiMYB46 in the graft healing process of Carya illinoinensis
莫正海1 2李风达1苏文川1曹 凡1彭方仁1*李永荣3
(1. 南京林业大学,南方现代林业协同创新中心,南京林业大学林学院,江苏 南京 210037; 2. 江苏省中国科学院植物研究所, 江苏 南京 210014; 3. 南京绿宙薄壳山核桃科技有限公司,江苏 南京 210007)
MO Zhenghai1 2 LI Fengda1 SU Wenchuan1 CAO Fan1 PENG Fangren1* LI Yongrong3
(1.Co-Innovation Center for the Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing 210037, China; 2. Institute of Botany, Jiangsu Province and Chinese Academy of Sciences, Nanjing 210014, China; 3. Green Universe Pecan Science and Technology Co., Ltd., Nanjing 210007, China)
薄壳山核桃 嫁接 基因克隆 CiMYB46基因 MYB转录因子
Carya illinoinensis grafting gene cloning CiMYB46 MYB transcription factor
Q943.2; S722
【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 with Arabidopsis 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.


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收稿日期:2018-11-26 修回日期:2019-05-21 基金项目:国家自然科学基金项目(31870672); 江苏省林业三新工程项目(LYSX[2016]44); 江苏高校优势学科建设工程资助项目(PAPD)。 第一作者:莫正海(mozhenghai@yeah.net)。*通信作者:彭方仁(frpeng@njfu.edu.cn),教授,ORCID(0000-0002-9001-2552)。引文格式:莫正海,李风达,苏文川,等. 薄壳山核桃嫁接愈合过程中CiMYB46基因的克隆与表达分析[J]. 南京林业大学学报(自然科学版),2019,43(5):156-162.
更新日期/Last Update: 2019-10-08