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薄壳山核桃嫁接愈合过程中CiMYB46基因的克隆与表达分析

Cloning and expression analysis of CiMYB46 in the graft healing process of Carya illinoinensis
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摘要 【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.
作者 莫正海 李风达 苏文川 曹凡 彭方仁 李永荣 MO Zhenghai;LI Fengda;SU Wenchuan;CAO Fan;PENG Fangren;LI Yongrong(Co-Innovation Center for the Sustainable Forestry in Southern China,College of Forestry,Nanjing Forestry University,Nanjing 210037,China;Institute of Botany,Jiangsu Province and Chinese Academy of Sciences,Nanjing 210014,China;Green Universe Pecan Science and Technology Co.,Ltd.,Nanjing 210007,China)
出处 《南京林业大学学报:自然科学版》 CAS CSCD 北大核心 2019年第5期156-162,共7页 Journal of Nanjing Forestry University:Natural Sciences Edition
基金 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)。
关键词 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
作者简介 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)。
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