薄壳山核桃嫁接愈合过程中CiMYB46基因的克隆与表达分析

doi:10.3969/j.issn.1000-2006.201811041

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南京林业大学学报（自然科学版） ›› 2019, Vol. 43 ›› Issue (5): 156-162.doi: 10.3969/j.issn.1000-2006.201811041

所属专题：薄壳山核桃专题

薄壳山核桃嫁接愈合过程中CiMYB46基因的克隆与表达分析

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

收稿日期:2018-11-26 修回日期:2019-05-21 出版日期:2019-10-08 发布日期:2019-10-08
通讯作者: 彭方仁
基金资助:
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)

Cloning and expression analysis of CiMYB46 in the graft healing process of Carya illinoinensis

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

Received:2018-11-26 Revised:2019-05-21 Online:2019-10-08 Published:2019-10-08
Contact: PENG Fangren

摘要/Abstract

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。

关键词: 薄壳山核桃, 嫁接, 基因克隆, CiMYB46基因, MYB转录因子

Abstract:

【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.

Key words: Carya illinoinensis, grafting, gene cloning, CiMYB46, MYB transcription factor

中图分类号:

Q943.2
S722

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.

MO Zhenghai, LI Fengda, SU Wenchuan, CAO Fan, PENG Fangren, LI Yongrong. 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(5): 156-162.DOI: 10.3969/j.issn.1000-2006.201811041.

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基因名称 gene name	正向引物(5'-3') forward primer(5'-3')	反向引物(5'-3') reversed primer(5'-3')
CiMYB46	CTTCAGCATGGCCTGAACA	TAACCCAGTATCGAGACCC
CAD	GAGGATGAGGCAATCAACAG	GGCTTATCAGGCAAACCGA
CCoAOMT	CGGTGGTTGATGTTGGTG	AATGGCGTCTCCTTTTGG
CesA1	TTCCACCAACGACCAACC	GCGAGCGGGCATACAAT
CesA2	CTTTCTTCCTCCGCTTCCG	TTGGTTCTCCCTCACGCT
IRX 10	GTGTACTTCCGGGGATTGTT	GTTGTTGGGTGCTCTGTGG

元件类别 element type	元件名称 name of element		正(+)/反(-) 链 sense(+) / anti-sense (-) strand		序列 sequence		拷贝数 copies of element		预测的功能 putative function
植物激素 hormone	ABRE		+ /		ACGTGGC/CACGTG/TACGTG		3		响应脱落酸
	CGTCA-motif		+ /		CGTCA		2		茉莉酸甲酯响应元件
	GARE-motif		+ /		TCTGTTG/AAACAGA		2		赤霉素响应元件
	TCA-element		+		TCAGAAGAGG/CAGAAAAGGA		2		水杨酸响应元件
胁迫 coercion	HSE		-		AGAAAATTCG		1		热胁迫响应元件
	LTR		-		CCGAAA		1		低温响应元件
	TC-rich repeats		+		ATTTTCTCCA		1		防御及逆境响应元件
光响应 light response	Box 4		+ /-		ATTAAT		4		光响应元件
	G-box		+		CACGTT/CACGTG/CACGTA		3		光响应元件
	TCCC-motif		+		TCTCCCT		2		光响应元件
元件类别 element type		元件名称 name of element	正(+)/反(-) 链 sense(+) / anti-sence (-) strand	序列 sequence		拷贝数 copies of element		预测的功能 putative function
基础元件 basic component		CAAT-box	+ /-	CAAAT/CAATT/CCAAT/CAAT/ CCAAT/GGCAAT/CAAT		10		启动子、增强子区域常见的顺式作用元件
		TATA-box	+ /-	TATA/ATATAT/TATATATA/ TTTTA/TAATA		31		转录起始位点-30核心启动元件
		5UTR Py-rich stretch	-	TTTCTCTCTCTCTC		29		具有高转录水平的顺式元件
其他 other		Box III	-	CATTTACACT		1		蛋白结合位点
其他 other		ARE	-	TGGTTT		1		厌氧诱导所必需的作用元件

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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