中国野生葡萄抗霜霉病相关基因VpPR4b及其启动子的克隆和功能分析

doi:10.16420/j.issn.0513-353x.2020-0343

摘要/Abstract

摘要：

以中国野生葡萄（Vitis piasezkii）‘留坝-8’（抗霜霉病）和欧亚种葡萄（V. vinifera）‘黑比诺’（易感霜霉病）为材料,克隆得到抗霜霉病相关基因VpPR4b和VvPR4b。序列分析发现VpPR4b和VvPR4b核苷酸序列相似性为98.61%。VpPR4b编码区为432 bp,编码143个氨基酸,含有BARWIN保守结构域,属于典型的Ⅱ型PR4蛋白。VpPR4b和VvPR4b均可在霜霉菌侵染后诱导表达。亚细胞定位表明VpPR4b定位于细胞膜上。分别从‘留坝-8’和‘黑比诺’中克隆得到了VpPR4b和VvPR4b的启动子,其序列相似性高达95.65%,并且含有相似的顺式作用元件。通过酵母单杂交验证了转录因子WRKY40和WRKY75可以结合VpPR4b启动子。本研究的结果表明PR4b可能在葡萄抵御霜霉病侵染过程中发挥一定作用。

关键词: 葡萄, 霜霉病, PR4b, 启动子

Abstract:

VpPR4b and VvPR4b were cloned form Vitis piasezkii‘Liuba-8’（resistant to downy mildew）and V. vinifera‘Pinot Noir’（susceptible to downy mildew）. Sequence analysis showed that the identity between VpPR4b and VvPR4b nucleotide was 98.61%. The coding region of VpPR4b is 432 bp,which encodes 143 amino acids and contains the BARWIN conserved domain,indicating it is a typical typeⅡPR4 protein. Both VpPR4b and VvPR4b are highly expressed after inoculation with Plasmopara viticola. Subcellular localization analysis indicated that VpPR4b was localized in the membrane. The promoters of VpPR4b and VvPR4b were cloned from‘Liuba-8’and‘Pinot Noir’,respectively. It showed that VpPR4b and VvPR4b promoter sequence similarity is as high as 95.65%,and contains similar cis-acting elements. It has been verified that the transcription factors WRKY40 and WRKY75 can bind the VpPR4b promoter in yeast. This study showed that PR4b may play a role in the resistance to downy mildew in grape.

Key words: grape, downy mildew, PR4b, promoter

中图分类号:

S663.1

刘兵, 李梦媛, 张娜, 尚博兴, 刘国甜, 徐炎. 中国野生葡萄抗霜霉病相关基因VpPR4b及其启动子的克隆和功能分析[J]. 园艺学报, 2021, 48(2): 265-275.

LIU Bing, LI Mengyuan, ZHANG Na, SHANG Boxing, LIU Guotian, XU Yan. Cloning and Functional Analysis of the CDS and Promoter of VpPR4b Gene Response to Downy Mildew in Chinese Wild Grape[J]. Acta Horticulturae Sinica, 2021, 48(2): 265-275.

图/表 7

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用途Usage	引物名称Name of primer	引物序列（5′-3′）Primer Sequence
PR4b基因扩增 Amplification of PR4b	PR4b-CDS-F	ACGGGGGACGAGCTCGGTACCATGGAGAGGAGAGGCATATGCAAGG
PR4b基因扩增 Amplification of PR4b	PR4b-CDS-R	CACCATGGTGTCGACTCTAGAGTCACCACAGTTCACAAACTGGTAATTGA
启动子扩增 Amplification of promoters	PR4b-pro-F	GTACTTTCTTATAGCTAAAAGTTATAGAGAAC
启动子扩增 Amplification of promoters	PR4b-pro-R	TTTTGTTGAATGATTTTTGCTTCATCACCTC
实时荧光定量PCR qRT-PCR	VvActin-qPCR-F	CCATCCTTCGTCTTGACCTTGCTG
	VvActin-qPCR-R	AGTGGTGAACATGTAACCCCTCTC
	VvPR4b-qPCR-F	GCCAGCAATGTGAGGGCCAC
	VvPR4b-qPCR-R	GCTGGCATCCCAAGTGGAGC
酵母单杂交 Yeast one hybrid	VpPR4b-pAbAi-F	GAGCTCGGTACCCGGGCTATGAAGCTTGAAGCTTG
酵母单杂交 Yeast one hybrid	VpPR4b-pAbAi-R	CAGAGCACATGCCTCGAGGAAGATTATGATCCAACG

用途Usage	引物名称Name of primer	引物序列（5′-3′）Primer Sequence
PR4b基因扩增 Amplification of PR4b	PR4b-CDS-F	ACGGGGGACGAGCTCGGTACCATGGAGAGGAGAGGCATATGCAAGG
PR4b基因扩增 Amplification of PR4b	PR4b-CDS-R	CACCATGGTGTCGACTCTAGAGTCACCACAGTTCACAAACTGGTAATTGA
启动子扩增 Amplification of promoters	PR4b-pro-F	GTACTTTCTTATAGCTAAAAGTTATAGAGAAC
启动子扩增 Amplification of promoters	PR4b-pro-R	TTTTGTTGAATGATTTTTGCTTCATCACCTC
实时荧光定量PCR qRT-PCR	VvActin-qPCR-F	CCATCCTTCGTCTTGACCTTGCTG
	VvActin-qPCR-R	AGTGGTGAACATGTAACCCCTCTC
	VvPR4b-qPCR-F	GCCAGCAATGTGAGGGCCAC
	VvPR4b-qPCR-R	GCTGGCATCCCAAGTGGAGC
酵母单杂交 Yeast one hybrid	VpPR4b-pAbAi-F	GAGCTCGGTACCCGGGCTATGAAGCTTGAAGCTTG
酵母单杂交 Yeast one hybrid	VpPR4b-pAbAi-R	CAGAGCACATGCCTCGAGGAAGATTATGATCCAACG

名称 Name	序列 Sequence	功能 Function		VpPR4启动子位置 Location of VpPR4 promoter			VvPR4启动子位置 Location of VvPR4 promoter
名称 Name	序列 Sequence	功能 Function		正链 Positive strand		负链 Negative strand	正链 Positive strand	负链 Negative strand
CAAT-box	CAAT	光响应元件 Light response element		391、399、761、 1 287		426、546、611、678、746、 1 059	380、388、750、1 281	16、170、322、415、535、600、667、735、1 052
CAAT-box	CAAAT	启动子和增强子区域共有顺式作用元件 Common cis-element in promoter and enhancer		109、118、369、433、534		131、340、393、487、666	109、118、358、422、523、888	329、382、476
MYC	CATGTG	干旱和ABA响应元件 Drought and ABA response element		665、1 227		521、534、861	1 221	850、888
ARE	AAACCA	厌氧诱导必需元件 Anaerobic induction essential element		470、857、1 272		731	459、846	720
Box 4	ATTAAT	参与光反应的部分保守DNA模块 Part of the conserved DNA module involved in light reactions		92、176、580			92、569
ABRE	ACGTG	脱落酸响应元件 ABA response element		1 184、1 185			508、510、511、1 178、1 179
LTR	CCGAAA	低温响应元件 Cold response element				407、1 353	396、1 347	396、1 347
TATA	TATAAAAT	转录起始位点上游核心元件Core promoter element of transcription start site		201		98	98	98
W-box	TTGACC	与WRKY转录因子特异结合 Specific binding to WRKY transcription factors		827		1 299	816	1 293
DRE core	GCCGAC	与DREB转录因子特异结合Specific binding to DREB transcription factors		627			616
名称 Name	序列 Sequence		功能 Function		VpPR4启动子位置 Location of VpPR4 promoter		VvPR4启动子位置 Location of VvPR4 promoter
名称 Name	序列 Sequence		功能 Function		正链 Positive strand	负链 Negative strand	正链 Positive strand	负链 Negative strand
MBS	CAACTG		参与干旱诱导的MYB结合位点 Drought-induced MYB binding site			815		804
MRE	AACCTAA		参与光响应的MYB结合位点 Light response MYB binding site		540		529
P-box	CCTTTTG		赤霉素响应元件 GA response element			793
STRE	AGGGG		胁迫响应元件 Stress response element		1 367		1 361
chs-CMA1a	TTACTTAA		光敏元件的一部分 Part of light response element		38
G-box	TACGTG/CACGTG		光响应顺式作用元件 Light response cis-element		1 184		1 178、510
GA-motif	ATAGATAA		光敏元件的一部分 Part of light-sensitive element		64		64

名称 Name	序列 Sequence	功能 Function		VpPR4启动子位置 Location of VpPR4 promoter			VvPR4启动子位置 Location of VvPR4 promoter
名称 Name	序列 Sequence	功能 Function		正链 Positive strand		负链 Negative strand	正链 Positive strand	负链 Negative strand
CAAT-box	CAAT	光响应元件 Light response element		391、399、761、 1 287		426、546、611、678、746、 1 059	380、388、750、1 281	16、170、322、415、535、600、667、735、1 052
CAAT-box	CAAAT	启动子和增强子区域共有顺式作用元件 Common cis-element in promoter and enhancer		109、118、369、433、534		131、340、393、487、666	109、118、358、422、523、888	329、382、476
MYC	CATGTG	干旱和ABA响应元件 Drought and ABA response element		665、1 227		521、534、861	1 221	850、888
ARE	AAACCA	厌氧诱导必需元件 Anaerobic induction essential element		470、857、1 272		731	459、846	720
Box 4	ATTAAT	参与光反应的部分保守DNA模块 Part of the conserved DNA module involved in light reactions		92、176、580			92、569
ABRE	ACGTG	脱落酸响应元件 ABA response element		1 184、1 185			508、510、511、1 178、1 179
LTR	CCGAAA	低温响应元件 Cold response element				407、1 353	396、1 347	396、1 347
TATA	TATAAAAT	转录起始位点上游核心元件Core promoter element of transcription start site		201		98	98	98
W-box	TTGACC	与WRKY转录因子特异结合 Specific binding to WRKY transcription factors		827		1 299	816	1 293
DRE core	GCCGAC	与DREB转录因子特异结合Specific binding to DREB transcription factors		627			616
名称 Name	序列 Sequence		功能 Function		VpPR4启动子位置 Location of VpPR4 promoter		VvPR4启动子位置 Location of VvPR4 promoter
名称 Name	序列 Sequence		功能 Function		正链 Positive strand	负链 Negative strand	正链 Positive strand	负链 Negative strand
MBS	CAACTG		参与干旱诱导的MYB结合位点 Drought-induced MYB binding site			815		804
MRE	AACCTAA		参与光响应的MYB结合位点 Light response MYB binding site		540		529
P-box	CCTTTTG		赤霉素响应元件 GA response element			793
STRE	AGGGG		胁迫响应元件 Stress response element		1 367		1 361
chs-CMA1a	TTACTTAA		光敏元件的一部分 Part of light response element		38
G-box	TACGTG/CACGTG		光响应顺式作用元件 Light response cis-element		1 184		1 178、510
GA-motif	ATAGATAA		光敏元件的一部分 Part of light-sensitive element		64		64