甜樱桃PavMYB10.1促进PavRiant表达和花青苷积累

doi:10.16420/j.issn.0513-353x.2021-0732

摘要/Abstract

摘要：

为了解谷胱甘肽S-转移酶（GST）参与甜樱桃花青苷积累的机制,以甜樱桃‘美早’为试材,克隆1个phi类型GST基因PavRiant（XM_021968160）。通过系统进化树分析、qRT-PCR、瞬时表达沉默、酵母单杂交、电泳迁移率试验和荧光素酶报告试验,探究PavRiant调控甜樱桃花青苷积累的作用。系统进化树分析表明,PavRiant与桃PpRiant蛋白序列相似度最高。果实发育期,PavRiant表达量与花青苷含量和花青苷调控基因PavMYB10.1表达量变化趋势一致。瞬时沉默试验证实PavRiant在甜樱桃花青苷积累调控中发挥重要作用。酵母单杂交和EMSA试验发现,PavMYB10.1结合PavRiant启动子的MRE序列。荧光素酶试验表明PavRiant启动子活性受PavMYB10.1正调控。因此,PavMYB10.1可能通过促进PavRiant的表达促进花青苷的积累。

关键词: 甜樱桃, 花青苷, PavMYB10.1, PavRiant, 谷胱甘肽S-转移酶

Abstract:

To understand how GST regulates anthocyanin accumulation in sweet cherry,a phi-type GST gene PavRiant（XM_021968160）was cloned from the sweet cherry cultivar‘Tieton’. The effects of PavRiant on anthocyanin accumulation in sweet cherry were investigated using phylogenetic tree analysis,real-time reverse transcription-polymerase chain reaction,transient silencing assay,yeast one-hybrid（Y1H）,electrophoretic mobility shift assay（EMSA）,and luciferase reporter assay. Phylogenetic tree analysis showed that PavRiant has the highest similarity to the PpRiant protein sequence of peach. During sweet cherry fruit development,the changing trends of PavRiant expressions were consistent with anthocyanin contents and PavMYB10.1 expressions. Further,the transient silencing assay showed that PavRiant plays an essential role in regulating anthocyanin accumulation. Y1H and EMSA experiments showed that PavMYB10.1 binds to the MRE sequence of the PavRiant promoter. Finally,the luciferase assay showed that PavMYB10.1 positively regulates the activity of the PavRiant promoter. Therefore,PavMYB10.1 can promote anthocyanin accumulation by positively regulating PavRiant expression in sweet cherry.

Key words: sweet cherry, anthocyanin, PavMYB10.1, PavRiant, GST

中图分类号:

S662.5

李丽仙, 王烁, 陈莹, 邬滢涛, 王雅倩, 房月, 陈学森, 田长平, 冯守千. 甜樱桃PavMYB10.1促进PavRiant表达和花青苷积累[J]. 园艺学报, 2022, 49(5): 1023-1030.

LI Lixian, WANG Shuo, CHEN Ying, WU Yingtao, WANG Yaqian, FANG Yue, CHEN Xuesen, TIAN Changping, FENG Shouqian. PavMYB10.1 Promotes Anthocyanin Accumulation by Positively Regulating PavRiant in Sweet Cherry[J]. Acta Horticulturae Sinica, 2022, 49(5): 1023-1030.

图/表 6

表1 引物序列列表

Table 1 The list of primer sequences

用途 Purpose	引物名称 Primer name	序列（5-3′） Sequence
瞬时沉默试验 Transient silencing assay	PavRiant-（TRV2）-F	GAATTCTCCCAACCTATTGGGAACAACC
瞬时沉默试验 Transient silencing assay	PavRiant-（TRV2）-R	GGTACCGTAGTCACTAGCAAGGCTCATTAG
荧光实时定量 PCR qRT-PCR	PavRiant-F	GCTATCTGGCTGGAGAAG
	PavRiant-R	GCTATCTGGCTGGAGAAG
	PavActin-F	CCAGGGCTGTGTTTCCTTCTATT
	PavActin-R	ATGATCTGCGTCATCTTTTCT
电泳迁移率试验 EMSA	PavMYB10.1-（PGEX4T）-F	GGATCCATGGCCAACAAGAAGATGGAGG
电泳迁移率试验 EMSA	PavMYB10.1-（PGEX4T）-R	GAATTCGTCCTTCTGAACATTGGTACACTGTC
荧光素酶报告基因试验 LUC	PavMYB10.1-（62-SK）-F	TGGATCCATGGCCAACAAGAAGATGGAGG
	PavMYB10.1-（62-SK）-R	AAGCTTGTCCTTCTGAACATTGGTACACTGTC
	PavRiant-（0800-LUC）-F	GTCGACTCGAATTGGAGTTTGTTCGAAA
	PavRiant-（0800-LUC）-R	GGATCCTTCTAGACATAAAAGTAAACATTAAATTATATAAACC
酵母单杂交试验 Y1H	PavRiant-（PHIS2）-F	GAATTCTCGAATTGGAGTTTGTTCGAAA
	PavRiant-（PHIS2）-R	GAGCTCTTCTAGACATAAAAGTAAACATTAAATTATATAAACC
	PavMYB10.1-（PGADT7）-F	CATATGATGGCCAACAAGAAGATGGAGG
	PavMYB10.1-（PGADT7）-R	GGATCCGTCCTTCTGAACATTGGTACACTGTC

图1 ‘美早’甜樱桃不同发育阶段果实（A）、花青苷含量（B）及PavMYB10.1与PavRiant的相对表达量（C） ** 表示与S1差异极显著（P < 0.01.）.

Fig. 1 Phenotype（A）,anthocyanin content（B）and relative expressions of PavMYB10.1 and PavRiant（C）in‘Tieton’sweet cherry fruit ** Indicate significant difference between S1（P < 0.01）.

图2 不同物种 GST蛋白系统进化树分析

Fig. 2 Phylogenetic tree analysis of GST proteins in different species

图3 瞬时沉默PavRiant‘美早’甜樱桃果实表型（A）、花青苷含量（B）、PavRiant和花青苷相关基因的表达量（C）

Fig. 3 Transient silence of PavRiant inhibits anthocyanin accumulation in fruit phenotype（A）,anthocyanin content（B） and relative expressions of PavRiant and anthocyanin related genes（C）in‘Tieton’sweet cherry fruit ** P < 0.01.

图4 PavMYB10.1与PavRiant启动子结合 A:酵母单杂验证PavMYB10.1可以结合PavRiant启动子;B：PavRiant启动子的MBS和MRE位点;C：EMSA验证PavMYB10.1可以结合PavRiant启动子的MRE位点。“+”代表添加探针或蛋白;“-”代表不添加探针或蛋白;代表增加竞争探针和突变探针的浓度。

Fig. 4 PavMYB10.1 binds to the promoter of PavRiant A：Y1H assay showing the interaction between PavMYB10.1 and the PavRiant promoter;B：MBS and MRE sites in the PavRiant promoter;C：Electrophoretic mobility shift assay（EMSA）showing that PavMYB10.1 can bind to the MRE site of the PavRiant promoter. The“+”and “-”indicate the presence and absence of the probe and protein. represents the increasing concentration of competition and mutation probes.

图5 PavMYB10.1激活PavRiant基因的启动子活性

Fig. 5 PavMYB10.1 activates the promoter activities of PavRiant **P < 0.01.

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