‘巨峰’葡萄细胞分裂素响应调节因子VlRR5的克隆与表达分析

doi:10.16420/j.issn.0513-353x.2020-0668

摘要/Abstract

摘要：

葡萄在生长发育过程中会出现3次落果高峰,‘巨峰’葡萄的落果现象更为严重。从‘巨峰’葡萄中克隆得到1个细胞分裂素响应调节因子VlRR5,该基因有1个磷酸受体结构域（REC）和1个MYB样DNA结合结构域,cDNA全长2 597 bp,编码693个氨基酸,定位在4号染色体上。亚细胞定位在细胞核中。酵母转化试验中,转入pGBKT7-VlRR5的酵母菌可以在SD/-Trp/-Ade/-His/+X-α-Gal缺陷型培养基上正常生长且显蓝色,证明VlRR5具有转录激活活性。荧光定量PCR分析显示,VlRR5的表达具有组织特异性,在叶和茎中表达量较高;在幼果发育阶段呈现先升高后降低的趋势;并且在外源细胞分裂素CPPU处理后表达量上升,在细胞分裂素合成抑制剂洛伐他汀处理后表达量下降。这些结果表明VlRR5可以响应外源细胞分裂素处理,在细胞分裂素介导葡萄坐果过程中发挥重要作用。

关键词: 葡萄, 细胞分裂素, CPPU, 细胞分裂素响应调节因子, VlRR5

Abstract:

There are three fruit-dropping peaks in grape growth and development. Grape fruit-dropping phenomenon is more serious in Kyoho grapevine. In this study,a cytokinin response regulator VlRR5 was cloned from Kyoho grapevine,which has a phosphoric acid receptor domain（REC）and a MYB-like DNA binding domain. Sequence analysis revealed that cDNA of VlRR5 was 2 597 bp in length,encoding 693 amino acids,and located on chromosome 4 of grape. Subcellular localization assay showed that VlRR5 was localized in the nucleus. The yeast transformed into pGBKT7-VlRR5 could grow normally and turn blue on SD/-Trp/-Ade/-His/+X-α-Gal deficient medium,which proved that VlRR5 was a transcription factor with transcriptional activation activity. By quantitative real-time PCR analysis,VlRR5 was characterized by tissue specific expression and showed high expression level in leaf and stem,which was increased and then decreased in the development stage of young fruit. The expression level increased after treatment with exogenous cytokinin,and decreased after treatment with the cytokinin synthesis inhibitor lovastatin. These results indicated that VlRR5 can respond to cytokinin and plays an important role in participating in cytokinin to improve the fruiting rate of grape.

Key words: grapevine, cytokinin, CPPU, cytokinin response regulators, VlRR5

中图分类号:

S663.1

边璐, 郭大龙, 于可可, 韦同路, 裴茂松, 刘海楠, 余义和. ‘巨峰’葡萄细胞分裂素响应调节因子VlRR5的克隆与表达分析[J]. 园艺学报, 2021, 48(8): 1437-1445.

BIAN Lu, GUO Dalong, YU Keke, WEI Tonglu, PEI Maosong, LIU Hainan, YU Yihe. Cloning and Expression Analysis of the Cytokinin Response Regulator VlRR5 in Kyoho Grapevine[J]. Acta Horticulturae Sinica, 2021, 48(8): 1437-1445.

图/表 9

表1 引物列表

Table 1 List of primers

引物名称基因 Primer	碱基序列（5'-3'） Primer sequence
QT-VlRR5	F：TGGGTATGCATTGCCTTTCT;R：GAAGAAGGAAGCTCTGCGTTT
VlRR5	F：ATGACAGTTGAACCGAGGGT;R：TTAATGGAAAAGAACTTGAT
Y1H-RR5	F：ATGGCCATGGAGGCCGAATTCATGACAGTTGAACCGAGGGT;
Y1H-RR5	R：ATGCGGCCGCTGCAGGAATTCTTAATGGAAAAGAACTTGAT
LS-RR5	F：AGAGAACACGGGGGACTCTAGAATGACAGTTGAACCGAGGGT;
LS-RR5	R：CTTCTCCCTTACCCATGGTACCTTAATGGAAAAGAACTTGAT

图1 VlRR5和其他物种RR氨基酸序列比对 A：磷酸接收结构域（REC）;B：MYB样DNA结合域。Pan：糙叶山麻黄;Md：苹果;Os：水稻;At：拟南芥。

Fig. 1 Amino acid sequence alignment of VlRR5 and other species RRs A：Phosphoric acid receptor domain（REC）;B：MYB-like DNA binding domain. Pan：Parasponia andersonii;Md：Malus × domestica;Os：Oryza sativa;At：Arabidopsis thaliana.

表2 VlRR5启动子区域的顺式作用元件

Table 2 The cis-acting elements in promoters of VlRR5

顺式作用元件 cis-acting element	功能 Function	序列 Sequence	数量 Amount
gGTAAAGAAA	脱落酸响应Abscisic acid responsiveness	ACGTG	1
ARE	厌氧诱导Anaerobic induction	AAACCA	3
AuxRR-core	生长素响应Auxin responsiveness	GGTCCAT	1
CAT-box	分生组织表达Meristem expression	GCCACT	1
CGTCA-motif	茉莉酸甲酯响应MeJA-responsiveness	CGTCA	2
G-box	光响应Light responsiveness	TACGTG	2
MBS	参与干旱诱导的MYB结合位点MYB binding site involved in drought-inducibility	CAACTG	9
Pc-CMA2a	光响应元件的一部分Part of a light responsive element	CAGCCAATCACAG	1
TATA-box	在转录开始-30左右的核心启动子元件 Core promoter element around-30 of transcription start	TATA	5
TCA-element	水杨酸响应Salicylic acid responsiveness	TCAGAAGAGG	1
TGACG-motif	茉莉酸甲酯响应MeJA-responsiveness	TGACG	2
CAAT-box	启动子和增强子区域的顺式作用元件 Common cis-acting element in promoter and enhancer regions	CAAAT	49

图2 VlRR5与其他物种RR蛋白质的进化树分析 St：马铃薯;Sl：番茄;Os：水稻;Tt：唐松草小银莲花;Nn：莲;At：拟南芥;Ns：蓝果树;Ac：中华猕猴桃;Cs：茶;Cm：香瓜;Cf：川黔千金榆;Pan：山黄麻;Zj：枣;Md：苹果;Pd：扁桃;Pa：欧洲甜樱桃。

Fig. 2 Phylogenetic tree of VlRR5 and RR proteins of other species St：Solanum tuberosum;Sl：Solanum lycopersicum;Os：Oryza sativa subsp. japonica;Tt：Thalictrum thalictroides;Nn：Nelumbo nucifera;At：Arabidopsis thaliana;Ns：Nyssa sinensis;Ac：Actinidia chinensis var. chinensis;Cs：Camellia sinensis;Cm：Cucumis melo;Cf：Carpinus fangiana;Pan：Parasponia andersonii;Zj：Ziziphus jujuba;Md：Malus × domestica;Pd：Prunus dulcis;Pa：Prunus avium.

图3 VlRR5的亚细胞定位

Fig. 3 Analysis of the localization of the VlRR5 protein

图4 VlRR5的转录激活活性检测

Fig. 4 Transcriptional activation assay of the VlRR5

图5 VlRR5的表达特异性分析

Fig. 5 Diverse expression patterns of VlRR5

图6 CPPU处理后葡萄表型（A）及VlRR5表达水平（B）

Fig. 6 Grape phenotype（A）and VlRR5 expression level（B）after CPPU treatment * α = 0.05.

图7 细胞分裂素合成抑制剂Lov处理后葡萄表型（A）及VlRR5的表达水平（B）

Fig. 7 Grape phenotype（A）and VlRR5 expression level（B）after inhibitors of cytokinin synthesis Lov treatment * α = 0.05.

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