桃TRM基因家族鉴定及与果形调控因子PpOFP1的互作筛选

doi:10.16420/j.issn.0513-353x.2025-0250

摘要/Abstract

摘要：

蟠桃因其特有的扁果形果实为解析果形调控机制提供了理想材料。前期研究证实PpOFP1是蟠桃扁果形形成的关键调控因子，但其分子调控网络尚未明晰。研究表明TRM（TONNEAU1 recruiting motif proteins）通过保守的M8结构域与OFP家族蛋白互作，在番茄果实形状发育中发挥重要调控作用。本研究中从桃中鉴定出12个TRM家族成员，系统进化分析显示其分属4个进化分支。蛋白保守基序（motif）分析发现，其中8个成员含有TRM与OFP互作必需的M8保守结构域。通过酵母双杂交实验、荧光素酶互补及双分子荧光互补分析证实，PpTRM5、PpTRM17、PpTRM18和PpTRM26与PpOFP1互作，且该互作发生在细胞质和细胞膜。转录组分析表明，PpTRM17在蟠桃和圆桃果实第1次快速生长期均高表达，这与该时期PpOFP1在蟠桃中高表达相一致，揭示二者可能共同调控蟠桃扁果形的形成。

关键词: 桃, 果形, PpTRM基因家族, 蛋白互作

Abstract:

Flat peach，with its unique flat fruit shape，provide an ideal material for analyzing the mechanism of fruit shape regulation. Previous studies have confirmed that PpOFP1 is a key regulator of flat fruit formation in peach；however，its molecular regulatory network remains unclear. Studies have shown that TRMs（TONNEAU1 Recruiting Motif proteins）interact with OFP family proteins though a conserved M8 domain，and this interaction plays a pivotal role in regulating tomato fruit shape. In this study，a total of 12 TRM family members were identified in peach，and phylogenetic analysis revealed their classification into four evolutionary clades. The analysis of protein conserved motifs revealed that 8 of these members contain the conserved M8 domain，which is essential for TRM-OFP interaction. In this study，we also found that PpTRM5，PpTRM17，PpTRM18，and PpTRM26 were confirmed to interact with PpOFP1 through yeast two-hybrid assay，luciferase complementation assay，and bimolecular fluorescence complementation analysis，and this interaction occurs in the cytoplasm and cell membrane. Transcriptome analysis indicated that PpTRM17was highly expressed during the first rapid growth phase of both flat and round peach fruits，which was consistent with the high expression of PpOFP1 in flat peaches during this period，revealing that the two might jointly regulate the formation of flat fruit shape in flat peaches.

Key words: peach, fruit shape, PpTRM gene family, protein interaction

苏艳艳, 王雨童, 荆彦付, 徐摇光, 于洋, 谢华. 桃TRM基因家族鉴定及与果形调控因子PpOFP1的互作筛选[J]. 园艺学报, 2025, 52(11): 2835-2845.

SU Yanyan, WANG Yutong, JING Yanfu, XU Yaoguang, YU Yang, XIE Hua. Identification of PpTRM Gene Family Members and Screening the Interacting PpTRMs with the Fruit Shape Regulator PpOFP1 in Peach[J]. Acta Horticulturae Sinica, 2025, 52(11): 2835-2845.

图/表 8

表1 本研究中使用的引物序列信息

Table 1 Primer sequence information used in this study

基因 Gene	上游引物（5′-3′） Forward primer	下游引物（5′-3′） Reverse primer
PpOFP1	ATGGGTAACCACAAGTTTAG	CTTGGACCTGAGGTCA
PpTRM5	ATGACGACAGGCATGGT	GAACACCAACTTCCTACAAG
PpTRM17	ATGAACGGGATGCAGAT	TGTCCTCACTGTCAAATCAA
PpTRM18	ATGGAATCTGCACCAGGTA	AGAACTCTCCGAGCTAACAAA
PpTRM26	ATGGGAGGAGGCTTATTG	TAACTGCATATCATCCAA
PpTRM19	ATGGAGAAGTTGCGGCCAA	CATAACCACTACCTCTGCTACTAGC
PpTRM13/14/15/33a	ATGGCAAAGAAGTCCCAG	CAAGTTCAAAATGCTATC
PpTRM13/14/15/33b	ATGGCAAAGATGACACC	AAATATGGTTTCCATGATCAG
PpTRM32	ATGGGAAAGCAATTGCAGC	AATACAAATTGCTTCATCTAATAGT
PpTRM16	ATGGGGAAGAATTTGCCAGA	GGAAGGAACCTGATCCATCC
PpTRM6/7/8	ATGGAGGTTGATAAAAAACG	CCAAAATCAGACACCAACTCATC
PpTRM1	ATGTCTGCAAAGATTTTGC	CCAAAGCCCCCTTTC
PpTRM4	ATGGCTGCAAAACTTCTG	TTTAGCAAACAGCTGCCTAC

表2 PpTRM家族成员的理化性质

Table 2 Physicochemical properties of PpTRM family members

进化分支 Evolutionary branch	基因 Gene	基因登录号 GeneBank No.	氨基酸数 Amino acid number	分子量/kD Molecular weight	等电点 pI	不稳定指数 Instability index	总平均疏水指数 Grand average of hydropathicity
Clade Ⅱ	PpTRM1	Prupe.3G252400	1 063	119.26	9.25	59.78	-0.88
	PpTRM4	Prupe.5G206500	1 082	120.25	9.32	57.44	-0.87
	PpTRM5	Prupe.8G233400	767	86.28	9.31	81.32	-0.80
Clade Ⅲ	PpTRM6/7/8	Prupe.6G315200	903	99.98	7.51	54.76	-0.73
Clade Ⅶ	PpTRM13/14/15/33a	Prupe.2G170700	956	108.55	5.24	54.73	-0.77
	PpTRM13/14/15/33b	Prupe.5G101000	915	103.31	5.50	56.34	-0.74
	PpTRM32	Prupe.6G078200	644	73.02	4.99	48.47	-0.73
	PpTRM16	Prupe.6G254700	904	100.83	5.73	50.10	-0.71
Clade Ⅵ	PpTRM17	Prupe.8G209800	981	108.94	5.74	59.38	-0.77
	PpTRM18	Prupe.2G227000	781	88.20	6.38	50.70	-0.70
	PpTRM19	Prupe.6G247900	970	108.29	6.18	65.72	-0.83
	PpTRM26	Prupe.7G183200	886	100.99	6.59	55.83	-0.84

图1 桃、番茄（Sl）和拟南芥（At）TRM家族成员系统进化分析

Fig. 1 Phylogenetic analysis of TRMs family members in peach，tomato（Sl）and Arabidopsis（At）

图2 PpTRM家族成员保守基序分析

Fig.2 Conserved motif analysis of PpTRM family members

图3 酵母双杂交（Y2H）筛选与PpOFP1互作的PpTRM蛋白分别以pGBKT7-53与pGADKT7和pGBKT7-Lam与pGADKT7共转AH109酵母菌株作为阳性对照和阴性对照

Fig. 3 Screening of PpTRM proteins interacting with PpOFP1 by yeast two-hybridization Co-transform AH109 yeast strains with pGBKT7-53 and pGADKT7，and with pGBKT7-Lam and pGADKT7，serving as the positive control and negative control，respectively

图4 LCA（A）和BiFC（B）验证PpTRM和PpOPF1互作 A中将cLuc与nLuc共同注射烟草，以及将它们分别与各融合蛋白混合后注射烟草，这两种处理方式均作为阴性对照；B中将YFPc和YFPn共同注射烟草，以及将它们分别与各融合蛋白混合后注射烟草，这两种处理方式均为阴性对照

Fig. 4 LCA（A）and BiFC（B）assay verify the interaction between PpTRM and PpOPF1 In Figure A，both the co-injection of cLuc and nLuc into tobacco，and the injection of each of them mixed with their respective fusion proteins into tobacco，were used as negative control；In figure B，both the co-injection of YFPc and YFPn into tobacco，and the injection of each of them mixed with their respective fusion proteins into tobacco，were used as negative control

图5 RNA-seq分析PpTRM5、PpTRM17、PpTRM18、PpTRM26和PpOFP1在蟠桃‘瑞油蟠1号’和圆桃‘大久保’果实中的表达模式

Fig. 5 RNA-seq analysis of expression patterns of PpTRM5，PpTRM17，PpTRM18，PpTRM26 and PpOFP1 in flat‘Ruiyoupan 1’and round‘Okubo’peach fruits

图6 RNA-seq分析PpTRM5、PpTRM17、PpTRM18、PpTRM26和PpOFP1在圆桃‘B-4’不同组织的表达模式

Fig. 6 RNA-seq analysis of the expression patterns of PpTRM5，PpTRM17，PpTRM18，PpTRM26，and PpOFP1 in different tissues of round peach‘B-4’ NCBI-SRA database（https://www.ncbi.nlm.nih.gov/bioproject/PRJNA694331）.

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