异源过表达森林草莓FveBBX32负调控拟南芥的叶绿素含量和开花时间

doi:10.13560/j.cnki.biotech.bull.1985.2025-0574

摘要/Abstract

摘要：

目的解析森林草莓转录因子FveBBX32在调节叶绿素积累和开花时间中的功能，为花期调控和培育高光效草莓新品种提供理论依据和候选基因资源。方法以森林草莓‘Ruegen’cDNA为模板克隆FveBBX32，并进行生物信息学分析和组织表达分析，利用农杆菌侵染获得转基因拟南芥株系，观察并记录转基因材料的叶片颜色和开花表型，并利用RT-qPCR检测叶绿素和开花途径中相关基因的表达量。结果 FveBBX32的CDS全长为795 bp，编码264个氨基酸，为亲水性蛋白，相对分子质量为28 638.63 Da，含有1个B-box结构域，属于BBX第Ⅴ亚组，与月季的亲缘关系较近。FveBBX32在森林草莓根、茎、叶、花、果中均有表达，其中，在花和叶中表达较高。过表达FveBBX32拟南芥表现出叶色变黄和开花时间延迟的表型，过表达株系中叶绿素合成相关基因AtGUN4、AtCHLH、AtCHL27和开花相关基因AtFT、AtSOC1、AtFUL的表达量均有不同程度的降低。结论森林草莓FveBBX32是植物叶绿素积累和开花时间的负调控转录因子。

关键词: 草莓, BBX32, 叶绿素, 开花, 转基因

Abstract:

Objective This study aims to clarify the function of transcription factor FveBBX32 in regulating chlorophyll accumulation and flowering time, providing theoretical basis and candidate gene resources for flowering period regulation and cultivation of high-efficiency strawberry (Fragaria×ananassa Duch.) varieties. Method The Fragaria vesca 'Ruegen' cDNA was used as template to clone FveBBX32 gene for bioinformatics analysis and tissue expression analysis. Transgenic Arabidopsis plants were obtained by Agrobacterium infection, the leaf color and flowering phenotype of the transgenic material were observed and recorded, and the expressions of related genes in chlorophyll and flowering pathways were detected by RT-qPCR. Result The CDS length of FveBBX32 genewas 795 bp, encoding 264 amino acids, the protein was hydrophilic with a relative molecular weight of 28 638.63 Da. It contained one B-box domain, belonging to subgroup V of the BBX family, showing close phylogenetic relationship with Rosa chinensis. FveBBX32 was expressed in the roots, stems, leaves, flowers, and fruits of F. vesca, with higher expressions in flowers and leaves. The overexpression of FveBBX32 in Arabidopsis thaliana resulted in yellowing of leaf color and delayed flowering time. The expressions of chlorophyll biosynthesis-related genes AtGUN4, AtCHLH, AtCHL27, and flowering-related genes AtFT, AtSOC1, and AtFUL were all reduced to varying degrees in transgenic lines. Conclusion FveBBX32 is confirmed as a negative regulatory transcription factor of chlorophyll accumulation and flowering time in plants.

Key words: strawberry, BBX32, chlorophyll, flowering, transgenic

缪百灵, 汪园园, 侯威威, 张怡如, 陈娟娟, 李亮杰, 张贺, 朱庆松, 董向向. 异源过表达森林草莓FveBBX32负调控拟南芥的叶绿素含量和开花时间[J]. 生物技术通报, 2025, 41(11): 293-300.

MIAO Bai-ling, WANG Yuan-yuan, HOU Wei-wei, ZHANG Yi-ru, CHEN Juan-juan, LI Liang-jie, ZHANG He, ZHU Qing-song, DONG Xiang-xiang. Hetero-overexpression of Fragaria vescaFveBBX32 Negatively Regulates the Chlorophyll Content and Flowering Time in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2025, 41(11): 293-300.

图/表 9

表1 引物信息

Table 1 Primer information

基因

Gene

引物名称

Primer name

引物序列

Primer sequence （5′‒3′）

用途

Usage

FveBBX32

FveBBX32-F

FveBBX32-R

TTGTTGTCGTCGTCCCTTGA

TGGACAAACCTTACCCGTCG

FveBBX32的扩增

Amplification of FveBBX32

FveBBX32-QF

FveBBX32-QR

TTGTTGTCGTCGTCCCTTGA

TGGACAAACCTTACCCGTCG

FveBBX32表达量分析

Analysis of FveBBX32 expression

AtACT2

AtACT2-QF

AtACT2-QR

AAGCTGGGGTTTTATGAATGG

TTGTCACACACAAGTGCATCAT

AtACT2表达量分析

Analysis of AtACT2 expression

Fve26S

Fve26S-QF

Fve26S-QR

TAACCGCATCAGGTCTCCAA

CTCGAGCAGTTCTCCGACAG

Fve26S表达量分析

Analysis of Fve26S expression

AtGUN4

AtGUN4-QF

AtGUN4-QR

GATACAGCGTGCAACGCAAA

TTCGTCAGGAAACGCTCTGT

AtGUN4表达量分析

Analysis of AtGUN4 expression

AtCHLH

AtCHLH-QF

AtCHLH-QR

GCTTACCTCGCTTCTTGGGT

CGCCCTGTAGAACTTGGTGT

AtCHLH表达量分析

Analysis of AtCHLH expression

AtCHL27

AtCHL27-QF

AtCHL27-QR

GGTGTCAAGACGAGAACCGT

GCAGAAGAAGCGAGACCAGA

AtCHL27表达量分析

Analysis of AtCHL27 expression

AtSGR1

AtSGR1-QF

AtSGR1-QR

TCAGTTGCAAGGATGGGCAA

AAGGAAATGGCCACCGCTTA

AtSGR1表达量分析

Analysis of AtSGR1 expression

AtFT

AtFT-QF

AtFT-QR

CTGGAACAACCTTTGGCAAT

AGCCACTCTCCCTCTGACAA

AtFT表达量分析

Analysis of AtFT expression

AtSOC1

AtSOC1-QF

AtSOC1-QR

AATTCGCCAGCTCCAATATG

CCTCGATTGAGCATGTTCCTA

AtSOC1表达量分析

AtSOC1 expression analysis

AtFUL

AtFUL-QF

AtFUL-QR

GGAGAAGAAAACGGGTCAGC

ACTCGTTCGTAGTGGTAGGAC

AtFUL表达量分析

Analysis of AtFUL expression

图1 不同物种FveBBX32蛋白序列比对Fve：森林草莓，XP_004303825.1；Rc：月季，XP_024182309.1；At：拟南芥，NP_188752.1；Md：苹果，XP_008342423.3；Pc：西洋梨，XP_068303896.1；Pd：扁桃，XP_034208713.1；Jr：胡桃，XP_018810913.1；Qr：夏栎，XP_050289181.1；Pa：银白杨，XP_034929259.1；Pc：灰牧豆树，XP_054781646.1；Cs：大麻，XP_030499327.2；Nn：莲，XP_010273594.1；Zj：枣，XP_015895718.1；Pe：胡杨，XP_011033038.1；Vr：河岸葡萄，XP_034695321.1

Fig. 1 Alignment of FveBBX32 protein sequences of different speciesFve: Fragaria vesca, XP_004303825.1; Rc: Rosa chinensis, XP_024182309.1; At: Arabidopsis thaliana, NP_188752.1; Md: Malus domestica, XP_008342423.3; Pc: Pyrus communis, XP_068303896.1; Pd: Prunus dulcis, XP_034208713.1; Jr: Juglans regia, XP_018810913.1; Qr: Quercus robur, XP_050289181.1; Pa: Populus alba, XP_034929259.1; Pc: Prosopis cineraria, XP_054781646.1; Cs: Cannabis sativa, XP_030499327.2; Nn: Nelumbo nucifera, XP_010273594.1; Zj: Ziziphus jujuba, XP_015895718.1; Pe: Populus euphratica, XP_011033038.1; Vr: Vitis riparia, XP_034695321.1

图2 不同物种FveBBX32蛋白的系统进化分析

Fig. 2 Phylogenetic analysis of FveBBX32 protein in different species

图3 FveBBX32在不同组织中的表达分析不同小写字母表示差异显著（P<0.05）。下同

Fig. 3 Expression analysis of FveBBX32 gene in different tissuesDifferent lowercase letters indicate significant differences (P<0.05). The same below

图4 过表达FveBBX32拟南芥的PCR鉴定（A）和基因表达量分析（B）M：DL 5000 marker；1：野生型拟南芥；2‒4：3个转基因株系

Fig. 4 PCR identification (A) and gene expression analysis (B) of A. thaliana with overexpressed FveBBX32M: DL 5000 marker; 1: wild type A. thaliana; 2‒4: three transgenic strains

图5 过表达FveBBX32拟南芥的叶色表型（A）及叶绿素含量测定（B）

Fig. 5 Leaf color phenotype (A) and chlorophyll content determination (B) of A. thaliana with overexpressed FveBBX32

图6 过表达FveBBX32拟南芥的开花表型（A）和抽薹时间（B）

Fig. 6 Flowering phenotype (A) and bolting time (B) of A. thaliana with overexpressed FveBBX32

图7 叶绿素合成和降解相关基因的表达量分析

Fig. 7 Analysis of the expressions of genes related to chlorophyll synthesis and degradation

图8 开花相关基因的表达量分析

Fig. 8 Analysis of the expressions of flowering-related genes

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