不结球白菜FRL基因家族鉴定、表达分析及BrcFRI1功能研究

doi:10.16420/j.issn.0513-353x.2024-1054

摘要/Abstract

摘要：

为探究不结球白菜FRIGIDA-Like（FRL）基因家族成员功能，对其基本性质、进化关系、表达特征以及BrcFRI功能进行分析。利用生物信息方法在不结球白菜‘NHCC001'基因组中对FRL基因家族进行鉴定和分析。利用转录组数据分析FRL基因在不同组织和ABA处理下的表达模式，并利用qRT-PCR分析了不结球白菜早晚花材料在不同时期、干旱胁迫以及野生型与转基因拟南芥三种开花基因的表达模式。结果表明不结球白菜共包含13个BrcFRL，可分为6个亚组，在进化过程中发生了不同程度的分化。十字花科FRL主要在根中高表达，不结球白菜BrcFRL成员间也存在组织表达差异性。BrcFRI1的过表达可使拟南芥开花延迟。BrcFRI在春化前期积累抑制开花，在春化过程中表达量降低，随着春化结束表达量再次上升。此外，11个BrcFRL成员对ABA信号均表现出不同程度的响应。干旱胁迫下，BrcFRL1、BrcFRI2和BrcFRL3-1有明显响应。BrcFRL1既受ABA处理正调控又响应干旱胁迫，说明FRL基因可能与不结球白菜激素应答反应有关，且ABA响应基因可能会被干旱胁迫所诱导。本研究中共鉴定获得了13个不结球白菜FRL家族基因，明确BrcFRL1推迟开花时间的功能，并筛选出多个与开花相关和在干旱胁迫中发挥作用的关键基因。

关键词: 不结球白菜, 植物分子学, 基因家族, 开花时间调控, 表达分析, 非生物胁迫

Abstract:

In order to understand the functions of the FRIGIDA-Like（FRL）gene family members in non-heading Chinese cabbage，their basic properties，evolutionary characteristics，and expression features at different times and under different abiotic stresses were analyzed. Bioinformatics methods were used to identify and analyze the FRL gene family in the genome of non-heading Chinese cabbage‘NHCC001'，and transcriptome data were used to analyze the expression patterns of non-heading Chinese cabbage FRL genes under ABA treatment，and qRT-PCR was used to analyze the expression patterns under drought stress and in different periods of early and late flowering materials and three flowering genes in wild-type and transgenic Arabidopsis thaliana. The results showed that the non-heading cabbage contained a total of 13 BrcFRLs randomly distributed on seven chromosomes，and the BrcFRLs could be classified into six subgroups. The covariance analysis of five cruciferous species revealed that the FRL genes were not completely on the covariance region，and different degrees of differentiation might have occurred during the evolutionary process. In addition，cruciferae FRLs were highly expressed mainly in roots compared to species of other families，and there was differential tissue expression of BrcFRLs. cis-acting element analysis showed that all BrcFRLs contained ABA response elements，and all 11 BrcFRL members showed different degrees of response to ABA signaling，which may be regulated by ABA. Under drought stress，BrcFRL1，BrcFRI2，and BrcFRI3-1 responded significantly. BrcFRL1 was both positively regulated by ABA treatment and responsive to drought stress，suggesting that the FRL genes may be related to the hormonal response of non-heading Chinese cabbage and that ABA-responsive genes may be induced by drought stress. Overexpression of BrcFRI1 delays flowering in Arabidopsis. However，the expression of FRI and FLC in Brassica was not consistent with that in Arabidopsis，with BrcFRI accumulating in the pre-vernalization period to inhibit flowering；its expression level decreases during the vernalization process and rises again after vernalization ends. In this study，a total of 13 non-heading Chinese cabbage FRL family genes were identified，clarified the function of BrcFRL1 in delaying flowering time，and screened out a number of key genes that are related to flowering and involved in drought stress response.

Key words: non-heading Chinese cabbage, plant molecular biology, gene family, flowering time regulation, expression analysis, abiotic stress

白雪滢, 唐曼芷, 渠天慧, 张利婷, 肖栋, 李英, 侯喜林, 刘同坤. 不结球白菜FRL基因家族鉴定、表达分析及BrcFRI1功能研究[J]. 园艺学报, 2026, 53(4): 1125-1142.

BAI Xueying, TANG Manzhi, QU Tianhui, ZHANG Liting, XIAO Dong, LI Ying, HOU Xilin, LIU Tongkun. Identification of FRL Gene Family，Expression Analysis and Functional Study of BrcFRI1 in Non-Heading Chinese Cabbage[J]. Acta Horticulturae Sinica, 2026, 53(4): 1125-1142.

图/表 13

表1 本研究中qRT-PCR分析所用引物序列

Table 1 Primer sequences used for qRT-PCR analysis in this study

基因 Gene	正向引物序列（5′-3′） Forward primer	反向引物序列（5′-3′） Reverse primer
ACTIN2	GTTCCAGCCCTCGTTTGTG	CAAGTGCTGTGATTTCTTTGCTC
At-FRI	GGTGCTTTGAAGCGGTCACAGT	GCCCTCTCAAATGACTCCTTGC
At-FLC	TACAGCTTCTCCTCCGGCGATA	TCCCACAAGCTTGCTATCCACA
At-FT	GGTTGGTGACTGATATCCC	GGAAGGCCGAGATTGTATAG
Brc-Actin	CTCAGTCCAAAAGAGGTATTCT	GTAGAATGTGTGATGCCAGATC
BrcFRI1	ATGGCCGTCCGTAATGGTTC	AAGACCGCATTGGAGGAATG
BrcFRI2	GGCCTTTCGTAATGGTTCTC	CAAAATCAGGTCTCCTCGTA
BrcFRL1	ATCACACCGCTCTCTCCTCT	ACCTAACTCAAACGCTGCGA
BrcFRL3-1	GGATATGGACTCAGCAGGGC	GACAATCCAAGAGACCGGCA
BrcFRL3-2	GCCTGGACAGCAACTCTCTT	GGAGTGGCTCAACTGGGTAC
BrcFRL3-3	GTGGAAGGAACTGGAGGAGC	GGTGGTGGTGACGACTCAAT
BrcFRL4-1	TCACCACGCTTGAGGAGAAC	AACGCTCGGAGTAACAAGCA
BrcFRL4-2	AGGAAGTCGCGGTTGATTGT	GCTGGCTCTGGTTCTCTTGT
BrcFLC	CCGCAAGCCTATCTCTAACC	CAGAGGAGTTTACAGAAAGCAGA
BrcFT	ACGAGAGTCCAAGGCCCAAC	TGGCGCCATCCTGGTTCATA

表2 不结球白菜FRL基因家族成员的理化性质

Table 2 Physicochemical properties of non-heading Chinese cabbage FRL gene family members

基因名称 Gene	基因号 Gene number	CDS长度/bp CDS length	氨基酸长度 Protein length	分子量 Molecular weigh	等电点 Isoelectric point	亲水性 Gravy	不稳定性指数 Instability Index
BrcFRI1	BraC03g015970.1	1 791	596	66 693.58	7.64	-0.48	70.28
BrcFRI2	BraC10g009270.1	1 710	569	63 390.88	9.14	-0.42	67.68
BrcFRL1	BraC10g024850.1	1 374	457	51 253.90	7.62	-0.50	52.23
BrcFRL2	BraC09g038630.1	1 386	461	51 418.23	9.04	-0.32	58.87
BrcFRL3-1	BraC02g041830.1	1 659	552	61 499.29	5.77	-0.41	52.56
BrcFRL3-2	BraC06g042450.1	1 140	379	41 594.13	6.42	-0.21	60.23
BrcFRL3-3	BraC09g003460.1	1 563	520	58 366.95	7.14	-0.56	58.92
BrcFRL4-1	BraC01g035560.1	1 605	534	59 035.71	6.53	-0.44	57.25
BrcFRL4-2	BraC05g030410.1	1 380	459	51 512.32	6.08	-0.40	52.40
BrcFRL5-1	BraC06g040730.1	2 595	864	96 375.21	7.73	-0.43	39.23
BrcFRL5-2	BraC09g004180.1	2 523	840	95 082.19	6.86	-0.48	37.41
BrcFRL6	BraC06g040740.1	3 471	1 156	131 268.90	5.65	-0.59	47.73
BrcFRL7	BraC09g004160.1	3 420	1 139	130 221.60	5.21	-0.57	49.36

图1 十字花科物种FRL基因家族的系统进化与多样性分析 A：基于最大似然法构建的FRL蛋白系统发育树。物种包括拟南芥（Arabidopsis thaliana）、不结球白菜（Brassica rapa ssp. chinensis）、大白菜（B. ssp. pekinensis）、甘蓝（B. oleracea）和甘蓝型油菜（B. napus）；B：5个物种的FRL基因在不同亚组（Ⅰ~ Ⅵ）中的分布；C：不同亚组间的核苷酸差异分析

Fig. 1 Systematic evolution and diversity analysis of the FRL gene family in cruciferous species A：Phylogenetic tree of FRL proteins constructed based on the maximum likelihood method. The species include Arabidopsis thaliana，Brassica rapa ssp. chinensis，B. rapa ssp. pekinensis，B. oleracea，and B. napus；B：Distribution of FRL genes in different subgroups（Ⅰ- Ⅵ） among the five species；C：Analysis of nucleotide differences between different subgroups

图2 5个十字花科物种间FRL基因的共线性关系

Fig. 2 Collinearity of FRL genes among five cruciferous species

图3 不结球白菜（Brc）与大白菜（Brp）中FRL基因的染色体定位

Fig. 3 Chromosomal localization of the FRL genes in non-heading Chinese cabbage（Brc）and Chinese cabbage（Brp）

图4 拟南芥、不结球白菜及大白菜FRL基因家族的综合分析 A：系统发育树；B：基因结构；C：保守基序分布；D：FRIGIDA结构域组成

Fig. 4 Comprehensive analysis of the FRL gene family in Arabidopsis，non-heading Chinese cabbage，and Chinese cabbage A：Phylogenetic tree；B：Gene structure；C：Distribution of conserved motifs；D：Characteristics of the FRIGIDA domain composition

图5 不结球白菜和大白菜FRL基因启动子顺式作用元件分析

Fig. 5 Analysis of cis-acting elements in the FRL gene promoter of non-heading and Chinese cabbage

图6 拟南芥、不结球白菜和大白菜中FRL基因在不同组织中及大白菜春化前后的表达分析

Fig. 6 Expression analysis of FRL genes in Arabidopsis thaliana，non-heading Chinese cabbage and Chinese cabbage in different tissues and before and after vernalization in Chinese cabbage

图7 早花与晚花不结球白菜品种中BrcFRI1和BrcFRI2在不同发育时期的表达模式不同小写字母表示差异显著（P < 0.05）。下同

Fig. 7 Expression patterns of BrcFRI1 and BrcFRI2 at different developmental stages in early-and late-flowering non-heading cabbage varieties The different lowercase letters indicate significant differences（P < 0.05）. The same below

图8 野生型（Col）和转基因型拟南芥植株形态比较（A）及开花时间（B）

Fig. 8 Comparison of plant morphology（A）and flowering time（B）between wild-type（Col）and transgenic Arabidopsis

图9 FRI、FLC和FT基因在野生型（Col）与BrcFRI1转基因型拟南芥中的表达水平

Fig. 9 Expression levels of FRI，FLC and FT genes in wild-type（Col）and BrcFRI1 transgenic Arabidopsis

图10 不结球白菜FRL基因响应ABA处理的时间表达模式

Fig. 10 Time-course expression pattern of FRL genes in non-heading Chinese cabbage in response to ABA treatment

图11 不结球白菜FRL基因在干旱胁迫处理下的时间表达模式

Fig. 11 Temporal expression pattern of the FRL gene in non-heading Chinese cabbage under different drought stress treatment

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