桂花OfbHLH89参与调控花开放的功能解析

doi:10.16420/j.issn.0513-353x.2023-0299

摘要/Abstract

摘要：

在桂花（Osmanthus fragrans）转录组数据库中筛选并克隆出可能参与花开放过程的候选基因OfbHLH89。该基因的开放阅读框（ORF）长744 bp，编码247个氨基酸。氨基酸序列和系统进化树分析表明，OfbHLH89与红豆VaBPE和辣椒CbBPE的亲缘相似性分别为54.1%和52.74%。亚细胞定位显示OfbHLH89定位于细胞核。时空表达分析表明OfbHLH89在花中高表达，且在花开放过程中的表达水平逐渐升高，盛开期达到峰值；19 ℃低温诱导下的花芽中，OfbHLH89的转录水平显著高于23 ℃对照。OfbHLH89过表达矮牵牛花瓣变大，表明OfbHLH89可能参与花开放过程的花瓣扩张；检测转基因矮牵牛下游与细胞扩张相关基因的转录水平发现，PhEXPA1、PhXTH24和PhXTH25均显著上调。此外，在瞬时过表达OfbHLH89的桂花花芽中，OfXTH5、OfXTH21、OfXTH32、OfXTH34、OfEXPA4和OfEXPA13的相对表达量也显著高于对照花芽。综上，OfbHLH89可能通过激活细胞扩张相关基因的转录使花瓣变大从而影响桂花开放。

关键词: 桂花, 花开放, bHLH, 细胞扩张

Abstract:

The flower opening process was investigated by screening and cloning gene OfbHLH89 from the Osmanthus fragrans transcriptome database. This gene has a coding frame（ORF）that is 744 bp long and encodes for 247 amino acids. Sequence alignment and phylogenetic tree analysis revealed that OfbHLH89 shared similarities of 54.1% and 52.74% with Vigna angularis VaBPE and Capsicum baccatum CbBPE，respectively. The subcellular localization results showed that OfbHLH89 was localized on the nucleus. Spatiotemporal expression analysis showed that OfbHLH89 was highly expressed in the flower，and its expression gradually increased from ball-shaped stage to full flowering stage，reaching a peak at the full flowering stage. Further analysis revealed that under low temperature induction，the relative expression level of OfbHLH89 in the flower buds was significantly higher than that of the control. In Petunia hybrida，overexpression of OfbHLH89 resulted in the enlargement of petals，suggesting that OfbHLH89 may be involved in petal expansion during flower opening in O. fragrans. Additionally，the expression of PhEXPA1，PhXTH24，and PhXTH25 were significantly up-regulated. Quantitative analysis of downstream cell expansion related genes by transient transformation of OfbHLH89 into O. fragrans flower buds revealed that the expression of OfXTH5，OfXTH21，OfXTH32，OfXTH34，OfEXPA4 and OfEXPA13 were significantly upregulated compared with empty vector. It is suggested that OfbHLH89 can impact the flower opening of O. fragrans by activating the transcription of genes related to cell expansion，leading to increase in petal size.

Key words: Osmanthus fragrans, flower opening, bHLH, cell expansion

李湘, 钟诗蔚, 王艺光, 邓金萍, 方遒, 赵宏波. 桂花OfbHLH89参与调控花开放的功能解析[J]. 园艺学报, 2024, 51(6): 1311-1320.

LI Xiang, ZHONG Shiwei, WANG Yiguang, DENG Jinping, FANG Qiu, ZHAO Hongbo. Functional Analysis of OfbHLH89 Gene in Regulating Flower Opening in Osmanthus fragrans[J]. Acta Horticulturae Sinica, 2024, 51(6): 1311-1320.

图/表 9

表1 实时荧光定量PCR特异性引物

Table 1 Specific primers for quantitative real-time PCR

基因 Gene	正向引物（5′-3′） Forward primer	反向引物（5′-3′） Reverse primer
OfbHLH89	ACAAAACTGTCCGAACCTCCA	TTTTTCTCTCCTTGCTCTTTCAGC
qOfXTH5	TTATCTTCTCAAGGAACTCAACACG	ATTGGTATGAACTGTATATGGCTGG
qOfXTH21	CTGTCACTGCTTTCTATCTGTCTTC	TTTGTCTCCCTTCCCTCCTGTGT
qOfXTH32	CAGGGAGCAGCAATTCTATCT	ATGCAACACCTAGAGACTCAGC
qOfXTH34	GTCAATGGAGTTGGCAATAGG	GTCTCATCGACCAAGAATACAAC
qOfEXPA4	TTTCCCAAACATAGTCCCATCA	AAAGAGTAAGCACGCCGCAG
qOfEXPA13	ACCAAACAATGCCCTCCCAA	ACCCTTCTGTACGCAACAGG
qPhXTH24	AAGGCAAAGGTAACAGAGAGCA	TGGAATAGGTGTGGAAATCAGCA
qPhXTH25	GCAACCTAAGTGGAGACCCT	ATTGGTGTCCCATCCACTGA
qPhEXPA1	CAGGGGGTTGGTGTAATCCT	CAACAGGGACAATTCCAGCTT
qOfACT	CCCAAGGCAAACAGAGAAAAAAT	ACCCCATCACCAGAATCAAGAA
qPhPCYP	AGGCTCATCATTCCACCGTGT	TCATCTGCGAACTTAGCACCG

图1 桂花OfbHLH89 PCR扩增条带

Fig. 1 PCR amplification band of OfbHLH89 from Osmanthus fragrans

图2 桂花OfbHLH89与其他植物bHLH蛋白的系统发育树分析 Bn：油菜；Bo：甘蓝；Br：白菜；Rs：萝卜；At：拟南芥；Va：红豆；Cb：辣椒；Cc：菜蓟；In：牵牛；Ha：向日葵；Cm：菊花；Ls：莴苣。

Fig. 2 Phylogenetic analysis of OfbHLH89 from Osmanthus fragrans and bHLH proteins of other plants Bn：Brassica napus；Bo：Brassica oleracea；Br：Brassica rapa；Rs：Raphanus sativus；At：Arabidopsis thaliana；Va：Vigna angularis；Cb：Capsicum baccatum；Cc：Cynara cardunculus；In：Ipomoea nil；Ha：Helianthus annuus；Cm：Chrysanthemum morifolium；Ls：Lactuca sativa.

图3 桂花OfbHLH89的亚细胞定位

Fig. 3 Subcellular localization of Osmanthus fragrans OfbHLH89

图4 OfbHLH89在桂花不同器官、不同开放时期和不同温度下的表达分析不同小写字母表示使用单因素方差（ANOVA）分析差异显著，P < 0.05。

Fig. 4 Expression analysis of OfbHLH89 at different tissues，stages of flowering and temperatures in Osmanthus fragrans Different lowercase letters indicate significant differences using one-way analysis of variance（ANOVA），P < 0.05.

图5 OfbHLH89转基因矮牵牛植株（OE）及其野生型（WT）验证和基因过表达分析

Fig. 5 Verification and overexpression analysis of OfbHLH89 transgenic line positive plant（OE）and wild type（WT）in Petunia hybrida ** P < 0.01.

图6 OfbHLH89转基因矮牵牛（OE）及其野生型（WT）花冠直径、花瓣长度和宽度观察

Fig. 6 Observation on corolla diameter，petal length and width of OfbHLH89 transgenic line（OE）and wild type（WT）in Petunia hybrida * P < 0.05，** P < 0.01.

图7 OfbHLH89转基因矮牵牛（OE）及其野生型（WT）花瓣中细胞扩张相关基因的表达量

Fig. 7 Expression level of cell expansion-related genes in petals of transgenic plants（OE）and wild type（WT）in Petunia hybrida

图8 细胞扩张相关基因在桂花花芽中的瞬时表达量

Fig. 8 Transient expression of cell expansion-related genes in flower buds of Osmanthus fragrans

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