Functional Analysis of OfbHLH89 Gene in Regulating Flower Opening in Osmanthus fragrans

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

Acta Horticulturae Sinica ›› 2024, Vol. 51 ›› Issue (6): 1311-1320.doi: 10.16420/j.issn.0513-353x.2023-0299

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Functional Analysis of OfbHLH89 Gene in Regulating Flower Opening in Osmanthus fragrans

LI Xiang, ZHONG Shiwei, WANG Yiguang, DENG Jinping, FANG Qiu, ZHAO Hongbo^*()

Key Laboratory of National Forestry and Grassland Administration on Germplasm Innovation and Utilization for Southern Garden Plants，Zhejiang Provincial Key Laboratory of Germplasm Innovation and Utilization for Garden Plants，School of Landscape Architecture，Zhejiang A & F University，Hangzhou 311300，China

Received:2023-11-27 Revised:2024-03-26 Online:2024-12-18 Published:2024-06-22
Contact: ZHAO Hongbo

Abstract

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

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.

Figures/Tables 9

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

Fig. 1 PCR amplification band of OfbHLH89 from Osmanthus fragrans

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.

Fig. 3 Subcellular localization of Osmanthus fragrans OfbHLH89

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.

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

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.

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

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

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Functional Analysis of OfbHLH89 Gene in Regulating Flower Opening in Osmanthus fragrans

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