Functional Study of RmEXPB2 Genein Rosa multiflora Based on the Identification of the Expansin Gene Family in Rosa sp.

doi:10.13560/j.cnki.biotech.bull.1985.2025-0216

Abstract

Abstract:

Objective This study is aimed to systematically identify the expansin gene family in Rosa chinensis ‘Old Blush’, dissect their sequence characteristics and expression patterns, screen genes associated with prickle development. Based on this result, the biological function of RmEXPB2 gene in Rosa multiflora was further explored in order to lay the foundation for in-depth identification of the function of expansin genes in prickle formation in Rosa sp. plants. Method Bioinformatics tools were used to identify RcEXP genes in R. chinensis ‘Old Blush’ genome. The amino acid residue range of their encoded proteins and chromosomal distribution were analyzed. A phylogenetic tree was constructed to analyze the evolutionary relationships among expansins from Arabidopsis thaliana (AtEXPs), R. chinensis ‘Old Blush’(RcEXPs), and Fragaria vesca (FvEXPs). RcEXP expression patterns in different tissues were detected, and its expression in the prickle transcriptome was analyzed. R. multiflora, a model for prickle research, served as the experimental material, the candidate gene RmEXPB2 was isolated, and its expression pattern during prickle development were studied. The biological function of RmEXPB2 was preliminarily explored by heterologous expression in Arabidopsis. Result A total of 29 RcEXP genes were identified in the genome of R. chinensis ‘Old Blush’. The encoded proteins ranged from 243 to 313 amino acids, and they were unevenly distributed on 7 chromosomes. Phylogenetic tree analysis divided the expansins AtEXPs, RcEXPs, and FvEXPs into 4 subgroups. Among different tissues and organs, 15 RcEXP genes showed diverse expression patterns, with RcEXPB2 and RcEXPA4 predominantly expressed in prickles. The 13 RcEXP genes were screened from the transcriptome data of prickles, which were significantly expressed at specific stages of prickle development. The gene RmEXPB2, preferentially expressed in the prickles, was isolated from R. multiflora, and its expression increased during prickle development, peaking at the late stage. Heterologous expression of RmEXPB2 significantly promoted leaf expansion and root hair growth in transgenic Arabidopsis. Conclusion A total of 29 RcEXPs were identified in the genome of R. chinensis ‘Old Blush’, of which 13 members were significantly expressed at specific stages of prickle development. RmEXPB2 is a prickle preferentially expressed gene in R. multiflora, which may play an active role in the process of prickle elongation.

Key words: Rosa chinensis ‘Old Blush’, expansin, Rosa multiflora, RmEXPB2, prickle development, heterologous expression, phylogenetic analysis, tissue expression pattern

LI Yu-zhen, LI Meng-dan, ZHANG Wei, PENG Ting. Functional Study of RmEXPB2 Genein Rosa multiflora Based on the Identification of the Expansin Gene Family in Rosa sp.[J]. Biotechnology Bulletin, 2025, 41(9): 182-194.

Figures/Tables 11

Table 1 Primer sequences for RT-qPCR

Gene name	Forward primer （5′‒3′）	Reverse primer （5′‒3′）
RmUBC	GCCAGAGATTGCCCATATGTA	TCACAGAGTCCTAGCAGCACA
AtEF1α	GCAAGATGGATGCCACTACCC	GCAAGATGGATGCCACTACCC
RmEXPB2	TCTAGAATGGCTAGTTTAAGAAGCAT	CCCGGGTAGATAATTGACCACGGATC
RmEXPB2-qRT	TGCTGCTTGTTTGAGCAGAG	AAGCTCCACATTCCCTGCC
RmEXPB2-eGFP	GAGAACACGGGGGACTCTAGAATGGCTAGTTTAAGAAGCAT	GCCCTTGCTCACCATCCCGGGTAGATAATTGACCACGGATC

Table 2 Basic information of expansin gene family members in R. chinensis'Old Blush'

基因ID Gene ID	基因 Gene	染色体位置 Chr location	等电点 pI	分子量 Molecular weight （Da）	蛋白长度 Protein length （aa）	亚细胞定位Subcellular localization	信号肽长度Signal peptie length （aa）
RchiOBHmChr5g0072761	RcEXPA1	Chr5	8.12	27 145.26	254	Cell wall	26
RchiOBHmChr7g0178811	RcEXPA2	Chr7	8.82	28 302.47	256	Cell wall	22
RchiOBHmChr7g0199891	RcEXPA3	Chr7	9.55	29 064.3	262	Chloroplast	28
RchiOBHmChr7g0181621	RcEXPA4	Chr7	8.64	26 524.79	250	Cell wall	20
RchiOBHmChr6g0281211	RcEXPA5	Chr6	6.93	27 816.75	253	Cell wall	23
RchiOBHmChr3g0488771	RcEXPA6	Chr3	7.55	29 302.32	272	Cell wall	24
RchiOBHmChr3g0470931	RcEXPA7	Chr3	9.00	28 950.9	260	Chloroplast	24
RchiOBHmChr4g0439541	RcEXPA8	Chr4	9.56	31 940.33	297	Mitochondrion
RchiOBHmChr3g0470941	RcEXPA9	Chr3	5.68	29 434.08	266	Cell wall	24
RchiOBHmChr5g0033911	RcEXPA10	Chr5	9.69	27 795.93	255	Cell wall	30
RchiOBHmChr1g0372131	RcEXPA11	Chr1	7.99	26 919.1	254	Cell wall	25
RchiOBHmChr3g0470961	RcEXPA12	Chr3	9.68	31 679.04	286	Chloroplast	25
RchiOBHmChr6g0245581	RcEXPA13	Chr6	5.22	28 332.98	260	Cell wall	27
RchiOBHmChr1g0367791	RcEXPA14	Chr1	9.53	27 917.86	259	Chloroplast	22
RchiOBHmChr3g0466461	RcEXPA15	Chr3	9.50	27 787.56	259	Cell wall	21
RchiOBHmChr2g0166091	RcEXPA16	Chr2	8.10	27 554.38	250	Chloroplast	22
RchiOBHmChr5g0065301	RcEXPA17	Chr5	9.68	27 890.68	260	Cell wall	22
RchiOBHmChr3g0460691	RcEXPA18	Chr3	8.36	27 487.08	255	Cell wall	24
RchiOBHmChr6g0303551	RcEXPA19	Chr6	8.87	27 306.82	257	Chloroplast	23
RchiOBHmChr2g0140121	RcEXPA20	Chr2	8.39	35 186.7	313	Cell wall	21
RchiOBHmChr2g0167701	RcEXPA21	Chr2	9.35	26 293.59	243	Cell wall	20
RchiOBHmChr2g0166111	RcEXPA22	Chr2	6.06	27 581.6	253	Cell wall	23
RchiOBHmChr5g0084081	RcEXPA23	Chr5	8.85	27 781.15	261	Nucleus	22
RchiOBHmChr2g0126231	RcEXPB1	Chr2	8.73	27 394.3	255	Cell wall	23
RchiOBHmChr1g0373921	RcEXPB2	Chr1	4.97	28 762.08	273	Chloroplast	23
RchiOBHmChr1g0348851	RcEXPB3	Chr1	6.40	30 068.99	287	Chloroplast	29
RchiOBHmChr6g0279781	RcEXLA1	Chr6	8.8	30 176.7	278	Chloroplast
RchiOBHmChr7g0205401	RcEXLB1	Chr7	6.87	27 333.67	250	Cell wall	22
RchiOBHmChr7g0208241	RcEXLB2	Chr7	4.70	28 099.57	258	Chloroplast	25

Table 2 Basic information of expansin gene family members in R. chinensis'Old Blush'

基因ID Gene ID	基因 Gene	染色体位置 Chr location	等电点 pI	分子量 Molecular weight （Da）	蛋白长度 Protein length （aa）	亚细胞定位Subcellular localization	信号肽长度Signal peptie length （aa）
RchiOBHmChr5g0072761	RcEXPA1	Chr5	8.12	27 145.26	254	Cell wall	26
RchiOBHmChr7g0178811	RcEXPA2	Chr7	8.82	28 302.47	256	Cell wall	22
RchiOBHmChr7g0199891	RcEXPA3	Chr7	9.55	29 064.3	262	Chloroplast	28
RchiOBHmChr7g0181621	RcEXPA4	Chr7	8.64	26 524.79	250	Cell wall	20
RchiOBHmChr6g0281211	RcEXPA5	Chr6	6.93	27 816.75	253	Cell wall	23
RchiOBHmChr3g0488771	RcEXPA6	Chr3	7.55	29 302.32	272	Cell wall	24
RchiOBHmChr3g0470931	RcEXPA7	Chr3	9.00	28 950.9	260	Chloroplast	24
RchiOBHmChr4g0439541	RcEXPA8	Chr4	9.56	31 940.33	297	Mitochondrion
RchiOBHmChr3g0470941	RcEXPA9	Chr3	5.68	29 434.08	266	Cell wall	24
RchiOBHmChr5g0033911	RcEXPA10	Chr5	9.69	27 795.93	255	Cell wall	30
RchiOBHmChr1g0372131	RcEXPA11	Chr1	7.99	26 919.1	254	Cell wall	25
RchiOBHmChr3g0470961	RcEXPA12	Chr3	9.68	31 679.04	286	Chloroplast	25
RchiOBHmChr6g0245581	RcEXPA13	Chr6	5.22	28 332.98	260	Cell wall	27
RchiOBHmChr1g0367791	RcEXPA14	Chr1	9.53	27 917.86	259	Chloroplast	22
RchiOBHmChr3g0466461	RcEXPA15	Chr3	9.50	27 787.56	259	Cell wall	21
RchiOBHmChr2g0166091	RcEXPA16	Chr2	8.10	27 554.38	250	Chloroplast	22
RchiOBHmChr5g0065301	RcEXPA17	Chr5	9.68	27 890.68	260	Cell wall	22
RchiOBHmChr3g0460691	RcEXPA18	Chr3	8.36	27 487.08	255	Cell wall	24
RchiOBHmChr6g0303551	RcEXPA19	Chr6	8.87	27 306.82	257	Chloroplast	23
RchiOBHmChr2g0140121	RcEXPA20	Chr2	8.39	35 186.7	313	Cell wall	21
RchiOBHmChr2g0167701	RcEXPA21	Chr2	9.35	26 293.59	243	Cell wall	20
RchiOBHmChr2g0166111	RcEXPA22	Chr2	6.06	27 581.6	253	Cell wall	23
RchiOBHmChr5g0084081	RcEXPA23	Chr5	8.85	27 781.15	261	Nucleus	22
RchiOBHmChr2g0126231	RcEXPB1	Chr2	8.73	27 394.3	255	Cell wall	23
RchiOBHmChr1g0373921	RcEXPB2	Chr1	4.97	28 762.08	273	Chloroplast	23
RchiOBHmChr1g0348851	RcEXPB3	Chr1	6.40	30 068.99	287	Chloroplast	29
RchiOBHmChr6g0279781	RcEXLA1	Chr6	8.8	30 176.7	278	Chloroplast
RchiOBHmChr7g0205401	RcEXLB1	Chr7	6.87	27 333.67	250	Cell wall	22
RchiOBHmChr7g0208241	RcEXLB2	Chr7	4.70	28 099.57	258	Chloroplast	25

Fig. 1 Chromosomal locations of expansin genefamily members in R. chinensis'Old Blush'

Fig. 2 Phylogenetic tree of expansin gene family in R. chinensis'Old Blush', F. avesca and ArabidopsisThe red five-pointed star indicates R.chinensis 'Old Blush', the blue circle indicates F. avesca, and the green box indicates Arabidopsis

Fig. 3 Analysis of conserved motif and gene structure of expansin gene family of R. chinensis'Old Blush'A: Conservative motif analysis. B: Conserved domain analysis. C: Gene structure analysis

Fig. 4 Analysis of the cis-acting elements on the promoters of expansin gene in R. chinensis'Old Blush'

Fig. 5 Expression pattern analysis of some expansin gene in various tissues of R. chinensis'Old Blush'

Fig. 6 Expression pattern analysis (A) and the gene number (B) of some expansin genes in different developmental stages of prickles in R. chinensis'Old Blush'Stage I: Soft and tender prickles that have not yet lignified. Stage Ⅱ: Prickles that are lignified to a certain extent and whose tips begin to bend toward the base. Stage Ⅲ: Prickles with relatively complete lignification and basically mature morphology. Stage IV: Prickles that have matured and initially begun to age

Fig. 7 Expression analysis of RmEXPB2A: Expressions of RmEXPB2 in different tissues of R. multiflora. B: Expressions of RmEXPB2 in the prickles at different developmental stages. C: RmEXPB2 expression levels in WT and RmEXPB2-OE (T1) Arabidopsis. D: RmEXPB2 expression levels in WT and RmEXPB2-OE (T2) Arabidopsis. Stage I: Starting period of R. multiflora prickles; Stage II: rapid growth period prickles; Stage III: transition period prickles; Stage IV: hardening period prickles; Stage V: maturation period prickles; line 1-10 indicates transgenic line 1‒10, WT indicates wild type. ns indicates P>0.05; * indicates P<0.05; ** indicates P<0.01; *** indicates P<0.001. The same below

Fig. 8 Leaf phenotypes of RmEXPB2-OE (T2) Arabidopsis plantsA: True leaf phenotypic diagram. B: Number of true leaves. n>20. C: The 1st, 3rd, 5th, 6th, 8th leaf length, width and area of the true leaf. Line1, 5, 6 indicates transgenic line1, 5, 6, WT indicates wild type. The same below

Fig. 9 Root phenotypic analysis of RmEXPB2-OE (T2) Arabidopsis plantsA: Roots of WT and RmEXPB2-OE (T2) transgenic Arabidopsis. B: Root hairs of WT and RmEXPB2-OE (T2) Arabidopsis. C‒D: The main root length and hypocotyl length of WT and RmEXPB2-OE (T2) transgenic Arabidopsis. E‒F: The root hair length and root hair density of RmEXPB2-OE (T2) Arabidopsis. n>20

References 36

[1]	Nakamura N, Hirakawa H, Sato S, et al. Genome structure of Rosa multiflora, a wild ancestor of cultivated roses [J]. DNA Res, 2018, 25(2): 113-121.
[2]	Zhang Y, Zhao MJ, Zhu W, et al. Nonglandular prickle formation is associated with development and secondary metabolism-related genes in Rosa multiflora [J]. Physiol Plant, 2021, 173(3): 1147-1162.
[3]	McQueen-Mason S, Durachko DM, Cosgrove DJ. Two endogenous proteins that induce cell wall extension in plants [J]. Plant Cell, 1992, 4(11): 1425-1433.
[4]	Arslan B, İncili ÇY, Ulu F, et al. Comparative genomic analysis of expansin superfamily gene members in zucchini and cucumber and their expression profiles under different abiotic stresses [J]. Physiol Mol Biol Plants, 2021, 27(12): 2739-2756.
[5]	Cosgrove DJ. Plant cell wall loosening by expansins [J]. Annu Rev Cell Dev Biol, 2024, 40(1): 329-352.
[6]	Wang ZZ, Cao JB, Lin N, et al. Origin, evolution, and diversification of the expansin family in plants [J]. Int J Mol Sci, 2024, 25(21): 11814.
[7]	Kök BÖ, Celik Altunoglu Y, Öncül AB, et al. Expansin gene family database: a comprehensive bioinformatics resource for plant expansin multigene family [J]. J Bioinform Comput Biol, 2023, 21(3): 2350015.
[8]	Liu WM, Lyu TQ, Xu LA, et al. Complex molecular evolution and expression of expansin gene families in three basic diploid species of Brassica [J]. Int J Mol Sci, 2020, 21(10): 3424.
[9]	Goh HH, Sloan J, Dorca-Fornell C, et al. Inducible repression of multiple expansin genes leads to growth suppression during leaf development [J]. Plant Physiol, 2012, 159(4): 1759-1770.
[10]	Yan A, Wu MJ, Yan LM, et al. AtEXP2 is involved in seed germination and abiotic stress response in Arabidopsis [J]. PLoS One, 2014, 9(1): e85208.
[11]	Feng X, Li CT, He FM, et al. Genome-wide identification of expansin genes in wild soybean (Glycine soja) and functional characterization of Expansin B1 (GsEXPB1) in soybean hair root [J]. Int J Mol Sci, 2022, 23(10): 5407.
[12]	Liu WM, Xu LA, Lin H, et al. Two expansin genes, AtEXPA4 and AtEXPB5, are redundantly required for pollen tube growth and AtEXPA4 is involved in primary root elongation in Arabidopsis thaliana [J]. Genes, 2021, 12(2): 249.
[13]	Jiang FL, Lopez A, Jeon S, et al. Disassembly of the fruit cell wall by the ripening-associated polygalacturonase and expansin influences tomato cracking [J]. Hortic Res, 2019, 6: 17.
[14]	Calderini DF, Castillo FM, Arenas-M A, et al. Overcoming the trade-off between grain weight and number in wheat by the ectopic expression of expansin in developing seeds leads to increased yield potential [J]. New Phytol, 2021, 230(2): 629-640.
[15]	Dai FW, Zhang CQ, Jiang XQ, et al. RhNAC2 and RhEXPA4 are involved in the regulation of dehydration tolerance during the expansion of rose petals [J]. Plant Physiol, 2012, 160(4): 2064-2082.
[16]	Lü PT, Kang M, Jiang XQ, et al. RhEXPA4, a rose expansin gene, modulates leaf growth and confers drought and salt tolerance to Arabidopsis [J]. Planta, 2013, 237(6): 1547-1559.
[17]	Raymond O, Gouzy J, Just J, et al. The Rosagenome provides new insights into the domestication of modern roses [J]. Nat Genet, 2018, 50(6): 772-777.
[18]	Han Y, Yu JY, Zhao T, et al. Dissecting the genome-wide evolution and function of R2R3-MYB transcription factor family in Rosa chinensis [J]. Genes, 2019, 10(10): 823.
[19]	由玉婉, 张雨, 孙嘉毅, 等. ‘月月粉’月季NAC家族全基因组鉴定及皮刺发育相关成员的筛选 [J]. 中国农业科学, 2022, 55(24): 4895-4911.
	You YW, Zhang Y, Sun JY, et al. Genome-wide identification of NAC family and screening of its members related to prickle development in Rosa chinensis old blush [J]. Sci Agric Sin, 2022, 55(24): 4895-4911.
[20]	Chen CJ, Chen H, Zhang Y, et al. TBtools: an integrative toolkit developed for interactive analyses of big biological data [J]. Mol Plant, 2020, 13(8): 1194-1202.
[21]	Hepler NK, Bowman A, Carey RE, et al. Expansin gene loss is a common occurrence during adaptation to an aquatic environment [J]. Plant J, 2020, 101(3): 666-680.
[22]	Krishnamurthy P, Hong JK, Kim JA, et al. Genome-wide analysis of the expansin gene superfamily reveals Brassica rapa-specific evolutionary dynamics upon whole genome triplication [J]. Mol Genet Genomics, 2015, 290(2): 521-530.
[23]	Liu XP, Dong SY, Miao H, et al. Genome-wide analysis of expansins and their role in fruit spine development in cucumber (Cucumis sativus L.) [J]. Hortic Plant J, 2022, 8(6): 757-768.
[24]	Sampedro J, Carey RE, Cosgrove DJ. Genome histories clarify evolution of the expansin superfamily: new insights from the poplar genome and pine ESTs [J]. J Plant Res, 2006, 119(1): 11-21.
[25]	Zhang W, Yan HW, Chen WJ, et al. Genome-wide identification and characterization of maize expansin genes expressed in endosperm [J]. Mol Genet Genomics, 2014, 289(6): 1061-1074.
[26]	Qiu DY, Xu SL, Wang Y, et al. Primary cell wall modifying proteins regulate wall mechanics to steer plant morphogenesis [J]. Front Plant Sci, 2021, 12: 751372.
[27]	Ono K. Signal peptides and their fragments in post-translation: novel insights of signal peptides [J]. Int J Mol Sci, 2024, 25(24): 13534.
[28]	Zhang JL, Dong TT, Zhu MK, et al. Transcriptome- and genome-wide systematic identification of expansin gene family and their expression in tuberous root development and stress responses in sweetpotato (Ipomoea batatas) [J]. Front Plant Sci, 2024, 15: 1412540.
[29]	Lou Y, Zhou HS, Han Y, et al. Positive regulation of AMS by TDF1 and the formation of a TDF1-AMS complex are required for anther development in Arabidopsis thaliana [J]. New Phytol, 2018, 217(1): 378-391.
[30]	Zhou NN, Simonneau F, Thouroude T, et al. Morphological studies of rose prickles provide new insights [J]. Hortic Res, 2021, 8(1): 221.
[31]	Swarnkar MK, Kumar P, Dogra V, et al. Prickle morphogenesis in rose is coupled with secondary metabolite accumulation and governed by canonical MBW transcriptional complex [J]. Plant Direct, 2021, 5(6): e00325.
[32]	Saint-Oyant LH, Ruttink T, Hamama L, et al. A high-quality genome sequence of Rosa chinensis to elucidate ornamental traits [J]. Nat Plants, 2018, 4(7): 473-484.
[33]	Xing SC, Li F, Guo QF, et al. The involvement of an expansin gene TaEXPB23 from wheat in regulating plant cell growth [J]. Biol Plant, 2009, 53(3): 429-434.
[34]	Zou HY, Wenwen YH, Zang GC, et al. OsEXPB2, a β-expansin gene, is involved in rice root system architecture [J]. Mol Breed, 2015, 35(1): 41.
[35]	Lin CF, Choi HS, Cho HT. Root hair-specific EXPANSIN A7 is required for root hair elongation in Arabidopsis [J]. Mol Cells, 2011, 31(4): 393-398.
[36]	Chen SK, Luo YX, Wang GJ, et al. Genome-wide identification of expansin genes in Brachypodium distachyon and functional characterization of BdEXPA27 [J]. Plant Sci, 2020, 296: 110490.