芦笋DUF247基因家族成员鉴定及表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0596

摘要/Abstract

摘要：

【目的】对芦笋全基因组DUF247家族进行鉴定与分析，为该基因家族特别是AoSOFF的性别决定的分子功能研究奠定基础。【方法】利用生物信息学方法对芦笋中的DUF247基因家族成员进行鉴定，开展其各成员编码蛋白理化性质、聚类、蛋白保守结构域、启动子中顺式作用元件及表达模式分析；利用实时定量PCR（RT-qPCR）和组织原位杂交技术验证芦笋性别决定基因AoSOFF的表达模式。【结果】芦笋中鉴定出24个DUF247基因家族成员，它们不均匀地分布在7条染色体上，且在不同器官中呈现差异表达。该家族成员编码蛋白的理化性质如氨基酸组成、分子量、理论等电点、不稳定系数和脂肪族系数等都存在差异。RT-qPCR和组织原位杂交结果表明，DUF247基因家族成员之一AoSOFF在芦笋雄花无性别分化早期的雌蕊、雄蕊以及花冠中均有较高表达，但在性别分化早期开始降低表达；在两性花中的表达量较低；而在雌性芦笋各组织中表达极低或检测不到，说明AoSOFF在雄性芦笋中特异性表达，与芦笋性别决定中的雌性抑制功能密切相关。【结论】芦笋DUF247家族成员具有明显的保守性，在芦笋不同性别的不同组织中的表达模式存在显著差异，特别是AoSOFF是一个雄性专一性基因，其同源基因参与植物的自交亲和调控，预示其以自交不亲和类似的反应导致雌性发育的抑制。

关键词: 芦笋, DUF247, AoSOFF, 性别分化, 雄性专一性, 雌性抑制, 原位杂交

Abstract:

【Objective】To establish a foundation for the molecular functional study of this gene family, particularly AoSOFF's role in sex determination, a comprehensive identification and analysis of the A. officinalis DUF247 family was conducted.【Method】The bioinformatics methods were used to identify the members of the DUF247 gene family in A. officinalis, and conducted analyses on their encoded protein physicochemical properties, clustering, protein conserved domains, cis-acting elements in the promoter, and expression patterns. Real-time quantitative PCR（RT-qPCR）and tissue in situ hybridization were used to validate the expression pattern of the A. officinalis sex determination gene AoSOFF.【Result】The 24 members of the DUF247 family were unevenly distributed on 7 chromosomes of A. officinalis, and showed differential expressions in various organs. The encoded proteins of these family members had differences in their physicochemical properties, including amino acid composition, molecular weight, theoretical isoelectric point, instability index, and aliphatic index. RT-qPCR and tissue in situ hybridization results demonstrated that AoSOFF had high expressions in the pistil, stamen, and corolla of male flowers during the early stage of sex differentiation but decreased its expression as sex differentiation progressed. Its expression in hermaphrodite flowers was low, and extremely low or undetectable in the female flowers, indicating that AoSOFF had male-specific expression in male asparagus, which was closely related to its female inhibition function in A. officinalis sex determination.【Conclusion】The members of the A. officinalis DUF247 family have obvious conservation and exhibit significantly different expression patterns in different tissues of A. officinalis of different sexes. In particular, AoSOFF is a male-specific gene, and its homologs are involved in regulating plant self-incompatibility, suggesting that its similar reaction to self-incompatibility may lead to the inhibition of female development.

Key words: Asparagus officinalis, DUF247, AoSOFF, sex differentiation, male specificity, female inhibition, in situ hybridization

李云宾, 黎玉萍, 成钦, 林春, 毛自朝, 刘正杰. 芦笋DUF247基因家族成员鉴定及表达分析[J]. 生物技术通报, 2025, 41(1): 198-209.

LI Yun-bin, LI Yu-ping, CHENG Qin, LIN Chun, MAO Zi-chao, LIU Zheng-jie. Identification and Expression Analysis of the Members of the Asparagus officinalis DUF247 Gene Family[J]. Biotechnology Bulletin, 2025, 41(1): 198-209.

图/表 8

图1 芦笋、文竹、大理天门冬和拟南芥DUF247蛋白系统进化树 V1：芦笋；OF：文竹；Ata：大理天门冬；AT：拟南芥

Fig. 1 Phylogenetic tree of DUF247 protein from A. officinalis, A. setaceus, A. taliensis and A. thaliana V1: A. officinalis; OF: A. setaceus; Ata: A. taliensis; AT: A. thaliana

图2 芦笋DUF247家族启动子顺式作用元件（A）及编码蛋白保守基序图（B）

Fig. 2 Cis-acting elements (A) and conserved motifs map (B) of encoded proteins of DUF247 family genes in A. officinalis

图3 芦笋DUF247家族基因染色体分布（A）以及共线性分析（B）

Fig. 3 Chromosome distribution（A）and collinearity analysis（B）of DUF247 family genes in A. officinalis

图4 芦笋DUF247家族基因成员不同性别和不同组织器官的表达量热图 MR：雄根；MS：雄茎；MF：雄花；FR：雌根；FS：雌茎；FF：雌花；HR：两性根；HS：两性茎；HF：两性花；R1、R2和R3表示为样品的3个重复；下同

Fig. 4 Heatmaps of expressions of gene members of A. officialis DUF247 family in different genders and tissues or organs MR: Male root; MS: male stem; MF: male flower; FR: female root; FS: female stem; FF: female flower; HR: hermaphrodite root; HS: hermaphrodite stem; HF: hermaphrodite flower; R1, R2, and R3 indicate the three replicates of the sample. The same below

图5 V1_01.231, V1_05.1843和V1_05.2202在不同性别的早期花蕾的表达量 ffs：雌花的雄蕊；mfs：雄花的雄蕊；fft：雌花的心皮；mft：雄花的心皮；ffp：雌花的雌蕊；mfp：雄花的雌蕊。不同小写字母代表差异显著（P<0.05），下同

Fig. 5 Different expressions of V1_01.231, V1_05.1843, and V1_05.2202 in early development buds of different genders ffs: Female flower'stamen; mfs: male flower'stamen; fft: female flower'tapal; mft: male flower'tapal; ffp: female flower'pistil; mfp: male flower'pistil. Different lowercase letters indicatet significant differences（P<0.05）, the same below

图6 芦笋AoSOFF基因编码蛋白质一级结构比较及其三维结构预测 A：AoSOFF-M蛋白的三维结构；B：AoSOFF-F蛋白的三维结构；C：AoSOFF-H蛋白的三维结构；D：3个蛋白在一级结构上的比较，M为雄株，F为雌株，H为两性株

Fig. 6 Primary structure comparison and 3D structure prediction of protein encoded by AoSOFF gene in A. officinalis A: 3D structure of AoSOFF-M protein; B: 3D structure of AoSOFF-F protein; C: 3D structure of AoSOFF-H protein; D: comparison of three proteins in primary structure, M for male, F for female, and H for hermaphroditic

图7 芦笋AoSOFF在不同样本中的RT-qPCR分析

Fig. 7 RT-qPCR analysis of A. officinalis AoSOFF in different samples

图8 芦笋AoSOFF在雄花、雌花和两性花中的原位杂交检测 A-C：雄花原位杂交结果；D-F：雄花对照；G-I：两性花原位杂交结果；J-L：两性花对照；M-O：雌花原位杂交结果；P-R：雌花对照。I，I，III：花发育的3个时期。白色箭头：胚珠；蓝色箭头：子房；红色箭头：柱头；黄色箭头：花粉囊外壁；Bar=500 μm

Fig. 8 Detection of A. officinalis AoSOFF in male, female and hermaphroditic flowers by in situ hybridization A-C: Male flower; D-F: male flower control; G-I: hermaphroditic flower; J-L: hermaphroditic flower control; M-O: female flower in situ hybridization results; P-R: female flower control. I, II, and III: Three stages of flower development. White arrow: Ovule; blue arrow: ovary; red arrows: stigma; yellow arrow: outer wall of pollen sac. Bar=500 μm

参考文献 36

[1]	Mistry J, Chuguransky S, Williams L, et al. Pfam: the protein families database in 2021[J]. Nucleic Acids Res, 2021, 49(D1): D412-D419.
[2]	Temple H, Mortimer JC, Tryfona T, et al. Two members of the DUF579 family are responsible for arabinogalactan methylation in Arabidopsis[J]. Plant Direct, 2019, 3(2): e00117.
[3]	Li XJ, Sun LJ, Tan LB, et al. TH1, a DUF640 domain-like gene controls lemma and palea development in rice[J]. Plant Mol Biol, 2012, 78(4/5): 351-359.
[4]	Yan DW, Zhou Y, Ye SH, et al. BEAK-SHAPED GRAIN 1/TRIANGULAR HULL 1, a DUF640 gene, is associated with grain shape, size and weight in rice[J]. Sci China Life Sci, 2013, 56(3): 275-283.
[5]	罗璇, 林正雨, 陈章, 等. 玉米DUF1685基因家族的鉴定和进化分析[J]. 热带作物学报, 2022, 43(12): 2431-2442. doi: 10.3969/j.issn.1000-2561.2022.12.005
	Luo X, Lin ZY, Chen Z, et al. Identification and evolutionary analysis of maize DUF1685 gene family[J]. Chin J Trop Crops, 2022, 43(12): 2431-2442.
[6]	Shinozuka H, Cogan NOI, Smith KF, et al. Fine-scale comparative genetic and physical mapping supports map-based cloning strategies for the self-incompatibility loci of perennial ryegrass(Lolium perenne L.)[J]. Plant Mol Biol, 2010, 72(3): 343-355. doi: 10.1007/s11103-009-9574-y pmid: 19943086
[7]	Zhang XJ, Jia Y, Liu Y, et al. Challenges and perspectives in the study of self-incompatibility in orchids[J]. Int J Mol Sci, 2021, 22(23): 12901.
[8]	Liang M, Cao ZH, Zhu AD, et al. Evolution of self-compatibility by a mutant Sm-RNase in citrus[J]. Nat Plants, 2020, 6(2): 131-142. doi: 10.1038/s41477-020-0597-3 pmid: 32055045
[9]	Ma L, Zhang CZ, Zhang B, et al. A nonS-locus F-box gene breaks self-incompatibility in diploid potatoes[J]. Nat Commun, 2021, 12(1): 4142.
[10]	Cheung AY. Self-incompatibility in Papaver rhoeas: a role for ATP[J]. New Phytol, 2022, 236(5): 1625-1628.
[11]	Gervais C, Awad DA, Roze D, et al. Genetic architecture of inbreeding depression and the maintenance of gametophytic self-incompatibility[J]. Evolution, 2014, 68(11): 3317-3324. doi: 10.1111/evo.12495 pmid: 25065256
[12]	Li C, Long Y, Lu MQ, et al. Gene coexpression analysis reveals key pathways and hub genes related to late-acting self-incompatibility in Camellia oleifera[J]. Front Plant Sci, 2023, 13: 1065872.
[13]	Silva NF, Goring DR. Mechanisms of self-incompatibility in flowering plants[J]. Cell Mol Life Sci, 2001, 58(14): 1988-2007. doi: 10.1007/PL00000832 pmid: 11814052
[14]	Hiscock SJ, McInnis SM. Pollen recognition and rejection during the sporophytic self-incompatibility response: Brassica and beyond[J]. Trends Plant Sci, 2003, 8(12): 606-613. pmid: 14659710
[15]	Herridge R, McCourt T, Jacobs JME, et al. Identification of the genes at S and Z reveals the molecular basis and evolution of grass self-incompatibility[J]. Front Plant Sci, 2022, 13: 1011299.
[16]	Lian XP, Zhang SL, Huang GF, et al. Confirmation of a gametophytic self-incompatibility in Oryza longistaminata[J]. Front Plant Sci, 2021, 12: 576340.
[17]	Harkess A, Zhou JS, Xu CY, et al. The asparagus genome sheds light on the origin and evolution of a young Y chromosome[J]. Nat Commun, 2017, 8(1): 1279. doi: 10.1038/s41467-017-01064-8 pmid: 29093472
[18]	刘江, 刘刚, 曾益春, 等. 桑树GATA基因家族的全基因组鉴定与分析[J]. 西南农业学报, 2022, 35(12): 2724-2731.
	Liu J, Liu G, Zeng YC, et al. Genome-wide identification and analysis of mulberry(Morus notabilis)GATA gene family[J]. Southwest China J Agric Sci, 2022, 35(12): 2724-2731.
[19]	陈曙, 张彧, 陈卓, 等. 玉米泛素连接酶U-box基因家族的全基因组鉴定及表达分析[J]. 西南农业学报, 2022, 35(3): 481-490.
	Chen S, Zhang Y, Chen Z, et al. Genome-wide identification and expression analysis of ubiquitin ligase U-box gene family in maize[J]. Southwest China J Agric Sci, 2022, 35(3): 481-490.
[20]	Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput[J]. Nucleic Acids Res, 2004, 32(5): 1792-1797. doi: 10.1093/nar/gkh340 pmid: 15034147
[21]	Stamatakis A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[J]. Bioinformatics, 2014, 30(9): 1312-1313. doi: 10.1093/bioinformatics/btu033 pmid: 24451623
[22]	Letunic I, Bork P. Interactive Tree Of Life(iTOL): an online tool for phylogenetic tree display and annotation[J]. Bioinformatics, 2007, 23(1): 127-128. doi: 10.1093/bioinformatics/btl529 pmid: 17050570
[23]	Bailey TL, Williams N, Misleh C, et al. MEME: discovering and analyzing DNA and protein sequence motifs[J]. Nucleic Acids Res, 2006, 34(Web Server issue): W369-W373.
[24]	Hu B, Jin JP, Guo AY, et al. GSDS 2.0: an upgraded gene feature visualization server[J]. Bioinformatics, 2015, 31(8): 1296-1297.
[25]	Wang YP, Li JP, Paterson AH. MCScanX-transposed: detecting transposed gene duplications based on multiple colinearity scans[J]. Bioinformatics, 2013, 29(11): 1458-1460. doi: 10.1093/bioinformatics/btt150 pmid: 23539305
[26]	Wen GZ. A simple process of RNA-sequence analyses by Hisat2, htseq and DESeq2[C]// Proceedings of the 2017 International Conference on Biomedical Engineering and Bioinformatics. Bangkok Thailand. New York: ACM, 2017: 11.
[27]	Liao Y, Smyth GK, Shi W. featureCounts: an efficient general purpose program for assigning sequence reads to genomic features[J]. Bioinformatics, 2014, 30(7): 923-930. doi: 10.1093/bioinformatics/btt656 pmid: 24227677
[28]	Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform[J]. Bioinformatics, 2009, 25(14): 1754-1760. doi: 10.1093/bioinformatics/btp324 pmid: 19451168
[29]	Robinson JT, Thorvaldsdóttir H, Winckler W, et al. Integrative genomics viewer[J]. Nat Biotechnol, 2011, 29(1): 24-26. doi: 10.1038/nbt.1754 pmid: 21221095
[30]	Delano W L. Pymol: An open-source molecular graphics tool[J]. Ccp4 Newslett Protein Crystallogr, 2002, 40(1): 82-92.
[31]	Waterhouse AM, Procter JB, Martin DMA, et al. Jalview Version 2—a multiple sequence alignment editor and analysis workbench[J]. Bioinformatics, 2009, 25(9): 1189-1191. doi: 10.1093/bioinformatics/btp033 pmid: 19151095
[32]	谷倩楠, 孔瑞文, 孙明哲, 等. 大豆混合盐碱胁迫应答基因GmDUF247-1的克隆及功能分析[J]. 植物遗传资源学报, 2024, 25(6): 978-989.
	Gu QN, Kong RW, Sun MZ, et al. Cloning and functional characterization of the GmDUF247-1 gene in soybean response to saline-alkaline stress[J]. J Plant Genet Resour, 2024, 25(06): 978-989.
[33]	Zhang FF, Liu YX, Liu F, et al. Genome-wide characterization and analysis of rice DUF247 gene family[J]. BMC Genomics, 2024, 25(1): 613.
[34]	Manzanares C, Barth S, Thorogood D, et al. A gene encoding a DUF247 domain protein cosegregates with the S self-incompatibility locus in perennial ryegrass[J]. Mol Biol Evol, 2016, 33(4): 870-884. doi: 10.1093/molbev/msv335 pmid: 26659250
[35]	Shin NR, Shin YH, Kim HS, et al. Function analysis of the PR55/B gene related to self-incompatibility in Chinese cabbage using CRISPR/Cas9[J]. Int J Mol Sci, 2022, 23(9): 5062.
[36]	Sun LH, Cao SY, Zheng N, et al. Analyses of Cullin1 homologs reveal functional redundancy in S-RNase-based self-incompatibility and evolutionary relationships in eudicots[J]. Plant Cell, 2023, 35(2): 673-699.

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