参薯DaKNOX16基因的特征分析及互作蛋白的筛选验证

Characterization of DaKNOX16 gene and screening and verification of its interacting proteins in Dioscorea alata

摘要

KNOX（KNOTTED1-like HOMEOBOX）转录因子参与植物器官的形态建成，调控植物的生长发育。本研究通过参薯不同组织的表达谱、同源比对等方法鉴定参薯块茎特异表达 DaKNOX16 基因，并通过生信分析和酵母双杂交实验，研究该基因的功能。通过 HMM search 和 BLAST 分析，共鉴定到 12 个参薯 KNOX 基因，与水稻、拟南芥、马铃薯 KNOX 基因构建系统发育树，发现 DaKNOX16 与拟南芥 AtSTM 亲缘关系较近。参薯转录谱数据表明 12 个 KNOX 基因中，DaKNOX16 具有块茎高表达的特征。DaKNOX16 包含保守的 KNOX1、KNOX2、Homeobox KN 和 ELK 结构域，是由 α−螺旋和无规则卷曲组成的一个酸性蛋白。通过酵母双杂交筛库和酵母点对点互作验证，发现DaKNOX16 可以与转录因子 D4ZF-HD 互作。本研究推测该基因可能通过与转录因子 D4ZF-HD 互作发挥功能。

Abstract

KNOX (KNOTTED1-like HOMEOBOX) transcription factors are involved in the morphogenesis of plant organs and regulate plant growth and development. The specific expression of DaKNOX16 gene in the tubers of Dioscorea alata was identified by the expression profiles of different tissues ofDioscorea and homologous alignment, and the function of the gene was analyzed by bioinformatics analysis and yeast two-hybrid experiments. A total of 12 Dioscorea KNOX genes were identified by HMMsearch and BLAST analysis. The phylogenetic tree was constructed with KNOX genes of rice, Arabidopsis and potato, and it was found that DaKNOX16 was closely related to Arabidopsis AtSTM. Transcriptome data showed that DaKNOX16 had the characteristics of high expression in tubers among the 12 KNOX genes. DaKNOX16 contains conserved KNOX1, KNOX2, Homeobox KN and ELK domains and is an acidic protein composed of ɑ-helix and random coil. Through yeast two-hybrid screening and yeast point-to-point interaction verification, it was found that DaKNOX16 could interact with the transcription factor D4ZF-HD. It is hence speculated that this gene may exert its function through interaction with the transcription factor D4ZF-HD, which provides an important theoretical basis for further understanding of the regulation of tuber development by KNOX genes.

关键词

参薯 / DaKNON16 / 特征分析 / 酵母双杂交

Key words

Dioscorea alata / DaKNON16 / characteristic analysis / yeast two-hybrid

参考文献

[1] Bredeson J V, Lyons J B, Oniyinde I O, et al. Chromo- some evolution and the genetic basis of agronomically important traits in greater yam[J]. Nature Communica- tions, 2022, 13(1): 2001. https://doi.org/10.1038/s41467- 022-29114-w

[2] Li J, Liang Q, Li C F, et al. Comparative transcriptome analysis identifies putative genes involved in dioscin bio- synthesis in Dioscorea zingiberensis [J]. Molecules, 2018, 23(2): 454. https://doi.org/10.3390/molecules23020454

[3] Jia L C, Yang Z T, Shang L L, et al. Genome-wide identi- fication and expression analysis of the KNOX family and its diverse roles in response to growth and abiotic toler- ance in sweet potato and its two diploid relatives[J]. BMC Genomics, 2024, 25(1): 572. https://doi.org/10.1186/ s12864-024-10470-4

[4] Hay A, Tsiantis M. KNOX genes: versatile regulators of plant development and diversity[J]. Development, 2010, 137(19): 3153-3165. https://doi.org/10.1242/dev.030049

[5] Cao X W, Du Q W, Guo Y H, et al. Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis[J]. Molecular Plant, 2023, 16(9): 1445-1459. https://doi.org/10.1016/j.molp.2023.09.005

[6] Fang S C, Chen J C, Chang P Y, et al. Co-option of the SHOOT MERISTEMLESS network regulates protocorm- like body development in Phalaenopsis aphrodite [J]. Plant Physiology, 2022, 190(1): 127-145. https://doi.org/10. 1093/plphys/kiac100

[7] Yang Q Q, Cong T C, Yao Y C, et al. KNOX genes were involved in regulating axillary bud formation of Chrysan- themum × morifolium [J]. International Journal of Mo- lecular Sciences, 2023, 24(8): 7081. https://doi.org/10. 3390/ijms24087081

[8] Rosin F M, Hart J K, Horner H T, et al. Overexpression of a Knotted-like homeobox gene of potato alters vegetat- ive development by decreasing gibberellin accumulation [J]. Plant Physiology, 2003, 132(1): 106-117. https://doi. org/10.1104/pp.102.015560

[9] Chen H, Banerjee A K, Hannapel D J. The tandem com- plex of BEL and KNOX partners is required for transcrip- tional repression of ga20ox1 [J]. The Plant Journal, 2004, 38(2): 276-284. https://doi.org/10.1111/j.1365-313X. 2004.02048.x

[10] Di Giacomo E, Laffont C, Sciarra F, et al. KNAT3/4/5- like class 2 KNOX transcription factors are involved in Medicago truncatula symbiotic nodule organ develop- ment [J]. New Phytologist, 2017, 213(2): 822-837. https: //doi.org/10.1111/nph.14146

[11] Sheng M H, Ma X L, Wang J Y, et al. KNOX II tran- scription factor HOS59 functions in regulating rice grain size[J]. The Plant Journal, 2022, 110(3): 863-880. https: //doi.org/ 10.1111/tpj.15709

[12] Wang S G, Yang H L, Mei J S, et al. Rice homeobox protein KNAT7 integrates the pathways regulating cell expansion and wall stiffness[J]. Plant Physiology, 2019, 181(2): 669-682. https://doi.org/10.1104/pp.19.00639

[13] Magnani E, Hake S. KNOX lost the OX: the Arabidopsis KNATM gene defines a novel class of KNOX transcrip- tional regulators missing the homeodomain [J]. The Plant Cell, 2008, 20(4): 875-887. https://doi.org/10.1105/tpc. 108.058495

[14] Mahajan A, Bhogale S, Kang I H, et al. The mRNA of a Knotted1-like transcription factor of potato is phloem mobile[J]. Plant Molecular Biology, 2012, 79(6): 595-608. https://doi.org/10.1007/s11103-012-9931-0

[15] Lin T, Sharma P, Gonzalez D H, et al. The impact of the long-distance transport of a BEL1-like messenger RNA on development [J]. Plant Physiology, 2013, 161(2): 760-772. https://doi.org/10.1104/pp.112.209429

[16] 叶明辉, 赵朋, 牛洋, 等. 马铃薯同源异形框基因家族 的鉴定和表达分析[J]. 农业生物技术学报, 2021, 29 (2): 224-239. https://doi.org/10.3969/j.issn.1674-7968. 2021.02.003

[17] Long J A, Moan E I, Medford J I, et al. A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis [J]. Nature, 1996, 379(6560): 66-69. https://doi.org/10.1038/379066a0

[18] LONG J, BARTON M K. Initiation of axillary and flor- al meristems in Arabidopsis [J]. Developmental Biology, 2000, 218(2): 341-353. https://doi.org/10.1006/dbio. 1999.9572

[19] Nidhi S, Preciado J, Tie L. Knox homologs shoot mer- istemless (STM) and KNAT6 are epistatic to CLAV- ATA3 (CLV3) during shoot meristem development in Arabidopsis thaliana [J]. Molecular Biology Reports, 2021, 48(9): 6291-6302. https://doi.org/10.1007/ s11033-021-06622-4

[20] Zhao H, Wang Y T, Zhao S, et al. HOMEOBOX PRO- TEIN 24 mediates the conversion of indole-3-butyric acid to indole-3-acetic acid to promote root hair elonga- tion[J]. New Phytologist, 2021, 232(5): 2057-2070. ht- tps://doi.org/ 10.1111/nph.17719

[21] Ferela A, Debernardi J M, Rosatti S, et al. Interplay among ZF-HD and GRF transcription factors during Ar- abidopsis leaf development[J]. Plant Physiology, 2023, 191(3): 1789-1802. https://doi.org/10.1093/plphys/ki- ad009

[22] Sánchez-gerschon V, Martínez-fernández I, González- bermúdez M R, et al. Transcription factors HB21/40/53 trigger inflorescence arrest through abscisic acid accu- mulation at the end of flowering[J]. Plant Physiology, 2024, 195(4): 2743-2756. https://doi.org/10.1093/plphys/ kiae234

[23] Lee Y K, Kumari S, Olson A, et al. Role of a ZF-HD transcription factor in miR157-mediated feed-forward regulatory module that determines plant architecture in Arabidopsis [J]. International Journal of Molecular Sci- ences, 2022, 23(15): 8665. https://doi.org/10.3390/ ijms23158665

[24] Li J Y, Li M C, Shen T T, et al. Molecular characteriza- tion of cassava zinc finger-homeodomain (ZF-HD) tran- scription factors reveals their role in disease resistance[J]. International Journal of Biological Macro- molecules, 2024, 279(Pt1): 134846. https://doi.org/10. 1016/j.ijbiomac.2024.134846

[25] Xu Y, Wang Y, Long Q, et al. Overexpression of Os- ZHD1, a zinc finger homeodomain class homeobox tran- scription factor, induces abaxially curled and drooping leaf in rice [J]. Planta, 2014, 239(4): 803-816. https:// doi.org/10.1007/s00425-013-2009-7

基金资助

国家自然科学基金项目(32160470)