菠萝AcKNOX1影响叶缘发育的功能研究

doi:10.16420/j.issn.0513-353x.2024-0304

摘要/Abstract

摘要：

以菠萝‘台农16’为材料，克隆得到基因AcKNOX1。AcKNOX1是同源异型盒基因，属于3个氨基酸环延伸蛋白超家族。AcKNOX1的开放阅读框为990 bp，编码329个氨基酸，编码蛋白定位于细胞核中。AcKNOX1启动子区域除核心启动子元件TATA-box和CAAT-box外，还包含许多与光响应、激素响应和非生物胁迫响应相关的元件。AcKNOX1在不同组织中均有表达，但具有明显的组织表达特异性，在茎的表达量最高，其次是叶片，且有刺叶片显著高于无刺叶片中。AcKNOX1响应GA₃、ABA和6-BA信号诱导其表达上调。异源表达拟南芥，叶缘裂刻加深。利用酵母单双杂进行互作蛋白筛选，发现AcKNOX1可以与AcCUC1和AcCUC2启动子区域结合激活其表达；与AcBLH1、AcBLH4可形成异源二聚体，在细胞核中互作，且荧光素酶互补和双分子荧光互补实验验证了其互作的准确性。研究结果表明AcKNOX1在菠萝叶缘发育中起重要作用。

关键词: 菠萝, 叶缘发育, AcKNOX1, 蛋白互作

Abstract:

In this study，AcKNOX1 was cloned from pineapple‘Tainong 16’，which is a homeobox gene，belongs to the three amino acid loop extension superfamily. The gene’s open reading frame was 990 bp with 329 amino acids. The protein was localized in the nucleus. In addition to the core promoter elements TATA-box and CAAT-box，the AcKNOX1 promoter region contained many elements related to light，hormonal and abiotic stress responses. AcKNOX1 was expressed in different tissues with the highest expression in the stem，followed by leaves，which suggested a certain tissue specificity. AcKNOX1 showed higher expression in spiny leaf materials than in spineless leaf materials. The expression of AcKNOX1 was up-regulated by short-term GA₃，ABA，and 6-BA stress. Ectopic overexpression of AcKNOX1 in Arabidopsis thaliana suggested that transformants showed larger and deeper serrations. Analysis of interaction proteins obtained by Yeast one and two hybrid system showed that AcKNOX1 could interact and activate AcCUC1 and AcCUC2 promoter regions. And AcKNOX1 interacted with AcBLH1 and AcBLH4 to form a heterodimer. And the inteaction was verified by Luciferase complementation imaging assay（LCI）and Bimolecular fluorescence complementation（BIFC）. The results revealed that AcKNOX1 gene played a vital role in the regulation of pineapple leaf margin development.

Key words: Ananas comosus, leaf margin development, AcKNOX1, protein-interation

荆永琳, 陈浪欣, 李俊国, 王加宾, 王小冰, 杨庆全, 孟春阳, 徐立. 菠萝AcKNOX1影响叶缘发育的功能研究[J]. 园艺学报, 2025, 52(2): 292-308.

JING Yonglin, CHEN Langxin, LI Junguo, WANG Jiabin, WANG Xiaobing, YANG Qingquan, MENG Chunyang, XU Li. Functional Studies of Pineapple AcKNOX1 on Leaf Margin Development[J]. Acta Horticulturae Sinica, 2025, 52(2): 292-308.

图/表 15

表1 AcKNOX1克隆与表达分析所用引物

Table 1 Primers for cloning and expression analysis of AcKNOX1

用途 Application	序列名称 Sequence name	上游引物序列（5′-3′） Forward primer	下游引物序列（5′-3′） Reverse primer
AcKNOX1克隆 Cloning of AcKNOX1	AcKNOX1	CCCTTTCCCAAAACCCTACC	CGCCTTCATCGATTGATCGT
AcKNOX1克隆 Cloning of AcKNOX1	AcKNOX1+	GGGGTACCCCCTTTCCCAAAACCCTACC	GCTCTAGACGCCTTCATCGATTGATCGT
亚细胞定位 Subcellular localization	pBI221-AcKNOX1	GACTCTAGAGGATCCATGGATGAGCAACTTTACCAAGTTA	CTCCGCGGCGGATCCTGCGCTTCTTGGTCACATCTATCG
启动子克隆 Cloning of promoter	AcKNOX1-promoter	CAAAATAATGAAATCTATGGCCAAA	AAAAGGAAACCACACCCGAAA
qRT-PCR	qPCR-AcKNOX1	TGCCGTTTTGATGTTCTCACTAAG	CGATTGATCGTATACCCAGTTTG

图1 AcKNOX1的扩增

Fig. 1 Amplification of the AcKNOX1

图2 AcKNOX1与拟南芥（At）KNOX蛋白氨基酸多序列比对

Fig. 2 Multiple sequence alignment of AcKNOX1 and KNOX proteins from Arabidopsis thaliana（At）

图3 AcKNOX1蛋白的进化分析 Ac：菠萝；At：拟南芥；Os：水稻；Pt：毛果杨；Zm：玉米

Fig. 3 Analysis of phylogenetic of AcKNOX1 Ac：Ananas comosus；At：Arabidopsis thaliana；Os：Oryza sativa；Pt：Populus trichocarpa；Zm：Zea mays

图4 AcKNOX1基因启动子PCR扩增

Fig. 4 PCR amplification of AcKNOX1 promoter

图5 AcKNOX1在菠萝‘台农16’不同组织中的表达分析不同小写字母表示具有显著性差异（P < 0.05）

Fig. 5 Expression analysis of AcKNOX1 in different tissues of pineapple‘Tainong 16’ Different lowercase indicate significant differences（P < 0.05）

图6 AcKNOX1在外源GA3、ABA和6-BA处理的菠萝组培苗中的表达量 * 表示在同一时间处理与对照在0.05水平差异显著

Fig. 6 Expression level of AcKNOX1 in pineapple seedlings after exogenous GA3，ABA and 6-BA treatments * Indicates a significant difference between treatment and control at the 0.05 level at the same time

图7 AcKNOX1蛋白在洋葱表皮细胞中的亚细胞定位

Fig. 7 Subcellular localization of AcKNOX1 protein in onion epidermal cells

图8 AcKNOX1转基因拟南芥分子鉴定 1：阳性对照；2：野生型拟南芥；3 ~ 5：转基因拟南芥

Fig. 8 Molecular identification of transgenic Arabidopsis plants 1：Positive control；2：Wild type；3-5：Transgenic Arabidopsis

图9 拟南芥野生型（WT）、转空载体（Vector）和转基因（AcKNOX1-OE）株系表型 A：子叶叶缘形态，红色三角形指缺口；B：子叶下胚轴，白色三角指下胚轴与根交界的位置；C：下胚轴长度；D：抽薹时叶片表型；E：第6枚叶片表型；F：第6枚叶片锯齿数；G：结荚后叶片表型。L1、L2、L3和L4为4个转基因株系

Fig. 9 The phenotype of wild-type（WT），trans-empty vector（Vector）and transgenic（AcKNOX1-OE）lines of Arabidopsis A：The leaf margin of cotyledon，the red triangle shows serration at the leaf margin；B：The hypocotyl of cotyledon，the white triangles indicate the junction of the hypocotyl and root；C：The hypocotyl length；D：The leaf phenotype during bolting；E：The phenotype of the sixth rosette leaf；F：Quantification of serration in the sixth rosette leaf；G：The leaf phenotype after podding. L1，L2，L3，L4 indicate four lines of transgenic plants

图10 酵母单杂交实验验证AcKNOX1与AcCUC启动子结合阴性对照：pAbAi-AcCUCpro + pGADT7，pAbAi + pGADT7

Fig. 10 Yeast one-hybrid assay of AcKNOX1 to the promoters of AcCUC Negative control：pAbAi-AcCUCpro + pGADT7，pAbAi + pGADT7

图11 双荧光素酶检测AcKNOX激活AcCUC启动子

Fig. 11 AcKNOX activates the AcCUC promoters in dual-luciferase assays

图12 酵母双杂交实验验证AcKNOX1与AcBLH1和AcBLH4互作

Fig. 12 Interaction between AcKNOX1 and AcBLH1，AcBLH4 validated by yeast two-hybrid experiments

图13 AcKNOX1蛋白与AcBLH1的BIFC互作验证

Fig. 13 BIFC assay between AcKNOX1 and AcBLH1

图14 AcKNOX1蛋白与AcBLH4的BIFC互作验证

Fig. 14 BIFC assay between AcKNOX1 and AcBLH4

参考文献 44

[1]	Bar M, Ori N. 2015. Compound leaf development in model plant species. Current Opinion in Plant Biology,23:61-69.
[2]	Bellaoui M, Pidkowich M S, Samach A, Kushalappa K, Kohalmi S E, Modrusan Z, Crosby W L, Haughn G W. 2001. The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals. Plant Cell,13:2455-2470.
[3]	Bharathan G. 2002. Homologies in leaf form inferred from KNOXI gene expression during development. Science, 296 (5574):1858-1860. doi: 10.1126/science.1070343 pmid: 12052958
[4]	Bilsborough G D, Runions A, Barkoulas M, Jenkins H W, Hasson A, Galinha C, Laufs P, Hay A, Prusinkiewicz P, Tsiantis M. 2011. Model for the regulation of Arabidopsis thaliana leaf margin development. Proceedings of the National Academy of Sciences, 108 (8):3424-3429.
[5]	Bolduc N, Hake S. 2009. The maize transcription factor KNOTTED 1 directly regulates the gibberellin catabolism gene Ga2ox1. Plant Cell,21:1647-1658.
[6]	Byrne M E, Barley R, Curtis M, Arroyo J M, Dunham M, Hudson A, Martienssen R A. 2000. Asymmetric leaves 1 mediates leaf patterning and stem cell function in Arabidopsis. Nature,408:967-971.
[7]	Chitwood D H, Sinha N R. 2016. Evolutionary and environmental forces sculpting leaf development. Current Biology, 26 (7):R297-R306.
[8]	Clough S J, Bent A F. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant Journal,16:735-743.
[9]	Cong Hanqing, Xin Caiyun, Zhang Yindong, Li Zhiying, Xu Li. 2013. Cloning of glutathione-S-transferase gene and primary expression analysis in Guzmania wittmackii‘Attila’induced by ethylene. Molecular Plant Breeding, 11 (3):365-370. (in Chinese)
	丛汉卿, 信彩云, 张银东, 李志英, 徐立. 2013. ‘阿蒂擎天’凤梨谷胱甘肽-S-转移酶基因的克隆与乙烯诱导表达特性的\初步分析. 分子植物育种, 11 (3):365-370.
[10]	Efroni I, Eshed Y, Lifschitz E. 2010. Morphogenesis of simple and compound leaves:a critical review. The Plant Cell, 22 (4):1019-1032. doi: 10.1105/tpc.109.073601 pmid: 20435903
[11]	Fang Z, Ji Y, Hu J, Guo R, Sun S, Wang X. 2019. Strigolactones and brassinosteroids antagonistically regulate the stability of D53-OsBZR1 complex to determine FC 1 expression in rice tillering. Molecular Plant, 13 (4):586-597.
[12]	Furumizu C, Alvarez J P, Sakakibara K, Bowman J L. 2015. Antagonistic roles forKNOX1 and KNOX2 genes in patterning the land plant body plan following anancient gene duplication. PLoS Genetics,11:e1004980.
[13]	Hake S, Smith H M, Holtan H, Magnani E, Mele G, Ramirez J. 2004. The role of KNOX genes in plant development. Annual Review of Cell and Developmental Biology,20:125-151.
[14]	Hasson A, Plessis A, Blein T, Adroher B, Grigg S, Tsiantis M, Boudaoud A, Damerval C, Laufs P. 2011. Evolution and diverse roles of the CUP-SHAPED COTYLEDON genes in Arabidopsis leaf development. Plant Cell,23:54-68.
[15]	Hay A, Tsiantis M. 2006. The genetic basis for differences in leaf form between Arabidopsis thaliana and its wild relative Cardamine hirsute. Nature Genetics, 38 (8):942-947.
[16]	Jasinski S, Piazza P, Craft J, Hay A, Woolley L, Rieu I, Phillips A, Hedden P, Tsiantis M. 2005. KNOX action in Arabidopsis is mediated by coordinate regulation of cytokinin and gibberellin activities. Current Biology,15:1560-1565.
[17]	Jia P, Xing L B, Zhang C G, Chen H, Li Y M, Zhang D, Ma J J, Zhao C P, Han M Y, Ren X L, An N. 2021b. MdKNOX15,a class I knotted-like transcription factor of apple,controls flowering and plant height by regulating GA levels through promoting the MdGA2ox7 transcription. Environmental and Experimental Botany,185:104411.
[18]	Jia P, Xing L B, Zhang C G, Zhang D, Ma J J, Zhao C P, Ming Y H, Xiao L R, Na A. 2021a. MdKNOX19,a class II knotted-like transcription factor of apple,plays roles in ABA signalling /sensitivity by targeting ABI 5 during organ development. Plant Science, 302 (1):110701.
[19]	Jia Y, Yu P, Shao W, An G, Chen J, Yu C, Kuang H. 2022. Up-regulation of LsKN1 promotes cytokinin and suppresses gibberellin biosynthesis to generate wavy leaves in lettuce. Journal of Experimental Botany, 72 (19):6615-6629.
[20]	Kim D, Cho Y H, Ryu H, Kim Y, Kim T H, Hwang I. 2013. BLH1 and KNAT 3 modulate ABA responses during germination and early seedling development in Arabidopsis. Plant Journal, 75 (5):755-766.
[21]	Kim J Y, Rim Y, Wang J, Jackson D. 2005. A novel cell-to-cell trafficking assay indicates that the KNOX homeodomain is necessary and sufficient for intercellular protein and mRNA trafficking. Genes Development, 19 (7):788-793.
[22]	Kumar R, Kushalappa K, Godt D, Pidkowich M S, Pastorelli S, Hepworth S R, Haughn G W. 2007. The Arabidopsis BEL1-LIKE HOMEODOMAIN proteins SAW1 and SAW 2 act redundantly to regulate KNOX expression apatially in leaf margins. The Plant Cell, 19 (9):2719-2735.
[23]	Ledford H. 2018. The lost art of looking at plants. Nature, 553 (7689):396-398.
[24]	Li G, Manzoor M A, Wang G, Song C. 2023. Comparative analysis of KNOX genes and their expression patterns under various treatments in Dendrobium huoshanense. Frontiers in Plant Science,14:1258533.
[25]	Liu Y Y, You S J, Taylor-Teeples M, Li W L, Schuetz M, Brady S M, Douglas C J. 2014. BEL1-LIKE HOMEODOMAIN6 and KNOTTED ARABIDOPSIS THALIANA7 interact and regulate secondary cell wall formation via repression of revolute. Plant Cell,26:4843-4861.
[26]	Long J A, Moan E, Medford J I, Barton M K. 1996. A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis. Nature,379:66-69.
[27]	Ma J, Mei G, Liu H, Li H. 2020. Overexpression of a novel LcKNOX transcription factor from Liriodendron chinense induces lobed leaves in Arabidopsis thaliana. Forests, 11 (1):33.
[28]	Piazza P, Bailey C D, Cartolano M, Krieger J, Cao J, Ossowski S, Schneeberger K, Fei H, Meaux J D, Hall N. 2010. Arabidopsis thaliana leaf form evolved via loss of KNOX expression in leaves in association with a selective sweep. Current Biology, 20 (24):2223-2228. doi: 10.1016/j.cub.2010.11.037 pmid: 21129970
[29]	Runions A, Tsiantis M. 2017. The shape of things to come:from typology to predictive models for leaf diversity. American Journal of Botany, 104 (10):1437-1441. doi: 10.3732/ajb.1700251 pmid: 29885230
[30]	Sakamoto T, Kamiya N, Ueguchi-Tanaka M, Iwahori S, Matsuoka M. 2001. KNOX homeodomainprotein directly suppresses the expression of a gibberellin biosynthetic gene in the tobacco shoot apical meristem. Genes & Development,15:581-590.
[31]	Shani E, Burko Y, Ben-Yaakov L, Berger Y, Amsellem Z, Goldshmidt A, Sharon E, Ori N. 2009. Stage-specific regulation of Solanum lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX proteins. The Plant Cell, 21 (10):3078-3092.
[32]	Shao J, Meng J, Wang F, Shou B, Chen Y, Xue H, Zhao J, Qi Y, An L, Yu F, Liu X. 2020. NGATHA-LIKEs control leaf margin development by repressing CUP-SHAPED COTYLEDON2 transcription. Plant Physiology, 184 (1):345-358.
[33]	Sluis A, Hake S. 2015. Organogenesis in plants:initiation and elaboration of leaves. Trends in Genetics, 31 (6):300-306.
[34]	Srinivasan C, Liu Z, Scorza R. 2011. Ectopic expression of class 1 KNOX genes induce adventitious shoot regeneration and alter growth and development of tobacco(Nicotiana tabacum L)and European plum(Prunus domestica L). Plant Cell Reports, 30 (4):655-664. doi: 10.1007/s00299-010-0993-7 pmid: 21212958
[35]	Tsukaya H. 2005. Leaf shape:genetic controls and environmental factors. The International Journal of Developmental Biology, 49 (5-6):547-555.
[36]	Tsukaya H. 2013. Leaf development. Arabidopsis Book,11:e0163.
[37]	Tsukaya H. 2018. Leaf shape diversity with an emphasis on leaf contour variation,developmental background,and adaptation. Seminars in Cell & Developmental Biology,79:48-57.
[38]	Uchida N, Kimura S, Koenig D, Sinha N. 2010. Coordination of leaf development via regulation of KNOX1genes. Journal of Plant Research, 123 (1):7-14.
[39]	Wang M, Lavelle D, Yu C, Zhang W, Chen J, Wang X, Michelmore R W, Kuang H. 2022. The upregulated LsKN1 gene transforms pinnately to palmately lobed leaves through auxin,gibberellin,and leaf dorsiventrality pathways in lettuce. Plant Biotechnology Journal, 20 (9):1756-1769.
[40]	Xu X M, Wang J, Xuan Z, Goldshmidt A, Borrill P G M, Hariharan N, Kim J Y, Jackson D. 2011. Chaperonins facilitate KNOTTED 1 cell-to-cell trafficking and stem cell function. Science, 333 (6046):1141-1144.
[41]	Yanai O, Shani E, Dolezal K, Tarkowski P, Sablowski R, Sandberg G, Samach A, Ori N. 2005. Arabidopsis KNOXI proteins activate cytokinin biosynthesis. Current Biology, 15 (17):1566-1571.
[42]	Yuan Jing, Zhou Bingying. 2021. The regulation of leaf margin serration development in plants:a review. Chinese Agricultural Science Bulletin, 37 (26):24-31. (in Chinese) doi: 10.11924/j.issn.1000-6850.casb2021-0477
	袁静, 周冰莹. 2021. 植物叶缘锯齿发育调控的研究进展. 中国农学通报, 37 (26):24-31. doi: 10.11924/j.issn.1000-6850.casb2021-0477
[43]	Zhang S, Pan Y, Zhi C, Zheng Y, Wang X, Li X, Cheng Z. 2021. Genomewide identification and characterization of KNOTTED-like homeobox (KNOX)homologs in garlic(Allium sativum L.) and their expression profilings responding to exogenous cytokinin and gibberellin. International Journal of Molecular Sciences, 22 (17):9237.
[44]	Zheng Jian, Pan Jihong, Yu Weilin, Song Yunlian, Bi Jue, Ling Mingwei, Wang Yuequan, Gao Xianyu, Zhang Huiyun, Luo Xinping. 2023. Research progress on the regulation of leaf margin serration development in plants. Journal of Plant Genetic Resources, 24 (4):927-936. (in Chinese) doi: 10.13430/j.cnki.jpgr.20221220002
	郑健, 潘继红, 余卫霖, 宋云连, 毕珏, 凌铭蔚, 王跃全, 高贤玉, 张惠云, 罗心平. 2023. 植物叶缘锯齿调控的研究进展. 植物遗传资源学报, 24 (4):927-936. doi: 10.13430/j.cnki.jpgr.20221220002