藜麦配子发育相关基因CqSTK的筛选及功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-0156

摘要/Abstract

摘要：

【目的】SEEDSTICK（STK）是MADS-box转录因子家族成员，在控制配子发育和种子大小形态方面起关键作用。探究STK 在藜麦配子发育过程中的作用。【方法】利用不同光周期处理下具有不同光周期特性的两种藜麦的转录组数据，筛选受光周期调控影响的、与配子发育相关的差异基因，克隆该基因，并对其进行生物信息学、表达模式、亚细胞定位分析和拟南芥异源表达验证基因功能。【结果】筛选到差异基因AUR62022366-RA。该基因的表达模式与植株的表型差异一致，在短日材料中，该基因在灌浆期表达量高时植株结实，表达量低时植株不结实。生物信息学分析表明，CqSTK的CDS全长为672 bp，编码223个氨基酸。STK同源基因进化树结果显示，CqSTK与同属物种菠菜、甜菜STK基因聚为一支，有较近的亲缘关系。此外，CqSTK与拟南芥STK具有相似的三级结构，单倍型分析表明，CqSTK的外显子序列在10个藜麦品种中完全一致，但在内含子区域存在SNP。该基因定位于烟草叶表皮细胞的细胞核和细胞膜上。荧光定量PCR显示，CqSTK在藜麦花器官形成和籽粒形成期高表达，尤其在籽粒形成期，其表达量达到最高。拟南芥过表达和突变体回补实验表明，过表达和回补植株开花时间远长于突变体和野生型，且过表达和回补植株果荚长度和荚果内种子数显著高于stk突变体和回补植株。【结论】CqSTK 高表达会延迟花期，同时正向调节拟南芥的种子长度和结实率。

关键词: 藜麦, SEEDSTICK, 亚细胞定位, 拟南芥异源表达

Abstract:

【Objective】SEEDSTICK（STK）, a member of the MADS-box transcription factor family, plays a key role in controlling gametophyte development and seed size and morphology. This study is aimed to investigate the role of STK in quinoa gametophyte development.【Method】Transcriptomic data from two quinoa varieties with different photoperiod characteristics under different photoperiod treatments were used to screen differentially expressed genes regulated by photoperiod and related to gametophyte development. The identified gene was cloned and subjected to bioinformatics analysis, expression pattern analysis, subcellular localization analysis, and functional verification through heterologous expression in Arabidopsis. 【Result】A differentially expressed gene, AUR62022366-RA, was identified. The expression pattern of this gene was consistent with phenotypic differences in plants. In short-day materials, high expression of the gene during the grain-filling stage resulted in fertile plants, while low expression led to infertile plants. Bioinformatics analysis revealed that the full-length coding sequence（CDS）of CqSTK was 672 bp, encoding 223 amino acids. Phylogenetic analysis of STK homologous genes showed that CqSTK clustered with STK genes from spinach and sugar beet, indicating a close evolutionary relationship. Furthermore, CqSTK shared a similar tertiary structure with Arabidopsis STK. Haplotype analysis indicated that the exonic sequences of CqSTK were completely conserved across ten quinoa varieties, while single nucleotide polymorphisms（SNPs）were present in the intronic regions. This gene was localized in the nucleus and cell membrane of tobacco leaf epidermal cells. Fluorescence quantitative PCR showed that CqSTK was highly expressed during flower organ formation and grain development, especially during grain development when its expression reached its peak. Overexpression and complementation experiments in Arabidopsis demonstrated that overexpressing and complementation plants had significantly delayed flowering time compared to mutants and wild-type plants, and their pod length and seed number per pod were significantly higher than those of stk mutants and complementation plants.【Conclusion】The overexpression of CqSTK delays flowering and positively regulates seed length and fruit set in Arabidopsis thaliana.

Key words: quinoa, SEEDSTICK, subcellular localization, heterologous expression in Arabidopsis

林彤, 袁程, 董陈文华, 曾孟琼, 杨燕, 毛自朝, 林春. 藜麦配子发育相关基因CqSTK的筛选及功能分析[J]. 生物技术通报, 2024, 40(8): 83-94.

LIN Tong, YUAN Cheng, DONG Chen-wen-hua, ZENG Meng-qiong, YANG Yan, MAO Zi-chao, LIN Chun. Screening and Functional Analysis of Gene CqSTK Associated with Gametophyte Development of Quinoa[J]. Biotechnology Bulletin, 2024, 40(8): 83-94.

图/表 8

表1 引物序列表

Table 1 Primer sequence

引物名称Primer name	引物序列Primer sequence（5'-3'）	用途Usage
CqSTK F	ATGGGGAGAGGGAAGATAGAG	基因克隆Gene cloning
CqSTK R	TTACCTTAGGTGAAATAGCTTCTTC	基因克隆Gene cloning
Cqactin F	GTCCACAGAAAGTGCTTCTAAG	内参基因Acting
Cqactin R	AACAACTCCTCACCTTCTCATG	内参基因Acting
CqSTKqpcr F1	ATAGAGAACACGACGAATCGT	RT-qPCR
CqSTKqpcr R1	AACCAAATACAGATGATGCAA	RT-qPCR

图1 藜麦CqSTK的鉴定与序列优化 A：藜麦和拟南芥MADS-box基因家族成员进化树（紫色五角星标注为藜麦MADS-box成员）；B:差异基因AUR62022366-RA与拟南芥STK聚为一支；C、D：MADS-box家族表达量热图；E、F：IGV可视化图；G：基因结构图。DVL为短日处理营养期叶，LVL为长日处理营养期叶；DVP为短日处理营养期花序，LVP为长日处理营养期花序；DFL为短日处理花期期叶，LFL为长日处理花期期叶；DFP短日处理花期花序，LFP为长日处理花期花序；DSL为短日处理灌浆期叶，LSL为长日处理灌浆期叶；DSP为短日处理灌浆期花序，LSP为长日处理灌浆期花序

Fig. 1 Identification and sequence optimization of the CqSTK in quinoa A: Phylogenetic tree of quinoa and Arabidopsis MADS-box gene family members（purple pentagrams indicate quinoa MADS-box members）. B: Differential gene AUR62022366-RA clusters with Arabidopsis STK gene. C, D: Heatmaps of MADS-box gene family expressions. E, F: IGV visualization plots. G: Gene structure diagrams. DVL refers to short-day treatment vegetative leaves, LVL to long-day treatment vegetative leaves, DVP to short-day treatment vegetative inflorescence, LVP to long-day treatment vegetative inflorescence, DFL to short-day treatment flowering phase leaves, LFL to long-day treatment flowering phase leaves. DFP refers to short-day treatment flowering phase inflorescence, LFP to long-day treatment flowering phase inflorescence. DSL refers to short-day treatment ripening phase leaves, LSL to long-day treatment ripening phase leaves; DSP to short-day treatment ripening phase inflorescence, LSP to long-day treatment ripening phase inflorescence

图2 10个藜麦种质表型及CqSTK等位基因信息 A：10个藜麦品种的种子表型统计，分为白、黄、红、黑四组；B：以QQ74为参考基因组，CqSTK在10品种中的序列信息，绿色方框（外显子）和黑色线段（内含子）组成CqSTK的基因结构。红色字体为参考基因组QQ74在特定位点的碱基，粉红色背景表示与参考基因组之间不一致，蓝色背景表示与参考基因组一致

Fig. 2 Phenotypic characteristics of 10 quinoa germplasms and the information on CqSTK allele genes A: Seed phenotype statistics of the 10 quinoa varieties, categorized into four groups: white, yellow, red, and black. B: Sequence information of CqSTK in the 10 varieties with QQ74 as the reference genome. The green boxes（exons）and black lines（introns）form the gene structure of CqSTK. Red font indicates the bases at specific loci of the reference genome QQ74, pink background denotes inconsistencies with the reference genome, and blue background signifies consistency with the reference genome

图3 STK同源基因的蛋白进化树

Fig. 3 Protein phylogenetic tree of homologous genes of STK

图4 CqSTK的克隆和时空表达 A：以cDNA为模板，CqSTK的全长CDS克隆；B：CqSTK在不同品种不同组织的表达量，*表示差异显著（P < 0.05），ns表示差异不显著

Fig. 4 Cloning and spatiotemporal expression of CqSTK A: Full-length CDS cloning of CqSTK using cDNA as a template. B: Expression levels of CqSTK in different varieties and tissues, * indicates significant difference（P < 0.05）, ns indicates no significant difference

图5 CqSTK的亚细胞定位 PCA1301-eGFP能在烟草细胞中瞬时表达绿色荧光蛋白GFP；PCA1301-CqSTK-GFP为CqSTK和GFP的融合表达载体，能瞬时表达CqSTK和GFP，通过检测绿色荧光位置来间接反映CqSTK的表达位置。叶绿体荧光为叶绿体在特定波长会显示红色荧光；叠加场为明场、叶绿体荧光和绿色荧光蛋白的3个视野叠加图

Fig. 5 Subcellular localization of CqSTK PCA1301-eGFP enables transient expression of green fluorescent protein（GFP）in tobacco cells. PCA1301-CqSTK-GFP is a fusion expression vector containing both CqSTK and GFP, allowing for the simultaneous expression of CqSTK and GFP. The localization of CqSTK expression can be indirectly inferred by detecting the position of green fluorescence. Chloroplast fluorescence refers to the red fluorescence emitted by chloroplasts at specific wavelengths. The overlay image consists of three fields: bright field, chloroplast fluorescence, and green fluorescent protein

图6 拟南芥及转基因材料的表型变化 A：野生型拟南芥、stk突变体、过表达T3代及stk突变体回补T3代植株的形态；B：荚果长度；C：每个荚果内种子数；*P<0.05，**P<0.01，***P<0.001

Fig. 6 Phenotypic variations in Arabidopsis and transgenic materials A: Morphology of wild-type Arabidopsis, stk mutant, overexpressing T3 generation, and stk mutant complementation T3 generation plants. B: Silique length. C: Number of seeds per silique. *P<0.05, **P<0.01, ***P<0.001, and ns indicates no significant difference

表2 野生型与转基因拟南芥的首次开花时间和莲座叶片

Table 2 First flowering time and rosette leaf number of wild-type and transgenic Arabidopsis

材料 Material	首次开花时间 First flowering time/d	首次开花莲座叶片 Number of first flowering lotus leaves/pieces
拟南芥野生型 Wild type	45	20
拟南芥stk突变体 stk mutant	49	16
回补CqSTK/stk Complementation CqSTK in stk mutant	72	28
过表达CqSTK Overexpress CqSTK in wild type	61	25

参考文献 45

[1]	Zurita-Silva A, Fuentes F, Zamora P, et al. Breeding quinoa(Chenopodium quinoa Willd.): potential and perspectives[J]. Mol Breed, 2014, 34(1): 13-30.
[2]	Hariadi Y, Marandon K, Tian Y, et al. Ionic and osmotic relations in quinoa(Chenopodium quinoa Willd.)plants grown at various salinity levels[J]. J Exp Bot, 2011, 62(1): 185-193. doi: 10.1093/jxb/erq257 pmid: 20732880
[3]	Ruales J, Nair BM. Content of fat, vitamins and minerals in quinoa(Chenopodium quinoa, Willd)seeds[J]. Food Chem, 1993, 48(2): 131-136.
[4]	Adolf VI, Shabala S, Andersen MN, et al. Varietal differences of quinoa's tolerance to saline conditions[J]. Plant Soil, 2012, 357(1): 117-129.
[5]	Jacobsen SE, Mujica A, Jensen CR. The resistance of quinoa(Chenopodium quinoaWilld.)to adverse abiotic factors[J]. Food Rev Int, 2003, 19(1/2): 99-109.
[6]	Alandia G, Rodriguez JP, Jacobsen SE, et al. Global expansion of quinoa and challenges for the Andean region[J]. Glob Food Secur, 2020, 26: 100429.
[7]	Losa A, Colombo M, Brambilla V, et al. Genetic interaction between AINTEGUMENTA(ANT)and the ovule identity genes SEEDSTICK(STK), SHATTERPROOF1(SHP1)and SHATTERPROOF2(SHP2)[J]. Sex Plant Reprod, 2010, 23(2): 115-121.
[8]	Dreni L, Kater MM. MADS reloaded: evolution of the AGAMOUS subfamily genes[J]. New Phytol, 2014, 201(3): 717-732. doi: 10.1111/nph.12555 pmid: 24164649
[9]	Mizzotti C, Mendes MA, Caporali E, et al. The MADS box genes SEEDSTICK and ARABIDOPSIS Bsister play a maternal role in fertilization and seed development[J]. Plant J, 2012, 70(3): 409-420.
[10]	Pinyopich A, Ditta GS, Savidge B, et al. Assessing the redundancy of MADS-box genes during carpel and ovule development[J]. Nature, 2003, 424(6944): 85-88.
[11]	Di Marzo M, Herrera-Ubaldo H, Caporali E, et al. SEEDSTICK controls Arabidopsis fruit size by regulating cytokinin levels and FRUITFULL[J]. Cell Rep, 2020, 30(8): 2846-2857.e3.
[12]	Paolo D, Orozco-Arroyo G, Rotasperti L, et al. Genetic interaction of SEEDSTICK, GORDITA and AUXIN RESPONSE FACTOR 2 during seed development[J]. Genes, 2021, 12(8): 1189.
[13]	Balanzà V, Roig-Villanova I, Di Marzo M, et al. Seed abscission and fruit dehiscence required for seed dispersal rely on similar genetic networks[J]. Development, 2016, 143(18): 3372-3381. doi: 10.1242/dev.135202 pmid: 27510967
[14]	Fischer A, Baum N, Saedler H, et al. Chromosomal mapping of the MADS-box multigene family in Zea mays reveals dispersed distribution of allelic genes as well as transposed copies[J]. Nucleic Acids Res, 1995, 23(11): 1901-1911. doi: 10.1093/nar/23.11.1901 pmid: 7596816
[15]	Schmidt RJ, Veit B, Mandel MA, et al. Identification and molecular characterization of ZAG1, the maize homolog of the Arabidopsis floral homeotic gene AGAMOUS[J]. Plant Cell, 1993, 5(7): 729-737. doi: 10.1105/tpc.5.7.729 pmid: 8103379
[16]	Ambrose BA, Lerner DR, Ciceri P, et al. Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots[J]. Mol Cell, 2000, 5(3): 569-579. doi: 10.1016/s1097-2765(00)80450-5 pmid: 10882141
[17]	Dreni L, Pilatone A, Yun DP, et al. Functional analysis of all AGAMOUS subfamily members in rice reveals their roles in reproductive organ identity determination and meristem determinacy[J]. Plant Cell, 2011, 23(8): 2850-2863.
[18]	Ocarez N, Mejía N. Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[J]. Plant Cell Rep, 2016, 35(1): 239-254.
[19]	Wang QJ, Dan NZ, Zhang XN, et al. Identification, characterization and functional analysis of C-class genes associated with double flower trait in carnation(Dianthus caryphyllus L.)[J]. Plants, 2020, 9(1): 87.
[20]	Yellina AL, Orashakova S, Lange S, et al. Floral homeotic C function genes repress specific B function genes in the carpel whorl of the basal eudicot California poppy(Eschscholzia californica)[J]. EvoDevo, 2010, 1: 13.
[21]	Serwatowska J, Roque E, Gómez-Mena C, et al. Two euAGAMOUS genes control C-function in Medicago truncatula[J]. PLoS One, 2014, 9(8): e103770.
[22]	袁加红, 刘正杰, 吴慧琳, 等. 111份藜麦种质资源农艺性状分析[J]. 云南农业大学学报: 自然科学, 2020, 35(4): 572-580, 650.
	Yuan JH, Liu ZJ, Wu HL, et al. The analysis of the main agronomic traits of 111 quinoa germplasm resources[J]. J Yunnan Agric Univ Nat Sci, 2020, 35(4): 572-580, 650.
[23]	Gramzow L, Theissen G. A hitchhiker's guide to the MADS world of plants[J]. Genome Biol, 2010, 11(6): 214. doi: 10.1186/gb-2010-11-6-214 pmid: 20587009
[24]	Smaczniak C, Immink RGH, Angenent GC, et al. Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies[J]. Development, 2012, 139(17): 3081-3098. doi: 10.1242/dev.074674 pmid: 22872082
[25]	Alvarez J, Smyth DR. CRABS CLAW and SPATULA, two Arabidopsis genes that control carpel development in parallel with AGAMOUS[J]. Development, 1999, 126(11): 2377-2386. doi: 10.1242/dev.126.11.2377 pmid: 10225997
[26]	Coen ES, Meyerowitz EM. The war of the whorls: genetic interactions controlling flower development[J]. Nature, 1991, 353(6339): 31-37.
[27]	Pelaz S, Ditta GS, Baumann E, et al. B and C floral organ identity functions require SEPALLATA MADS-box genes[J]. Nature, 2000, 405(6783): 200-203.
[28]	Schneitz K. The molecular and genetic control of ovule development[J]. Curr Opin Plant Biol, 1999, 2(1): 13-17. doi: 10.1016/s1369-5266(99)80003-x pmid: 10047571
[29]	Theissen G, Kim JT, Saedler H. Classification and phylogeny of the MADS-box multigene family suggest defined roles of MADS-box gene subfamilies in the morphological evolution of eukaryotes[J]. J Mol Evol, 1996, 43(5): 484-516. pmid: 8875863
[30]	Nguyen LT, Schmidt HA, von Haeseler A, et al. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies[J]. Mol Biol Evol, 2015, 32(1): 268-274.
[31]	Robinson JT, Thorvaldsdottir H, Turner D, et al. Igv. js: an embeddable JavaScript implementation of the Integrative Genomics Viewer(IGV)[J]. Bioinformatics, 2023, 39(1): btac830.
[32]	张东亮. 藜麦MADS-box基因家族的鉴定与分析及发根农杆菌介导的根转化研究[D]. 烟台: 烟台大学, 2021.
	Zhang DL. Identification and analysis of quinoa MADS-box gene family and study on Agrobacterium-mediated root transformation[D]. Yantai: Yantai University, 2021.
[33]	Malabarba J, Buffon V, Mariath JEA, et al. The MADS-box gene Agamous-like 11 is essential for seed morphogenesis in grapevine[J]. J Exp Bot, 2017, 68(7): 1493-1506. doi: 10.1093/jxb/erx025 pmid: 28369525
[34]	Colombo L, Franken J, Van der Krol AR, et al. Downregulation of ovule-specific MADS box genes from petunia results in maternally controlled defects in seed development[J]. Plant Cell, 1997, 9(5): 703-715. pmid: 9165748
[35]	Costantini L, Battilana J, Lamaj F, et al. Ber1ry and phenology-related traits in grapevine(Vitis vinifera L.): from quantitative trait loci to underlying genes[J]. BMC Plant Biol, 2008, 8: 38. doi: 10.1186/1471-2229-8-38 pmid: 18419811
[36]	Ocarez N, Mejía N. Suppression of the D-class MADS-box AGL11 gene triggers seedlessness in fleshy fruits[J]. Plant Cell Rep, 2016, 35(1): 239-254.
[37]	Kobayashi Y, Kaya H, Goto K, et al. A pair of related genes with antagonistic roles in mediating flowering signals[J]. Science, 1999, 286(5446): 1960-1962. doi: 10.1126/science.286.5446.1960 pmid: 10583960
[38]	Zicola J, Liu LY, Tänzler P, et al. Targeted DNA methylation represses two enhancers of FLOWERING LOCUS T in Arabidopsis thaliana[J]. Nat Plants, 2019, 5(3): 300-307. doi: 10.1038/s41477-019-0375-2 pmid: 30833712
[39]	Suárez-López P, Wheatley K, Robson F, et al. CONSTANS mediates between the circadian clock and the control of flowering in Arabidopsis[J]. Nature, 2001, 410(6832): 1116-1120.
[40]	Samach A, Onouchi H, Gold SE, et al. Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis[J]. Science, 2000, 288(5471): 1613-1616. doi: 10.1126/science.288.5471.1613 pmid: 10834834
[41]	Gregis V, Andrés F, Sessa A, et al. Identification of pathways directly regulated by short vegetative phase during vegetative and reproductive development in Arabidopsis[J]. Genome Biol, 2013, 14(6): R56.
[42]	Simonini S, Roig-Villanova I, Gregis V, et al. Basic pentacysteine proteins mediate MADS domain complex binding to the DNA for tissue-specific expression of target genes in Arabidopsis[J]. Plant Cell, 2012, 24(10): 4163-4172.
[43]	Matias-Hernandez L, Battaglia R, Galbiati F, et al. VERDANDI is a direct target of the MADS domain ovule identity complex and affects embryo sac differentiation in Arabidopsis[J]. Plant Cell, 2010, 22(6): 1702-1715.
[44]	Paolo D, Rotasperti L, Schnittger A, et al. The Arabidopsis MADS-domain transcription factor SEEDSTICK controls seed size via direct activation of E2Fa[J]. Plants, 2021, 10(2): 192.
[45]	He SC, Min YC, Liu ZJ, et al. Antagonistic MADS-box transcription factors SEEDSTICK and SEPALLATA3 form a transcriptional regulatory network that regulates seed oil accumulation[J]. J Integr Plant Biol, 2024, 66(1): 121-142. doi: 10.1111/jipb.13606