Screening and Functional Analysis of Gene CqSTK Associated with Gametophyte Development of Quinoa

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

Abstract

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

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.

Figures/Tables 8

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

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

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

Fig. 3 Protein phylogenetic tree of homologous genes of STK

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

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

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

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

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