C-terminal Truncation of CmoNST1 Leads to Hull-less Seeds in Cucurbita moschata Duch.

doi:10.13560/j.cnki.biotech.bull.1985.2026-0220

Abstract

Abstract:

Objective This study aimed to investigate the molecular basis of the hull-less seed trait in Cucurbita moschata by comparing the sequence, expression, and protein localization differences between the NST1 alleles (CmoNST1 and Cmonst1) from wild-type and mutant lines, with the goal of elucidating their functional divergence and providing a theoretical foundation for molecular breeding of hull-less C. moschata. Method The CmoNST1 and Cmonst1 genes were cloned from the hulled inbred line HS-A and the hull-less line HLS-B, respectively. Sequence alignment was performed to identify variant characteristics, and protein domain architecture was predicted. Subcellular localization was predicted using bioinformatics tools. Tissue-specific expression patterns of both genes were compared via RT-qPCR. To investigate gene function, we knocked out the cucumber ortholog CsaV3_6G008900 using a CRISPR/Cas9 system with dual guide RNAs. After Agrobacterium-mediated transformation, positive mutants were identified by PCR. The gene’s role was further evaluated by examining seed coat cellular structures with 3D micro-CT imaging and measuring lignin, cellulose, and hemicellulose content. Result The wild-type mediated transformation, positive mutants were identified by PCR. CmoNST1 (1 185 bp) and mutant Cmonst1 (1 072 bp) encode 394 and 112 amino acids, respectively. Both proteins contain a conserved NAM domain and lack transmembrane domains or signal peptides; their secondary structures are mainly composed of random coils and α-helices. Physicochemical analysis indicated that CmoNST1 is an unstable acidic protein, whereas Cmonst1 is a stable basic protein. Subcellular localization revealed that CmoNST1 is predominantly nuclear, while Cmonst1 distributes in both the nucleus and cytoplasm. Expression analysis showed that transcript levels of both genes are significantly higher in the seed coat than in other tissues. Phylogenetic analysis indicated that CmoNST1 is most closely related to homologs from other Cucurbitaceae species. Knockout of the cucumber ortholog CsaV3_6G008900 using CRISPR/Cas9 resulted in thinner seed coats, significantly reduced lignin, cellulose, and hemicellulose content, and loosely arranged seed coat cells with visible cavities. Conclusion These results demonstrate that CmoNST1 is a key regulator of seed coat development in C. moschata. Although the mutant allele Cmonst1 retains the NAM domain, its C-terminal truncation eliminates phosphorylation regulatory potential and alters physicochemical properties, which likely disrupts its transcriptional activation function and leads to the hull-less phenotype.

Key words: Cucurbita moschata, hull-less, CmoNST1, NAC, seed coat development, seed coat composition, CRISPR/Cas9, loss-of-function mutation

SHEN Qiong, LIU Ya-nan, MENG Hong-yu. C-terminal Truncation of CmoNST1 Leads to Hull-less Seeds in Cucurbita moschata Duch.[J]. Biotechnology Bulletin, 2026, 42(10): 198-206.

Figures/Tables 5

Fig. 1 Gene cloning and sequence analysis of CmoNST1 and Cmonst1 in pumpkinA: Electrophoresis analysis of full-length CDS amplification products of CmoNST1 and Cmonst1. B: Nucleotide sequence alignment of CmoNST1, Cmonst1, and the reference nucleotide sequence. C: Protein sequence alignment of CmoNST1 and Cmonst1. Ref: Reference sequence

Fig. 2 Phylogenetic tree analysis of CmoNST1

Fig. 3 Subcellular localization of CmoNST1 and Cmonst1From left to right: GFP green fluorescent protein, bright field, and merge. Scale bar = 20.0 μm

Fig. 4 Tissue‑specific expression analysis of CmoNST1 (wild type) and Cmonst1 (mutant)Different lower letters indicate significant differences (P<0.05)

Fig. 5 Effect of CsaV3_6G008900knockout on seed coat developmentA: Characterization of the vector. B: Contents of lignin, cellulose, and hemicellulose in the seed coat. C: Seed structure of the control cucumber. D: Seed structure of the mutant cucumber. M: DNA marker. 1‒3: Characterization of the vector. WT (Control): Wild-type cucumber seed. KO: Mutant cucumber seed with CsaV3_6G008900 knockout

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