CmoNST1基因C端截短导致南瓜裸仁表型

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

摘要/Abstract

摘要：

目的探究南瓜裸仁性状形成的分子基础，通过比较野生型与突变体中NST1等位基因（CmoNST1与Cmonst1）的序列、表达及蛋白定位差异，解析其功能差异，为南瓜分子育种提供理论依据。方法以有种皮自交系HS-A和裸仁自交系HLS-B为材料，克隆CmoNST1与Cmonst1基因，通过序列比对分析其变异特征，结合生物信息学方法预测蛋白结构与亚细胞定位，并利用RT-qPCR技术比较二者在各组织的表达模式。基于同源关系利用CRISPR/Cas9双靶点系统敲除黄瓜直系同源基因CsaV3_6G008900，经农杆菌转化、PCR鉴定获得阳性突变体。进一步利用显微CT三维成像比较种皮细胞结构，并测定种皮木质素、纤维素和半纤维素的含量，综合评价基因功能。结果野生型CmoNST1（1 185 bp）及其突变等位基因Cmonst1（1 072 bp）被成功克隆，二者分别编码394与112个氨基酸。两者均含有典型的NAM保守结构域，无跨膜结构域与信号肽，二级结构主要由无规则卷曲和α-螺旋构成。CmoNST1为不稳定酸性蛋白，而Cmonst1为稳定碱性蛋白。亚细胞定位显示，CmoNST1主要定位于细胞核，Cmonst1分布于细胞核与细胞质。表达分析表明，二者在种皮中表达量显著高于其他组织。系统进化分析表明，CmoNST1与葫芦科物种亲缘关系最近。CRISPR/Cas9敲除黄瓜同源基因CsaV3_6G008900后，黄瓜种皮变薄而且种皮木质素、纤维素和半纤维素的含量显著降低，细胞排列松散并出现空洞。结论 CmoNST1是调控南瓜种皮发育的关键基因，其突变体Cmonst1虽保留NAM结构域，但因C端截短导致磷酸化调控潜力丧失，蛋白理化性质改变，可能使其丧失转录激活功能，进而导致裸仁表型。

关键词: 南瓜, 裸仁, CmoNST1, NAC, 种皮发育, 种皮成分, CRISPR/Cas9, 功能丧失型突变

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. Methods 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. Results 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

申琼, 刘亚楠, 孟洪玉. CmoNST1基因C端截短导致南瓜裸仁表型[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2026-0220.

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

图/表 5

图1 南瓜CmoNST1和Cmonst1基因克隆和序列分析A：CmoNST1和Cmonst1全长CDS扩增产物电泳检测；B：CmoNST1、Cmonst1与参考核苷酸序列比对；C：CmoNST1和Cmonst1蛋白序列比对；Ref：参考序列

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

图2 CmoNST1的系统进化树分析

Fig. 2 Phylogenetic tree analysis of CmoNST1

图3 CmoNST1和Cmonst1的亚细胞定位从左到右分别为GFP绿色荧光蛋白、明场和叠加图。比例尺=20.0 μm

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

图4 CmoNST1（野生型）和Cmonst1（突变体）组织特异性表达分析不同小写字母表示差异显著（P<0.05）

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

图5 CsaV3_6G008900基因敲除对种皮发育的影响A：载体鉴定；B：种皮木质素、纤维素和半纤维素含量；C：对照黄瓜种子结构；D：突变体黄瓜种子结构；M：DNA marker；1‒3：载体鉴定；WT（Control）：对照野生型黄瓜种子；KO：敲除CsaV3_6G008900的突变体黄瓜种子

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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