| 摘要: |
| 【目的】评价低温下苦瓜种子的发芽能力，鉴定芽期低温响应基因并分析其表达模式，为探明苦瓜种子低温发芽分子调控机制提供理论指导。【方法】以6份苦瓜种质种子为材料，设置15 ℃的发芽温度，调查发芽率、发芽势、发芽指数、胚根长度、活力指数、平均发芽时间、平均发芽速度和种子萌发指数的变化，利用隶属函数法和聚类分析进行发芽能力综合评价及分类，选取发芽能力强和弱的种质进行转录组测序，获得差异基因后进行GO功能注释和KEGG富集分析，从中挖掘低温响应相关候选基因，利用荧光定量PCR进行验证，同时分析候选基因在苦瓜不同组织（芽、根、茎、叶）和不同发芽时期（0，7，14 d）的表达差异，探讨低温对苦瓜发芽的影响及相关基因的调控作用。【结果】低温处理下，不同种质苦瓜种子发芽指标出现差异，且各指标受影响程度不同；利用综合隶属函数值和聚类分析发现，苦瓜种质MC15和CH02发芽能力较强，ZK54和ZK90发芽能力中等，BG19和YW1发芽能力较弱。对MC15和BG19进行转录组测序，共获得3 062个差异基因，GO功能注释发现参与基因较多的有与胁迫、应激与防御响应，质膜和胞外区，DNA结合以及酶和蛋白活性相关的条目等;KEGG富集分析发现“植物激素信号转导”“淀粉和蔗糖代谢”“光合作用 天线蛋白”“二萜生物合成”等途径的差异基因数较多，富集因子较高；结合转录组测序、基因克隆、序列比对和荧光定量PCR分析得出，基因McLEAL和McDOG1L-3可能参与芽期低温响应，两者编码的氨基酸序列与葫芦科作物的序列相似性较高，且均在芽中特异性表达，表达趋势与转录组结果一致。在发芽0，7和14 d 3个时期中，2个基因的表达量在发芽能力较强的苦瓜种质MC15和CH02中呈下降趋势，在发芽能力较弱的苦瓜种质BG19和YW1中呈先降后升趋势。【结论】低温导致苦瓜种子发芽出现种质间及指标间差异，MC15和CH02的发芽能力较强，可用于后续种质创新和生产利用；2个低温响应相关基因McLEAL和 McDOG1L-3的表达存在组织特异性，且随发芽进程出现种质间差异和时间效应，推测两者可能协同调控低温下苦瓜的延迟发芽。 |
| 关键词: 苦瓜 低温 种子发芽 转录组测序 基因表达差异 |
| DOI：10.13207/j.cnki.jnwafu.2025.10.009 |
| 分类号: |
| 基金项目:福建省公益类科研院所专项(2021R10310014);福建省自然科学基金项目(2023J01375);特色蔬菜育种攻关与产业化开发项目(zycxny2021009);国家大宗蔬菜产业体系福州综合试验站项目(CARS-23-G-53) |
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| Evaluation of seed germination difference and identification of related genes in bitter gourd(Momordica charantia L.) under low temperature |
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QIU Boyin, LIN Hui, ZHANG Qianrong, CHEN Mindong, LI Yongping, WEN Qingfang, ZHU Haisheng, LI Dazhong
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Crops Research Institute,Fujian Academy of Agricultural Sciences,Fujian Key Laboratory of Vegetable Genetics and Breeding,Fujian Engineering Research Center for Vegetables,Fuzhou,Fujian 350013,China
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| Abstract: |
| 【Objective】This research aimed to evaluate the germination ability of bitter gourd seeds under low temperature,and to identify the low temperature response genes at germination stage and to further analyze their expression patterns,in order to provide theoretical guidance for exploring the molecular regulation mechanism of low temperature germination.【Method】Seeds of six bitter gourd germplasms and the germination temperature of 15 °C were prepared to investigate the changes of germination rate,germination vigor,germination index,embryo length,vigor index,mean germination time,average germination speed and seed germination coefficient.Membership function method and cluster analysis were used for comprehensive evaluation and classification of germination ability.Germplasms with strong and weak germination ability were selected for transcriptome sequencing(RNA-seq),and GO functional annotation and KEGG enrichment analysis were carried out after obtaining differentially expressed genes.Candidate genes related to low temperature response were explored and further verified by real time quantitative PCR,and differences in spatial and temporal expression were also analyzed,aiming to explore the effect of low temperature on the germination of bitter gourd and the regulation of related genes.【Result】Under low temperature treatment,the germination indexes of bitter gourd seeds showed differences among germplasms, and the effect showed on the indexes was also different.The comprehensive membership function value and cluster analysis result showed that germination ability of MC15 and CH02 was strong,ZK54 and ZK90 was moderate,BG19 and YW1 was weak.A total of 3 062 differentially expressed genes were obtained by RNA-seq of MC15 and BG19,and GO functional annotation showed that the functional items of stress,stress and defense response,plasma membrane and extracellular region,DNA binding,enzyme and protein activity were observed in numerous genes involved.KEGG enrichment analysis showed that more differential genes and higher enrichment factors were found in ‘plant hormone signal transduction’,‘starch and sucrose metabolism’,‘photosynthesis antenna protein’,and ‘diterpene biosynthesis’ pathways.With the combination of transcriptome sequencing,gene cloning,sequence alignment and fluorescence quantitative PCR analysis,it was concluded that McLEAL and McDOG1L-3 may be involved in the low temperature response at the germination stage.The amino acid sequences encoded by McLEAL and McDOG1L-3 were highly similar to the sequences of cucurbit crops.Both were specifically expressed in the germ,and the expression trend was consistent with the results of RNA-seq.In the germination 0.7 and 14-day three periods,expression levels of the two genes showed a downward trend in the germplasms with strong germination ability,and showed a trend of decreasing first and then increasing in the germplasms with weak germination ability.【Conclusion】Low temperature leads to differences in seed germination among germplasms and indicators.MC15 and CH02 have strong germination ability and can be used for subsequent germplasm innovation and production.Tissue specificity can be observed in the expression of two low temperature response-related genes McLEAL and McDOG1L-3,showing variety differences and time effects with the germination process,hence it is speculated that the two may synergistically regulate the delayed germination of bitter gourd at low temperature. |
| Key words: bitter gourd (Momordica charantia L.) low temperature seed germination transcriptome sequencing(RNA-seq) gene expression difference |
