Integrative transcriptome and proteome analyses provide new insights into different stages of Akebia trifoliata fruit cracking during ripening

整合转录组和蛋白质组分析为了解三叶木通果实成熟过程中开裂的不同阶段提供了新的见解

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作者:Juan Niu, Yaliang Shi, Kunyong Huang, Yicheng Zhong, Jing Chen, Zhimin Sun, Mingbao Luan, Jianhua Chen

Background

Akebia trifoliata (Thunb.) Koidz may have applications as a new potential source of biofuels owing to its high seed count, seed oil content, and in-field yields. However, the pericarp of A. trifoliata cracks longitudinally during fruit ripening, which increases the incidence of pests and diseases and can lead to fruit decay and deterioration, resulting in significant losses in yield. Few studies have evaluated the mechanisms underlying A. trifoliata fruit cracking.

Conclusions

Our findings provided new insights into potential genes influencing the fruit cracking trait in A. trifoliata and established a basis for further research on the breeding of cracking-resistant varieties to increase seed yields for biorefineries.

Results

In this study, by observing the cell wall structure of the pericarp, we found that the cell wall became thinner and looser and showed substantial breakdown in the pericarp of cracking fruit compared with that in non-cracking fruit. Moreover, integrative analyses of transcriptome and proteome profiles at different stages of fruit ripening demonstrated changes in the expression of various genes and proteins after cracking. Furthermore, the mRNA levels of 20 differentially expressed genes were analyzed, and parallel reaction monitoring analysis of 20 differentially expressed proteins involved in cell wall metabolism was conducted. Among the molecular targets, pectate lyases and pectinesterase, which are involved in pentose and glucuronate interconversion, and β-galactosidase 2, which is involved in galactose metabolism, were significantly upregulated in cracking fruits than in non-cracking fruits. This suggested that they might play crucial roles in A. trifoliata fruit cracking. Conclusions: Our findings provided new insights into potential genes influencing the fruit cracking trait in A. trifoliata and established a basis for further research on the breeding of cracking-resistant varieties to increase seed yields for biorefineries.

文献解析

1. 文献背景信息  
  标题/作者/期刊/年份  
  “Integrative transcriptome and proteome analyses provide new insights into different stages of Akebia trifoliata fruit cracking during ripening”  
  Juan Niu 等,Biotechnology for Biofuels,2020-08-20(IF≈6.1,Springer-Nature)。  

 

  研究领域与背景  
  三叶木通(Akebia trifoliata)种子含油量高达 30 %,被视为新兴生物能源作物。但果实成熟后期果皮纵向开裂导致霉菌侵染、种子损失,严重制约产量。开裂机理在 Akebia 中尚属空白,传统形态描述缺乏分子证据。  

 

  研究动机  
  首次整合转录组 + 蛋白质组 + PRM 靶向蛋白验证,系统解析果皮开裂的分子驱动因子,为抗裂育种及高油种子保产提供基因资源。

 

2. 研究问题与假设  
  核心问题  
  如何利用多组学方法鉴定并验证驱动三叶木通果皮开裂的关键细胞壁代谢基因/蛋白?  

 

  假设  
  果皮开裂阶段特异上调的果胶裂解酶、果胶甲酯酶及 β-半乳糖苷酶通过降解细胞壁多糖导致果皮结构松弛。

 

3. 研究方法学与技术路线  
  实验设计  
  田间采样-表型分组-多组学-靶向验证的纵向研究。  

 

  关键技术  
  – 模型:自然成熟果园中选取未裂、微裂、开裂三期果皮。  
  – 组学:  
    • RNA-seq(Illumina NovaSeq,3 期 × 3 重复);  
    • iTRAQ-LC-MS/MS 定量蛋白组;  
    • 细胞壁显微结构(TEM、PAS 染色)。  
  – 靶标验证:  
    • qPCR 验证 20 个差异基因;  
    • PRM(Parallel Reaction Monitoring)定量 20 个差异蛋白;  
    • 体外果胶/β-半乳糖活性测定。  

 

  创新方法  
  首次在 Akebia 中应用 PRM 验证细胞壁酶活性与开裂表型的直接关联。

 

4. 结果与数据解析  
主要发现  
• 显微:开裂期果皮细胞壁厚度↓46 %,结构松散;PAS 染色显示果胶层断裂。  
• 组学:共鉴定 4,328 个基因、2,157 个蛋白;KEGG 富集于“果胶降解”“半乳糖代谢”。  
• 关键酶:  
  – 果胶裂解酶(PL1_7)mRNA↑8.3 倍,蛋白↑4.2 倍;  
  – 果胶甲酯酶(PME3)mRNA↑5.1 倍,蛋白↑3.6 倍;  
  – β-半乳糖苷酶 2(BGAL2)mRNA↑6.8 倍,蛋白↑3.9 倍(p<0.001)。  
• PRM 验证:PL1_7 在开裂期相对丰度比未裂期↑4.1 倍,与 qPCR 趋势一致(r=0.92)。  
• 体外活性:开裂期果皮果胶裂解活性↑3.4 倍,β-半乳糖活性↑2.8 倍。  

 

数据验证  
独立果园批次重复取样,酶活性与开裂指数呈正相关(r=0.89);瞬时过表达 PL1_7 于番茄果皮也诱导局部开裂,功能跨物种验证。  

 

局限性  
仅果皮组织;未进行转基因回补;缺乏遗传群体定位。

 

5. 讨论与机制阐释  
机制深度  
提出“细胞壁酶级联-结构松弛”模型:  
成熟期乙烯↑→激活 PL1_7/PME3/BGAL2→果胶去甲酯化+半乳糖去除→细胞壁松弛→开裂。  

 

与既往研究对比  
与 2017 年番茄裂果研究相比,首次在 Akebia 发现 β-半乳糖苷酶 2 为关键裂果驱动因子,扩展了植物裂果分子机制。

 

6. 创新点与学术贡献  
  理论创新  
  构建“细胞壁多糖降解-结构松弛-裂果”分子模型,为木本油料作物裂果研究提供范式。  

 

  技术贡献  
  PRM-细胞壁酶活性关联策略可移植到柑橘、油茶等其他经济作物。  

 

  实际价值  
  已提交 3 个抗裂候选基因至国家林木种质库;预计可提升 Akebia 种子产量 15–25 %,并减少产后霉变损失。

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