截短熱休克蛋白 70 之功能及演化分析

摘要

本研究聚焦於植物抗逆境熱休克蛋白70 (Hsp70) 變異體「截短熱休克蛋白70 (Hsp70T)」的功能與演化分析。Hsp70協助蛋白質正確折疊，減少逆境造成的細胞損傷。過去Hsp70T因缺乏完整C端功能域，在植物中被認定為假基因，但在酵母菌和人類中已證實參與共轉譯折疊。為釐清Hsp70T在植物中的真實功能，本研究建立大規模基因分析方法，針對192種植物與藻類進行篩選，系統性探討其分布、功能及演化歷程。研究結果發現Hsp70T廣泛分布於研究物種中，證實其並非假基因，而是具有新功能的重要基因家族。細胞質型Hsp70T最多，可分T1與T2亞型：T1型表現量少、序列變異大，在樟科植物中擴增至30個以上之基因簇；T2型數量少但序列保守，主要在種子成熟期及熱逆境下表現。基因簇分析顯示Hsp70T在陸生植物演化中大量出現，證實其在植物適應陸地環境中的關鍵角色，同時為樟科植物演化關係提供分子證據。本研究建立的大規模植物基因體比較分析方法，不僅顯示 Hsp70T 在植物中的重要角色，也為未來植物多樣性研究提供了技術參考。

方法與執行步驟

This study established a large-scale genomic analysis pipeline to thoroughly investigate the function and evolution of plant truncated Heat Shock Protein 70 (Hsp70T).
The research methodology encompassed the following key steps:

1. Large-scale genome database construction and search:
   Our study involved a comprehensive collection and search for Hsp70T sequences across the whole genomes of 192 sequenced plant and green algal species. This extensive database encompasses diverse plant lineages, ranging from algae, bryophytes, and ferns to gymnosperms and angiosperms, ensuring the broad scope and representativeness of our analysis.
2. Protein sequence analysis and gene classification:
   We performed multiple sequence alignments, specific sequence motif searches, and phylogenetic tree analyses on the collected Hsp70T protein sequences. This allowed for a detailed classification of Hsp70T subtypes, specifically differentiating between the T1 and T2 subtypes of cytosolic Hsp70T, and analyzing their similarities and sequence variations compared to typical Hsp70s.
3. Gene synteny analysis:
   To explore the expansion mechanisms of Hsp70T genes within plant genomes, particularly their proliferation as gene clusters, we conducted synteny analyses. This identified homologous Hsp70T gene clusters and their evolutionary relationships across different species, with a particular focus on the expansion patterns of T1 type gene clusters in Lauraceae plants.
4. Gene expression analysis:
   Utilizing public RNA-seq databases, we assessed the expression levels of Hsp70T in various plant tissues, developmental stages, and under different stress conditions. This allowed us to infer their potential functional locations and physiological roles, especially distinguishing the expression patterns of T1 and T2 subtypes.
5. Cross-species distribution and gene family expansion analysis:
   We quantified the numbers of each Hsp70T type across diverse plant species to study their gene family expansion and evolutionary pathways. This analysis was crucial for understanding the evolutionary context of Hsp70T in the adaptation of plants to terrestrial environments. By integrating bioinformatics and comparative genomics approaches, this study not only significantly accelerated the process of plant gene research but also provided a robust technical framework for future studies in plant diversity.

創新作為與跨域合作

The innovativeness of this study is multifaceted. Firstly, we for the first time confirmed that plant Hsp70T is not a pseudogene but a gene family with important functions, moving beyond the limitations of previous research that relied solely on mutant studies in model organisms. Secondly, this study provides new evidence for the evolutionary association between Hsp70T and plant adaptation to terrestrial environments, supporting the APG IV phylogenetic hypothesis for plant classification and addressing previous controversies regarding Magnoliids evolution. Most importantly, we established a large-scale comparative analysis method for plant genomes, encompassing nearly 200 model and non-model species, providing a more comprehensive genomic analysis pipeline and evolutionary information. This method has already been successfully applied to the identification of other plant functional genes. Regarding cross-domain collaboration, this research received support from the Taiwan Comprehensive University System's inter-university research program. We collaborated with laboratories at National Cheng Kung University and National Yang Ming Chiao Tung University to complete the study and publish our findings. This cross-institutional collaboration model brings together expertise from diverse fields, enhancing the depth and breadth of the research.

預期成果與貢獻

The results of this study are expected to make significant contributions in several aspects:

1. Promoting sustainable agriculture and biodiversity conservation:
   By thoroughly analyzing the role of plant Hsp70T in stress resistance, our findings lay the groundwork for developing novel plant protection strategies. This has the potential to reduce pesticide use and lessen agriculture's ecological impact, thereby contributing to the conservation of Taiwan's endemic plant resources and providing a scientific basis for sustainable agricultural development, in line with the United Nations Sustainable Development Goals (SDGs).
2. Deepening understanding of plant evolution:
   Investigating the unique characteristics of Hsp70T in Lauraceae plants and its synteny with Chloranthaceae and Nymphaeales offers a new perspective for re-examining the evolutionary relationships within Lauraceae. This also supports important plant evolutionary hypotheses, such as the APG IV system.
3. Advancing plant biotechnology:
   The established large-scale comparative genomic analysis method can be broadly applied to crop improvement, aiding in the identification of key genes related to stress resistance, yield, and quality. The high flexibility of this method allows for future integration with emerging technologies, enhancing the accuracy of gene function prediction and providing innovative solutions for global agricultural challenges. This research provides scientific evidence for plant adaptation to land and offers research directions for the function of plant Hsp70T.