悬钩子属浆果黑莓转色后硬度降低的细胞壁降解分子机制研究

Blackberry (Rubus L.) is an excellently cultivated nonwood forest tree of the Rubus genus in the Rosaceae family. It is an important horticultural source of fresh fruits and produces beneficial to human health. However, the intrinsic thin skin and fruit tenderness traits of blackberry bring about much inconvenience for its fruit harvest, postharvest transport and storage, which has currently become the key limiting factor for effective utilization of blackberry fruits at home and abroad. Till now, limited data are available on revealing the firmness formation mechanism of the blackberry fruit. At the initial stage of this project, we found that from the turning stage to fruit ripening, hardness of the blackberry genotypes with lower fruit firmness decreased rapidly and the cell wall degradation of the mature fruit pulp was particularly evident. Regarding this respect, the cell wall degrading enzymes and its encoding genes may play a crucial role in blackberry fruit firmness formation. This project thus aimed to further compare the changing patterns of various cell wall degrading enzyme activities, components and contents of cell wall substances and fruit micro-morphological structures in blackberry cv. Boysenberry (lower firmness genotype) and Arapaho (higher firmness genotype) during the turning and ripening stage. The determination relationship between fruit firmness and cell wall degrading enzymes during the fruit ripening can thus be confirmed. Afterwards, by transcriptome sequencing analysis the related genes were identified and expression patterns of the key cell wall degrading enzyme-encoding genes are analyzed correspondingly and the critical candidate genes controlling blackberry fruit firmness formation can be determined. Finally, full length cDNA of the target genes can be obtained and used for generation of transgenic blackberry through Agrobacterium-mediated transformation with the antisense vector. The verification will be able to clarify the key roles of the cell wall degrading enzymes genes in revealing the regulation mechanism of blackberry fruit firmness formation. These results will provide potent theoretical proof and basis for firmness improvement of berry fruits like blackberry and other Rubus genus types.

黑莓为蔷薇科悬钩子属的一种重要经济林果，因其果实兼具营养价值和保健功能而日益受到重视，但其成熟果实十分柔软使得极不耐贮藏，目前关于黑莓硬度形成的机制鲜见报道。项目前期发现低硬度黑莓果实转色之始硬度急剧下降，成熟果实果肉细胞壁降解尤为彻底，推论细胞壁降解酶对黑莓果实硬度形成具有关键作用。本研究拟进一步对低硬度黑莓品种Boysenberry和高硬度品种Arapaho果实转色至成熟进程细胞壁降解酶活性、细胞壁物质组分与含量及果实内在形态结构的变化规律进行比较，明确果实硬度与细胞壁降解的关系。继而，依据转录组测序分析得到的相应基因序列，对其在果实转色至成熟进程中的表达模式进行比较分析，确定控制黑莓果实硬度下降的关键细胞壁降解酶基因，通过构建目标基因的反义载体和实现遗传转化验证其功能。本项目拟在探索细胞壁降解酶基因调控黑莓多汁浆果硬度形成的特异机制，为悬钩子柔软小浆果硬度改良提供理论依据。

非呼吸跃变类小浆果果实成熟软化及硬度形成受细胞壁降解调控的研究相对较少。作为悬钩子属的一种新型保健特色经济林果树，黑莓浆果果实有着极高的营养保健价值和日益增长的市场需求，但因其果实柔软多汁，加上成熟时正值夏季高温多雨季节，采后极其容易褐变和腐烂，大大降低了经济价值和市场供应潜力，成为制约国内外黑莓良性发展的限制因素。本项目以硬度差异明显但生育期相似的两个黑莓品种Boysenberry（低硬度）和Arapaho（高硬度）为材料，在硬度形成的转色至成熟时期对各类细胞壁降解酶活性、细胞壁物质组分与含量及果实微形态结构的变化规律进行了比较，揭示了转色后果实硬度即开始下降，花后33d后硬度即急剧下降，果实细胞细胞壁成分降解与表面细胞形态下降时期一致。不仅明确了果实硬度形成与6种细胞壁降解酶活性变化的关系，揭示PG和Cellulase细胞壁降解酶在黑莓果实转色后的花后30d之后成熟软化进程中具重要作用，Boysenberry果实软化较明显与果胶和半纤维素等细胞壁物质降解程度更彻底有关。依据现有测序分析结果得到的细胞壁降解酶基因序列，对其在黑莓果实转色至成熟进程进行表达分析验证，确定控制黑莓果实硬度形成的2个关键纤维素酶基因成员，构建了目标基因的正义和反义载体并通过转化烟草和黑莓验证了基因在降低纤维素含量中的功能。本项目的实施初步细致解析了多汁浆果果实硬度形成与细胞壁代谢的关系，阐明关键降解酶纤维素酶对多汁浆果类果实硬度的调控作用，为悬钩子类浆果果实的成熟软化机理揭示提供了理论依据。