Fatima Iqra, Wakade Geetanjali, Ahmad Niaz, Daniell Henry
Department of Basic & Translational Sciences, School of Dental Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
National Institute for Biotechnology and Genetic Engineering College, Pakistan Institute of Engineering and Applied Sciences (NIBGE-C, PIEAS), Faisalabad, Pakistan.
Plant Biotechnol J. 2025 May;23(5):1437-1451. doi: 10.1111/pbi.14596. Epub 2025 Feb 18.
Lettuce (Lactuca sativa) is a popular leafy vegetable with global production of ~28 million Mt, cultivated >1 million hectares, with a market value of US$ 4 billion in 2022. However, lettuce is highly susceptible to fungal pathogens that drastically reduce biomass and quality due to spoilage/rot. Therefore, in this study, we investigated the expression of chitinase genes via the lettuce chloroplast genome to enhance biomass and disease resistance. Site-specific integration of the expression cassette into chloroplast genomes was confirmed using two sets of PCR primers. Homoplasmy in transplastomic lines was confirmed in Southern blots by the absence of untransformed genomes. Maternal inheritance of transgenes was confirmed by the lack of segregation when seedlings were germinated in the selection medium. Chitinases expressed in chloroplasts are active in a broad range of pH (5-9) and temperatures (20-50 °C). Exochitinase expression significantly increased the number of leaves, root or shoot length and biomass throughout the growth cycle. Endochitinase expression reduced root/shoot biomass at early stages but recovered in older plants. Plant extracts expressing endochitinase/exochitinase showed activities as high as purified commercial enzymes. Antifungal activity in Candida albicans cultures inhibited growth up to 87%. A novel Carbotrace 680™ Optotracer binding to the ß-1,4 linkages of chitin, evaluated for the first time in plant systems, is highly sensitive to measure chitinase activity. To the best of our knowledge, this is the first report of chitinase expression via the chloroplast genomes of an edible plant, to confer desired agronomic traits or for biomedical applications.
生菜（Lactuca sativa）是一种广受欢迎的叶菜类蔬菜，全球产量约为2800万吨，种植面积超过100万公顷，2022年的市场价值为40亿美元。然而，生菜对真菌病原体高度敏感，这些病原体由于腐败/腐烂会大幅降低生物量和品质。因此，在本研究中，我们通过生菜叶绿体基因组研究了几丁质酶基因的表达，以提高生物量和抗病性。使用两组PCR引物确认了表达盒在叶绿体基因组中的位点特异性整合。通过Southern印迹法确认转基因系中的纯合性，即未检测到未转化的基因组。当幼苗在选择培养基中萌发时，由于缺乏分离现象，确认了转基因的母系遗传。在叶绿体中表达的几丁质酶在广泛的pH值（5-9）和温度（20-50°C）范围内具有活性。外几丁质酶的表达在整个生长周期中显著增加了叶片数量、根或茎的长度以及生物量。内几丁质酶的表达在早期阶段降低了根/茎生物量，但在老龄植株中恢复。表达内几丁质酶/外几丁质酶的植物提取物显示出与纯化的商业酶一样高的活性。白色念珠菌培养物中的抗真菌活性抑制生长高达87%。一种新型的Carbotrace 680™ Optotracer与几丁质的β-1,4键结合，首次在植物系统中进行评估，对测量几丁质酶活性高度敏感。据我们所知，这是通过可食用植物的叶绿体基因组表达几丁质酶以赋予所需农艺性状或用于生物医学应用的首次报道。
