Creating New Tomato (Solanum lycopersicum) Germplasm Resistance to Gray Mold Using Eucommia ulmoides Laccase 1 (EuLAC1) Gene
YANG Jin-Yu1,3, WANG Zuo-Ri1,3, ZHAO De-Gang1,3,4, ZHAO Yi-Chen1,2,3,*
1 College of Life Sciences/Key laboratory of Plant Resource Conservation and Germplasm Innovation in Mountainous Region (Ministry of Education), Guizhou University, Guiyang 550025, China; 2 College of Tea Sciences, Guizhou University, Guiyang 550025, China; 3 National-Local Joint Engineering Research Center of Karst Region Plant Resources Utilization &Breeding, Guiyang 550025, China; 4 Guizhou Key Laboratory of Agricultural Biotechnology/Plants Conservation Technology Center, Guizhou Academy of Agricultural Sciences, Guiyang 550025, China
Abstract：Laccase (LAC) is a glycoprotein oxidase that influences lignin synthesis, regulates the levels of phenolic compounds in plants, and participates in plant defense mechanisms against pests and diseases. In order to obtain a new tomato (Solanum lycopersicum) germplasm with high resistance to Botrytis cinerea and in compliance with transgenic safety, in this study, based on the pGM626-Act1 vector constructed in the laboratory in the previous stage, a gene-deleter system with the fruit-specific E8 promoter driving the FLP gene and the Act1 promoter driving the Eucommia ulmoides laccase 1 (EuLAC1) gene in a plant expression vector was designed and constructed. Agrobacterium-mediated transformation technology was used to genetically transform the tomato variety 'Micro-Tom' and 5 positive tomato transgenic plants overexpressing EuLAC1 were successfully obtained. The results of resistance analysis showed that the onset time of EuLAC1 overexpressing tomato plants was significantly delayed after inoculation with B. cinerea, which was in sharp contrast with wild type and empty vector control plants. Additionally, the lesion diameter in tomato plants overexpressing the EuLAC1 gene was significantly smaller than that in the control group (P<0.05), which comprised the wild type and empty vector tomato plants. The result showed that overexpressing the EuLAC1 gene in tomato plants markedly improved their resistance to B. cinerea. Overexpression of the EuLAC1 gene also increased the activity of protective enzymes and the expression level of disease process-related protein (PR) genes in tomato plants. Deletion analysis of exogenous genes in transgenic tomato fruits revealed that no exogenous genes were detected in 22 out of 40 tomato fruits, with an exogenous gene deletion efficiency of 55%. This study provides a new technical method for the development of new disease-resistant tomato germplasm that meets transgenic safety standards.
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