Preliminary study on Agrobacterium rhizogenes-mediated gene overexpression system in watermelon

Abstract: 【Objective】Watermelon (Citrullus lanatus) is a worldwide horticultural crop with a long history and extensive planting areas. China is the country with the largest watermelon planting area and yield in the world and also at the forefront of the world in the study on watermelon genomics and functional gene mining. Agrobacterium tumefaciens meditated transformation of watermelon has been successfully conquered by Beijing Academy of Agricultural and Forestry Sciences. However, the watermelon transgenic technology is still difficult for most researchers, which is an important factor that restricts the studying progress on watermelon genes. In this study, a rapid method of inducing adventitious roots in watermelon was established. Through the mediation of Agrobacterium rhizogenes, the exogenous genes could overexpress in adventitious roots.【Methods】The wild type A. rhizogenes strain K599 and watermelon variety'Sugar Baby'were chosen to explore the operation method. Seedlings with two just unfolded cotyledons were inoculated. A microsyringe needle was used to pick up a piece of A. rhizogenes K599 colony and pierce through the cotyledon node. After inoculation, the seedlings were covered with plastic cups to maintain high air humidity and then cultured at 22 ℃ in dark for 8-12 h. After dark incubation, the bottoms of the cups were cut off, and then the cups were placed upward on the seedlings. Vermiculite was used to fill the cups until the seedling inoculation sites were covered. Pour the vermiculite through with a spray pot. Seedlings were cultured in the light incubator with a 12 h light/12 h darkness photoperiod, and the day and night temperatures were set to 28/25 ℃. Vermiculite was added and seedlings were watered daily to keep the inoculation site moist and in darkness. About 20 days after inoculation, the adventitious roots appeared at the inoculation sites. The main roots and hypocotyls were cut off. Then, the plants were buried in the nutrient soil and continued to grow. With this method,the overexpression vectors pCAMBIA3301 and pCAMBIA1300-ProSuper containing GUS and EGFP genes were introduced into A. rhizogenes K599 to infect'Sugar Baby'. The effect of the overexpression system was evaluated by common PCR. The reaction system of PCR was as follows: 1 μL DNA template, 0.5 μL of each upstream and downstream primers, 5 μL 2×PCR Master Mix, and 3 μL ddH2O.The PCR reaction procedure for pCAMBIA3301 vector detection was pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 s, annealing at 50 ℃ for 30 s, and extension at 72 ℃ for 50 s for 35 cycles, and finally extension at 72 ℃ for 8 min and preservation at 4 ℃. The PCR reaction procedure for pCAMBIA1300-ProSuper vector detection was pre-denaturation at 95 ℃ for 3 min, denaturation at 95 ℃ for 30 s, annealing at 50 ℃ for 50 s, and extension at 72 ℃ for 1 min for 35 cycles, and finally extension at 72 ℃ for 10 min and preservation at 4 ℃. PCR products were detected by 1% agarose gel electrophoresis. To analyze the expression of GUS and EGFP in adventitious roots, qRT-PCR was conducted. First strand cDNA was synthesized using a PrimeScriptTM RT reagent Kit with gDNA Eraser (Takara, RR047 A) following the manufacturer's protocol. PCR reactions were performed on a Roche LightCycler®480 RT-PCR System using the SYBR Premix Ex TaqTM Kit (Takara, RR420 A). Expression levels were analyzed following the 2-ΔΔtmethod. The expression of GUS and EGFP was also detected by GUS staining and confocal laser microscopy.【Results】After seedlings were inoculated by the wild type A. rhizogenes strain K599, adventitious roots were successfully induced at the inoculation site in'Sugar Baby'. This indicated the operation method of inducing adventitious roots in watermelon by A. rhizogenes K599 was feasible. Following this method, A. rhizogenes K599 carrying pCAMBIA3301 and pCAMBIA1300-ProSuper vectors successfully induced the adventitious roots in'Sugar Baby'. The induction rate of adventitious roots was 70%-90%. PCR verification results showed that the positive rate of adventitious roots was 100%. GUS and EGFP genes were detected to be highly expressed by qRTPCR and verified by GUS staining and confocal laser microscopy detection.【Conclusion】Genetic transformation is an important means to analyze gene function, but the research progress on cucurbita genetic transformation is relatively slow. Although the transgenic technology of watermelon has been conquered by Beijing Academy of Agricultural and Forestry Sciences, it is still an important factor restricting many researchers to study the gene function of watermelon. In this study, A. rhizogenes K599-mediated gene overexpression system was established in watermelon. This system has the advantages of short cycle, easy operation, high transformation rate, and can realize the rapid verification of gene function in watermelon roots. It will provide technical support for the research on the mechanism of genes that specifically play roles in watermelon roots.