Evaluation of Citrus-derived Constitutive Promoters in Citrus, Nicotiana benthamiana, and Grapevine

Citrus is affected by a plethora of biotic and abiotic challenges. The long juvenile phase in citrus (varying from 5 to 20 years) makes rapid cultivar improvement through conventional breeding difficult. The development of new cultivars via genetic engineering can provide a much faster alternative for citrus improvement. The production of genetically modified (GM) crops containing viral and/or bacterial sequences is however controversial due to potential but unsubstantiated environmental and health risks. Development of GM citrus by utilizing solely citrus DNA can also help build confidence in the evaluation and acceptance of GM foods/plant by both regulatory agencies and the general public. We have identified several constitutive promoters from Citrus clementina cv. Nules that regulate transgene expression at high levels in dicotyledonous plants including Citrus sinensis X Poncirus trifoliata cv. Carrizo, Nicotiana benthamiana and Vitis vinifera cv. Thomson Seedless. The genomic sequence upstream of the transcription start site of seven Ubiquitin and one Rubisco small subunit gene was identified from the citrus genome database and characterized using the Vector NTI® sequence analysis software. A 1 kb upstream sequence was cloned from genomic DNA and verified by Sanger sequencing. Two sets of transformation vectors (one set containing the respective promoter driving the gus gene and the other set driving a VvmybA1 reporter gene, that induces the anthocyanin biosynthesis) were produced and incorporated into each of the three cultivars via Agrobacterium mediated transformation.  Gene expression generally followed a similar trend in all three plant species studied. In citrus, plants containing the UBQ3 promoter had the highest gene activity followed by the UBQ9, RBCS1A and the UBQ10 promoter. The remaining promoters were functionally weak in their ability to drive the gus gene.  In Nicotiana benthamiana, plants containing the UBQ9 promoter had the highest gene activity followed by the UBQ3, RBCS1A and the UBQ10 promoter. In Vitis vinifera the UBQ9 promoter had the highest overall gene activity followed by the UBQ3, UBQ10 and the RBCS1A promoter. Transient grapevine embryos weakly expressed both the gus and the VvmybA1gene(s) driven by the RBCS1A promoter but activity improved on transfer to light. Both UBQ3 and UBQ9 promoters were significantly superior to the control d35S promoter. Our results demonstrate the utility of using endogenous promoter sequences to drive transgenes for the genetic improvement of citrus and other crops.