Citrus Transformation Of Poncirs Trifoliata Cold-responsive Genes ERF109 And ZFP1 And Analysis Of Transgenic Plants

Citrus takes up a very important economic status in the fruit industry of China.However,the periodical freezing injury seriously affects the yield and quality of citrus,which makes it important to carry out cold resistance breeding of citrus so as to improve the cold resistance of citrus.Genetic transformation is one way to get the stress resistant germplasms.In this research ERF 109 gene and ZFP1 gene cloned from Poncirus trifoliata were transformed into cold sensitive lemon via Agrobacterium-mediated method Meanwhile,cold tolerance of the transgenic plants was assessed.The main results of the experiment are as follows:1.Transformation and regeneration.A total of 800 lemon epicotyls were used for transformation of ERF109,from which 200 regenerating buds were obtained,while 300 regenerating buds were produced from 600 lemon epicotyls transformed with ZFP1.2.Molecular identification of the regenerating buds.Based on the results of PCR analysis,22 positive lines were verified as transgenic plants overexpressing ERF109,while 6 transgenic plants were confirmed from the ZFP1-transforming lines.The expression levels of the ERF 109 and ZFP1 were higher than those of the wild type plants,as revealed by semi-quantitative PCR and the qRT-PCR.3.The cold resistance analysis of transgenic lines.After cold treatment,the leaves of WT plants suffered from more serious wilting and had a higher relative electrolyte leakage than the transgenic lines.The staining with DAB and NBT suggests that transgenic lines overexpressing ERF109 and ZFP1 exhibited less accumulation of ROS after cold treatment.Collectively,these results indicate that overexpression of ERF109 and ZFP1 may greatly improve cold tolerance of lemon by maintaining a low level of ROS.