Functional Characterization Of Nitrogen-responsive LBD Transcription Factors In Tea Plant (Camellia Sinensis L.)

Tea plant is a perennial leaf-harvesting crop.To increase tea yields,people often apply large amount nitrogen fertilizers.This not only raises production costs and causes huge waste of resources,but also causes serious threats to the ecological environment.Although some pollution can be reduced through deep fertilizer application and slow-release fertilizer,it cannot fundamentally solve the problem.With the development of molecular biology,exploiting nitrogen-efficient germplasm resources are an important direction of tea tree biology.The LBD family is a class of plant-specific transcription factors.They regulate plant growth and development,and also plays an important role in the regulation of plant carbon/nitrogen metabolism.In this study,25 Cs LBDs genes were selected from the transcriptomic data of tea plant roots treated with five nitrogen forms.Subsequently,the function of Cs LBDs genes was studied through a series of molecular biology.Preliminary research results show that Cs LBDs gene plays an important role in the regulation of nitrogen metabolism and nitrogen utilization efficiency in tea plants.The main results are summarized as follows.1.Twenty-five Cs LBDs were screened from transcriptomic data of nitrogen treated tea plant roots.These Cs LBDs contain the specific LOB domain,are hydrophilic proteins,and have multiple phosphorylation sites.The ORFs of Cs LBD38,Cs LBD1＿1,Cs LBD37＿2 and Cs LBD39＿1 were cloned from the tea variety "Shuchazao".2.The expression patterns of Cs LBDs in roots under different nitrogen treatments,in different tissues,and in different developmental stages of different varieties were detected by real-time quantitative PCR.The results showed that Cs LBDs have tissue expression specificity and respond differently to nitrogen forms.Cs LBDs expression also varied in different developmental stages and in different varieties.3.Subcellular localization vectors were constructed and the nuclear localization of four Cs LBDs was verified by transient expression of tobacco.The results clearly showed that these Cs LBDs are localized in nucleus.4.Cs LBDs were cloned into the expression vector p GBKT7 and expressed in yeast.Cs LBDs were proved to have transcriptional activation activity by transcription activation assay.5.Prokaryotic expression vectors were constructed and induced by IPTG.Cs LBD-GST fusion proteins were purified.The polyclonal antibody of Cs LBD1＿1 was prepared.EMSA and Ch IP experiments were preliminarily carried out.This provided important basis for further research on the molecular mechanism of Cs LBDs.6.The overexpression vectors of Cs LBDs was constructed and transformed into Arabidopsis thaliana,and overexpression lines of four Cs LBDs were obtained.Preliminary results showed that the overexpression of Cs LBDs in Arabidopsis thaliana had different effects on growth,development and response to nitrogen deficiency stress.Among these lines,Cs LBD38 overexpression lines showed promoted root development and resistance to nitrogen deficiency.