Cloning And Functional Study Of Blue Light Receptor Gene ZTL In Camellia Oleifera

Camellia oleifera Abel.(C.oleifera)is an important woody oil tree species in China,and the influencing factors and regulatory mechanism of its flowering and fruiting have attracted much attention.ZTL is a blue light receptor and circadian clock regulatory protein,as well as an important component of the E3 ubiquitin ligase complex.It can be directly or indirectly involved in a variety of physiological processes in plants,such as flowering,light morphogenesis,and Circadian clock signal input.In order to understand the effect of the CoZTL gene of C.oleifera on photomorphogenesis and flowering time of C.oleifera,the expression mode and function of ZTL gene in C.oleifera were preliminarily understood by means of bioinformatics analysis,qRT-PCR and heterologous expression,and the potential interaction proteins were screened out in C.oleifera by using yeast two-hybrid technique,which provided a theoretical basis for exploring and improving the light regulation mechanism of C.oleifera.The main results were as follows:1.Cloning of CDS of CoZTL gene in C.oleifera and bioinformatics analysis of its encoded protein.In this study,the CoZTL gene was isolated and translated into protein sequences.Online websites Expasy,SOPMA and NCBI were used to analyze the physical and chemical properties,domain and protein structure of CoZTL protein,as well as the homologous sequence analysis of CoZTL and the construction of evolutionary tree.The results showed that the CDS fragment of CoZTL gene was 1 827 bp,and it could encode 609 amino acids;the molecular formula encoding protein was C2947H4616N826O872S27,the relative molecular weight was 66 435.70 Da,and the PI was 5.78.CoZTL was a hydrophilic and unstable intracellular protein,the protein primary amino acid sequence included PAS,F-box,and Kelch repeat domains,it was highly similar and conserved in the evolution process with ZTLs protein from other species.CoZTL was closely related to InZTL(Ipomoea nil)and NaZTL(Nicotiana attenuata).2.The expression pattern of CoZTL in C.oleifera and the effect of CoZTL heterologous overexpression on Arabidopsis thaliana(A.thaliana)growth and development.The relative expression levels of CoZTL in different tissues and different flower bud stages of C.oleifera were determined by qRT-PCR technology,and CoZTL was heterologous transformed into A.thaliana by flower dip method to obtain transgenic A.thaliana plants.The hypocotyl growth and flowering times of the transgenic A.thaliana plants and Col-0 and ztl mutant plants were compared.The results showed that the tissue expression analysis showed that CoZTL had the highest expression in the fruit shells of C.oleifera and the expression at different flower bud stages was the highest in the middle of September.Compared with Col-0,the flowering time of the three CoZTL overexpressed A.thaliana lines was delayed by 2 d,2 d and 0.9 d,and the number of rosette leaves was increased by 2.1,2 and 1.6 under long day condition.Moreover,under dark condition,the hypocotyl lengths of A.thaliana lines were almost the same,while under weak light condition,overexpression of CoZTL increased the hypocotyl lengths of A.thaliana by 2.2 mm,2.85 mm and 1.8mm relative to Col-0.Therefore,overexpression of CoZTL could delay the flowering time of A.thaliana under long day condition and light-dependently promote of hypocotyl elongation.3.Detection of self-activating activity of CoZTL protein and screening of potential interaction proteins from C.oleifera.The self-activating activity of CoZTL was detected and the proteins that interact with CoZTL were screened using the yeast twohybrid.The results showed that the self-activating activity of CoZTL was inhibited by the addition of 20 mM/L 3-AT in the medium.Seven interaction protein fragments are obtained by yeast two-hybrid screening library of C.oleifera,they are:aspartate aminotransferase cytoplasmic(AAT),beta-galactosidase(β-GAL),anther-specific protein LAT52-like(LAT52-like),C2-DOMAIN ABA-RELATED 4-like(CAR4-like),L-ascorbate oxidase(AO),ubiquinol-cytochrome-c reductase complex assembly factor 1(UQCC1)and actin-depolymerizing factor 2(ADF2).The results of this studies indicated the expression pattern of CoZTL and its role in plant growth and development,we obtained seven protein fragments that directly interacted with CoZTL protein,providing a theoretical basis for the function of CoZTL gene in the growth and development of C.oleifera.