Cloning And Preliminary Functional Analysis Of AfCER2-LIKE1,A Gene Related To Wax Synthesis In Allium Fistulosum

Welsh onion(Allium fistulosum),which originats in China,is a traditional vegetable and condiments in China,Japan,South Korea and other East Asian countries.Wax is the outermost organic mixture of plants,which is composed of long-chain fatty acids with different carbon chain lengths and derivatives such as alkanes,alcohols,aldehydes,carboxylic acids and so on,forming a hydrophobic protective layer.It is commonly found in the surface layer of aboveground organs,injured tissues and cork of terrestrial plants.Wax can prevent the non-stomatal loss of water in the internal tissue of the plant or the plant from being damaged by harmful light,and has the functions of avoiding the damage of bacteria,it plays a certain role in reducing the impact of external stress on plants.Meanwhile,wax is associated with plant attraction and rejected to insects.The synthesis and transport of wax in the epidermis of Allium fistulosum is regulated by a series of enzymes and genes related to enzyme synthesis,and has a complex molecular mechanism.At present,the genes related to wax synthesis have been studied in Arabidopsis thaliana,maize and other plants,and the regulatory network has been roughly clear.Based on the data of transcriptome sequencing of the wax-deficient mutant glbg,it was found that there was a single nucleotidebase,polymorphismmutation of Af CER2-LIKE1 gene in the wax-deficient mutant of Allium fistulosum.It was speculated that the frameshift mutation led to the occurrence of the wax-deficient mutant of welsh onion.The function of Af KCS6 has been preliminarily understood in welsh onion.Studies have shown that Af KCS6 might mainly promote the synthesis of long-chain fatty acids and the synthesis of alkanes,alcohols and aldehydes in the process of wax synthesis,and indirectly inhibit the formation of esters.It has been confirmed that there is an interaction between KCS6 and CER2-LIKEs in rice and other plants.However,the molecular mechanism of Af CER2-LIKE1 gene regulating wax synthesis and metabolism in welsh onion epidermis was unclear,and whether there was an interaction between Af CER2-LIKE1 and Af KCS6 remains to be studied.In this study,in order to clarify the function of Af CER2-LIKE1 gene,bio-informatics technology was used to analyze the gene to understand its physicaland chemical properties,and then sub-cellular localization technique was used to identify the expression position of Af CER2-LIKE1 gene in cells.Finally,yeast two-hybrid technique,LUC and Bi FC technique were used to study the interaction between Af CER2-LIKE1 and Af KCS6 proteins.The main results of this study are as follows:(1)The Af CER2-LIKE1 gene was cloned from the c DNA of bg.The CDS sequence of the gene was 1260 bp and encodes 419 amino acids.Through multiple alignment analysis of amino acid sequences,it was found that there is a HXXXD motif in Af CER2-LIKE1.BLASTp alignment results showed that Af CER2-LIKE1 has more than 50% homology with asparagus and pineapple.Phylogenetic tree analysis shows that Af CER2-LIKE1 and Ao CER26-LIKE are on the same branch.The genetic relationship between them is the most similar.(2)Using sub-cellular localization technique,tobacco protoplast cells were transformed by polyethylene glycol induction,and it was found that Af CER2-LIKE1 was localized in the cytoplasm.(3)Using yeast two-hybrid technique,the full-length CDS of Af KCS6 and Af CER2-LIKE1 genes were constructed in p GBKT7 vector and p GADT7 vector respectively,and then co-transformed into Y2 HGold yeast strain.The results showed that bg Af KCS6 and Af CER2-LIKE1 could interact in yeast in bg,but there was no interaction between Af KCS6 and Af CER2-LIKE1 in glbg.(4)Af KCS6 and Af CER2-LIKE1 were sub-cloned into p SAT6-c EYFP-C1 and p SAT6-n EYFP-C1 vectors by Bi FC technique,and then they were co-transformed into Nicotiana.Benthamiana protoplasts by PEG induction transformation.The results showed that Af KCS6 and Af CER2-LIKE1 could be observed in protoplasts when using bg as materials,and there was an interaction between Af KCS6 and Af CER2-LIKE1 in plants.Signal could not be detected in protoplasts and there was no interaction between the two proteins in glbg.(5)Af KCS6 and Af CER2-LIKE1 were subcloned into p CAMBIA-n LUC and p CAMBIA-c LUC vectors by double luciferase reporter gene system,and Agrobacterium tumefaciens was injected into the back of Nicotiana.Benthamiana leaves by syringe injection.Later,through the observation of plant in vivo imaging system,strong fluorescence signal could be observed in bg experimental group,but there was no fluorescence signal in glbg.By observing whether there was fluorescence signal or not,we could judge whether there was interaction between proteins or not,which further proved that there was interaction between Af KCS6 and Af CER2-LIKE1 in plants.