Cloning And Expression Analysis Of Wax Genes AfCER1 And AfKCS6 In Welsh Onion

Welsh onion is one of the traditional vegetables and condiments,and the leaves surface is covered clearly with the wax layer.In this study,a mutant welsh onion with fewer wax was found,and this mutant is considered to be the better material to investigative the mechanism of the waxy biosynthesis.We analyzed the transcriptome of welsh onion and screened out two genes AfCER1 and AfKCS6,which are related to waxy biosynthesis.In this research,we used the wild-type and mutant welsh onion as experimental materials,and combined the morphology,Bioinformatics,physiology and biochemical analysis to explore the characteristics of waxy biosynthesis,aiming to provide a theoretical basis for the molecular mechanism of waxy biosynthesis in welsh onion.The main results are as follows:1.It was observed the surface of leaves in wild-type welsh onion is covered with pointed crystals by scanning electron microscope,while no wax crystals was found in mutant welsh onion.Compared with wild-type,the mutant welsh onion had lower height,finer stem diameter,slower transpiration rate and less stomas of per unit area,and had higher chlorophyll content and fater net photosynthesis rate.2.The AfCER1 gene full sequences were obtained by RT-PCR(reverse transcription-polymerase chain reaction)and race(rapid amplification of cDNA ends)technology,and then analyzed the gene sequence by bioinformatics.The spatial expression pattern of AfCER1 in wild-type and mutant welsh onion was analyzed by RT-PCR and Real-time PCR.The data showed that the AfCER1 full-length cDNA was 2144 bp,and contained an 1857 bp open reading frame(ORF)which encoded 618 amino acids,the protein structure showed that AfCER1 had a fatty acid hydroxylase region and a WAX2-C domain,which was similar to the CER1 functional domains of other species.RT-PCR results indicate that AfCER1 gene was expressed in root,leaf sheath and leaf,and there were differences in the expression of wild and mutant plants.AfCERl was less expressed in the root,leaf sheaths and leaves of mutant,but higher expressed in the lighted leaves of mutant plants.3.The base sequences of AfKCS6 have some difference between wild-type and mutant welsh onions,but predicted amino acid sequences were identical.Full-length gene contained a 1491 bp open reading frame(ORF)and encoded 497 amino acids.AfKCS6 gene was obviously expressed in leaf,and the expression have significant differences in wild type and mutant welsh onions,AJKCS6 was slower expressed in mutant plants.4.We cloned the waxy biosynthesis related gene AfKCS6 into the prokaryotic expression vector PET-28a,and the recombinant plasmid was renamed as PET28a-AJKCS6.