Cloning And Functional Verification Of The Wax Synthesis Gene BoCER1 In Cabbage(Brassica Oleracea L.var.capitata L.)

Cabbage（Brassica oleracea L.var.capitata L.）,belonging to Brassica of Cruciferous,is an important cultivated vegetable in China.Leaf color is an important trait in cabbage breeding.Compared with common cabbage,wax-less cabbage shows bright green color,which displays good economic characteristics.In this study,combined with fine mapping,transcriptomics and third-generation resequencing analysis,it was determined that the wax-deficient trait of the mutant was caused by the mutation of Bo CER1..The function of Bo CER1 gene was analyzed and verified,which laid a foundation for analyzing the molecular mechanism of dominant wax deficiency in cabbage.The main results are as follows:1.The gene expression of wax-less mutant g974 and wild type（WT）was analyzed by transcriptome sequencing.A total of 2360 genes were differentially expressed between g974 and WT.Some key genes of wax synthesis including Bo CER1,KCS2,CER2,CER3,MAH1,ACE1,CYP86A2and HIC1,were significantly down-regulated in g974.Expression analysis of genes in the mapping interval showed that only the expression of Bo CER1 is significantly different between g974 and WT,so Bo CER1 was regarded as an important candidate gene for the wax deficiency of g974.Expression analysis with q RT-PCR showed that Bo CER1 was significantly decreased in g974 and F₁compared with WT.2.The third generation re-sequencing showed that two structural variants were detected in Bo CER1gene of g974,including a 20bp deletion and a 210bp insertion.Moreover,there are two regions on the Bo CER1 gene of g974 that are not covered by reads.These results suggested that the Bo CER1 gene in g974 has undergone complex structural changes.The mutated Bo CER1 gene has new biological functions and interferes with the biosynthesis of cabbage wax.3.Expression analysis with q RT-PCR showed that Bo CER1 was expressed in various tissues including stems,leaves,flowers,and siliques,but not in the roots of the cabbage.The expression level in siliques was the highest,followed by that in the flowers and leaves.Moreover,the in situ hybridization showed that Bo CER1 was expressed in the epidermis and mesophyll of cabbage leaves.4.To determine the function of Bo CER1 in cabbage,the gene editing vector CRISPR-Bo CER1 was constructed and transformed into the cabbage inbred line‘YL-1’.Three plants with edited genomes were obtained:CW1-1,CW1-2 and CW1-3.The leaves color of three plants showed bright green compared with the WT.Scanning electron microscopy and gas chromatography mass spectrometry analyses of CW1-3 showed that the wax components,including alkanes,alcohols and ketones,were significantly reduced compared with the WT.The results showed that Bo CER1 plays an important role in the biosynthesis of cuticular wax in cabbage.