Cloning And Functional Analysis Of BoGL4 And BoGL1 Involved In Cabbage Wax Reduction

Cabbage?Brassica oleracea L.var.capitata L.?,originated from the Mediterranean Coast,is one of the important vegetables belonging to Brassica genus in Cruciferae.It plays important roles in the domestic supply and export trade.With a layer of wax covering leaves and stems,appearance of normal cabbage turns to be green,dark green,or grayish green.The glossy waxless cabbages owning attractive appearance are mutants from normal ones.In the present study,the genes in two cabbage glossy mutants were finely mapped,and the target gene in mutant LD10 was cloned and functionally verified.The main research results are as follows:1. Compared with the wild type materials,glossy green trait is exhibited by both the two cabbage waxless mutants LD10 and 10Q-974,which shows better commodity character of the glossy character.Wax crystals are severely reduced in two mutants and decreased content of different wax components in two mutants was detected,LD10 shows a drastic reduction of C22,C24,C26,C28 and C30 alcohols,while 10Q-974 shows a drastic reduction of C29 and C33 alkane.Hybridized combinations experiment results showed that the glossy green trait owns great breeding application value.2.Six generation populations were constructed through crossing,self and back-crossing of LD10 and Chinese Kale material M36.All the individuals in F₁ population and BC₂population show the normal waxy phenotype.The ratio of normal to waxless plants in F₂population is 3:1,and it is 1:1 in BC₁ population.All the ratios show that the glossy wax-less trait in cabbage LD10 is controlled by one single recessive gene.Inheritance of glossy waxless trait in 10Q-974 was analyzed with the same method and the results showed that the glossy waxless trait in cabbage mutant 10Q-974 is controlled by one single dominant gene.3.Based on primer screening and gene mapping result,the mutant gene in LD10 was finely mapped to a region of 170 kb on cabbage chromosome 1 between markers C01g SSR147 and C01g SSR150 with a genetic distance of 0.2 c M.Through sequence analysis of the genes in the mapped region,a gene named Bol013612 homologous to Arabidopsis CER4 was defined as the candidate gene for Bo GL4.With primer screening and gene mapping,the mutant gene in 10Q-974 was finely mapped to a 177kb region on the end of cabbage chromosome 8 by flanking markers.Gene Bol018504 homologous to CER1?ECERIFERUM 1?was selected as the candidate gene for Bo GL1.4.The candidate gene for Bol GL4 and Bo GL1 were cloned and sequenced respectively.Compared with wild type,there existing a base substitution in g DNA of Bol013612 and the base substitution brought an insertion of six nucleotides to c DNA.But out of our expection,no nucleotide variance was found both in c DNA and g DNA of gene Bol018504in 10Q-974,which implys that Bol018504 is not the gene for Bo GL1.5.The phenotypic defect of LD10 was confirmed by a functional complementation test with Arabidopsis mutant cer4-1,which shows that the glossy green trait in cer4-1 could not be restored to glaucous with gene Bol013612 from LD10 but it could be restored to glaucous phenotype with gene Bol013612 from WT.The functional complementation test verified the loss of function mutation of gene Bol013612 in LD10 and further implies that it is the mutation of gene Bol013612 causes glossiness in LD10.