Cloning And Identification Of BnCLG1 Gene In Brassica Napus

The leaf surface of most terrestrial plants' is covered with a layer of natural hydrophobic barrier-the cuticle,a special membrane structure secreted by the epidermal cell.The main components of cuticle are cutin and wax.It plays an important role in plant resistance to environmental stresses such as drought,ultraviolet radiation and pathogen infection.The wax biosynthesis has been extensively studied previously in Arabidopsis,but it still remains elusive in Brassica napus.In this study,we cloned two copies of a wax biosynthesis related gene,named BnCLG1,and their promoters in B.napus,through the homology cloning technology.We used the GUS staining,semi quantitative PCR and fluorescence quantitative PCR and genetic transformation technologies to study preliminarily the function of BnCLG1 gene in waxy cuticle,and aimed to explore the basic mechanism of cuticle wax synthesis in B.napus.The main results are resumed as followings:(1)Two copies of BnCLGl gene,named BnCLG1 A and BnCLGIC,respectively,have been identified in B.napus genome by bioinformatics analysis.They are orthologous to the Arabidopsis CER9 gene.They contain both 9 exons and 8 introns.BnCLG1A encodes a protein of 1110 amino acids in length and BnCLG1C encodes a protein of 1108 amino acids.The two proteins contain both 14 transmembrane domains and belong to the category of hydrophobic membrane proteins.They contain each a RING finger domain,and belong to the E3 ubiquitin protein ligase family.Analysis of BnCLGl promoter regions showed there existed not only the basic core components such as TATA-box and CAAT-box,but also the light regulatory elements,anaerobic cis regulatory elements,plant hormone regulatory elements,fungal elicitor responsive elements,and a variety of stress related components.(2)The GUS histochemical staining analysis showed that the pBnCLG1A and pBnCLG1C were continuously expressed in most of examined tissues,but only weakly expressed at the base of the stem.(3)RT-PCR analysis showed that BnCLG1A and BnCLGIC genes were consecutively expressed in roots,stems and leaves,flowers,buds and pods,and expressed at a higher level in stems,buds and siliques tissues.Meanwhile,the expression patterns of these two genes were the same among the different examined tissues.(4)The results of fluorescence quantitative PCR showed that,under the 200mM mannitol treatments(0?48h),the expression levels of BnCLGl genes increased firstly,and reached the highest point at 6 hours,under the 200mM NaCl treatments(0?48h),the expression levels of BnCLGl genes were down regulated,under the 200?M GA3 treatments(0?24h),the expression levels of BnCLGl genes increased sharply and reached the highest value at 24 hours,under the 100?M ABA treatments(0?24h),the expression levels of BnCLG1 genes were down regulated.It is worth noting that the expression of BnCLGl genes was completely inhibited from 3 to 24 hours by the 200?M SA treatments.So,BnCLG1 A and BnCLGIC genes could be induced by both drought and salt stresses and by phytohormones GA3,ABA and SA treatments.The expression patterns of these two genes were the same during the above stresses or treatments.(5)By using the scanning electron microscopy to observe the phenotype of overexpression of BnCLG1A and BnCLG1C genes in Arabidopsis,we found that the form of the cuticle morphology was more smooth in transgenic plants than in wild controls on both the leaf adaxial and abaxial surfaces.An obvious difference was also observed at the level of stem wax,the overexpression plants showed less wax particles than the wild controls.Our results suggested that the BnCLGl genes may negatively regulate the synthesis of cuticle wax in Arabidopsis and Brassica napus.