Cloning And Functional Analysis Of NnCER26 Genes Involved In Very Long Chain Fatty Acids Elongation In Sacred Lotus

Cuticular waxes that cover the aerial organs of land plants have many functions,such as protecting plants against pathogens and insects,protecting plants from desiccation and so on.Therefore,waxes play an important role in plants growth and development as well as adaption to the environment.In plants,very long chain fatty acids(VLCFAs)are essential precursors of various lipids,including the cuticular waxes,the sphingolipids and phospholipids in cell membranes,the aliphatic suberin,and the triacylglycerols(TAGs)that accumulate in seeds.VLCFAs and their derivatives can act as protective barriers between plants and the environment,provide energy that stored in seeds,and function as signaling molecules in membranes.There have been many studies about wax synthesis and VLCFAs elongation in Arabidopsis so far,whereas the research on lotus wax is still limited to component analysis,leaving genes involved in its wax synthesis and VLCFAs elongation unreported.Using the expression levels of genes in lotus leaves as an reference,we screened out two candidate genes that involved in VLCFAs elongation in sacred lotus by sequence alignments,which were named NnCER26LIKE1 and NnCER26LIKE2.After cloning the genes,we analyzed their functions through genetic transformation intoArabidopsis and heterologous expression in yeast assays.This study enriches the theoretical basis of wax synthesis and provides a basis for further studies on the VLCFAs elongation mechanism of lotus.The main results are as follows:1?Scanning electron microscope was used to observe the cuticular waxes of different varieties of lotus.The results showed that there were slight differences in their wax structures.Then we used Thin-layer chromatography(TLC)to separate their wax compounds.The results showed that the TLC separation strips were also different between different varieties,and the separation strips of the 'Chinese Antique Lotus 543 ' were relatively less,which suggests its wax component is relatively simple.2?By sequence alignments,we screened out two genes that are homologous to CER26\gene in Arabidopsis,which are named NnCER26LIKE1 and NnCER26LIKE2.Tissue specific expression analysis showed that NnCER26LIKE1 and NnCER26LIKE2 are highly expressed in petal,anther,floating leaves and erect leaves.And their expression patterns are consistent with that of the genes which have been reported to relate to the VLCFAs elongation.Subcellular localization experiments showed that the NnCER26LIKE1 protein was localized to the endoplasmic reticulum,consistent with the localization results of proteins related to wax synthesis.3?Overexpression of NnCER26LIKE1 or NnCER26LIKE2 in Arabidopsis affected the carbon chain composition of waxes,indicating that two genes are involved in the VLCFAs elongation in lotus.And NnCER26LIKE1 participates in the C30-C32 elongation process,while NnCER26LIKE2 has a role in the production of C30 acid.Analysis of results of heterologous expression in yeast assay suggested that NnCER26LIKE2 and NnCER6 together contribute to the synthesis of C28 acid and C30 acid.