Screening And Function Identification Of Candidate Genes Involved In Diketone Biosynthesis Of Cuticular In Wheat

Cuticle wax is the outmost layer of plants,it is a protective barrier to prevent the interaction of plant and environment.Waxes are composed of very-long-chain fatty acids and their derivatives,they play an important role in plant’s growth and development,they can effectively stop the non-stomatal water loss,against the UV-B damage,maintain a sanitary and also prevent the erosion of insect.MAH1 is a key enzyme in the decarbonylation pathway,which mainly catalyzes the consecutive oxidation from alkanes to secondary alcohols,and then from secondary alcohols to ketones.Wheat is an important food crop,it’s epidermal waxes are consist of alkanes,primary alcohols,fatty acids,fatty aldehyde,and also a mass of β-diketones.In this study,we analyzed the contents and compositions of cuticle waxes on spikes from 17 wheat varieties(lines)by using GC-MS、GC-FID in order to understand the characteristics of the wax of wheat.We also utilized BLAST to search the database of wheat to find the high homology genes,and used 310 as the DNA template cloned 8 homologous genes.Then we transformed the genes into “Micro Tom” to detect the compositions and contents of leaves of positive tomatoes for proving genes’ function.The results are as follows:1.The results had shown that the compositions were no obvious difference but significant variance in the total wax and the contents of each component among the 17 wheat varieties(lines).The total waxes were significant higher in the 16 glaucous varieties(lines)than the glossy one(Chinese Spring).The β-diketones were the major component of the epicuticular wax and account for 70.68%-88.29% in the glaucous varieties,while the β-diketones and alkanes were the dominant components in the spike waxes of the Chinese Spring(accounted for 38.36% and 38.14%,respectively).The β-diketones could be detected in both glaucous and glossy wheats,but the derivatives OH-β-diketones could only be detected in the glaucous wheat varieties(lines).2.We filtered 45 genes from the database of wheat,by using bioinformatics analysis we successfully selected 10 candidate genes,and named as TaMAH1-10,and the genomic sequences are between 1300 bp and 1500 bp.According to phylogenetic analysis,we found TaMAH2 and TaMAH5 encoded the same protein,TaMAH1,TaMAH2,TaMAH3,TaMAH4,TaMAH5 and TaMAH9 had close kinship to AtMAH1,however,TaMAH6,TaMAH7,TaMAH8 and TaMAH9 they are far with AtMAH1.Finally,8 wheat diketones candidate genes were cloned and the plant expression vectors were constructed.3.8 TaMAH genes were successfully introduced into the tomato "Micro Tom" by Agrobacterium-mediated method and obtained several transgenic plants.Based on the PCR identification,54 positive transgenic plants were selected.By the detection of wax of positive plants,the results showed that the compositions of wax in positive transgenic plant had no difference with the control,but the total wax load,alkanes,alcohols,fatty acids of TaMAH1B、TaMAH3 increased.Among them the content of alcohols had significantly increased to the control,and the carbon distribution had also changed.The odd carbon alcohols were detected in leaves of positive transgenic plants,in addition,the content of C26:0-OH、C27:0-OH、C29:0-OH、C31:0-OH elevated obviously.The terpenoid had no obvious difference compared to the control.