Genetic Analysis And Functional Verification Of Candidate Genes Of Blue Aleurone Traits In Triticale

Triticale is a new species that is composed of Triticum and Secale through intergeneric hybridization and hybrid chromosome number doubling.The color of Triticale kernel is diverse.The blue grain is caused by the anthocyanins in the aleurone layer,and the blue grain trait can be used as a special genetic marker,which has practical breeding value.But its molecular genetic mechanism is still unclear.In this study,transcriptome sequencing technology was used to analyze the expression levels of genes involved in anthocyanin biosynthesis pathway in blue-white aleurone layer to find differentially expressed genes,and to screen out candidate genes for the regulation of anthocyanin synthesis in Triticale aleurone.Bioinformatics was used to analyze the biological characteristics of candidate genes,and the coding sequence of the candidate gene was isolated and verified by transient expression.The main results are as follows:1.The blue and white aleurone layers were separated for transcriptome sequencing,and the raw data of blue grain was 11.08 Gb,the white grain were 9.74 Gb,the effective data by Trinty was 20.82 Gb,and the average length of genes wasbp,there producing total 73886 unigenes.The homologous alignment of the protein indicated that the proteins in the aleurone layer had the highest homology with Aegilops tauschii,followed by barley and wheat.Compared with the white grain,there were 7588 unigenes were up-regulated in the blue granule layer.There are25,319 unigenes were down-regulated.There are 808 unigenes involved in secondary metabolism in the GO functional classification;KEGG enrichment analysis found that there were about 100 differential unigenes in anthocyanin biosynthesis;86homologous genes were screened using structural genes in the anthocyanin biosynthesis pathway Among them,most of the structural genes in the anthocyanin biosynthesis pathway in the blue grain were higher than the white grain,indicating that the anthocyanin biosynthetic pathway in the blue grain was activated.In addition,two MYC and two MYB transcription factors were obtained.The expression level of MYC transcription factor Unigene5672_All(BcMYC1)was 42 times in blue graincompared with white grainand MYB transcription factor Unigene12228_All was expressed 2 times in blue grain than white grain.,Unigene5672_All(BcMYC1),were not expressed in the white grain and the MYB transcription factor Unigene12228_All was expressed in the white grain.Therefore,the MYC transcription factor BcMYC1 was selected as a candidate gene for regulating anthocyanin biosynthesis in the blue aleurone layer.2.The BcMYC1 gene CDS sequence was 1683 bp and encoded 560 amino acids.The results of ExPaSy-Protparam software showed a molecular weight of 61,070 Da,an isoelectric point of 4.71,an instability index of 50.58,and a fat index of 78.71.The hydrophilicity coefficient was-0.400,which confirmed that the BcMYC1 protein possessed a hydrophilic function.According to SOPMA online analysis,the secondary structure of BcMYC1 gene had ?-helix accounting for 39.64%,irregular coiling accounting for 43.93%,extended chain accounting for 11.96% and ?-turn accounting for 4.46%.Analysis using TMHMM-2.0 software showed that BcMYC1 protein did not pass through the transmembrane region and acted outside the membrane.Phylogenetic tree analysis revealed that BcMYC1 protein is clustered with the bHLH transcription factor involved in the regulation of anthocyanin biosynthesis metabolism in wheat,petunia and rice,indicating that BcMYC1 and bHLH transcription factors are homologous.Amino acid alignment showed that BcMYC1 protein was similar to the protein ThMYC4 E controlling blue grain trait wheat and TaMYC1 controlling purple grain trait There were contained bHLH-MYC_N domain,HLH domain and ACT-like domain,suggesting that BcMYC1 likely was a functional gene that regulated the biosynthesis of anthocyanins.Quantitative analysis by RT-PCR showed that the BcMYC1 gene was expressed in blue granules higher than white granules.The transient expression vector pBract214: BcMYC1 was constructed using Gateway cloning technology.Using the bHLH transcription factor ZmR as a control vector,the expression vector pBract214:BcMYC1 was mixed with the MYB transcription factor ZmC1 then bombarded into the white coleoptile in the wheat‘Opata'.And the mixed vectors produced several red cells while independently bombarding pBract214: BcMYC1,ZmR and ZmC1 failed to induce red cells.This proved that BcMYC1 was the candidate gene regulating anthocyanin biosynthesis in blue aleurone.