Identification and Analysis of Amylase Genes Related to Pre-harvest Sprouting Resistance in Wan 1329 Based on Transcriptome Sequencing

WANG Zhen; ZHANG Bin; SHI Lichao; LIU Rui; LI Jinxiu; ZHOU Ran; YANG Ling; ZHAO Qian; LI Jinbang

doi:10.15933/j.cnki.1004-3268.2025.12.004

Abstract：Using Jimai 22 with high sensitivity to pre-harvest sprouting(PHS)as the control,the PHS resistance and α-amylase activities of the new white-grain wheat variety Wan 1329 and its parent Lianmai 2 were analyzed.During the critical period when α-amylase activity changed,transcriptome sequencing,KEGG and GO enrichment analysis were employed to identify differentially expressed amylase genes,and real-time fluorescence quantitative PCR(qRT-PCR)was used for verification their expression patterns.In addition,AlphaFold was employed to analyze the effects of coding sequence variations on protein structure.The results showed that both Wan 1329 and Lianmai 2 exhibited moderate resistance to PHS,while Wan 1329 had a lower relative germination index,indicating stronger resistance.At 24 h after seed imbibition,the α-amylase activities of Wan 1329 and Lianmai 2 were markedly lower than that of Jimai 22,and α-amylase activities at 20 h,24 h,and 72 h were highly positively correlated with the relative germination index.Transcriptome sequencing,KEGG and GO enrichment analysis revealed that two α-amylase genes located on the long arm of chromosome 6D,TraesCS6D02G313300 and TraesCS6D02G313500,were candidate α-amylase genes.The third exon of TraesCS6D02G313500 in Wan 1329 was elongated by 138 bp,extending into the 3'UTR region.qRT-PCR validation confirmed that the expression patterns of both genes were consistent with transcriptome analysis,and the cumulative relative expression level of TraesCS6D02G313500 in Wan 1329 was significantly lower than that in Lianmai 2.AlphaFold prediction suggested that this variation caused a C-terminal extension of α-amylase,resulting in decreased structural stability and increased conformational flexibility.Collectively,TraesCS6D02G313300 and TraesCS6D02G313500 are key genes related to PHS resistance in Wan 1329,among which the structural variation of TraesCS6D02G313500 may further enhance resistance to PHS.