Cloning of γ-Gliadin 1 Gene and Screening of Its Interacting Proteins in Wheat

WANG Shasha; HE Ning; HUANG Chao; WANG Gang; WANG Qingchang; CHAO Yueen

doi:10.15933/j.cnki.1004-3268.2024.11.003

Abstract：In order to further study the molecular mechanism of γ-gliadin 1 gene in regulating flour quality of wheat,the γ-gliadin 1 gene was cloned using Zhengmai 158(low protein content and high dough strength)as materials,bait vector pGBKT7-γ-gliadin 1 was constructed,the interacting proteins of wheat γ-gliadin 1 were screened from Zhengmai 158 cDNA library by the yeast two-hybrid system,and rotary test was used to validate the interaction between key candidate proteins and γ-gliadin 1.The results showed that the γ-gliadin 1 gene was successfully cloned with the cDNA length of 1 020 bp.A total of 117 blue clones were screened by the yeast two-hybrid system.Ten possible candidate interacting proteins of γ-gliadin 1 were obtained through colony PCR detection,sequencing,and homology analysis using the BLAST on NCBI,which were cysteine-rich and transmembrane domain-containing protein 1-like(XM_044546254.1),alpha-amylase/trypsin inhibitor-like(XM_044509330.1),lichenase 2(XM_044550285.1,XM_044594213.1),sucrose 1-fructosyltransferase-like(XM_044554537.1),pullulanase 1(XM_044577151.1),ervatamin-B-like(XM_044577994.1),cell number regulator CNR8-like(XM_044483337.1,XM_044491902.1)and CNR2-like(XM_044468782.1)related to plant reproductive growth(especially development of fruit or grain),and ubiquitin domain-containing protein DSK2b-like(dominant suppressor of KAR 2b like,XM_044546840.1)and CIP73(CCa MK-interacting protein of 73 ku,XM_0445 54473.1),respectively.The rotation validation assays of candidate interacting proteins indicated that four candidate proteins were probably interacted with γ-gliadin 1,which were cysteine-rich and transmembrane domain-containing protein 1-like,alpha-amylase/trypsin inhibitor-like,lichenase 2 and ervatamin-B-like,respectively.Therefore,it is speculated that γ-gliadin 1 may mainly interact with these candidate proteins to be involved in the synthesis and degradation of storage proteins(such as cysteine)and starch in wheat grains.