HvGBSSI Mutation At The Splicing Receptor Site Affected RNA Splicing And Decreased Amylose Content In Barley

Barley(Hordeum vulgare L.)is an excellent source of complex carbohydrates.The barley grain is an important source of starch,and accounts for 60%-75% of the grain weight.Granule-bound starch synthase I(Hv GBSSI)is encoded by the barley waxy(Wx-1)gene and is the sole enzyme in the synthesis of amylose.Hv GBSSIa is expressed in the endosperm,whereas Hv GBSSIb is expressed in the pericarp and the embryo.Ethyl methane sulfonate(EMS)is a commonly used chemical that induces mutations and has been widely used in crops,such as maize,rice,and wheat.The mutant lines caused by EMS can be used directly for breeding without any gene-modification controversies.Here,a Wx-1 mutant was identified from an ethyl methane sulfonate(EMS)-mutagenized barley population.EMS-mutagenized barley population,and identified a mutant,M2-1105,with a base mutation at fourth intron splicing receptor site,leading to a near-waxy phenotype with decreased levels of amylose.There were two single-base mutations G1086 A and A2424 G in Wx-1 in the mutant(M2-1105).The G1086 A mutation is located at the 3′ splicing receptor(AG)site of the fourth intron,resulting in an abnormal RNA splicing.The A2424 G mutation was a synonymous mutation in the ninth intron.The pre-m RNA of Wx-1was incorrectly spliced and transcribed into two abnormal transcripts.The type I transcript had a 6 bp deletion in the 5′ of fifth exon,leading to a translated Hv GBSSI protein lacking two amino acids with a decreased starch-binding capacity.In the type II transcript,the fourth intron was incorrectly cleaved and retained,resulting in the premature termination of the barley Wx-1 gene.The mutations in the Wx-1 decreased the enzymatic activity of the Hv GBSSI enzyme and resulted in a decreased level in amylose content.This work sheds light on a new Wx-1 gene inaction mechanism.