Abstract: 【Objective】 The objective of the experiment is to study the polymorphism of the Amy32b gene in Chinese barley and dissect its relevance to the α-amylase activity 【Method】 Three pairs of specific primers covering the whole sequence of the gene were designed to locate single nucleotide polymorphism (cSNP) and other In/Del mutations. The complete cDNA of Amy32b was cloned from barley via RT-PCR. Full-length cDNA carrying alleles（G/A）and truncated cDNA of Amy32b were cloned into expression vector pET-28a(+). Their expression products were purified, characterized, and compared. 【Result】 According to the result of amplified DNA sequences of Amy32b (x05166), there was a single nucleotide polymorphism cSNP(G/A) and an insertion/deletion of A were found in the coding region of the gene, located in 2 269 bp and 2 403 bp, respectively. The SNP(G/A) caused an E355K amino acid substitution in the enzyme. And the In/Del variation of A insertion formed an in-frame stop codon, resulting in the deletion of a 48 bp fragment in cDNA as well as a β5 sheet in carboxyl terminal of the α-amylase. The full-length of Amy32b cDNA is 1 314 bp, while the truncated cDNA is 1 266 bp in length. The enzymatic activity assay showed that rAmy32b_A and rAmy32b_G exhibited normal and similar amylase activities. But for the truncated Amy32b gene, the mutant enzyme (rΔAmy32b) did not produce detectable actvity. 【Conclusion】 The In/Del mutation formed an early translation stop codon, resulting in the losses of 48 bp sequence fragment and a β-strand in C-terminal end of α-amylase, and seriously affected enzymatic activity, while the G/A transition only resulted in amino acid substitution and did not alter enzymatic activity.

Key words: barley, Amy32b, coding region single nucleotide polymorphism, α-amylase activity