Genetic diversity, functional properties and expression analysis ofNnSBEgenes involved in starch synthesis of lotus (Nelumbo nuciferaGaertn.)

Abstract

BackgroundStarch branching enzyme (SBE) is one of the key enzymes in starch biosynthetic metabolism, determining amylopectin structure.MethodsFull length coding sequences (CDS) ofSBEgenes were cloned using reverse transcription PCR (RT-PCR) technology, and neighbor-joining (NJ) tree was used for phylogenetic analysis. Single nucleotide polymorphisms (SNPs) were determined to assess the genetic polymorphisms and variation indexes between individuals and clusters. Quantitative real time PCR (qRT-PCR) was performed to analyze the spatial and temporal expression ofNnSBEgenes. The effect ofNnSBEgenes on amylopectin’s fine structures was explored using affinity and the enzyme activity analysis of two isoforms in amylopectin and amylose.ResultsIn this study, two SBE family genes,NnSBEIandNnSBEIII, were identified in lotus (Nelumbo nuciferaGaertn.). Phylogenetic analysis sorted NnSBEI into SBE family B and NnSBEIII into SBE family A. UPGMA phylogenetic tree divided 45 individuals of lotus into three classes. The homozygous haplotype (A G G A G) ofNnSBEIIIwas observed in seed lotus. During the seed embryo development stage,NnSBEIIIreached the peak in the middle of the development stage, whileNnSBEIincreased in the mid-late developmental stage. The different affinity activity of the two isozymes binding amylopectin and amylose assay indicated NnSBEI has higher activity and wider affinity.DiscussionGenetic diversity showed thatNnSBEgenes received artificial selection during the process of cultivation and domestication in lotus seeds. Furthermore, the expression pattern and affinity activity analysis indicated thatNnSBEgenes were related to the chain length of amylopectin.