Engineering high amylose and resistant starch in maize by CRISPR/Cas9-mediated editing of starch branching enzymes

To improve the amylose content(AC)and resistant starch content(RSC)of maize kernel starch,we employed the CRISPR/Cas9 system to create mutants of starch branching enzyme I(SBEI)and starch branching enzyme IIb(SBEIIb).A frameshift mutation in SBEI(E1,a nucleotide insertion in exon 6)led to plants with higher RSC(1.07%),lower hundred-kernel weight(HKW,24.71±0.14 g),and lower plant height(PH,218.50±9.42 cm)compared to the wild type(WT).Like the WT,E1 kernel starch had irregular,polygonal shapes with sharp edges.A frameshift mutation in SBEIIb(E2,a four-nucleotide deletion in exon 8)led to higher AC(53.48%)and higher RSC(26.93%)than that for the WT.E2 kernel starch was significantly different from the WT regarding granule morphology,chain length distribution pattern,X-ray diffraction pattern,and thermal characteristics;the starch granules were more irregular in shape and comprised typical B-type crystals.Mutating SBEI and SBEIIb(E12)had a synergistic effect on RSC,HKW,PH,starch properties,and starch biosynthesis-associated gene expression.SBEIIa,SS1,SSIIa,SSIIIa,and SSIIIb were upregulated in E12 endosperm compared to WT endosperm.This study lays the foundation for rapidly improving the starch properties of elite maize lines.

Cloning and Expression of the Promoter of Maize Starch-Branching Enzyme sbeⅡb Gene

[Objective] The aim was to construct promoter of maize starch-branching enzyme sbe Ⅱb gene specifically expressed in seed.[Method] The promoter sequence of maize starch-branching enzyme sbe Ⅱb gene was amplified by LA-PCR and then cloned into pMD18-T vector.Subsequently,the promoter was cloned into pBI121 vector to construct plant expression vector pBI121-sbe Ⅱb.Recombinant plasmid pSBE-GUS was constructed by connecting GUS gene into pBI121-sbe Ⅱb and transformed into tobacco mediated by Agrobacterium tumefaciens,before detection by PCR and Southern blot.The biochemical analysis and fluorescence detection of GUS activity were performed in different parts of the tobacco by Gene Gun Method and Agrobacterium tumefaciens transformation respectively.[Result] The homology between promoter sequence cloned in this experiment and the corresponding sequence announced in the GenBank reached 98.52%.Four transformed tobaccos were obtained by PCR and Southern blot after co-culture and selective culture.A small number of blue spots appeared in leaves,and the spots could barely been seen in the stems and roots,while a large number of blue spots were found in seeds.So,it could be concluded that the promoter of sbeⅡb gene was specifically expressed in seeds.[Conclusion] Promoter of maize starch branching enzyme sbe Ⅱb specifically expressed in seeds was successfully cloned.

Characterization and Expression Analysis of Starch Branching Enzymes in Sweet Potato

Spatial and temporal expression patterns of Sbel and Sbe2 that encode starch branching enzyme （SBE） Ⅰ and Ⅱ, respectively, in sweet potato （Ipomoea batatas L.） were analyzed. Expression of both genes in Escherichia coli indicate that both genes encoded active SBE. Analysis with real-time quantitative polymerase chain reaction technique indicates that IbSbel mRNA was expressed at very low levels in leaves but was the predominant isoform in tuberous root while the reverse case was found for lbSbe2. The expression pattern of IbSbel, closely resembles that of AGPase S, a gene coding for one of the subunits ofADP-glucose pyrophosphorylase, which is the key regulatory enzyme in the starch biosynthetic pathway. Western analysis detected at least two isoforms of SBE I in tuberous roots, those two isoforms showed adverse expression patterns with the development of the tuberous roots. Expression of the two IbSbe genes exhibited a diurnal rhythm during a 12-h cycle when fed a continuous solution of sucrose. Abscisic acid （ABA） was aother potent inducer of IbSbe expression, but bypassed the semidian oscillator.

Temperature Stress at Grain Filling Stage Mediates Expression of Three Isoform Genes Encoding Starch Branching Enzymes in Rice Endosperm

An early-maturity indica rice variety Zhefu 49, whose grain quality and starch structure are sensitive to environmental temperature, was subjected to different temperatures （32℃ for high temperature and 22℃ for optimum temperature） at the grain filling stage in plant growth chambers, and the different expressions of three isoform genes （SBEI, SBEIII and SBE/V） encoding starch branching enzyme （SBE） in the endosperms were studied by the real-time fluorescence quantitative PCR （FQ-PCR） method. Effects of high temperature on the SBE expression in developing rice endosperrns were isoform-dependent. High temperature significantly down-regulated the expressions of SBEI and SBEIII, while up-regulated the expression of SBEIV. Compared with SBEIV and SBEIII, the expression of SBEI gene in Zhefu 49 rice endosperms was more sensitive to temperature variation at the grain filling stage. This study indicates that changes in weather/climate conditions especially temperature stress influence rice grain formation and its quality as evidenced by isoform expression.

Effects of Weak Light on Starch Accumulation and Starch Synthesis Enzyme Activities in Rice at the Grain Filling Stage

Dynamic changes of starch, amylose, sucrose contents and the activities of starch synthesis enzymes under shading treatments alter flowering were studied using two rice varieties IR72 （indica） and Nipponbare （japonica） as materials. Under shading treatments, the starch, amylose and sucrose contents decreased, while ADP-glucose pyrophosphorylase （ADPGPPase） activity only changed a little, soluble starch synthase activity and granule bound starch synthase activity decreased, soluble starch branching enzyme （SSBE, Q-enzyme） activity and granule bound starch branching enzyme （GBSBE, Q-enzyme） activity increased, and starch debranching enzyme （DBE, R-enzyme） activity varied with varieties. Correlation analyses showed that the changes of starch content were positively and significantly correlated with the changes of sucrose content in the weak light. Both ADPGPPase activity and SSBE activity were positively and significantly correlated with starch accumulation rate. It was implied that the decline of starch synthase activities was related to the decrease of starch content and the increase of the activity of starch branching enzyme played an important role in the decrease of the ratio of amylose to the total starch under the weak light.

Origin and evolution of the main starch biosynthetic enzymes

Starch,a semi-crystalline energy storage form primarily found in plant plastids plays a crucial role in various food or no-food applications.Despite the starch biosynthetic pathway’s main enzymes have been characterized,their origin and evolution remained a subject of debate.In this study,we conducted the comprehensive phylogenetic and structural analysis of three types of starch biosynthetic enzymes:starch synthase(SS),starch branching enzyme(SBE)and isoamylase-type debranching enzyme(ISA)from 51,151 annotated genomes.Our findings provide valuable insights into the possible scenario for the origin and evolution of the starch biosynthetic pathway.Initially,the ancestor of SBE can be traced back to an unidentified bacterium that existed before the formation of the last eukaryotic common ancestor(LECA)via horizontal gene transfer(HGT).This transfer event likely provided the eukaryote ancestor with the ability to synthesize glycogen.Furthermore,during the emergence of Archaeplastida,one clade of SS was transferred from Deltaproteobacteria by HGT,while ISA and the other clade of SS originated from Chlamydiae through endosymbiosis gene transfer(EGT).Both these transfer events collectively contributed to the establishment of the original starch biosynthetic pathway.Subsequently,after the divergence of Viridiplantae from Rhodophyta,all three enzymes underwent multiple duplications and N-terminus extension domain modifications,resulting in the formation of functionally specialized isoforms and ultimately leading to the complete starch biosynthetic pathway.By shedding light on the evolutionary origins of key enzymes involved in the starch biosynthetic pathway,this study provides important insights into the evolutionary events of plants.

Preparation of high-quality resistant dextrin through pyrodextrin by a multienzyme complex

As a soluble food raw material with a low calorie content,resistant dextrin (RD) has broad application prospects in the food industry.Branching enzymes (BEs),as a key enzyme for RD preparation,can break the α-1,4 glycosidic bonds of donor chains and reconstruct the cleaved chains to acceptor chains through the α-1,6 glycosidic bonds.BEs with high transglucosidic activity toward amylopectin and short-chain substrates are urgently needed to increase the quality of RD.Herein,BE derived from Thermuobifida fusca (TfBE) was mined and characterized.The optimal temperature and pH of the TfBE were 40 ℃ and 6.5,respectively.A total of 1500 U/g substrate TfBE reacted with 20% (w/v) pyrodextrin for 12 h,the ratio of α-1,4 to α-1,6 glycosidic bonds was changed from 3.52:1 to 2.33:1,and the content of enzyme-resistant components notably increased from 44.0% to 53.8%.Furthermore,to make full use of receptor chains and small molecular sugars in the reaction system,a multienzyme complex of TfBE with T.fusca α-cyclodextrin glucosyltransferase (TfCGTase),TfBE with TfCGTase and Aspergillus nidulans α-glucosidase (AnGS) was used to further increase the enzyme resistance of RD from 44.0% to 65.3% and 70.6%,respectively.The developed multienzyme complex method could effectively contribute to improving the production quality and efficiency of RD preparation.