Structural Properties And Directional Regulation Of Sorghum Starch

Starch is a versatile carbohydrate that has many applications beyond its use as an energy source in the human diet,such as paper and textiles,adhesives,biodegradable plastics,pharmaceutics and personal care products.Its application in various fields is directly related to its physicochemical properties,which are determined by the starch source and multi-scale structure.Therefore,clarifying the multi-scale structure of starch is a prerequisite for achieving precise application of various types of starch.Native starch has some shortcomings,such as poor solubility,limited gel properties,poor viscoelasticity,and susceptibility to aging and digestion.These limitations can be overcome by modifying the structure of starch using various methods,including chemical,physical,and biological methods.Biological methods can be considered"green"modification methods because they are typically more environmentally friendly than other methods.Biological methods can regulate the structure and output of starch from plant.Researchers around the world are actively investigating the production of new types of starch that are more tailored to the needs of industry and society.Sorghum（Sorghum bicolor（L.）Moench）is a C4 plant that is widely planted in tropical,subtropical,and temperate regions.It is known for its strong environmental adaptability,particularly to conditions of saline-alkali,drought,and heat stress.Sorghum has become the fifth most important grain crop in the world after wheat,corn,rice and barley.Starch is the main component of sorghum grains（65%-70%）,and thorough research on the structural properties of sorghum starch is of great significance for expanding the sorghum,sorghum starch,and starch industry.However,there are few reports on the study of regulating starch structure from the source of sorghum starch synthesis.On the basis of the current sorghum germplasm resources,it is of great significance to clarify how to obtain new sorghum starch types through genetic transformation to expand the application of starch.This study is focused on analyzing the multiscale structural growth evolution of starch granules in different lines of sorghum with different amylose contents,and"reverse order"method was used.The study examines the physicochemical properties of the starch in sorghum and how it changes at different growth stages.The types and physiological functions of genes involved in starch multiscale structure synthesis were discussed by sequencing transcriptome.Different types of genetically modified sorghum starch and its related enzymes were obtained through genetic engineering technology,through transcriptome sequencing;three key enzymes that regulate starch synthesis were obtained and purified in vitro.Furthermore,a network of key genes,structures,and properties for starch synthesis was constructed;providing a theoretical basis to achieve a targeted regulation of the multi-scale structure of sorghum starch.The main results are as follows:（1）Different varieties of sorghum starch multiscale structure and its physicochemical properties at different growth stages was analyzed.The results showed that the amylose content distribution range of waxy,stick and H37 sorghum at different growth stage were4.80%-6.25%,5.82%-9.76%and 17.87%-27.47%,respectively.The intermediate fraction existed in the early stages of development,and in the later stages of development of waxy sorghum,amylose could be converted into branched starch through intermediate fraction.Waxy sorghum starch with 19 days of development has the highest B1 chain content,which opposite to stick sorghum.The internal chain length of H37 sorghum starch was higher than that of waxy sorghum.The semi-crystalline layered structure thickness（DBragg）of starch ranged from 9.4 nm-10.7 nm,which belongs to the typical A-type crystal structure.With the growth and development of sorghum grains,the relative crystallinity of sorghum starch granules of the three varieties increased gradually.The ¹³C CP/MAS NMR patterns of waxy,stick and H37 sorghum starch at different developmental stages showed three independent peaks at 100-106 ppm（C1）.The sorghum starch granules developed in 10-19 days were in regularly shape,but their size and smoothness were not uniform.Micropores with pore diameters of 178-298 nm were observed on the surface.With the extension of growth time,the surface of the sorghum starch granules became irregular polygons due to extrusion,and the depth of the micropores pathway deepened.In addition,the enthalpy and resistance to hydrolysis of starch granules of all sorghum varieties increased with the extension of the growing period,and the hydrolysis rate of H37 sorghum was significantly lower than that of waxy ones.（2）The pathway of starch and sucrose synthesis metabolic pathway（Ko00500）in sorghum endosperm has been identified by transcriptome sequencing analysis,which involves a total of 184 genes.22 genes played physiological functions in starch synthesis in sorghum,but only 13 genes directly related to starch synthesis enzymes and highly expressed in waxy,stick&H37 sorghum.Their main functions are summarized as follows:（1）AGPLS1gene determines grain yield.（2）GBSSI gene is mainly responsible for amylose synthesis and participates in ultralong branched chain synthesis of amylopectin.（3）SSSI gene is essential for amylopectin synthesis,and SSSIIa&SSSIIIa are responsible for the synthesis of amylopectin side chains of DP>25 and 12<DP<25,respectively,and（4）SBEI,SBEIIa and SBEIIb genes are responsible for the synthesis of long-,short-and medium-long-chain branching points of amylopectin,respectively.（5）ISA1 and PUL genes act on different objects,and both are continuously expressed in the process of starch synthesis.（3）Mutant GBSSI gene sorghum plants were obtained through genetic transformation.The order of expression levels of GBSSI genes was as follows:overexpression sample>control group>knockout sample.GBSSI knockout sorghum grain starch had an amylose content of 4.5%-11.2%;the amylose content in sorghum grain starch overexpressing GBSSI was 38.3%-52.1%.The knockout of the GBSSI gene directly reduced the synthesis of B2 chains,while the overexpression of this gene significantly increased the content of B1 and B2.The gelatinization temperature and viscosity order of genetically modified starch were:GBSSI knockout<control<GBSSI overexpression,and the digestibility order was GBSSI knockout>control>GBSSI overexpression.（4）Genetically engineered bacteria p ET-28a（+）containing SSSIIa,SBEIIb,and GBSSI were obtained through transgenic methods.After induction and purification,the three enzymes with higher activity were obtained.By reacting the above three enzymes with starch,it was found that there were significant changes in the CLD,crystal structure,thermodynamics,and digestibility of starch.SBEIIb hydrolyzes chain lengths with different degrees of polymerization to A;SSSIIa uses ADPG as the substrate to extend the length of each polymerization degree B chain,with the B1 chain being the main chain;GBSSI uses ADPG as a substrate to extend the A-chain and form the B1 chain.（5）According to the relationship between the types/functions of genes regulating sorghum starch synthesis,the multi-scale structure of starch and the physical and chemical properties of starch in the above contents,a relationship network of directional regulation of sorghum starch structure and properties was constructed,in order to obtain new sorghum starch by biological methods.