Molecular Mechanism Of NnSBEⅡ Regulation On Amylopectin Synthesis In Nelumbo Nucifera

Lotus(Nelumbo nucifera Gaertn.)is a perennial aquatic herb of the lotus family,which is widely grown in our country.Lotus root is rich in starch,protein,vitamins and other nutrients,deeply loved by the majority of consumers.Starch is the main storage substance of lotus root,the synthesis of starch is closely related to yield and quality.The content of Amylopectin is considered to be the main factor determining the quality of food.Many studies have reported that starch branching enzyme gene SBEⅡ plays an extremely important role in the synthesis of amylopectin in crops.However,the role and mechanism of NnSBEH in lotus root amylopectin synthesis are still unclear.In this paper,the function and mechanism of NnSBEII are identified and analyzed by taking the main lotus root variety ’MRH’ as the test material.,so as to explore the regulation effect of NnSBEⅡ on the synthesis of amylopectin in lotus root,and to provide theoretical reference for improving the quality of lotus root starch.1.SBEs analysis of amylopectin branching enzyme genes in lotus rootThe contents of total starch,amylose and amylopectin increased during rhizome enlargement.The accumulation rate of amylopectin was significantly higher than that of amylose,and the ratio of amylose to amylopectin content decreased significantly in the middle stage of rhizome expansion.The activity of SBE increased first and then decreased during rhizomes enlargement of lotus root.We found three SBE genes named NnSBEⅡ,NnSBEⅡ and NnSBEⅢ in the genome of lotus root variety ’MRH’.The quantitative analysis showed that the expression of NnSBEⅠ and NnSBEⅡ was significantly higher than that of NnSBEⅢ in the process of rhizome enlargement.The enzyme activity test showed that the enzyme activity of NnSBEⅡ was significantly higher than that of NnSBEⅠ and NnSBEⅢ.2.Functional identification of NnSBEⅡ in regulating amylopectin synthesisHere,we identified the function of NnSBEⅡ by functional complementation assay in defective Arabidopsis thaliana,which showed that NnSBEⅡ compensated for the low content of starch in the mutant sbe2.2.It was found by measuring the starch content that the content of total starch and amylopectin in the transgenic lines was significantly higher than that in the mutant.And the SBE enzyme activity of the transgenic lines was significantly higher than that of the mutant.In addition,we constructed NnSBEⅡ overexpression lines using wild-type Arabidopsis thaliana as the receptor,and the results showed that NnSBEⅡ gene could promote the biosynthesis of amylopectin in Arabidopsis thaliana.The SBE enzyme activity of the overexpression lines was significantly higher than that of the wild-type lines,and the contents of total starch and amylopectin were significantly higher than those of the wild-type lines.3.Molecular mechanism analysis of NnSBEⅡ in the synthesis of amylopectin in Nelumbo nuciferaY1H test showed that NnNAC100 protein could directly bind to NnSBEⅡ promoter and promote NnSBEⅡ gene expression.We overexpressed NnNAC100 in wild-type Arabidopsis thaliana,the contents of total starch and amylopectin in the NnNAC100 overexpressing Arabidopsis thaliana WT lines were significantly higher than those in the WT lines.At the same time,the SBE enzyme activity,total starch and amylopectin contents of the overexpression lines were significantly higher than those of the WT lines.The results of Y2H and BiFC complementation experiments showed that NnSBEⅡ and NnSS2 could interact at the protein level.Transient expression of NnSBEⅡ and NnSS2 in tobacco leaves showed that coconversion of NnSBEⅡ and NnSS2 significantly increased amylopectin content in tobacco leaves.