Effect Of MeCHS Gene On Postharvest Decay Functional Analysis Of Cassava Storage Roots

Cassava（Manihot esculenta Crantz）is perennial shrub of Euphorbiaceae,widely cultivated in tropical and subtropical regions.Cassava is grown because of its starch-rich in storage roots,which can be used as staple food,animal feed and industrial raw materials.Postharvest physiological deterioration（PPD）caused by injury is one of the important factors restricting the development of cassava industry.The inevitable injury in the process of harvest and treatment will cause a series of physiological reactions within 24 to 48 hours,resulting in the deterioration and decay of the stored roots and can not be processed and utilized.In the process of PPD reaction,a large number of phenylpropane compounds,especially scopoletin and its glycosides,were accumulated in the roots.It has been found that candidate genes and metabolites of flavonoid biosynthesis pathways may be involved in the regulation of PPD.Here,our aim is to study the effect of changing the synthesis of flavonoids in cassava root on PPD reaction.Chalcone synthase is a key rate-limiting enzyme gene in flavonoid biosynthesis.The purpose of this study was to analyze the cassava Me CHS gene to regulate the PPD reaction by regulating the content of flavonoids,so as to provide a theoretical basis for the creation of cassava germplasm with PPD tolerance.In this study,the main variety SC9 was selected as the experimental material,and the correlation between flavonoid content and PPD was preliminarily determined by observation and determination of physiological indexes;the structure of Me CHS gene encoding chalcone synthase and the physicochemical properties of its protein were analyzed by bioinformatics;the expression differences of cassava Me CHS gene under different variables were analyzed by quantitative PCR.The regulatory effect of Me CHS gene on PPD reaction was further verified by transgenic technology.The main results are as follows:1.The relative expression of CHS gene in different varieties,tissues and storage time was analyzed by Control variates method.The results showed that Me CHS1,Me CHS2,Me CHS3 and Me CHS4 were all expressed in cassava;The expression level of Me CHS gene in cassava tissue is in the order of stem>bud>flower>leaf>root;During the storage of cassava,the relative expression levels of Me CHS1,Me CHS2 and Me CHS3 significantly increased as the degree of PPD increased.2.Bioinformatics analysis showed that the structure of Me CHS gene family was relatively conservative,consisting of two exons and one intron;The clustering branches composed of Me CHS1 and other Me CHS proteins are far apart;Me CHS proteins exhibit differences in physical and chemical properties.Me CHS1,Me CHS2 and Me CHS3 are stable proteins,while Me CHS4 and Me CHS5 are unstable proteins.It is speculated that the instability of Me CHS4 and Me CHS5 proteins is the main reason for their gene non expression during cassava storage.3.The subcellular localization results of Me CHS protein showed that the three Me CHS-GFP fusion proteins were mainly distributed in the cytoplasm of tobacco leaf cells.4.The measurement results of physiological indicators indicate that the total antioxidant capacity of stored roots gradually weakens,the content of reactive oxygen species increases,and the trend of changes in flavonoid content is consistent with the trend of changes in CAT activity and H₂O₂ content.It is speculated that during cassava storage,flavonoids cooperate with CAT to clear H₂O₂.5.The overexpression vector and RNAi vector of Me CHS were constructed,and the callus tissue was transformed and cultured.The VIGS vector of Me CHS gene was constructed to infect cassava plants.Compared with the blank control group,the storage roots of the experimental group were more prone to decay during storage.After measurement,it was found that the flavonoid content and total antioxidant capacity of the blank control group were significantly higher than those of the experimental group plants.This experiment can draw the following conclusion:Me CHS is a key gene in the PPD resistance process of cassava.The occurrence of cassava PPD is caused by the explosion of reactive oxygen species and the weakening of total antioxidant capacity.The Me CHS gene cooperates with CAT to participate in the clearance of reactive oxygen species through the synthesis of flavonoids,thereby resisting the PPD process of storage roots.