Abstract:
Salicylic acid(SA) is a key signaling molecule in the process of plant defense reaction. The ICS pathway is the main pathway for SA synthesis, and isochorismate synthase(ICS) is the rate-limiting enzyme in the synthesis pathway of isochoridate. It catalyzes the allosteric transformation of chorismate to isochoridate, which is then decomposed by pyruvatelyase to form salicylic acid. In this study, Lycium barbarum leaves were used as materials.RACE(rapid amplification of c DNA ends) technology was used to clone the wolfberry ICS gene, and Blastn and Blastp were used to analyze the similarity of nucleic acid and amino acid sequences and build an evolutionary tree.ExPaSy online analysis was used to predict protein secondary structure and construct a 3 D structural model. Using fluorescence quantitative qPCR to determine the expression characteristics of LbICS in different tissues and organs,and further analyze the relationship between the expression profile of LbICS and the synthesis of salicylic acid during aphids feeding on the leaves. The results showed that the LbICS(GenBank accession number MK457455) has an open reading frame of 1 731 bp, encodes 577 amino acids, and has multiple phosphorylation sites. The constructed protein 3 D model is a compact spherical structure. Analysis by Signal P4.0 Server and others(online software) found that the LbICS protein contains a typical conserved chorionic acid binding domain, and the N-terminus contains a 37-amino acid residue chloroplast transmembrane signal peptide, with the highest similarity to pepper(Capsicum annuum XM_016722852) ICS, followed by tobacco(Nicotiana sylvestris XM_019 374845) ICS, morning glory(Ipomoea nil XM_019342500) ICS, orange(Citrus sinensis XM_006476586) ICS and other close relationships. Real time PCR results showed that the expression of LbICS in different organs of Lycium barbarum was tissue-specific, and the expression level in the leaves reached the highest value. Real time qPCR results showed that the expression of LbICS is tissue-specific, with the highest expression in leaves, followed by stems, and lowest in roots. The expression of LbICS gradually increased at 1~3 days after aphid feeding and reached the maximum at 3 days the value gradually decreased afterwards, which was consistent with the change trend of SA content in the leaves of aphids after feeding. The above results showed that LbICS of wolfberry regulated the synthesis of wolfberry SA under aphids feeding. The results of the study are of great significance for elucidating the synthetic pathway of SA in Lycium barbarum and the physiological function of LbICS.