Cloning And Functional Identification Of MsPSY2a Gene

Banana(Musa nana Lour.)is a large perennial monocot which is important food security source in Africa,Asia and Latin America.Due to the varieties of banana,the nutritional components of different varieties have much difference,most varieties lack carotenoids.Therefore,it is urgent to improve the banana germplasm resources,improve the yield and nutritional value of banana.In this study,‘Pilose’ and ‘Karat’(Musa troglodytarum)(High content of carotenoids in fruits)was used as the experimental material,and the MsPSY2a(Phytoene synthase)gene,related to carotenoid synthesis in banana,was screened through transcriptome data analysis.The MsPSY2 a gene was successfully cloned in the experiment,the bioinformatics and expression characteristics of the target gene were analyzed.The expression vector was constructed and transformed into Arabidopsis thaliana,banana embryonic suspension cells for functional identification.The main research results are as follows:1.MsPSY2 a gene was successfully cloned from ‘Karat’(High carotenoid content in fruit),with a total length of 1 551 bp.2.Bioinformatics analysis results showed that MsPSY2 a protein was mainly composed of α-helix and random coil,belonging to hydrophilic protein without highly hydrophobic structure.The prediction of subcellular localization and transmembrane domain showed that the protein was located in mitochondria without transmembrane structure.Phylogenetic tree analysis showed that the MsPSY2 a amino acid sequence of banana shared more than 70% homology with pineapple,strawberry,cherry and hemp,indicating that PSY protein has a highly conserved region,but its functional expression may be different.The relationship between banana MsPSY2 a and pineapple Ac PSY2 is similar,so it is speculated that MsPSY2 a has similar function with pineapple Ac PSY2 protein.3.The q RT-PCR results showed that the expression of MsPSY2 a gene in pulp of‘Karat’ was much higher than that of ‘Pilose’.Since ‘Karat’ pulp is rich in lycopene and the carotenoid accumulation is extremely high,it is speculated that the high level of MsPSY2 a gene expression in ‘Karat’ pulp promotes its carotenoid synthesis,which eventually leads to the high accumulation of carotenoid content.The relative expression of MsPSY2 a gene of ‘Pilose’ in roots was generally lower than that in other tissues at different growth stages.MsPSY2 a gene was expressed at different levels in the root,stem,leaf,pulp and peel of banana during fruit ripening.It is speculated that MsPSY2 a gene plays an important role in the growth and development of fruits,but there is tissue specificity.4.The 35S::MsPSY2 a overexpression vector was successfully constructed,and transformed into the model plant Arabidopsis thaliana to obtain,screen the positive T3 generation plants.Compared with the wild type,transgenic Arabidopsis plants cultured for14 days showed dwarfing situation.During the growth cycle of Arabidopsis,the plant phenotype did not change significantly.However,during the ripening period,it was found that the transgenic Arabidopsis seeds changed from yellow-brown to yellow.The results of carotenoid content determination showed that overexpression of MsPSY2 a gene in Arabidopsis led to the increase of carotenoid content by 18.75%.GUS staining in transgenic Arabidopsis plants showed that GUS staining signals were detected in all tissues.GUS staining signal was the most obvious in seeds,followed by floral organs,and extremely weak in roots.5.The antibiotic gradient screening results of banana ‘Pilose’ suspension cells showed that hygromycin could effectively inhibit the growth of suspension cells;The lethal concentration of hygromycin was 30 mg/L,which could be used for subsequent genetic transformation experiments.The overexpression vector was transformed into suspension cells.GUS staining results showed that 35S::MsPSY2 a overexpression vector had been successfully transformed into suspension cells and this system was suitable for the genetic transformation of suspension cells.This study provides a theoretical basis for exploring the biological function of MsPSY2 a gene in banana,lays a foundation for further exploring the molecular mechanism of carotenoid biosynthesis and nutritional strengthening breeding of banana.