Effects Of Different Shading Intensities On Ascorbate Synthetase Activity And Expression Of Related Genes In Black Currant

In this experiment,the Ascorbic acid(As A)variety "Adelinia" and the low As A variety "Heifeng" were used as test materials.Plants are shaded to varying degrees(shading intensity: 0(CK),40%(T1),60%(T2)).The effects of shading on As A,glutathione(GSH)content and related enzyme activities during As A synthesis and regeneration were analyzed.The relative expression of L-galactose dehydrogenase(Gal DH)gene in different fruit development stages(young,expansion,half-veraison,veraison and maturity stages)under different shading strengths during As A synthesis was analyzed by real-time PCR.The effects of different shading intensity on the fruit Gal DH gene were studied.The influence of illumination on As A anabolism was analyzed on the molecular level,which provided a scientific basis for revealing the inherent reasons for the full accumulation of As A in black currant fruit.It provides theoretical support for As A anabolism and molecular-assisted breeding techniques in black currant,and also provides some theoretical support for actual production.The main results are as follows:(1)The shade extended the development period of the two varieties of black currant fruit,and the shading period of the two varieties was extended by 5-10 days compared with the control.Shading affects the quality of the fruit,and shading treatment increases the fruit firmness of the mature fruits of the two varieties.The hardness of the "Adelinia" fruits under 40% and 60% shading was 1.97 and 2.36 times of unshaded(CK),respectively,and "Heifeng" was 1.70 and 2.21 times,respectively.The soluble solids in the mature fruit of "Adelinia" decreased with the increase of shading intensity,but the soluble solids in the "Heifeng" mature fruit were not affected.(2)Under different shades,the contents of As A,DHA and T-As A in the fruits of the two varieties showed a decreasing trend during the whole fruit growth and development,but the decline and time of different treatments were different.Shading significantly reduced the As A content in the mature fruits of the two varieties,on the 40% shading and 60% shading treatment,the As A content in the "Adelinia" mature fruit decreased by 41.06% and 64.71% compared with the unshadowed.The As A content in the "Heifeng" mature fruit decreased by 55.02% and 77.89% compared with the control.(3)Shading has little effect on the content of GSH in mature fruits of black currant,but the GSH contents of different varieties in the early stage of fruit development(from young fruit to expansion)is different.Under the shade,the "Adelinia" GSH content decreased significantly in the early stage of fruit development,but the unshaded treatment showed an upward trend.Under the treatment of severeshading(T2),the "Heifeng" fruit showed an upward trend in the early stage of fruit development,and decreased under the other two treatments.(4)Mild shading(T1)had little effect on the activity of As A synthesis and regeneration related enzymes during the whole fruit development period,but under severe shading(T2),the enzyme activity decreased with the development of the fruit.Shading reduced the activity of enzymes involved in the synthesis and regeneration pathway of As A.And on mild shading(T1)treatments,the Gal DH activity in the mature fruits of "Adelinia" and "Heifeng" decreased by 11.11% and 14.29%,respectively.The two varieties under severe shading decreased by 88.89% and 86.29% respectively.The activities of key enzymes APX,DHAR,MDHAR and GR of As A regeneration pathway decreased with the increase of shading intensity.(5)Gal DH and DHAR activities were positively correlated with As A content under the CK,T1 and T2 treatments,and the correlation coefficients were above 0.8.This indicated that Gal DH and DHAR in Ascorbic acid synthesis pathway and As A-GSH cycle pathway played a key role in As A content in black currant fruit.There was a high correlation between GSH content and activities of Gal DH,DHAR,MDHAR,GR and APX in blackcurrant fruit.Especially,the correlation between APX activity and GSH content was strong,all above 0.9,which indicated that APX had a great influence on GSH content in black currant fruit.Shading treatment had no significant effect on the correlation between As A and GSH contents and related enzymes activities in fruits.(6)Screening for the black currant Gal DH(Rn Gal DH)gene from transcriptome sequencing data.The length of open reading frame was 1470 bp,encoding 476 amino acids.The relative molecular weight of protein encoded by Rn Gal DH gene of blackcurrant was 52.12 k Da and theoretical isoelectric point PI was 5.42.It was a hydrophilic protein with no signal peptide and no transmembrane structure.Protein subcellular location is predicted in chloroplasts.It contains the conserved domain of aldosterone reductases(AKRs).It has close relationship with sweet cherry,tomato and Actinidia arguta.(7)The results of real-time PCR showed that the expression of Gal DH gene was higher in young fruit and mature fruit,indicating that the gene expression was related to the developmental stage of the fruit.Shading reduced the relative expression of Gal DH gene.In T1 treatment,the gene expression in fruit of "Adelinia" fruit decreased by 27.21% compared with CK,and the other periods don't have much different.On the contrary,"Heifeng" had little effect on the development of shady fruit,and the color change and maturity of fruit decreased by 36.17% and 55.84% compared with CK.In the T2 treatment,the expression of Gal DH gene in both varieties decreased more.