Selection And Indentification Of MYB Negative Regulators In Anthocyanin Biosynthesis Of Litchi Fruit Peel

Litchi(Litchi chinensis Sonn.)is an evergreen tree of the genus Liriodendron which is an important southern subtropical fruit tree in China.It is rich in germplasm resources,and its fruit color,aroma and taste are good that is widely loved by consumers.Color is an important part of the quality of litchi fruit.When the litchi is mature,the coloration of the peel is mainly caused by the accumulation of anthocyanins.Studies have shown that MYB regulators play a very important role in the synthesis of anthocyanins in litchi pericarp.In this study,the results of litchi genome sequencing were used to perform local Blast with anthocyanin MYB negative regulatory genes reported in other species,and 6 litchi MYB anthocyanin synthesis negative regulation related candidate genes were Selected to color the good litchi variety cv.Guiwei.The litchi cDNA development stage was mixed as a template,and the DNA sequences of four MYB genes were cloned.The gene sequence characte ristics and its spatiotemporal expression in different samples were analyzed,and the gene function was further verified by subcellular localization,Agrobacterium tumefaciens-mediated tobacco genetic transformation,yeast double hybrid and double fluores cent molecule complementation experiments(BiFC).The obtained research results make up the blank of yttrium anthocyanin MYB negative regulators,and provides a theoretical basis for the regulation of color and the improvement of appearance quality of litchi fruits.The main findings are as follows:1.Based on the sequencing results of the litchi genome database,four litchi MYB anthocyanin synthesis negative regulatory genes were cloned from cv.Guiwei,named LcMYBC2-L1,LcMYBC2-L2,LcMYB4 and LcMYBx,respectively,with lengths of 708 bp,600 bp,669 bp and 264 bp.The number of encoded amino acids is 235,199,222,87,respectively.All of them belong to the SANT family.2.Real-Time PCR analysis of the expression of four MYB genes in ’Guiwei’ peel and leaf development stage revealed that LcMYBC2-L1,LcMYBC2-L2,LcMYB4 genes along with leaf color during the development of ’Guiwei’ litchi leaves The expression level increased,while the anthocyanin synthesis decreased and the expression increased in LcMYBx at the late stage of leaf development,which was presumed to be negatively correlated with the synthesis of leaf anthocyanins.In the development stage of the pericarp,LcMYBC2-L1,LcMYBC2-L2,and LcMYB4 all had peak expression at 45 d of ’Guiwei’ peel,and LcMYBC2-L2 and LcMYB4 showed a sharp increase,suggesting that they participate in other regulatory pathways,and the LcMYBx gene is There was a peak of expression at 50 days and a decrease in expression at a later stage.3.The subcellular localization expression vector of p CAMBIA1300-35S::e GFP-candidate gene was constructed,and the leaves of N.benthamiana were injected by Agrobacterium infiltration.The fluorescence microscopy showed that LcMYBC2-L1,LcMYBC2-L2,LcMYB4 and LcMYBx were mainly located in plasma membrane.4.Through yeast two-hybrid and BiFC experiments,it was found that LcMYBx interacted with LcWDR1 and Lcb HLH3.5.The p BI-121-candidate gene expression vector was constructed respectively.It was found by heterologous transgene verification that LcMYBC2-L1 and LcMYBx significantly inhibited the anthocyanin synthesis of tobacco K326 flower,and the inhibition effect of LcMYBC2-L1 was stronger than that of LcMYBx.