The Regulation Mechanism Of MYBA1 Gene Related To Peel Coloring Bud Sports In Grape

Grapevine is a perennial fruit crop which belongs to genus Vitis.Bud sport is related to somatic mutation which frequently occured in fruit trees.Somatic mutation occurres in the form of chimera.'Benitaka' is a deep red bud sport of‘Italia'grapevine.‘Brasil'is a black bud sport of 'Benitaka' grapevine.At present,multiple earlier ripening and coloring bud sports were found in 'Summer Black' grapevine.These bud sports plants have great significance in the improvement and breeding of early-ripening varieties of grapevines.In this study,we cloned the VvMYBAl gene and it's promoter region of white grape cultivar'Italia' and it's bud sport cultivars;we analyzed the agronomic traits and also studied the effect of ABA on fruit ripening and coloring,and it's influence on genes such as VvMYBA1;we carried out the functional analysis of VvMYBAl gene and it's alternative splicing via a transient expression experiment using strawberry as transforming material;we performed whole genome re-sequencing analysis,endogenous ABA content and ripening and coloring related genes expression were analyzeed between 'Summer Black' and each early ripening and coloring bud sport plants.The contents and conclusions of this study are as under:1.In the coloring cultivars such as 'Benitaka',‘Brasil'and‘Jigo',there is a 513 bp fragment sequence in the promoter region(32 bp upstream of VvMYBA1)of VvMYBA1,the sequence is a transposon structure and there are two imperfect(6 bases different)151 bp positive repeat sequence at the each end of the sequence.Due to the existing this fragment,the genotype of these three coloring cultivars is heterozygous.This fragment was not found in white 'Italia' and‘Rosario Bianto'cultivar,their genotype is homozygous.After comparing with grape reference genome,we infer a replication and insertion event.Our previous study suggested that,this 513 fragment in promoter region had the ability to show the color in callus.We speculate that the structure of this transposon might be a key loci which can regulate berry coloring.We found two response elements after analyzing the sequence of VvMYBA1.2.In order to verify the function of two ABA response element in upstream region of VvMYBA1 gene,during veraison period.'Benitaka' was treated with ABA@200 mg/L while Benitaka' and 'Brasil' grape treated with water were considered as control.The result of anthocyanins contents indicated that the color of ABA treated group were deeper than control group at three week post treatment.The peel color become significantly deeper at three weeks post treatment;the content of anthocyanin in 'Brasil' was mainly due to the expression of peonidin-3-O-monoglucoside,malvi din-3-O-monoglucoside,and malvidin-3-O-coumaryl glucoside.The main changes in the contents of anthocyanins in the'Benitaka' group were mainly due to the elevated level of cyanidin-3-O-monoglucoside and peonidin-3-O-mono glucoside.The qRT-PCR results demonstrated that the relative expression of the VvMYBA1 gene was higher in treated berries as compared to 'Benitaka'and 'Brasil' at two weeks and three weeks post ABA treatment.ABA treated 'Benitaka'showed highest relative gene expression level compared to other anthocyanin biosynthesis genes at three weeks post treatment,and it is up-regulated about 300 times more in ABA treated group.3.To verify the lack of transposon structure sequence that inhibited the expression of VvMYBA1 gene in 'Italia'.The nest PCR of VvMYBA1 gene was carried out using 'Italia''Benitaka' and 'Brasil'as templates.The results showed that,VvMYBA1 gene and it's alternative splicing which containing all two introns were cloned from 'Benitaka' and'Brasil',but none of them were found in 'Italia' grape.VvMYBA1 gene and its alternative splicing were transiently infected by agrobacterium in strawberry,the results showed that the VvMYBA1 gene contribute to coloring in grape peel,only injected alternative splicing can't promote coloring in strawberrywhile both VvMYBA1 gene and it's alternative splicing insertions showed deeper color than VvMYBA1 gene.4.The whole genome re-sequencing was performed using 'Summer Black'(CK1,CK2)and its multiple bud sport(Sum1,Sum2,and Sum3),multiple plant endogenous hormones like ABA,GA,IAA and BR were measured in the berries of CK1 and Sum1.The results showed that the content of ABA was higher than other hormone in CK1 vs Suml comparison;the NECD6 gene which might related to ABA biosyntheses pathway and had a 15 bp deletion in Suml genome;the ERF5 gene had a 8818 bp deletion region in Sum1.Based on the part of 513 bp which was elucidated from our previous study and the same initial concentration(0.1 ?g)of whole genome re-sequencing on CK2,Sum2,and Sum3,the results showed that comparing with CK2(123),the higher Reads number of sequence part of 513 bp in Sum2(159)and Sum3(161)was found.In conclusion,a 513 transposon sequence which contains two ABA response elements were formed in 'Benitaka' and 'Brasil' grape.This sequence probably indirect respond to ABA,elevated the expression of VvMYBA1 gene and ultimately contribute in the coloring of grape peel.The above mentioned factors might be due to 'Italia' grape was mutated to'Benitaka' grape;the increase in ABA contents,the occurance of InDel of NECD6 and ERF5 gene,the Reads number in the upstream of VvMYBA1 gene,all these factors probably related to the earlier ripening and coloring bud sport in 'Summer Black' grape.