Isolatioiin And Functional Analysis Of A Specific Fragment In Delicious' Spur Mutants

The frequency and variety of apple bud mutation plays an important role in apple's breeding work,one type of bud mutation is spur mutation,but the mechanism of spur mutation is not clear.In recent years,some studies about retrotransposons induce bud mutation of apple are reported,retrotransposons are being identified as the molecular bases of somatic mutation.Previous research showed that retrotransposon atr1 was involved in apple's spur mutation,a specific4345 bp fragment in “Delicious” spur sports ‘Chinese Marshal',‘Meiguihong',‘Show Red',‘Oregon Spur ‘Delicious' ' was isolated by using IRAP based on primers designed from the long terminal repeat sequence of atr1(atr1-LTR)and genome walking.Base on the previous research,we identify mutation site in four ‘Red Delicious' spur mutants by genome walking and bioinformatics method,analyze structure of mutation site in‘Red Delicious' spur mutation and non spur mutation varieties of ‘Red Delicious',and determine the function of the specific insertion by ectopic expression,this provides a research basis for analysis of apple spur mutant.1.Tail-PCR and IPCR technology were utilized to separate flanking sequence of4345 bp in ‘Red Delicious' spur mutation ‘Chinese Marshal',‘Meiguihong',‘Show Red',‘Oregon Spur ‘Delicious' '.Finally,we obtain a 6758 bp and a 8329 bp fragments in ‘Red Delicious',and analyze these two fragments,we find a 1536 bp fragment in 8329 bp but not in 6758 bp;we obtain a 8329 bp and a 10504 bp fragments in ‘Chinese Marshal',“Meiguihong”,‘Show Red',we find a 2190 bp fragment in 10504 bp but not in 8329 bp;we obtain a 6758 bp and a 10504 bp fragments in ‘Oregon Spur ‘Delicious' ',we find a 2190 bp and 1536 bp in 10504 bp but not in 6758 bp,and 2190 bp insert in the 1038 th nucleotide of 1536 bp.Structure analysis of 2190 bp and 1536 bp finds they have a typical structure of LTR,and they are solo LTR.2.Using PCR technique to detect the structure of 2190 bp insertion site in in‘Red Delicious',its three non-spur mutants,namely ‘Richared',‘Starking',and ‘Early Red Delicious',and five spur mutants,namely ‘Chinese Marshal 1',‘Meiguihong',‘Show Red',‘Oregon spur Delicious',and ‘Jujiadian spur starking'.The results show that the result of PCR are same in ‘Red Delicious' and its three non-spur mutants‘Richared',‘Starking',and ‘Early Red Delicious',the site of 1536 bp solo LTR is heterozygous,one chromosome contain the 1536 bp and another not.Compared with‘Red Delicious',in ‘Chinese Marshal 1',‘Meiguihong',‘Show Red' and ‘Jujiadian spur starking',one chromosome contain the 1536 bp and another cotain 2190 bp and1536 bp insertion in the chromosome that don't cotain 1536 bp.Compared with ‘Red Delicious',in ‘Oregon spur Delicious',one chromosome don't contain the 1536 bp and another contain a 2190 bp insertion in the 1038 th nucleotide of 1536 bp.Finally,the 2190 bp insertion only in ‘Chinese Marshal 1',‘Meiguihong',‘Show Red',‘Oregon spur Delicious',and ‘Jujiadian spur starking' may lead to the spur mutant.3.The bidirectional transcription of the 2190-bp and 1536-bp fragments were analyzed using Agrobacterium rhizogenes-mediated transformation of soybean.GUS expression in the 2190-bp-sense and 2190-bp-antisense lines were prominent in stems.4.Predict the genes in 150 kb domain on each side of the 2190-bp insertion site by bioinformatics methods.23 genes were predicted.Some of these genes had predicted functions that could affect shoot development.they are acyl-coenzyme A oxidase 1(ACX1),one ethylene-responsive transcription factor(ERF),a1-aminocyclopropane-1-carboxylate oxidase(ACO),several auxin-induced proteins,an auxin-responsive protein SAUR,several MYB transcription factors,,several cyclin-dependent kinases,a CBL-interacting protein kinase.