Comparative Analysis Of Expression And Sequence Of Pigment-related Genes In White-flesh Mutant Of’sachinoka’and Its Wild Type

Fruit color is an important index for evaluation of the appearance quality of strawberry fruit, but the molecular mechanism of strawberry fruit color formation is not very clear. In the experiment, we used the white-flesh mutant and wild type plants as test materials, through detecting the content of anthocynins, sequencing analysis with RNA transcriptome sequencing, testing the expression of fruit anthocyanin synthesis related gene and analysis the seqences. Cloned alignment of related difference genes were transient expression analysis. The main results are as follows.1.’Sachinoka’white-flesh mutant content of anthocyanin is28.01%and46.94%of wild type in pulp and fruit, consistent with the phenotype observed. The total RNA was extracted from the white-flesh mutant and wild type with transcriptome sequencing. White-flesh mutant and wild-type transcriptome data which contains40779835and47879074effective clean reading sequence, after clustering, assembly and analysis, obtained25704Unigene were identified from white-flesh mutants,26606transcripts in the wild type, and identified4790significantly different genes in white-flesh mutant and wild type, in which1242Unigene is up regulated in the white-flesh mutant, down regulation Unigene are3548.2. Analysis the transcriptome sequencing the found that ANS, CHS, F3H, MYB10, bHLH78, bHLH143, WD40in anthocyanin synthesis pathway are down regulation expressed in white-flesh mutant fruit. To verify the transcriptome data by using qRT-PCR technology, the results showed that ANS, CHS, F3H, MYB10, bHLH143, WD40gene expression consistent with the transcriptome data.3. qRT-PCR analysis showed that bHLH78gene in strawberry fruit specific expression. Along with fruit development and maturation, the expression of bHLH78gradually increased.4. Cloned the full-length sequence of genes ANS, CHS, F3H, MYB10, bHLH78and bHLH143from the white-flesh mutant and wild type fruit, is1152bp,1170bp,1095bp,702bp,1653bp and1083bp respectively. Analysis and comparison of nucleotide and amino acid sequences of the six genes, They are in the mutant and wild-type similarity were98.98%,98.96%,99.00%,98.20%,99.11%,98.67%and98.32%,99.74%,98.96%,97.30%,98.73%,97.58%. There is no significant difference of nucleotide and amino acid sequences of the six genes in the white-flesh mutant and wild type.5. Constructed the overexpression vector of ANS, CHS, F3H, MYB10, bHLH78, bHLH143. The transient expression of bHLH78and MYB10gene over expression of pRI-bHLH78and pRI-MYB10were introduced into the big green stage and white stage of the white-flesh mutant, the white-flesh mutant fruits with bHLH78gene over expression have no obvious color change. There is phenomenon that anthocynin accumulation increasing in white-flesh fruits which injecting in over expression vector pRI-MYB10.