Molecular Mechanism Of Auxin Related Genes Regulating Peach Fruit Ripening

Flesh texture of peach was classified into three types:melting(MF),non-melting(NMF),and stony hard(SH)according to the characteristics of fruit ripening,YUCCA encodes flavin monooxygenase-like enzyme,and which can convert indole-3-pyruvic acid to IAA.To elucidate the reason that the different expression of YUCCA11(ppa008176m)in MF and SH peach,the amplification of upstream regions of YUCCA11 promoter in two peach cultivars were performed by PCR.And Aux/IAA protein is the center of IAA signal;therefore,the Aux/IAA gene family in the peach genome was identified and analyzed by bioinformatics.Combining with transgenic technology to verify the function of Aux/IAA genes,and constructioning yeast cDNA library of peach fruit and screening the binding IAA-related protein with PpACS1 promoter.The results of the study are as follows:1.Transposable element in a YUCCA11 gene promoter is associated with the stony hard phenotype in peach1)YUCCA11 showed increasing expression in the MF fruits during fruit ripening,and peaked at S4 III,but the expression was very low in the SH fruits.Unlike in the fruits,the expression of YUCCA11 was high both in the MF and the SH seeds.2)To explore the reason that leading to the difference of the expression in the MF and the SH fruits,we cloned the upstream predicted promoter region of the YUCCA11 gene in the MF(‘Zhongyoutao 13'and‘Gold honey 3')and the SH(‘Zhongyoutao 16'and‘Yumyeong')according to peach genome sequence.Compared with MF peaches,a large insertion was found in the 2.1 kb upstream from the start codon of the YUCCA11 gene in the SH peaches.NCBI blast found that the insert fragment was the CACTA type DNA transposon,with terminal inverted repeats(CAAGAAAAAA)and conserved sequence(CACTA),which could clearly distinguish SH and non-SH types and the insertion of transposons may result in the loss of function of the fruit specific elements.2.Identification and function profiling of Aux/IAA family in peach1)There were 22 members of the PpAux/IAA in the peach genome with bioinformatics analysis.They were not evenly distributed on the 5 chromosomes.qRT-PCR suggested that the expression level of 10 PpAux/IAA genes in MF peach fruits were higher than in SH peach fruits,and the expressions of ppa011935 m,ppa020369m,ppa010303 m and ppa010871 m.These results indicated that the PpAux/IAA gene family members are involved in regulating peach fruit ripening.2)Tomato(Solanum lycopersicum cv.‘Micro-Tom')was used as a model for investigating the phenotypic and molecular changes associated with over-expression of ppa011935m(PpIAA19).This revealed that PpIAA19 was involved in regulating tomato lateral root number,stem elongation,parthenocarpy,and fruit shape.Molecular analyses of transgenic tomato plants over-expressing PpIAA19 indicated that the observed phenotypic changes were partially mediated by regulating the expression of auxin response genes.3.Construction of a normalized yeast hybridization cDNA library and screening the binding protein of PpACS1 promoter1)A normalized full-length yeast hybridization cDNA library was constructed by DSN(duplex-specific nuclease)normalization method in combination with SMART(switching mechanism at 5'end of RNA transcript)technique.The titer of library was about 3.2×10~6 cfu/ml,which contained 3.5×10~7 independent clones.The size of cDNA inserts was 750 to 2000 bp with a recombination rate of 83.3%.The results suggested that the normalized yeast hybridization cDNA library has been successfully established with high quality,which is convenient to screen transcription factors and interaction protein regulated during peach fruit development.2)We used 1784 bp PpACS1 promoter sequence construct the bait to screen the yeast hybrid library.50 positive clones were screened and sequenced,33 single sequences were finally obtained.The binding protein mainly contained ribosomal protein,transport protein,cytochrome related protein,and hormone related protein.Two auxin related protein(Prupe.4G050800 and Prupe.7G100000),three disease resistance protein(Prupe.2G301600,Prupe.6G141100 and Prupe.7G049200),and one ABA related protein(Prupe.8G034100)will be used to further investigate.