CDNA-AFLP Screening Of Differentially Expressed Genes In New Shoots Of Semi-dwarf Peach During Different Growth Stages

Peach is the third largest deciduous fruit tree in China, whose planting area and production are the top of the world. But excessive vegetative growth of peach not only increases management difficulties, pruning labor and production costs, but also enormous waste of photosynthate, which is one of the key problems need to be solved urgently in peach industry. However, the ideal cultivar of peach suitable for facilitation management is still deficient now. The"SD9238", a semi-dwarf mutation from seedlings, was found in breeding nursery of Zhengzhou Fruit Research Institute, the Chinese Academy of Agricultural Sciences. The tree height is only 1/2-2/3 of standard, with short internode, small trim, stout fruiting branches and moderate length, which is the ideal genetic resource to control peach tree growth vigor and limite the canopy diameter. It grows slowly at the early stage and turns fast after Mid-May. This character is apparently different from standard type.We investigated the differentially expressed genes in new shoots of"SD9238"at four growth stages using cDNA-AFLP approach. The differential fragments were screened, cloned and sequenced. Bioinformatic analysis and functional identification was followed. The result was verified by real-time quantitative RT-PCR. We hope the study could promote further understanding of the molecular mechanism of growth regulation in semi-dwarf peach. The main results are as follows:(1) Established and optimized the RNA extraction and cDNA-AFLP protocol for new shoots of semi-dwarf peach.(2) cDNA-AFLP screening of differentially expressed genes in new shoots of semi-dwarf peach during four growth stages. Obtained and function identified the differentially expressed genes related to growth regulation. Two hundred and fifty-six primer combinations were used for selective amplification, and 1987 transcript derived fragments (TDFs) were selected for their differential expression, of which 497 target genes were cloned and sequenced. The function of target genes was identified by BlastX homology analysis and Blast2go software.BlastX homology analysis showed that there were 254 genes with high homology. In these genes, 51 genes are unknown function, and the other gene's homologous proteins include: growth regulators, such as cinnamoyl-CoA reductase (CCR), F-box family protein, Tubby-like protein (TLP), IAA-amino acid hydrolase, cytochrome P450, brassinosteroid receptor, senescence-associated protein and cytokinin oxidase and so on; transcription factors; key enzymes of metabolic pathway; heat shock protein. In addition, none similar sequence for 80 differentially expressed genes was found in the GenBank database, which may be new genes.The results gotten from Blast2go database were as follows: 132 genes have no similar sequence, might be new genes; other 365 target genes with homologous proteins were divided into 50 categories according to function. The reactions that the genes might be involved in mainly include: metabolic pathway, such as glyoxylate metabolic, starch metabolism and sucrose metabolism process; biosynthetic process, such as auxin, phylloquinone and ubiquinone biosynthetic process; stress reaction to light, thermal stimulus and oxidative stress; cellular localization; photosynthesis; transmembrane transport and transcriptional regulation and so on.(3) Twenty-five genes, from differentially expressed fragments separated by cDNA-AFLP approach, were selected for real-time fluorescence quantitative PCR experiments. Relative quantitative analysis of the target genes were tested with a standard of reference gene (RPâ…¡). The results were consistent with cDNA-AFLP polyacrylamide gel electrophoresis displayed.