Investigation Of Genes Expression Of Calyx Survival And Shedding Of Pear By Digital Gene Expressionand Functional Analysis Of PsIDA And PsJointless

Fruit quality is becoming more and more attractive, and it is always the key factor of fruit value. However, some pear fruit with calyx, usually shows high content of stone cells, bad flavor and appearance, thus the worse quality, compared with the fruit without calyx. In this study,’Kuerlexiangli’, the pear variety possess high rate of calyx survival is selected as material. Firstly, we studied physiological respects on this phenomenon. We then analyzed genome-wide the differentially expressed genes related to calyx development by the methods of high-flux RNA-Seq sequencing on transcriptional level. Based on the analysis from transcription profiles between calyx persisting and removing processes, as well as by using the reverse transcription PCR method, two full-length cDNA related to abscission genes were cloned from ’Kuerlexiangli’, and their characters and functions were analyzed. The main results achieved in this work were summarized as follows:1. Spraying Flusilazole, ethephon and PBO significantly increased the calyx abscission rate. The calyx abscission rate increased 24.07-33.58%. Calyx abscising treatments decreased the cellulase and polygalacturonase activities in calyx than that of BA, GA3 and control treatments. Meanwhile, Auxin and gibberellin (GA3) levels in the calyx were higher in calyx preserving treatments than that in abscising treatments. Another striking point is the decreasing IAA+GA3/ABA with calyx abscising treatments.2. Through comparison among the expression profiles of seven different experimental samples in calyx after calyx abscising treatment, calyx persisting and control treatment using RNA-seq, we obtained a lot of differentially expressed genes. These genes mainly involved in photosynthesis (PSI, PSII activity and photosynthetic electron transport in photosystem), plant hormone signal transduction(hormone synthesis, perception, an signal transduction), cell wall modification (polygalacturonases, celluloses, pectate lyases and expansin), transcriptional regulation(MYB, MADS-box, WRKY,) and carbohydrate metabolism(starch and sugar metabolizing). Real-time PCR confirmation showed a consistent tendency of gene expression with the digital gene expression data. This indicates that DGE was reliable. A large number of genes involved in calyx abscission. Some of them have been found to function in abscission. We also found a lot of novel candidate genes that may play important roles in the processes. Our results provide a basis for further analysis of calyx abscission.3. A cDNA was identified by analyzing the previous digital gene expression data of the persisting and removing calyx treatments of’Kuerlexiangli’. We isolation and functional characterization of this gene, designated PsIDA (Pyrus Inflorescence Deficient in Abscission). We isolated PsIDA gene, the gene contained a 312 open reading frame (ORF), and encodes a 104 amino acids polypeptide of molecular weight 11.312 kDa. The PsIDA-GFP fusion protein was localized to the nucleus and cell membrane in a transient expression assay.The full length PsIDA was subcloned into the expression vector pCAMBIA1301 downstream of the 35S-CaMV promoter. The constructs were introduced into Agrobacterium tumefacies GV3101 by the freezing transformation methods. The sense transgenic tomato plants were verified by PCR and semi-quantitative RT-PCR. It was indicated that PsIDA had been recombined into tomato genome as well as the transgenic were obtained. In transgenic tomato plants overexpression PsIDA, the inflorescences were added, and some fruit were unnormal in 35::PsIDA.4. Another cDNA clone was isolated, designated PsJOINTLESS from’Kuerlexiangli’. PsJOINTLESS encodes a MADS-box family gene which contains a conserved domain. The nucleotide sequence of PsJOINTLESS contains a 672 bp open reading frame, which encoding a protein of 224 amino acid with a mass of 25.439 kDa. Expression of PsJOINTLESSwas strongly induced by removing calyx treatment. The PsJOINTLESS-GFP fusion protein was localized to the nucleus in a transient expression assay. To understand function of PsJOINTLESS in plants related organ abscission, we generated transgenic plants in tomato over-expressing PsJOINTLESS under control of the CaMV 35S promoter. In transgenic tomato plants overexpression PsJOINTLESS, the abscission zone cell layers increased. In addition, the abscission zone of tomato transgenic lines showed thinner flower pedicels, more cell number, small pedicel cell size and displayed a fracture surface composed by irregular cells. The cellulase activity in pedicel abscission zone of transgenic plants was higher than that of wild type. Meanwhile, overexpression of PsJOINTLESS gene activated the expression of encoding transcription factors and cell wall hydrolase genes.These results indicated that PsJOINTLESS may effect the pedicel abscission zone development.