| Chinese cherry (Prunus pseudocerasus) is one of the most important fruit trees, which belongs to Rosaceae family. People enjoy Chinese cherry due to its beautiful appearance, delicate taste and abundant nutrition. Like other fruit trees, it has typical natural dormancy. Dormancy is a way that fruit trees survive in cold temperate. If dormancy of floral bud could not be broken properly, abnormal blooming and low fruit-bearing will appear, which directly affect the fruit quality and yield. Dormancy of deciduous fruit trees is a very complex process. Sufficient chilling accumulation is one of the key factors response for dormancy release. Due to lack of Chinese cherry genome, molecular mechanism triggering dormancy release in flower buds remained unclear.Therefore, transcriptome and DGEs of flower buds in different dormant stages of P. pseudocerasus. cv’Duanbing’, an elite landrace of Chinese cherry, was constructed using RNA-Seq technology. Lots of differentially expressed genes (DEGs) were screened. The transcriptome MADS-box transcription factors having full-length sequence were analyzed. Three DAM (Dormancy Associated MADS-box) genes were cloned. These results laid the foundation for the molecular biological mechanism of Chinese cherry flower bud dormancy release which induced by low temperature.The main results were summarized as followings,(1) By sequencing the homogenization of the cherry dormant flower bud, approximately 47 million clean reads were assembled into 50,604 sequences, with an average length of 837 bp. A total of 16,186 unigenes were successfully annotated by biological process, molecular function and cellular component. One hundred and twenty-eight pathways (21,143 unigenes) were annotated by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Metabolic (KO 01100:5077 Unigenes, 24.01%), biosynthesis of second metabolite (KO 01110:2518 Unigenes, 11.91%),plant-pathogen interaction (KO 04626:1381 Unigenes,6.53%) and plant hormone signal transduction (KO 04075:1048 Unigenes,4.96%) accounted for higher percentage in flower buds.(2) Digital gene expression profiles in deep dormancy stage (FBO),50%(FB365) and 100% (FB724) stage of dormancy release were performed using RNA-Seq sequencing. With multiple comparison among the differentially expressed genes (DEGs) in FBO, FB365, FB724 profiles, it was found that total clean reads was higher than 6 M. There are 259 up-regulated and 1,385 down-regulated DEGs among FB0-VS-FB365; 1103 up-regulated and 1,853 down-regulated among FB0-VS-FB724; 940 up-regulated and 513 down-regulated among FB365-VS-FB724 respectively. Ten Gene Ontology (GO) terms are enriched in FB0-VS-FB365. The most significant category related to molecular function was photosynthetic membrane. Extracellular region was the sole term among FB365-VS-FB724.In FB0-VS-FB724, there was 4 terms. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis demonstrated that 889, 909and 1712 DEGs were annotated to 111,11 land 120 metabolic pathway among FB0-VS-FB365, FB365-VS-FB724 and FB0-VS-FB724, respectively, which involved in various metabolic processes.572 (64.3%),771 (84.8%),1385 (80.9%) DEGs were annotated to 13,16,17 metabolic pathways dicovered from transcriptional profiles of FB0-VS-FB365, FB365-VS-FB724 and FB0-VS-FB724. The random selection of Q-PCR DEGs data domain RNA-Seq data basically showed a similar trend, with a very significant level, which shows that the results of gene expression profile based on RNA-Seq analysis is credible.(3) 18 MADS-box genes (PpcMADS) with full open read frame were identified from transcriptome generated from flower bud of Chinese cherry. In order to reveal the functions of PpcMADS, physicochemical property, secondary structure, subcellular localization and conserved motifs of amino acid were analyzed using bioinformatic methods. The results showed that 16 members of PpcMADS belong to MIKC family, while KP347550 and KP347552 were M-type. Most of them were unstable proteins with hydrophilcity. The secondary structure of PpcMADS protein included alpha helix, beta turn, extended strand and random coil, in which alpha helix was major part. Most of PpcMADS proteins located in nucleus, while KP347545 and KP347552 were found in plasma membrane. Four motifs were identified from PpcMADS protein, and Motif 1 was a conserved MADS domain. KM243368 and KM243374 belong to AP3 subclass. KM243373 and KP347551 were PI subclass and AG subclass, respectively. KM243369 and KM243372 belong to SHP subclass. KM243377 and KP347546 were AP1M243371. KM243376 and KP347549 belong to SEP subclass. KM243375, KP347545 and KP347547 were SOC subclass. KP347548 was SVP subclass. Expression analysis showed that KM243377 and KP347550 did not expressed in flower and fruit, while KM243368, KM243373, KM243375, KP347546, KP347547, KP347551 and KP347552 only expressed in flower.(4) Three dormancy-associated MADS-box (PpcDAM) genes were clustered together with MADS transcription factor DAM4, DAM5 and DAM6, respectively, which were related to the dormancy of peach and plum, and the relationship between MADS-box gene from Prunus was very closely. With the release of enddormancy, the expression was down-regulated, maintaining a low level during the ecodormancy. The peak of three genes occurred in endodomancy, subsequent down-regulation. It is speculated that these three genes may have a regulatory effect on the dormant state. The promoter of PpcDAM4 gene was found to contain specific function elements of promoter in higher plant and CRT/DRE recognition sites. By transient expression of double luciferase, C-repeat binding transcription factor (CBF) was a regulatory effect on the promoter, which indicated that low temperature response is probably mediated through the CBF pathway. In addition, seed germination inhabitation was found in the PpcDAM6 transformed Arabidopsis thaliana, which indicated that PpcDAM6 in the flower bud was induced by low temperature and could inhabit the germination of flower buds. According to the above results, it is suggested that the PpcDAM gene probably involved in the regulation of dormancy and its release of Chinese cherry flower bud. However, much more study should be performed on the regulation mechanism of flower bud dormancy. |
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