2016YFD0400103), the National Natural Science Foundation of China (grant no. during ripening, especially in bananas, is largely unknown. In this study, 38 genes encoding starch degradation\related proteins were recognized and characterized from banana fruit. Expression analysis revealed that 27 candidate genes were significantly induced during banana fruit ripening, with concomitant conversion of starch\to\sugars. Furthermore, iTRAQ\based proteomics experiments identified 18 starch degradation\associated enzymes bound to the surface of starch granules, of which 10 were markedly up\regulated during ripening. More importantly, a novel bHLH transcription factor, MabHLH6, was identified based on a yeast one\hybrid screening using promoter as a bait. Transcript and protein levels of MabHLH6 were also increased during fruit ripening. Electrophoretic mobility shift assays, chromatin immunoprecipitation and transient expression experiments confirmed that MabHLH6 activates the promoters of 11 starch degradation\related genes, including MaMaMaMaMaMaMaMaMaand by recognizing their E\box (CANNTG) motifs present in the promoters. Collectively, these findings suggest that starch degradation during banana fruit ripening may be attributed to the complex actions of numerous enzymes related to starch breakdown at transcriptional and translational levels, and that MabHLH6 may act as a positive regulator of this process via direct activation of a series of starch degradation\related genes. leaves where starch accumulates in chloroplasts during the day and is broken down at night to supply substrates for local use and export (Santelia leaves at night is initiated through reversible glucan phosphorylation mediated by glucan water dikinase (GWD) and phosphoglucan water dikinase (PWD) to disrupt the semi\crystalline starch structure at the granule surface (Hejazi leaves point out that this pathway of starch degradation is usually highly complex and is mediated by the changes of putative enzymes at transcript and protein levels. However, the regulation of these processes in other plant organs such as fleshy fruits is largely unknown, especially in banana fruits. Banana is the world’s most important fruit crop and one of the top 10 10 crops by production (Paul and in banana (Xiao via multiple mechanisms in apple (Li transcription (Han MaGWD2MaLSF1MaAMY2BMaAMY2CMaAMY3MaAMY3AMaAMY3Band and and MapGlcT2\1MapGlcT2\2MapGlcT4\1and MaGWD2MaPWD1MaSEX4MaLSF1MaLSF2MaDPE1MaDPE2MaMEX1MaMEX2MapGlcT1MapGlcT2\1MapGlcT2\2MapGlcT4\1and (Table?S1). As shown in Physique?3 and Determine?S1, during ripening in all three different treatments, transcripts of 27 genes (MaPWD1MaSEX4MaLSF1MaLSF2MaBAM1\MaBAM4MaBAM6\MaBAM8MaBAM10MaAMY2BMaAMY2CMaAMY3MaAMY3AMaAMY3CMaISA2MaISA3MaPHS2MaMEX1MaMEX2MapGlcT2\1MapGlcT2\2MapGlcT4\1and MaAMY2AMaBAM9MaPHS1MaDPE1MaDPE2and were decreased (Physique?3 and Determine?S1). However, 3 genes (MaBAM5and leaves (Hejazi values 0.01. Open in a separate window Physique 5 Accumulation of the MaGWD1 transcript and protein during banana fruit ripening. (a) The transcript levels of are expressed as a ratio relative to the harvest time (0 day of control), which was set as 1. Each value represents the mean??SE of three replicates. (b) Preparation of polyclonal antibody against MaGWD1 and specificity test of the antibody by Western blot with four antibody gradient concentrations ranging from 1?:?5000 to 1 1?:?100?000. (c) Western blot T338C Src-IN-2 analysis of protein level of MaGWD1 from the starch granule T338C Src-IN-2 surface in banana fruits with three different ripening behaviours. Equal weight of starch was used to normalize the loading proteins. Identification of a promoter\interacting protein MabHLH6 Based on the transcript and protein expression of MaGWD1 during ripening, it seems that like AtGWD1, MaGWD1 might also play an important role in banana fruit starch degradation. To investigate the potential regulators of MaGWD1promoter was isolated and its expression pattern was tested using a GUS reporter (Physique?6a) in a transient expression assay. As shown in Physique?6b, in banana pulp transfected with GUS reporter driven by the promoter, GUS signals were observed in pulp with ethylene treatment. As a positive control (35S::GUS), GUS signals were constitutively expressed in banana pulp with or without ethylene treatment, while no E.coli monoclonal to V5 Tag.Posi Tag is a 45 kDa recombinant protein expressed in E.coli. It contains five different Tags as shown in the figure. It is bacterial lysate supplied in reducing SDS-PAGE loading buffer. It is intended for use as a positive control in western blot experiments GUS signal was detected in unfavorable control (empty::GUS) (Physique?6b). Open in a separate window Physique 6 promoter activity in response to ethylene and binding of MabHLH6 to promoter by Yeast one\hybrid assay. (a) GUS reporter constructs made up of the promoter (pro::GUS), the CaMV 35S promoter (35S::GUS, positive control) and the GUS without promoter (empty::GUS, unfavorable control). (b) Three reporters were transiently transformed into banana pulp using promoters. Left: No basal activities of MaGWD1 promoter were detected in yeast produced on SD medium lacking Leu in the presence of aureobasidin A (AbA). Right: Yeast growth assay after the Y1H reporter strains was transformed with plasmids carrying cassettes constitutively expressing MabHLH6 effectors. Conversation was determined based on the ability of the T338C Src-IN-2 transformed yeast to grow on SD medium lacking Leu in the presence of AbA. Then, we performed a yeast one\hybrid library screening using the promoter as a bait and a banana ripening associated cDNA library as prey. After high\stringency screening, a cDNA encoding a bHLH TF was identified and was designated as MabHLH6 (“type”:”entrez-protein”,”attrs”:”text”:”XP_009408340.1″,”term_id”:”695041466″,”term_text”:”XP_009408340.1″XP_009408340.1) after the names of previous five genes (Peng promoter was further confirmed by yeast one\hybrid assay (Physique?6c,d). Molecular characterization of cDNA contains an Open Reading Frame (ORF) of 1437?bp in length, encoding a polypeptide of T338C Src-IN-2 478 amino acids with a.
2016YFD0400103), the National Natural Science Foundation of China (grant no