| Cyclobalnopsis gilva belongs to the family Fagaceae and it is native to East Asia countries i.e.China and Japan.Due to its low germination rate of its seeds,it is a good plant material for studying molecular mechanism of low seed germination.Transcriptional and proteomics analysis of C.gilva germination mechanisms at mRNA and protein levels were performed using RNA-seq,2DE and Label-free methods.In addition,the results of transcriptome and proteome were confirmed through the physiological and biochemical measurements.The seed transcriptome of C.gilva was analyzed by using the Illumina HiSeq 2500 platform.After matching the sequence in the public database including Nr,SwissProt,GO and KEEG,our transcriptome results showed that 54,130 unigene were annotated in this manner.We found a large number of unigenes were enriched in many process including starch and sucrose metabolism,glycolysis,phytohormone signal transduction,ABC transporters,carbohydrates metabolism,post translation modification,ribosome structure and biosynthesis by analyzing the annotated unigenes using GO and KEEG pathway data base,.These transcriptome data showed many seed germination genes which could be used as a reference data base for analyzing differentially expressed seed germination in C.gilva.Difference of genes was analyzed in germinated and non-germiated seeds of C.gilva and this analysis revealed that a total of 26,003 unigenes with at least 2-fold changes were differentially expressed were identified in germinated and non-germinated condition.Among them 9683 genes were up regulated and 16,320 genes were down regulated in germinated seed.Additionally,a total of 23,685 unigenes with at least 2-fold changes were differentially expressed were identified in non-germinated and control seeds,among which 14,260 unigenes were up regulated and 9425 unigenes down regulated in non-germinated seeds.Differentially expressed genes in function annotation and metabolic pathways were comprehensively analyzed.The results showed that up-regulated differentially expressed genes were focused on the pathways including starch and sucrose metabolism and glycolysis pathway containing enzymes like PK,PGK,and PFK in germinated seed.The 13 differentially expressed genes involved in glycolysis and TCA cycle were confirmed by qRT-PCR,which were consistent with the gene expression patterns of transcriptome result.These findings indicated that non-germinated seeds can't provide sufficient energy and substances due to a significant lower respiratory metabolism compared to germinated seeds.A pragmatic comparative proteomic approach was carried out to investigate this feature on germinated and non-germinated seeds of C.gilva.More than 700 proteins were detected through two-dimensional electrophoresis(2-DE),among which 26 proteins had 2-fold changes in abundance(control,non-germinated and germinated seeds).The 24 differential proteins were identified through Matrix-Assisted Laser Desorption Ionization-Time of Flight/Time of Flight Mass Spectrometry(MALDI-TOF/TOF-MS).Most of the identified differential proteins were found up-regulated and were involved in starch metabolism.P-amylase,in particular,its abundance and mRNA level were both up-regulated in germinated seeds of C.gilva by proteomics and qRT-PCR analyses,respectively.The enzyme activities of a-and ?-amylases indicated that P-amylase activity was higher in germinated seeds than those in the control and non-germinated seeds.Interestingly,a-amylase did not show significant changes in terms of protein abundance and enzyme activity between the germinated and non-germinated seeds.However,starch and sugar contents were observed higher in non-germinated and control seeds as compared to the germinated ones.These findings provide new insights into the understandings of C.gilva seed germination problems.Label free,a comparative proteomics approach was carried out to investigate the protein changes in germinated and non-germinated seeds of C.gilva.With this technique,3078 proteins were identified,among which 603 differential abundance proteins had fold changes>2.0 in abundance with P<0.05(control,non-germinated and germinated seeds).Most of the identified differential proteins were observed up-regulated in germinated seeds and these proteins are mainly involved in energy metabolism,suggesting low energy supply blocked seed germination in C.gilva.The enzyme activities related to energy metabolism found in transcriptome and proteome data were analyzed.The results showed that the enzyme activities of HK,PFK,PK,PGK,CS,IDH and MDH increases under germinating condition.The results of physiological analysis in energy production were consistent with the data relative mRNA expression levels and the label free,which reinforce each other to demonstrate the reason of low seed germination rate in C.gilva.In conclusion,the comprehensive analysis of transcriptome,proteome,and physiological revealed several important seed germination characteristics from C.gilva.Systematics investigation at the three levels of mRNA,protein(enzyme)and metabolic products was conducted to study molecular mechanism in non-germinated and germinated seeds of C.gilva.In the present study,we found that most key genes or protein involved in energy metabolism were up regulated in germinated seeds,but no changes or down-regulations were found in non-germinated seeds after integrating transcriptome and proteome data.Therefore,we proposed that the problem of starch degradation and the shortage of energy and substance supply in non-germinated seeds are the major causes of lower rate seed germination in C.gilva.In addition,the seed germination is regulated at transcriptional,translational,and physiological levels.This study will provide a better understanding to low seed germination rate in C.gilva,and a novel idea to improve seed germination of the plant,and a theoretical foundation for using genetic engineering technique to breeding high seed germination rate of C.gilva in the future. |
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