Transcriptome Sequencing Of Fruits Of Chinese Dwarf Cherry [Cerasus Humilis (Bge.) Sok.] And Cloning And Expression Analysis Of Key Enzyme Genes Involved In Malic Acid Accumulation

Chinese dwarf cherry [Cerasus humilis (Bge.) Sok.], a peculiar shrubby fruit tree resource only found in China, is resistant to cold, drought and barrenness and have good capacity of soil and water conversation. The fruits of Chinese dwarf cherry, which have bright color and varied types, are rich in nutrients and bioactive substances such as organic acid, amino acid, anthocyanin, tannin, flavanone and flavonol, indicating abundant potential genes. However, present parents selection for its hybridization is mainly based on appearance characters and physiological indexes, and is of low accuracy. So, excavating excellent germplasm genes using modern biotechnologies is very important for directional breeding of Chinese dwarf cherry. Fruits of Chinese dwarf cherry are high in acidity. Acidity is the leading factor affecting fruit quality and an important quality index in breeding work. Malic acid, the main organic acid in Chinese dwarf cherry fruits, varies in contents in different varieties and plays an important role in the formation of fruit acidity.In this study, three Chinese dwarf cherry varieties (’Nongda 3’,’Nongda 4’ and’ Nongda 5’) with different fruit color and malic acid content characteristics were selected for experiment. Transcriptome sequencing of fruits in late developmental phases was conducted and gene expression differences of the varieties were analyzed in order to excavate excellent genes. Based on RT-PCR and transcriptome sequencing information, three key enzyme genes involved in malic acid accumulation were cloned and their sequences were analyzed. Changes in gene expression during fruit development were tested and relationships among gene expression, enzyme activity and malic acid accumulation were analyzed. This work lay a foundation for the in-depth study of molecular mechanism of malic acid accumulation in Chinese dwarf cherry fruits.The main results are as follows:1. Transcriptome sequencing of fruits in late developmental phases from the three varieties were conducted through the Illumina Hiseq2000 platform. Data of 19282809960 nt were obtained in total. Clean reads were assembled using Trinity, and 37222,37856 and 38390 Unigenes were obtained respectively in ’Nongda 3’,’Nongda 4’ and ’Nongda 5’. After further sequence assembly and redundancy removal,39347 non-redundant all-Unigenes with length from 300 bp to 8531 bp were obtained. The length of 2505 all-Unigenes was longer than 3000 bp. Sequence of the Unigenes were aligned with 6 databases (evalue<0.00001) and 31726 Unigenes were annotated. The amount of Unigenes annotated in NR, NT, Swiss-Prot, KEGG, COG and GO were 30332,29410,20093,18230,12562 and 22844, respectively.2. NR-based similarity analysis showed that 18784 proteins (61.93% of the total) have similarity of 95%～100% with registered ones. Analysis of species distribution demonstrated that 26991 Unigenes (88.99% of the total) share high homology with genes from peach (Amygdalus persica).18230 Unigenes obtained by transcriptom sequencing were annotated in KEGG, including 4084 (22.4%) in metabolism,1807 (9.91%) in biosynthesis of secondary metabolites and 1428 (7.83%) in interaction between plants and pathogens. Abundant types of SSRs were found in the transcriptomes. Among the SSRs, monanucleotide, dinucleotide and trinucleotide repeats are the main types, accounting for 11.94%,54.60% and 27.76%, respectively. SSR with 6 repeats appeared most frequently and counted for 20.98%.3. Expressions of 3766 and 3537 Unigenes were demonstrated to be up-regulated respectively in ’Nongda 4’ and ’Nongda 5’ compared to ’Nongda 3’.3008 Unigenes showed higher expression in’Nongda 5’than in ’Nongda 4’. Corresponding annotated Unigenes of MDH, ME and VHA were found in the transcriptome database. Gene expression analysis showed that the highest expression of Unigenes responsible for malic acid synthesis was in ’Nongda No.4’ while the highest expression of Unigenes responsible for malic acid degradation was in’Nongda 3’, which could be an important cause of malic acid accumulation differences between the two varieties.4. GO analysis showed that in comparison between ’Nongda 3’ and ’Nongda 4’, differentially expressed genes were mostly enriched in cell periphery of cellular component, in tetrapyrrole combination and anthocyanin-3-O-glucosy- ltransferase activity of molecular function, and in oxygenated compounds reaction, ion transport and injury response of biological process. In comparison between ’Nongda 4’ and ’Nongda 5’, differentially expressed genes were mostly enriched in extracellular region of cellular component, in oxidoreductase activity of molecular function, and in far-red light reaction of biological process. In comparison between’Nongda 3’and’Nongda 5’, differentially expressed genes were mostly enriched in extracellular region of cellular component, in tetrapyrrole combination and heme combination of molecular function, and in chitin reaction, lignin metabolism and respiratory burst of biological process.5. Pathway analysis of differentially expressed genes showed that the most significantly enriched Unigenes were concentrated on Biosynthesis of Secondary Metabolites and further concentrated on Flavonoid Biosynthesis. In Flavonoid Biosynthesis pathway, there were 77, 53 and 55 differentially expressed genes respectively between ’Nongda 3’ and ’Nongda 4’, between ’Nongda 4’ and ’Nongda 5’ and between ’Nongda 3’ and ’Nongda 5’. Expressions of CHS, F3H, F3’5’H, ANR, LAR and UFGT were higher in ’Nongda 4’ than in ’Nongda 3’. Expressions of CHS, ANR and UFGT were lower and expression of LAR was higher in ’Nongda 5’ than in ’Nongda 4’. Expressions of CHS, F3’5’H, DFR, ANR and LAR were higher and expression of UFGT was lower in ’Nongda 5’ than in ’Nongda 3’.6. Primers were designed according to transcriptome data and CDS sequences of malic acid metabolism-related ChMDH, ChME, ChVHA were cloned. Sequence analysis demonstrated that the genes were highly homologous with corresponding genes in peach, apple and pear. Physical and chemical properties of proteins encoded by the three genes were analyzed and secondary structure and three-dimensional structure of the proteins were predicted and analyzed. Further study showed that expression changes of ChMDH and ChVHA, which were involved in synthesis and storage of malic acid, were similar with activity changes of MDA and VHA. The increase in expressions of ChMDH and chVHA and in activities of MDA and VHA in maturation stage promoted the rapid accumulation and storage of malic acid. The expression of ChME was not directly related with the activity of ME. In late developmental phases, the continuous increase of ChME expression was related with the degradation of malic acid. Differences in expression of ChME and ChMDH and activity of ME and MDH before fruit maturation were important reasons for the varied fruit acidities in different varieties.