Transcriptome And MiRNAs Sequencing Analysis The Mechanism Of Aroma Weakened In ‘Nanguo' Pear After Cold Storage

Nangguo pear is Pyrus ussuriensis Maxim.,and a specialty of southern Liaoning.The unique aroma is the main characteristic of the fruit,and it is also an important index for evaluating the quality.Nanguo pear is without aroma when it is picked from the tree,it reaches the optimum taste period and loved by people after about 10 d,with beautiful colour,soft flesh and rich aroma,but the fruit pulp becomes soft and rotten,the aroma is weakened after 15 days,and lost its edible value.In recent years,cold storage is a common way to extent the storage time of Nanguo pear,but the fruit senescence process accelerated,and the aroma weakened observably after cold storage.These have seriously affected the quality of the fruit.Therefore,research on the mechanism of aroma weakening and find out the main factors causing the aroma deterioration in Nanguo pear after cold storage,which has important theoretical and practical significance for improving the quality of the fruit.With the development of molecular biology technology,the application of high throughput sequencing technology is becoming more and more widely.With high-throughput sequencing,this study established the transcriptome database of Nanguo pear on the first day and the optimum taste period during shelf life at room temperature natural ripening and after 150 d cold storage.Then the differentiallly expressed genes?DEGs?in the fruit after cold storage were find out,and carried on the GO function annotation analysis and KEGG metabolic pathways analysis.To find out the main metabolic pathways that affected the aroma weakening of cold storage fruit.The miRNAs library of Nanguo pear was establishment on the first day and the optimum taste period during shelf life at room temperature natural ripening and after 150 d cold storage by high-throughput sequencing.The differentially expressed miRNAs of cold storage Nanguo pear were identified,and predict the target genes of these miRNAs were predicted.Then these miRNAs and their target genes were analyzed by the biological information analysis,and the expression pattern were analyzed by RT-PCR to find out the main miRNAs that affected the aroma weakening of cold storage fruit.Based on the above research,we further studied the mechanism of aroma weakening in cold storage Nanguo pear from fatty acid synthesis pathway.We investigated the contents of total aroma eaters and unsaturated fatty acid,the activity and gene expression of lipase,the key genes in short chain saturated fatty acid synthesis pathway,unsaturated fatty acid synthesis pathway and the ester aroma synthesis by unsaturated fatty acid metabolism pathway.The main results of this paper are as follows:Compared to the without being cold storing Nanguo pear,3137 differentially expressed genes were found that might include in the aroma forming in the Nanguo pear during shelf life after cold storage.The GO function analysis of DEGs showed that they are mainly involved in the “metabolic process”,“cell process”,“stimulus response” and “catalytic activity”.KEGG pathway analysis showed that the DEGs mainly included in lipid metabolism: fatty acid biosynthesis,alpha-linolenic acid metabolism,biosynthesis of unsaturated fatty acid,fatty acid degradation and linoleic acid metabolism,and plant hormone signal transductionin,which influenced the formation of aroma after cold storage Nanguo pear.314 known miRNAs were identified in Nanguo pear after miRNAs sequencing analysis,176 and 135 differentially expressed miRNAs were detected on the day of remove of cold storage and the optimum taste period,respectively.Prediction the target genes of these differentially expressed miRNAs revealed that most of these target genes were DGEs in the transcriptome results,including LOX2 S,LOX1₅,HPL,ADH1,which were directly related to the aroma formation.The main regulation miRNAs were mdm-miR156a-o,mdm-miR172a-h,mdm-miR159a/b/c,mdm-miR395a-i,mdm/ppe-miR535a/b,mdm/ppe-miR399 a,mdm-miR408b/c/d and mdm-miR7120a/b.The expression pattern of miRNAs and their target genes by RT-PCR further verified that mdm-miR172a-h,mdm-miR159a/b/c,mdm-miR395a-i,mdm/ppe-miR535a/b,mdm/ppe-miR399a/b/c and mdm-miR7120a/b are the key regulated miRNAs of aroma forming in Nanguo pear after cold storage.The differentially expressed genes enriched in the fatty acid metabolism pathway from the results of transcriptional sequence.This study analyzed the content of aroma ester,the content of fatty acid,the change of key enzyme activity and key genes expression in fatty acid metabolism cold stored Nanguo pear for different time and unrefrigerated fruit.The results showed that the total amount of ester aroma was much lower on the OTP during shelf life in the fruit of cold storage for 90 d than that unrefrigerated fruit?P<0.05?,and was more lower in the fruit of cold storage for 150 d?P<0.05?.At the same time,the content of unsaturated fatty acids decreased significantly?P<0.05?,and the expression of fadD,fadE,fadJ,atoB,fabF,SCD,FAD2,LOX2 S and LOX1₅ was also down-regulated significantly in the fruit of cold storage for 150 d,and all were lower than that in the fruit of cold storage for 90 d and the unrefrigerated fruit.With the extend of cold storage time,the total content of the aroma ester on the OTP during the shelf life declined more seriously,with the lower content of the unsaturated fatty acid and the more lower expression of key genes fadD,fadE,fadJ,atoB,fabF,SCD,FAD2,LOX2 S and LOX1₅ in the fatty acid metabolism pathway.It can be seen that cold storage inhibited the metabolism of fatty acid in a certain extent and reduced the production of unsaturated fatty acids,which was the main cause of the aroma weakening in the cold stored Nanguo pear.The content of all ester aroma was significantly higher on the OTP during shelf life at room temperature in the low temperature condition?LTC?treatment cold stored Nanguo pear than that the directly cold stored fruit for 150 d.The content of palmitic acid and stearic acid decreased,and the content of oleic acid,linoleic acid and linolenic acid increased in the LTC treatment fruit.The key genes of fadD,fadE,fadJ,atoB,fabF and fabG in the short chain fatty acid synthesis pathway were up-regulated,the key genes of SCD and FAD2 in the unsaturated fatty acid synthesis pathway were up-regulated,and the key genes of LOX2 S,LOX1₅ and HPL were obviously up-regulated in the synthesis pathway of ester aroma by linoleic acid and linolenic acid in the fruit during shelf life at room temperature after LTC treatment.Thus,the LTC treatment alleviated the problem of aroma weakening in the cold stored Nanguo pear at a certain extent by regulating the expression of the key genes in the long chain saturated fatty acids degraded to the short chain saturated fatty acids pathway,and the short chain saturated fatty acids to unsaturated fatty acids pathway,and the synthesis of ester aroma by unsaturated pathway.The results further verified that the aroma weakening were caused by the block of fatty acid metabolism pathway in the cold stored Nanguo pear.