Analysis of genes related to glycolysis in the leaves and roots of Malus sieversii under NaCl stress based on transcriptome sequencing

Abstract:【Objective】Malus sieversii belongs to the genus Malus of Rosaceae, which has played an important role in the history of apple cultivation in the world. It is widely used as a good rootstock for ap- ple production in Northwest China because of its excellent characters of cold resistance, drought resis- tance, pest resistance and barren resistance. Therefore, in-depth study on these precious germplasm re- sources can not only promote rational development and utilization of M. sieversii resources, but also provide abundant gene resources for genetic improvement of Malus genus. Salt tolerance is a complex trait controlled by multi-genes, which involves many physiological and biochemical processes, such as growth, photosynthesis and energy metabolism, osmoregulation and cell signaling. At present, studies on Malus sieversii are mainly seen in the distribution, origin, evolution, and characteristics of physiolo- gy and biochemistry. The research on M. sieversii under NaCl stress is mainly seen in the determination of leaf anatomical structure, proline, malondialdehyde, chlorophyll and soluble sugar content, and the research on molecular mechanism is rarely reported. This study analyzed the genes related to glycolysis pathway of M. sieversii under NaCl stress by transcriptome sequencing technology, in order to provide reference for the mining of salt tolerance genes of apple. 【Methods】M. sieversii seeds were purchased  from Yili, Xinjiang. The seedlings were obtained by tissue culture. After rooting, the uniform seedlings with fine roots were transferred into the water culture boxes with continuous supplyof the Japanese Gar- den Nutrient Solution. When the 6th to 8th leaves of hydroponic seedlings were fully expanded, the following treatments were carried out: (1) CK, the Japanese Garden Nutrient Solution; (2) NaCl treatment, adding 150 mmol∙L-1 NaCl to the Japanese Garden Nutrient Solution. In order to avoid salt shock reac- tion, the concentration of NaCl gradually increased with a gradient of 50 mmol·L-1 per day, and all the treatments reached the target concentration on the same day, which was set as 0 h for NaCl treatment. The leaves and roots of the seedlings were taken 48 hours after the treatment, weighed 0.1 g, and put in- to 1.5-2.0 mL cryopreservation tube. The leaves and roots of the control were recorded as LCK48h and RCK48h; the leaves and roots treated with NaCl were recorded as LNa48h and RNa48h. They were quickly frozen in liquid nitrogen and stored in the refrigerator at -80 ℃ for standby. Each treatment was repeated three times. Total RNA was extracted from the leaves and roots of the seedlings 48 hours after NaCl treatment. RNA sequencing was completed by Nanjing paisennuo Gene Technology Co., Ltd. af- ter qualified quality inspection, the libraries were constructed and sequenced based on Illumina Hiseq Sequencing Platform.HISAT2 was used to match the filtered high-quality sequence (clean data) to ap- ple’s reference genome. Deseq was used to analyze the difference of gene expression. The condition of differential gene expression was: the multiple of expression difference was |log2FoldChange| > 1, corrected P-value < 0.05. On this basis, the expression difference analysis, GO and KEGG enrichment anal- ysis were further carried out of the samples, and the differential genes were classified according to the annotation results, and the differential genes were screened out and verified by qRT-PCR【. Results】Among the 39 million to 48 million filtered clean reads, 82% of the total sequence of the comparative reference genome was clean reads. After 48 hours of NaCl treatment, there were 3 364 differentially ex- pressed genes in the leaves of the seedlings, 1 745 of which were up-regulated in response to salt stress and 1 619 were down regulated in response to salt stress. And among the 3808 differentially expressed genes in the roots of the seedlings, 1 057 were up-regulated in response to salt stress, and 2 751 were down regulated. According to GO enrichment analysis of the differentially expressed genes, molecular functions mainly included catalytic activity, oxidoreductase activity, etc.; cell components mainly in- cluded membrane, thylakoid, photosystem II, etc.; biological processes mainly included oxidation- reduction process, photosynthesis, etc.According to KEGG analysis, 2 095 differential genes in the leaves and roots of M. sieversii had been annotated. These 2 095 differential genes involved 44 pathways. The most significant pathways were glycolysis/gluconeogenesis, pentose phosphate pathway, fructose and mannose metabolism, pyruvate metabolism, etc. Therefore, further analysis of the differential genes re- lated to glycolysis and pyruvate metabolism showed that after NaCl treatment, Enolase (ENO), Pyru- vate dehydrogenase E1 component (aceE), 2,3-bisphosphoglycerate-dependent phosphoglycerate mu- tase (gpmA), NADP- dependent malic (NADP- ME) and Phosphoenolpyruvate carboxylase (PEPC) genes were up-regulated in the leaves. Alcohol dehydrogenase (ADH), Phosphoenolpyruvate carboxyki- nase (pckA), Triosephosphate isomerase (TPI), Fructose 1,6-bisphosphatase (FBPase), Malate synthase, glyoxysomal (MS), Pyruvate, phosphate dikinase (PPDK) genes were down-regulated expression in the leaves. Phosphoenolpyruvate carboxykinase (ATP) (pckA), Triosephosphate isomerase (TPI), Malate synthase, glyoxysomal (MS) genes were up-regulated in the roots, while the rest were down-regulated. These differentially expressed genes in the leaves and roots showed that the metabolism of sugar and en- ergy in M. sieversii was very active and complex during salt stress.【Conclusion】Through transcrip- tome sequencing and qRT-PCR, TPI, FBPase, pckA, PPDK and other related genes in M. sieversii were significantly changed during glycolysis, Indicating that the glycolysis pathway plays a certain role in the response to NaCl stress in M. sieversii.