Identification And Expression Analysis Of RALF Genes In Mulberry

Plants live in a very complex living environment facing various biological stress(insects, fungi, bacteria, and viruses) and abiotic stress. Plant peptide hormones are important intercellular signaling molecules that regulate plant defense and development. Rapid alkalinization factor(rapid alkalinization factor, RALF) widely exists in plants as plant peptide hormone. RALF causes the extracellular p H rapidly increasing and participates in the regulation of plant development. The changes of extracellular p H are important responses of plants to regulate cell growth and environmental stimuli involved in serval physiological processes including ion absorption, solute transport, and cell wall growth. Transient changes in and extracellular H+ concentration associate with the polarization and depolarization of cell membrane in order to rapid response to environmental stimuli. These environmental stimulations include changes in turgor pressure and gravity, the pathogen infection, and abiotic stresses.Mulberry is a perennial woody plant. Mulberry leaves can not only provide food for silkworm, but also have important edible value, medicinal and ecological protection functions. For example, the studies of modern pharmacology indicated that mulberry leaf is abundant in polyphenols, flavonoids, polysaccharides, a variety of vitamins and other bioactive substances, which can reduce the blood sugar, delay aging, have antimicrobial and antioxidant effect. As an excellent ecological and economic tree species, mulberry has notable resistance to high temperature, drought, saline-alkali soil, and cold damage. With the completion of the mulberry genome sequencing, the research on the resistance of mulberry has been developed from the selection of germplasm resources, physiological and biochemical development to the molecular level. In the present study, using bioinformatics methods, 19 RALF related genes were identified from Sichuan mulberry(Morus notabilis) genome database. The relationship between the gene structure, evolution, conserved amino acid sequences, upstream transcriptional regulatory sites were also investigated. Meanwhile, the expression of mulberry RALF genes in root, stem and leaf were studied by q RT-PCR. In addition, the expression pattern of mulberry RALF genes after treated by mechanical damage, silkworm biting, and methyl jasmonate have been detected. The results of this study are as follows:Identification and bioinformatics analysis of mulberry RALF genesUsing the method of bioinformatics, 19 RALF genes were identified in the genome of M. notabilis, which were named Mn RALF1 ~ 19. The length of amino acid residues ranged from 74 aa to 164 aa with the molecular weight ranged from 7 to 19 KD. Except for Mn RALF4, Mn RALF7, Mn RALF10, Mn RALF11, and Mn RALF12, other mulberry RALF proteins have predicted signal peptides. The prediction of subcellular localization revealed that Mn RALF4 mainly distributed in the chloroplast, Mn RALF12 in the cytoplasm, and other RALF members are mainly distributed in the extracellular matrix. Gene structure analysis showed that the number and location of mulberry RALF genes were consistent with the results of the corresponding genes reported in other plants. Multiple sequence alignment analysis of RALF amino acids in different plants showed that the C-terminal sequence was highly conserved. RALF gene mainly contains three conserved motifs including Motif1, Motif2, and Motif3. It was predicted that the C-terminal of the mulberry precursor RALF protein mainly was alpha helix and a small amount of beta folding structure formed between the conservative amino acids. Since the conservative region rich in charged amino acids may affect the tertiary structure of mature protein and the C-terminal extension regions of precursor protein were changeable, it suggested that the conserved regions may and related to the interaction of peptides with different receptor molecules.Seventy RALF protein sequences from five species including mulberry were used for phylogenetic tree construction. The results showed that the phylogenetic tree was divided into A, B, C, and D four groups. The mulberry RALF proteins were in the fourth group. The upstream transcription regulatory element analysis indicted that these elements were mainly divided into three categories: light responsive, stress response, and hormone response, suggesting that mulberry RALF genes can response to stress and hormone involved in different aspects of mulberry defense including pest damage, fungal infection, drought resistance and may have important functions.Cloning and expression analysis of RALF genes in mulberry tissuesSix mulberry RALF genes were cloned. The results showed that these six genes in two mulberry germplasm resources(M. notabilis and M. atropurpurea) were successfully cloned with the predicted sizes. By sequence analysis, it was found that there were only several SNPs in the sequences between related two genes from different germplasm resources. Specific primers were designed to detect the expression of 19 mulberry RALF genes in M. atropurpurea in root, stem, and leaf using q RT-PCR. The results indicated that seven RALF genes expressed relatively high in root and stem, whereas five RALF genes were abundantlyexpression in leaves. Differences in patterns of gene expression suggested that RALF genes in different tissues of M. atropurpurea may have different physiological roles.Analysis of expression patterns of mulberry RALF genes under different stress treatmentsIn order to study the expression patterns of mulberry RALF genes in response to stresses, we extracted the RNAs from the M. atropurpurea leaf tissues after being treated by mechanical injury, silkworm biting, and methyl jasmonate for 0h, 0.5h, 1H, 6h, and 12 h. The expressions of 19 mulberry RALF genes were detected using q RT-PCR technique. The results revealed the 19 mulberry RALF genes in response to trauma reaction and chemical induced stress, suggesting they may play important physiological roles. Our result provided basic information of mulberry RALF genes.