Cloning And Functional Study Of AkRALFs Family Genes In Amorphophallus Konjac

Amorphophallus is rich in glucomannan and has become a special economic crop in China.It is planted in the southwestern region in large areas.A.konjac and A.albus of main cultivated Amorphophallus.Glucomannan,as a small amount of polysaccharide in most plants,is a structural component of cell wall.However,glucomannan is synthesized in large amounts and transported into the vacuoles of glucomannan idioblasts in konjac.Amorphophallus provides an ideal model system for studying polysaccharide glucomannan synthesis and regulation in cell wall.These mechanisms are unknown,how the cell wall senses the glucomannan content of the polysaccharide member,which signals direct glucomannan synthesis in large amounts and why it is transported into the vacuole in a specific type of cells called glucomannan idioblasts in Amorphophallus.Plant polypeptide signals(peptide hormones)are important receptors that mediate intercellular messengers and regulate plant defense and development processes.Rapid Alkalinization Factor(RALF)is a new type of protein family.As a key regulator of cell growth,it cooperates with various receptors at various stages of plant growth and development,participating in cell integrity and regulating cell wall expansion.In this study,three AkRALFs family genes were were cloned from the corm cDNA of A.konjac,and their bioinformatics and gene function were analyzed.The results are as follows:1.Cloning and bioinformatics analysis of AkRALFs geneThe AkRALF1,AkRALF4 and AkRALF19 genes in the RALF family were successfully cloned from Amorphophallus konjac.The full length of the AkRALF1 CDS region is 366 bp and encodes 121 amino acids.The full length of the AkRALF19 CDS region is 405 bp and encodes 134 amino acids.The full length of the AkRALF4 CDS region is 390 bp and encodes 129 amino acids.The analysis of amino acid sequence structure all contained the N-terminal signal peptide.The proteins encoded by the three genes all contained four highly conserved cysteine residues,YISY motifs and a pair of arginine residues(Arg-Arg;RR)conserved binary site structure.The gene structure analysis showed that no introns conformed to the structural characteristics of the plant RALF family.Subcellular localization is predicted to be localized on the plasma membrane.Analysis of the biological evolutionary tree constructed with the Arabidopsis RALF family shows that AkRALF1 is similar to the AtRALF1 gene,AkRALF4 is near the AtRALF4 gene,and AkRALF19 is close to the AtRALF19 gene.2.Analysis of AkRALFs gene expression by q RT-PCRThe expression patterns of AkRALF1,AkRALF4 and AkRALF19 genes were analyzed by quantitative real-time PCR technology,and the results showed that the overall expression of AkRALF4 in all parts showed a downward trend;the expression of AkRALF1 in the corms showed a downward trend,but it showed an "inverted S" type with the whole;AkRALF19 expressed in the roots showed an "inverted S" type,but the overall expression showed a downward trend.The overall expression levels of the three genes in the corm decay and new corm formation stages were higher than those in other stages and all were highest in petioles;the three genes in the rapid growth period of leaves and new corms are still highly expressed in petioles.The second expression site of AkRALF4 is the corm,AkRALF1 is the root,and AkRALF19 is the leaf.When the new corms continued to expand and fill stages,the highest expression of AkRALF 1and AkRALF19 was the corms,and AkRALF1 was the petiole;the growth of the above-ground part of the corm during the dormant period fell sharply and even stagnated.Atthis time,all three genes were at low expression levels,and AkRALF4 was hardly expressed in the leaves.All three genes were highly expressed during the initial rapid growth cycle,especially during the rapid growth of petioles.3.Prokaryotic expression analysis of AkRALFs geneAkRALF4,AkRALF1 and AkRALF19 genes to construct a prokaryotic expression vector.Prokaryotic expression analysis revealed that all three genes could induce the expression of the fusion protein in E.coli BL21(DE3).Among them,AkRALF4 and AkRALF1 could purify the target protein,and AkRALF1 protein could inhibit the growth of Arabidopsis roots.4.Isolation and characterization of AkRALFs promoterThe length of AkRALF1 pro was 1092 bp,AkRALF4 pro was 941 bp and AkRALF19 pro was 1752 bp in length by FPNI-PCR.In addition to the common characteristic promoter elements such as TATA-box,CAAT-box,all of the promoters contain cis-acting regulatory elements that participate in light response,substitution acid reaction,Me JA response,and MYB binding sites involved in remission induction,etc.Among them,AkRALF19 pro has cis-acting elements that participate in low-temperature response.The cloned promoter fragment and p BI121 plasmid were ligated into a GUS fusion expression vector,and transformed into Arabidopsis thaliana for GUS staining.The results show that AkRALF4 pro,AkRALF1 pro and AkRALF19 pro can drive the expression of downstream reporter genes.5.Heterologous overexpression of AkRALFs gene in ArabidopsisAkRALFs gene was overexpressed in Arabidopsis,and the homozygous lines OE-AkRALF1-1～OE-AkRALF1-5,OE-AkRALF4-1～OE-AkRALF4-5,OE-AkRALF19-1～ OE-AkRALF19-6 were obtained.Semi quantitative RT-PCR was used to evaluate the expression of each strain,and OE-AkRALF1-4,OE-AkRALF4-3,OE-AkRALF19-6 were selected for subsequent experiments.The results showed that the OE-AkRALF1-4 plant was short,the number of lateral roots was decreased and the sensitivity to salt stress was increased,which could counteract the effect of exogenous Brassinolide on the plant and was more sensitive to abscisic acid.The lateral roots of OE-AkRALF4-3 plants increased;the rosette leaves of some OE-AkRALF19 plants were large and dark green,and bolting time was delayed;both of them could respond to exogenous brassinolide treatment,but could not respond to salt stress.