Cloning The Key Genes Ea-δ-oat And Ea-p5cs Involved In Proline Metabolism Of Erianthus Arundinaceum And Their Function Analysis

Drought is one major abiotic factor that adversely affects the crop production and distribution and account for the reduction in the yields of crops.At present,to improve the crop's quality of water stress resistant ability trough breeding anti-water stress plants or cultivars is one of the main methods to solve this problem.Proline is an important osmoprotectant.Proline seems to have diverse roles under stress conditions,such as stabilization of proteins,membranes and subcellular structures,and protecting cellular functions by scavenging reactive oxygen species.Understanding the molecular responses of proline metabolism under different abiotic stresses is of much importance as they give hope for genetically modifying crops to cope with these stresses in a better way.Erianthus arundinaceum,a close genera of sugarcane(Saccharum L.),posseses various desirable characters,especially excellent vigor and good adaptability to environmental stresses, and has always been the most important germplasm being used to broaden the genetic base of sugarcane.In this study,we use reverse-transcript-polymerase-chain-reaction(RT-PCR) and rapid amplification of cDNA end(RACE) technologies to clone the key genes during proline biosynthesis.Using real-time PCR technology,the expression of key genes obtained from Erianthus arundinaceum treated with polyethylene glycol(PEG),NaCl and abscisic acid(ABA) was analyzed.By our study,the Ea-δ-OAT(EU113256) gene was 1720bp in full length,encoding 454 amino acid residues.The blast-p results of the studied Ea-δ-OAT gene with that from mammal, higher plant and microorganism show that Ea-δ-OAT gene shares the highest homology(87%) with sugarcane,the lower homology(70%) with other higher plants and the lowest homology(60%) with animals.Same as that from sugarcane,No N-terminal mitochondrial transit peptide(MTP) was found in the amino acid sequence encoded by Ea-δ-OAT gene.Complete domain of OAT,rocD, was included in Ea-δ-OAT gene.The obtained Ea-P5CS gene was 2563 bp in full length,including 5'UTR 103 bp,one open reading frame(ORF) 2148 bp encoding 716 amino acids,and 3'UTR 312 bp.There is one set of AATAAA at the upstream of poly(A) site.The blast-p result of the encoded amino acid sequence shows that the studied sequence encoded by Ea-P5CS gene shares the highest homology(97%) with sugarcane,lower homology(87%) with Oryza.Sativa.As bi-functional enzyme,P5CS contains a glu-5-kinase(G5K) domain-Pro Bin the N end and a glutamyl 5-phosphate reductase(G5PR) domain-Pro A in the C end.The study about the relationship of the two pathways of Proline metabolism under different treatments shows that 1) With the treatment of 30%PEG,Glutamic acid pathway and Ornithine pathway both work at the first stage during the course of the accumulation of Proline,but at the later stage,Glutamic acid pathway plays the senior part gradually.2) Under the treatment of 250mMNaCl,in the first 6hours,the accumulation of Proline is regulated by Glutamic acid pathway and thereafter be mainly regulated by Ornithine pathway.3) With 10mg/L ABA,during the course of Proline in Erianthus arundinaceum,Glutamic acid pathway and Ornithine pathway work together in the former phase,but the Ornithine pathway plays the senior part in the later phase.4) There are also two genes coding P5CS in E.arundinaceus,one ABA-dependence,the other non-ABA-dependence.The Ea-P5CS gene is one non-ABA-dependence in this study.