Functions Of Retrotransposon OsRTPP1 And Its Mechanism To Dought Stress Toleranse In Rice

Transposons and retrotransposons widely exist in the genomes of both plants and animals.They are a kind of special DNA fragments that can randomly transfer from one site to another in genomes by self-replicating or self-splicing and therefore play a crucial role in genome evolution,leading to structure modification,composition variation,function change and expression regulation of the genomes.Recently,it was found that transposons and retrotransposons also participate in stress tolerance in plants,but the functional mechanisms are largely unknown.Asian cultivated rice（Oryza sativa）which was domesticated in 8,000-10,000 years ago is one of the four major food crops.It belongs to the last evolutionary AA genome.Due to exist of transposons and retrotransposons,natural selection,artificial domestication and other reasons,rice species have produced rich genetic diversity during the long evolutionary differentiation process.35%of rice genome sequences are transposons,including 19.4%retrotransposons.Most of the transposons in plant genome are stationary,but certain regulatory elements can be activated under specific biological processes or abiotic stresses.Previously,we found that a SINE type retrotransposon gene,OsRTPP1 was greatly up-regulated in a rice NADPH oxidase gene Os NOX2 knockout mutant.Os NOX2 is a major producer of rice H₂O₂ and its expression was significantly upregulated by drought,indicating that OsRTPP1 might play an important role in ROS metabolism or signaling during stress response of rice.In the present study,we cloned OsRTPP1 gene and its functions were fully investigated by employing a series of molecular and cellular research methods.The major results obtained are as follows:（1）OsRTPP1 gene was cloned in rice and its expression profiles were investigated:Based on the 741-bp long sequence from MSU database,the full length cDNA of OsRTPP1gene was cloned.Then bioinformatics and expression analysis for the gene were conducted.According to an insertion of a fragment like member r3024 of p SINE-3 family found in AA genome of rice which derived from p SINE-1 in chromosome 11 at the 3’end of OsRTPP1,we speculated that OsRTPP1 is a new gene produced by the insertion of a sequence similar to r3024 in rice chromosome 11,encodes a protein located in the nucleus,the molecular weight is 28.1 k Da and the isoelectric point is 4.13.OsRTPP1 has the highest similarity to the DUF1668 domain of the DUF1668 protein superfamily.The DUF1668 superfamily exists only in the rice genus,and its members are predicted to be functional unknown proteins.OsRTPP1 was found to be expressed in various tissues of rice at every growth stage with the highest transcripts in roots.OsRTPP1 responds to many abiotic stresses such as drought,high salt,high temperature,low temperature,oxidative stress and also hormone treatments such as ABA,SA and GA.The expression level of OsRTPP1 was significantly up-regulated under high temperature stress,while under oxidative stress caused by MV treatment,its expression was greatly down-regulated.（2）The transgenic plants of both the OsRTPP1-overexpressed and knocked down（RNAi）were generated:The OsRTPP1-overexpressing and knocked down（RNAi）transgenic lines were generated using Agrobacterium-mediated transformation with cv.Nipponbare-derived calli.The T₂ generation was used for further analysis.Seed germination experiments showed the increase of numbers of root hairs of overexpression plants and the decrease of RNAi plants.We found that the RNAi transgenic plants of OsRTPP1 exhibited developmental retardation,lower plant height,more tillers,and lower seed-seting rate with higher thousand seed weight whereas the overexpression transgenic lines showed lower thousand seed weight.In addition,the RNAi plants had lower drought tolerance whereas the overexpression lines are opposite in drought tolerance.The overexpression transgenic plants also showed higher survival rate under drought stress treatment as compared to the wide type（WT）and RNAi plants.The contents of H₂O₂,enzyme activity of APX、CAT、POD and SOD were lower in overexpression lines.Under artificial drought condition,contents of H₂O₂ were increated in both WT and overexpression lines,but it was more significantly in WT than in the overexpression lines.When drought stress ended,the contents of H₂O₂ decreased more obvious in the overexpression lines than in WT.ABA treatment,H₂O₂treatment and artificial drought treatment demonstrated that the overexpression lines of OsRTPP1 were more capable to keep the low H₂O₂level,so that the overexpression lines showed increased tolerance to stress.（3）Screening of proteins interacted with OsRTPP1 and the verification:For getting further insights in OsRTPP1 functions,proteins which might interact with OsRTPP1 were screened with the Yeast Two-hybrid System and total 14 proteins were found to be potentially interacted with OsRTPP1 such as CATs and GS1 protein.Further studies showed that OsRTPP1 can form homodimers in cells and it can interact with the three members of catalase（CAT）family OsCATA,OsCATB and OsCATC.Except the domain of inversely parallel eight-strandβbarrel structure,all the other domains of OsCATB can interact with OsRTPP1.These results suggest that OsRTPP1 regulates the function of OsCATs and therefore participates in ROS–mediated stress tolerance of rice.In addition,OsRTPP1 also can interact with a cytosol located glutamine synthetase OsGS1.（4）A gene networks of OsRTPP1 regulation was established by using a RNA-Seq method:As compared with WT,365 genes are upregulated in the OsRTPP1-overexpressing transgenic lines whereas,611 genes are downregulated.These differential genes are mainly belonging to protein kinases,transporters and transferases,transposons and retrotransposons,stress-related transcription factors,hormone response proteins,stress related proteins,etc,including rice respiratory burst oxidase homologs.However,446 genes are upregulated in the OsRTPP1-knocked down transgenic lines while 225 genes are downregulated as compared to WT,which are mainly belonging to receptor protein kinase,transporters and transferases,transposons and retrotransposons,transcription factors,hormone response proteins,stress related proteins,etc,including atalases.43 genes which significantly changed in expression in both type transgenic plants were selected for RT-PCR experiments to verify their expression profiles obtained in RNA-Seq analysis.These results suggest that a complicated network might be involved in the regulation of OsRTPP1 to rice development and stress responses.In summary,in this study a novel SINE-like retrotransposon gene was cloned and its functions in rice development and stress response were clarified.OsRTPP1 is a nuclear located protein and it can interact with OsCATs.It plays an important role in rice development regulation and drought stress tolerance,probably by regulating ROS signaling.