Analysis Of The Characteristics Of Autophagy-related Genes In Physcomitrella Patens

Autophagy is a very conserved intracellular material degradation mechanism in eukaryotes.Usually refers to the process in the signal-induced cells by the engulfinent of cytoplasmic composition in de novo form a double-membrane vesicles called autophagosomes which subsequently deliver their cargo to the lysosome/vacuole for degradation.In the past study,although autophagy has been shown to play an important role in plant development and response to various stresses in several plants,the molecular mechanisms,regulation pathways and potential roles of autophagy in plants are still largely unknown.Physcomitrella patens is a bryophyte which has a relatively simple structure and a very special status in the evolutionary of the transition from aquatic to terrestrial plants.It has developed into a model plant for studying plant development and trait evolution.In this study,based on the whole genome sequence of P.patens,using bioinformatics and modern molecular biology research methods,the structural characteristics and expression patterns of autophagy related genes(ATG)were comprehensive investigated,so as to provide basic data for understanding the evolution and biological functions of autophagy related genes in plant.The main results of this study are as follows:1.The PpATG genes contain 38 homologues could be classified into 19 ATG classes,among which number of members was uneven.Among them,PpATG2,PpATG3,PpATG5,PpATG7,PpATG10,PpATG12,PpATG16,PpATG20,PpTOR and PpVPS34 had only a single member each,PpATG4,PpATG6,PpATG9 and PpVPS15 each contained two members,PpATG1,PpATG18 and PpVTI12 contained 3,4 and 5 members respectively,whereas PpATG8 contains 6 members.The results of chromosome localization and gene replication analysis showed that there were 8 pairs of segmental replication events and 2 pairs of tandem replication events which happened in Chll,Chr2,Chr7,Chr9,Chrll,Chr14,Chr17,Chr18,Chrl9,Chr21,these events indicating that gene replication plays an important role in the amplification and evolution of ATG genes.Sequence alignment and phylogenetic analysis with C.reinhardtii and A.thaliana homologous proteins showed that the PpATG protein sequences exhibit higher homology with AtATG protein sequences than CreATG protein sequences.Similarly,by subcellular localization prediction,it was found that the cellular distribution of PpATG proteins was more similar to AtATG proteins than CreATG proteins.The comparison of exon/intron gene structure revealed that the intron number and exon length of PpATG genes were higher conserved with AtATG genes than CreATG genes.These results suggest that autophagy-related genes undergo a conservative function and structural differentiation before and after plant landing.2.The expression differences of PpATGs in two different growth stages under four stresses was investigated by RT-qPCR at the temporal and spatial levels.In the protonema stage,there were 8,7,20,and 21 ATG genes were up-regulated or down-regulated under N-free,high salt,dark and drought stress,respectively,while 9 genes did not respond to any of these stresses.In the gametophyte,there were 7,20,14,and 6 ATG genes were up-regulated or down-regulated under N-free,high salt,dark and drought stress,respectively,while 10 genes did not respond to any of these stresses.Only PpATG5 was found did not respond to any stress at both developmental stages.The differences in expression of these genes indicate that not only different classes of ATG genes have different temporal and spatial expression patterns,but also the members of the same class also have differences in temporal and spatial expression patterns,which may play a multiple role in the response of coping with different stress conditions at different stages of development of P.patens.3.We found that the autophagy inducer-rapamycin-treated plants showed the increase of yellowing and withered symptoms comparing with normal drought plants,indicating that autophagy may be related to drought capacity of P.patens.The fluorescence signal was observed in the protoplasts of P.patens by constructing the plant transient expression vector of the ubiquitin-like conjugate enzyme PpATG3 gene,which confirmed the occurrence of autophagy in cytoplasm in P.patens.Through the homologous recombination knockout experiment by constructing and transferring the homologous recombination knockdown vector of the PpATG3 gene,we screened amutant strain ATG3-1 of PpATG3 deletion,the mutant showed a very serious yellowing and senescence phenomenon under normal culture conditions after one month,indicating that the autophagy is related to the nutrient stress-induced senescence process in P.patens.The results of this study not only expanded the understanding of the roles of autophagy in the stress-resistance process of P.patens,but also provided a new direction and possibility for the further study of the roles and molecular mechanismsof autophagy in the process of adaption to stresses during plant evolution.