Identification And Analysis Of The TLP Family In Apple And The Role Of MdTLP7 In Abiotic Stress Response

Tubby-like proteins?TLPs?,which have a highly conserved tubby domain,have been found to be associated with some animal-specific characteristics,for example,obesity,blindness and deafness.In the plant,encoding TLP homologous genes also were identified,some members have been reported response biotic and abiotic stress.In this study,the bioinformatics method is used to identify TLP gene families in apple genome and to systematically analyze their sequence features,promoter regions and stress response.In addition,the resistance function of MdTLP7 was further investigated in transgenic Arabidopsis.This study provided more bases for the function research of the TLP family in response in stress.The main results were showed as following:?1?9 MdTLPs were identified in apple genome.Two strategies,BLASTp and Hidden Markov Model?HMM?profile were used to find candidate TLP sequence in apple genome.And the Interpro and SMART database are used to screen candidate TLP sequence.As a result,9 TLP coding gene of apple genome are identified and named as MdTLP1-9.The MdTLPs were mapped to six different chromosomes.When combined with the TLPs from other plant species,the TLPs were divided into three groups?group A,B,and C?,group A were further divided into the four subgroups A1–A4.7 MdTLPs distributed in 3 subgroups in group A and 2 MdTLPs are in group B.Gene structure analysis proform that the MdTLPs in the same group shared similar distribution characteristics of exons,but the characteristics were different between the groups.?2?Analysis of protein domain and motif characteristics of MdTLPs family.In addition to the tubby domain in C-terminal of MdTLPs,6 MdTLPs in the group A contain an additional F-box domain.Some studies have reported that F-box protein is related to plant response to abiotic stress.Further analysis of the tubby domain,three types of conserved motifs were identified,Motif 1 and 2,the signature motifs in the confirmed TLPs;Motif 3was concerned for the first time in plant TLP.The homology modeling of MdTLPs result shows that MdTLPs'tubby domain are the same as the animal TLP which present as a12-stranded?barrel with acentral hydrophobic?helix.Four MdTLPs members formed typical?barrel and?helix,some members without the central?helix,neither the central?helix or incomplete?barrel.?3?MdTTLPs were involved in abiotic stress response.Promoter analysis of MdTLPs showed that there are some stress response elements and transcription factor binding elements,including ABRE,DRE,LTRE,MYB and MYC.Further investigate the relationship between the MdTLPs and the stress response by qRT-PCR,the expression of MdTLPs in a variety of stress conditions and exogenous ABA was detected.Most MdTLPs were induced to varying degrees in leaves and roots under abiotic stresses.During PEG treatment,some members were up-regulated by approximately 35-fold compared with the control condition in levels,and were up-regulated by about 30 fold in roots.Under cold stress,almost all MdTLPs in leaves and in roots shows significantly up-regulated transcript levels.In response to H₂O₂ or ABA treatment,the response of each gene in roots and leaves was different.Five members?MdTLP1-5?are significantly up-regulated in leaves,while in roots,except MdTLP4,the other 4 members did not significantly increase,and the other 3 members?MdTLP6,MdTLP8and MdTLP9?show a significant increase.Expression profiling revealed that the MdTLP genes were expressed in multiple organs and were abundant in roots,stems,and leaves but low in flowers.?4?Overexpression of MdTLP7 in Arabidopsis enhances the plant tolerance to stress.Construction plant expression vectors of MdTLP7 full-length gene(OE-_FL)and tubby domain(OE-_Tub),transforming them into Arabidopsis and screening for homozymous transgenic plants.The OE-_FL,OE-_Tub and wild type Arabidopsis?WT?seedlings are treated with high salt,drought and cold stress respectively.In high salt and drought treatments,compared with WT,the OE-_FL and OE-_Tub lines significantly enhanced plant salt tolerance and drought resistance.Under cold and high salt stresses,the growth of OE-_FL plants was significantly better than WT.It is further demonstrated that the MdTLP7 can enhance the plants osmotic stress both the full-length MdTLP7 and the tubby domain,and F-box domain of MdTLP7may play an important role in the response to cold stress.?5?The basic amino acid site?K190XR192?of MdTLP7.plays an important role in the anti stress function.The amino acid sites?KXR?directly affect the function of TLP in animals,and the corresponding sites of MdTLP7 are K190XR192.Using the constructed MdTLP7prokaryotic expression plasmid,the K190XR192 amino acid sequence encoding were mutated,and then transformed into E.coli.Under salt stress,the results showed that compared with the the empty plasmid,the MdTLP7 recombinant strain significantly enhanced the E.coli resistance.The MdTLP7 mutant strain reduces the E.coli resistance,especially,the mutants MdTLP7_K190E,MdTLP7_R192A and MdTLP7_K190ER192E190ER192E significantly reduced the resistance function of the MdTLP7.These results indicate that the K190XR192 amino acids play an important role in the anti stress function of MdTLP7.