Identification Of Telomere-related Genes From Old Platycladus Orientalis And Their Involvement In Abiotic Stress Tolerance

Telomeres are closely related to aging,and telomere-related proteins regulate the activity of telomerase,which affects telomere length.However,the mechanism of telomere and its related proteins on regulating plant growth and stress response is unclear.In this study,the leaves from 20-,500-,1 000-and 1 700-year-old individuals of Platycladus orientalis were analyzed by RNA-seq,then the telomere-related genes(PoWHY1,PoKub3,PoTRF1 and PoPOT1)were discovered.Quantitative Real-time PCR(qRT-PCR)was used to study the changes of telomere-related genes under the treatments of NaCl and H2O2 stresses and exogenous abscisic acid(ABA).Based on the full-length cDNA sequences of PoWHY1,PoKub3 and PoTRF1 genes from 500-year-old P.orientalis,the expression of these genes in response to several abiotic stresses were analyzed.The potential mechanism of PoWHY1,PoKub3 and PoTRF1 genes in response to high salt and drought stresses in the transgenic plants of Arabidopsis thaliana were explored by overexpressed these genes,respectively.The main conclusions were as follows:(1)The transcriptome from the pool of the leaves of four different ages of P.orientalis was generated by RNA-seq with Illumina HiSeqTM 2000.A total of 48 044 unigenes were obtained by de novo assembly,with an average length of 896 bp and N50 value of 1 714 bp.Of all unigenes,24 080,8 380,18 483,18 546,20 029,9 877 and 7 783 unigenes were annotated against the NR,NT,SwissProt,PFAM,GO,COG and KEGG databases,respectively.In addition,1 256 SSR loci with 2-6 nucleotide repeat types were identified by using MISA software.Four telomere-related genes(PoWHY1,PoKub3,PoTRF1 and PoPOT1)were screened from the transcriptome.The analysis of qRT-PCR showed that,with the increasing age of P.orientalis,PoWHY1 and PoTRF1 expression first rose and then fell,while PoPOT1 expression was the opposite trend,and PoKub3 expression gradually increased.These indicate that the expression of four telomere-related genes is closely related to age and provide a reference for the study of the function of genes related to the senescence of P.orientalis.(2)Based on the physiological indexes of reactive oxygen metabolism,expression patterns of genes involved in reactive oxygen metabolism(Cu/Zn-SOD,CAT,GR,APX,MDAR and GST)and telomere(WHY1,Kub3,TRF1 and POT1)were studied in P.orientalis seedlings under the treatments of NaCl and H2O2 stresses and exogenous ABA.The results showed that,Na Cl stress caused the increasing expression levels of genes related to reactive oxygen metabolism and telomere except for POT1.However,the application of ABA altered the effect of NaCl stress on the expression levels of these genes.H2O2 stress caused the changes of expression levels of genes related to reactive oxygen metabolism and telomere,but the presence of 0.5 ?mol/L ABA at 48 h could further enhance their expression.These suggest that telomere-related genes may be closely related to the stress response of P.orientalis.(3)A 1 295-bp full-length cDNA of PoWHY1 was cloned using the RACE method from the leaves of 500-year-old P.orientalis,and bioinformatics was analyzed,together with the full-length genes of PoKub3 and PoTRF1.The results showed that,the cDNA of PoWHY1 contained an open reading frame(ORF)of 723 bp,and the encoded protein(240 amino acids)had a molecular mass of 26.47 kDa and a pI of 9.19,mainly composed of random coil with a Whirly domain,and had a high homology with Sesamum indicum.PoKub3 protein had a molecular mass of 20.92 kDa and a pI of 6.05,mainly composed of alpha helix and random coil with the Peptidase_M76 domain,and had high homologies with A.thaliana,Arabidopsis lyrata,Populus trichocarpa and Theobroma cacao.PoTRF1 protein had a molecular mass of 34.06 kDa and a pI of 9.07,contained SANT_TRF and H15 domain,and had a high homology with Amborella trichopoda.qRT-PCR result demonstrated that the treatments of NaCl,heat,PEG6000,UV-C and ABA led to the different changes of PoWHY1,PoKub3 and PoTRF1 expression levels in leaves and roots.(4)The coding sequences of PoWHY1,PoKub3 and PoTRF1 were respectively cloned into pCAMBIA1304 under the control of the 35 S promoter,and then transformed into A.thaliana mediated by Agrobacterium to test their functions in abiotic stresses of salinity and drought.The results showed that,compared with wild-type(WT)and vector control(VC)plants,overexpression of PoWHY1 increased the germination rate of Arabidopsis seeds under salt stress,decreased MDA levels and improved proline contents,accompanied with the lower expression levels of JAZ1,LOX1,ABI1 and ABI2,and the higher expression levels of RAB18,APX1,GSTF6 and DREB2 A in A.thaliana under salt stress,which provided the strong evidence that salt tolerance was improved in the overexpressed PoWHY1 plants.In addition,overexpression of PoWHY1 in A.thaliana increased the tolerance to drought stress.Similarly,overexpression of PoKub3 improved the tolerance to salt and drought stresses in A.thaliana and enhanced ABA sensitivity.The above results indicate that the higher expression levels of PoWHY1 and PoKub3 in the leaves of P.orientalis probably contribute to their adaptability to the adverse environments.However,PoTRF1 overexpression reduced the germination rate of Arabidopsis seeds under salt stress,while it had not yet been able to demonstrate the resistance to salt stress,which suggests that the PoTRF1 in plants may have different roles at the different stages.In summary,the mechanism of three telomere-related genes from old P.orientalis on resisting stresses was studied in the present work,focusing on the theme that how telomere-related proteins adjust the dynamic balance of old trees telomeres to affect their longevity mechanism,which would provide the basis for the study of the function of telomere-related genes in plant resistance and play a positive role in promoting the study of telomeres in old trees.