Isolation And Function Research Of PbCIPK1 And PbNHXs Genes In Pyrus Betulaefolia

Pyrus spp.is one of the most important fruit trees in China that is a genus of Pyrus and belongs to the subfamily of Rosaceae,which has a long history of cultivation,has important economic value and edible value.As the main rootstock of Pear,Pyrus betulaefolia Bunge is resistant to salt-alkali,cold,waterlogging,drought and so on,it is strong adaptability widely used in pear production.In a large environment where the degree of salinization is increasing,the resistance of P.betulaefolia is very important.In order to explore the response mechanism of P.betulaefolia to salt stress,In this study,sixteen Na+/H+ antiporter proteins were identified from P.betulaefolia.Their gene structure,sequence characteristics,expression characteristics and ion content were studied.The function of two Na+/H+antiporter genes in salt stress response was investigated.The main results of this paper are as follows:(1)CIPK(CBL-interacting protein kinases)transcription factors are widely involved in plant growth and development,as well as biological and abiotic stresses.It is important to clone and identify the CIPK genes for the research on salt stress response and thus salt tolerance improvement in pyrus.In this study,a special ragment,named PbCIPK1 was selected for further research,and the sequence characteristics of this gene and its expression patterns under salt treatment was analyzed using bioinformatics and quantitative RT-PCR analysis.The PbCIPK1 gene was 1368 bp in length,with an open reading frames containing 1365 bp,which encodes 455 amino acids.Real-time PCR analysis revealed that PbCIPK1 transcript was expressed highly in leaf and down-regulated under salt stress.(2)Identification of NHX gene family members of Pyrus betulaefolia with bioinformatics,combining published pear genome and Arabidopsis related data.Using the MEGA7.0 software to sequence alignment and phylogenetic analysis,Pfam,SMART and GSDS software were used to analyze gene structure.The plantlets propagated in Pyrus was cultured in Hoagland inoculum and were treated with 250 mmol·L-1 NaCl and 10%PEG6000 for 0 h,1 h,8 h,16 h,24 h,48 h,72 h and 84 h,respectively.Plant material in Hoagland inoculum without NaCl and PEG6000 was used as control.The total RNA was extracted from roots,stems and leaves of the plantlets with different treatments,and the expression level of the gene family under abiotic stress was analyzed systematically.qRT-PCR was further used to analyse the expression of PbNHXs under PEG6000 and NaCl stress.Determining Na+ content and K+ content in different tissues of NaCl stress by flame graphite furnace atomic absorption spectrometry.Sixteen candidate genes were successfully identified,of which four genes belong to NHX gene family,four genes belong to CHX gene family and eight genes belong to KEA gene family.The longest length NHX protein(PbNHX13)encodes 857 amino acids,the shortest protein(PbNHX12)encodes 401 amino acids,theoretical PI range from 5.14(PbNHX10)to 9.92(PbNHX12).All proteins except PbNHX7 and PbNHX10,contain 8-13 transmembrane structures.In the PbNHX1-PbNHX16 group of genes,the number of introns spans a little bit,PbNHX12 has only one intron,and PbNHX3 has 25 introns.The predicted candidate genes contain Na+/H+exchange domain,PbNHX7 and PbNHX10 contain TrKA_N domain,also.Under NaCl stress,the predicted expression trend of 16 genes in leaves was fluctuating,but all of them were negative regulated after 8 hour.Eleven genes(PbNHX1-4,PbNHX6,PbNHX7,PbNHX11-15)appeared to peak at 8 h and 48 h in the stem,the expression of PbNHX1,2,3,6,11 was the highest at 48 h and the lowest at 1 h;similar expression patterns of PbNHX2,PbNHX3,PbNHX4,PbNHX7,PbNHX10,PbNHX11,PbNHX14,PbNHX15 in leaves,reached the highest expression level at 1 h,decrease to minimum expression at 1 h-8 h.The expression patterns of PbNHX5 and PbNHX12 are very similar,the expression level decreased gradually at 0 h-16 h,it tends to level off after 16 hours.PbNHX1-16 in the roots,it is more vulnerable to the positive regulation of PEG6000 stress compared with NaCl stress.The trend expression of PbNHX7 and PbNHX10 in stem is very similar,take "M"shape,and a low peak at 16 h.The expression of PbNHX5,PbNHX8 and PbNHX9 genes in NaCl treatment was higher than that in PEG6000 treatment at the same time,only in 8 h.Under the treatment of PEG6000,the expression of PbNHX1 in stem increased significantly,and was higher than that under NaCl stress in the same period,and was lower than that under NaCl stress in the same period only at 48 h.In leaves,the expression patterns of PbNHX2-5,PbNHX7,PbNHX9-11 and PbNHX13-15 genes are very similar,there were two peaks of PbNHX2 and PbNHX13 at 8 h and 72 h,two peaks of high expression of the other eight genes occurred at 1 h and 72 h.In roots,PbNHX12、PbNHX13、PbNHX14 and PbNHX15 were negatively regulated at any time after NaCl stress and PEG6000 treatment.Determination of Na+ content and K+content in three tissues of P.betulaefolia under Salt stress(0 h-84 h)showing,K+content in leaves increased slowly,K+ content in stem showed a trend of "W" change and slight downward trend in the root at 24 h,Na+ content in leaves has a inflection point at 16 hour,Na+ content rises linearly after 16 hours,Na+ content in stem is also increasing,but the increase was more moderate after 48 hours,Na+ content in the root increased slowly with a wave-like trend,but it showed a downward trend at the 72 h-84 h.PbNHX1 and PbNHX11,which closely related to the evolution of NHX gene in Arabidopsis thaliana,show a negative regulatory pattern during salt stress.PbNHX10 and PbNHX7,which are closely related to the evolution of Arabidopsis KEA gene,may be involved in K+ transport during salt stress.(3)In order to investigate the response of Na+/H+ antiporter(NHX)to abiotic stress,PbNHX7 and PbNHX11,were selected for further study among the 16 identified genes.The two genes were successfully cloned from Pyrus betulaefolia by designing primers for PCR amplification;Bioinformatics analysis of these two genes;Successful ligation of the target gene into the over-expression vector pRI201-AN-GUS was transformed into Arabidopsis thaliana;The germination rate and root growth of Arabidopsis thaliana seeds treated with different salt stress were studied.The results show that the base number of the open reading frame of PbNHX7 is 2298 bp,which encodes 765 amino acids,and the base number of the open reading frame of PbNHX11 is 1659 bp,which encodes 552 amino acids.Results of PCR and GUS staining showed,PbNHX7 and PbNHX11 have been successfully transferred into Arabidopsis thaliana.After different degrees of salt stress treatment,the germination rate of transgenic Arabidopsis thaliana seeds with PbNHX7 and PbNHX11 was higher than that of wild type Arabidopsis thaliana.The roots of Arabidopsis thaliana seeds transformed with PbNHX7 and PbNHX11 genes were also better than those of wild type Arabidopsis thaliana.These results suggest that PbNHX7 and PbNHX11 genes improve the salt tolerance of Arabidopsis thaliana.