Functional Verification Of Key Genes Of CBL-CIPK Pathway In Pyrus Betulaefolia

Pyrus spp.is one of the main fruit trees in China that is a genus of Genus Pyrusa and belongs to the subfamily of Rosaceae,which has a long history of cultivation,was cultivated widely,has important economic value and edibleness.As one of the main rootstock in China,Pyrus betulaefolia Bunge is resistant to cold,drought,salt,alkali,waterlogging,barren and so on,is widely used in pear production.At present,the degree of soil salinization is becoming increasingly serious and the area of salinization is increasing in China,which has become one of the important factors that restrict the development of the pear industry.In order to discuss the molecular response mechanism of P.betulaefolia to salt stress,in this study,to verify the salt tolerance of the two calcineurin B-like protein genes in response to salt stresses;and the three Na+/H+antiporter genes were cloned and isolated from P.betulaefolia seedlings,their sequence characteristics,expression characteristics,and stress resistance were preliminarily studied.The main results of this paper are as follows:(1)Bioinformatics analysis of PbCBLl and PbCBL2 genes of P.betulaefolia;Identification of transgenic PbCBLl and PbCBL2 N.benthamiana strains by PCR and GUS staining;The expression of CBL-CIPK pathway was studied under 200 mmol·L-1 NaCl salt stress;The phenotypes were identified for transgenic tobacco under the stress of different concentration of NaCl solution;we studied the changes of osmoregulatory substances and anti-oxidation system under the 200 mmol·L-1 NaCl stress of transgenic tobacco.The results showed that the DNAs of PbCBL1 and PbCBL2 were 2969 bp and 1927 bp in length,respectively,the cDNAs of PbCBL1 and PbCBL2 were 642 and 681 bp in length,separately,and they encoded 213 and 226 amino acids,both consisted of 8 exons and 7 introns.PCR and GUS staining results showed that PbCBL1 and PbCBL2 genes have been successfully transferred into Nicotiana benthamiana.Studies on the expression of CBL-CIPK signaling pathway show that the expression of part related genes in the downstream were up-regulated.After 30 d treatment with salt stress,it was obvious that the growth of tobacco plants was weakening in turn;when treated with 300 mmol·L-1 NaCl and 500 mmol·L-1 NaCl,the growth of tobacco plants was significantly inhibited by salt stress;the height,root length,leaf number,weight and fresh dry weight of transgenic lines were significantly higher than those of ck.After 3 days with 200 mmol·L-1 NaCl stress,the activity of superoxide dismutase(SOD),peroxidase(POD)and catalase(CAT)in leaves of transgenic tobacco plants was increased first and then weakened,and osmotic adjustment was observed;The content of soluble sugar,soluble protein and proline of osmoregulation substance were increased significantly,the content of(MDA)was increased significantly,and the content of reactive oxygen species(O2-)was also reduced significantly.reduced the degree of lipid peroxidation and effectively relieve the damage of tobacco leaves caused by salt stress;The content of Na+ and K+were significantly higher than those of ck.These results indicate that the PbCBLl and PbCBL2 genes increase the salt tolerance of tobacco.(2)To isolate a Na+/H+antiporter gene PbNHXl from Pyrus betulaefolia Bunge and analyze its sequence characteristics,expression patterns and transport functions.The cDNA and DNA sequences were isolated using RT-PCR and PCR techniques,and the characteristics of nucleotide sequences and its protein structure was analyzed by bioinformatics.The expression patters of PbNHX1 under the stress were studied by qRT-PCR and the transport function was revealed by the yeast complementation.The cDNAs of PbNHX1 was 1704 bp,and the DNA sequence was 3594 bp,which included 13 exons and 12 introns,encoding a protein containing 567 amino acids.The phylogenetic analysis showed that PbNHX1 is located in the branch of vacuolar Na+/H+antiporter,and was closely related to poplar vacuolar Na+/H+exchange protein PtNX1.3.When treated by 200 mM NaCl,10%w/v PEG6000 or 100 ?M ABA,the transcription level of PbNHX1 continued to rise in the leaves;PbNHX1 transcription abundance increased firstly and then decreased in the roots under the same condition.Moreover,its expression peak appeared at 6 h,3 h or 6 h once the plantlet suffered under 200 mM NaCl,10%it,/v PEG6000 or 100?M ABA,respectively.Transform of PbNHX1 can recover the growth inhibition of NaCl,KC1 and hygromycin B to the nhxl mutant yeast strain AXT3.Furthermore,the accumulation of Na+ and K+ions was increased significantly in the recombinant yeast cells carrying PbNHX1.The results of our experiments show that the PbNHX1 genes belong to the NHXs gene family of P.betulaefolia ad its transcription response to NaCl,osmotic and ABA stresses.PbNHX1 transform can increase the tolerance of the nhxl mutant yeast strain AXT3 to salt stress and partly recover its ion transport capacity and improved accumulation of the Na+and K+ ions.(3)The vacuolar Na+/H+antiporter NHX is a salt tolerance determinant in higher plants.Pyrus betulaefolia,a popular rootstock in Asia,can improve pear salt tolerance through grafting.In this study,two novel NHX genes were identified from the transcriptome database of P.betulaefolia NaCl-treated seedlings.After cloning and sequencing,they were named PbNHX2.1 and PbNHX2.2,respectively.Gene structure similarly,phylogenetic classification,the presence of functional domains conserved in PbNHX2s,and prototypical vacuolar plant Na+/H+antiporters led to them to be classified as the same type of NHX.Similar to AtNHX2,both PbNHX2s are localized to the plant cell tonoplast.The PbNHX2.2 mRNA was the prevalent transcript in seedling roots,stems,and leaves,but was mainly found in the roots.However,lower amounts of PbNHX2.1 mRNA were also present in all three tissues.The seedling steady-state mRNA levels of PbNHX2.1 and PbNHX2.2 increased in a similar way after treatment with NaCl,or polyethylene glycol(PEG),but PbNHX2.2 transcript abundance increased more than PbNHX2.1 in the roots,stems,and leaves.Both of the transcripts accumulated in response to abscisic acid(ABA),which indicated that the salt and osmotic responsiveness of these genes may be ABA-dependent.Finally,the yeast recombined experiments revealed that PbNHX2.1 or 2.2 restored,with different efficacy,the Na+-sensitive phenotype of a yeast mutant,AXT3,by deleting endosomal/vacuolar Na+/H+antiporter ScNHXl.The ion accumulation data suggested that these PbNHX2s proteins,especially PbNHX2.2,facilitated Na+ion compartmentalization and maintained intracellular K+status.Together,these results suggest that PbNHX2.2 was salt tolerance determinant and has a major function in the vacuolar compartmentalization of Na+.