The Transcription Response Of Pyrus Betulaefolia To Salt Stress And The Research On Physiological Regulation Under Salt Treatment

Pyrus spp.is one of the main fruit trees in China.In China,the degree of soil salinization is aggravated year by year.This condition severely limits the development of pear industry in our country.Pyrus betulifolia Bge.is a deciduous tree belonging to Rosaceae that is widely used as a pea rootstock in China.This species can adapt to most types of soil,making it popular in the orchards.However,little is known about its molecular and physiology mechanisms for salt stress tolerance.To explore the molecular response and physiological regulation mechanism of P.betulifolia to salt stress,the transcriptome analysis was preformed between the salt treatment and the control of the leaves of P.betulifolia to find different expression genes.Then,two calcineurin B-like protein(CBLs)genes were isolated from salt-treated leaves of this species.Subsequently,their sequence features,expression characteristics and tolerance functions under different stresses were compared.Finally,the effects on main physiological indexes were detected after exogenous substances(CaCl2 or riboflavin)application in hydroponic when the seedlings were exposed to salt stress.And the possibility of the alleviating role of CaCl2 or riboflavin for the salt toxicity of P.betulaefolia was discussed.The main results were shown as follows:(1)The transcriptome sequencing(RNA-Seq)technology was used to analyze the differentially expressed genes between the NaCl-treated leaves and the control leaves of P.betulaefolia seedlings.The results displayed that 92 differentially expressed genes were found in two kinds of leaves.Among them,82 genes belonged to down-regulated groups and 10 genes were divided into up-regulated groups,respectively.The GO enrichment analysis of over 92 genes indicated that 18,7 and 5 DEGs were classed as the biological process,molecular function and cellular component,respectively,which were also among the 30 most enrichment GO terms.KEGG enrichment analysis found that the number of significant difference pathways was 17,which consisted of 15 up-regulated pathways and 2 down-regulated pathways.(2)The open-reading frame(ORF)of cDNA and DNA sequences was isolated by EST sequences splice,RT-PCR and PCR techniques step by step.Bioinformatics methods were employed in the analysis of nucleotide sequences and their deduced protein structure.Semi-quantitative RT-PCR was used to reveal the expression modes of the above genes via cross-introns primers and prokaryotic expression was applied in the research of stress tolerance of E.coli cells mediated by the above genes apart.The results showed:The cDNAs of PbCBLl and PbCBL7 were 642 bp and 639 bp in length,respectively,and encoded 213 and 212 amino acids.The DNAs of PbCBL1 and PbCBL7 were 2969 and 1788 bp in length,separately,and both consisted of 8 exons and 7 introns.The mRNA abundance of either of these genes was induced in leaves and roots by the stress of salt,drought,osmotic and ABA dramatically.The tolerance of E.coli BL21(DE3)cells conferred PbCBL1 or PbCBL7 was enhanced under either salt stress or osmotic stress compared to the control cells.It was found that the stress tolerance ability of E.coli mediated by PbCBL1 was stronger than that of PbCBL1.(3)The effect of exogenous different concentration CaCl2 or riboflavin on the antioxidant system was analyzed when the seedlings of P.betulaefolia under salt stress.The results showed that the activity of SOD was decreased,the activity of POD,CAT,GR,GSH-Px and APX was enhanced,the contents of GSH and AsA were reduced,and the accumulation of O2·-,H2O2 and MDA was increased in leaves after plants growing with 200 mmol·L-1 NaCl for 3 days.At the same time,CaCl2 or riboflavin was added into the Hoagland solution together with NaCl.Then,the activity of SOD,POD,CAT,GR,GSH-Px and APX was improved,the contents of GSH and AsA were increased,the production of O2·-and H2O2 was decreased,and the degree of lipid peroxidation was falling down.Considering the various indexes,the effect of 10 μmol·L-1 CaCl2 or riboflavin was the most significant.