Identification And Functional Analysis Of PmSUSs Gene In Masson Pine(Pinus Massoniana Lamb)

Masson pine（Pinus massoniana Lamb）is one of the most widespread conifers in southern China.As an important timber species,it occupies more than half of the forest stock in southern provinces.Plant sucrose metabolism is not only involved in growth and development,but also responds to a variety of abiotic stress.Therefore,the response role of sucrose metabolism to stress is of great significance for improving plant stress tolerance.So far,there have been few reports on the genes related to sucrose metabolism in masson pine.In the present study,12 sucrose metabolism-related genes were identified and their expressions was carried out in response to abiotic stresses.In addition,the function of the genes were then genetically verified by transformation.The main results are as follows:1.Identification of genes related to sucrose metabolism in masson pineBy analyzing the high and low-yield oleoresin transcriptome sequencing results of Pinus massoniana,we identified 12 sucrose metabolism-related genes in three categories,i.e,SUS,INV,and SPS.And it was verified by expression analysis that its expression level was basically the same as that of the transcriptome.2.The sucrose synthase gene was cloned from masson pineThe full-length ORFs of two masson pine sucrose synthase genes,named PmSUS1and PmSUS2,were cloned,and their ORF sequences were 2,454 bp and 2,460bp,encoding 817 and 819 amino acids,respectively.The products encoded by the genes all contain a typical conserved domain of sucrose synthase（sucrose synthase,PLN00142）.Their molecular weights were predicted to be 93.21k D and 93.79k D,and their theoretical isoelectric points were 6.02 and 6.04.The results of phylogenetic analysis showed that PmSUS1,PmSUS2 had the highest homology with the SUS protein of Pinus taeda,and the relationship was the closest.3.Response of PmSUS1 and PmSUS2 genes to abiotic stressesThe expression of PmSUS1 and PmSUS2 genes under abiotic stresses were analyzed by q PCR.Both genes were up-regulated under low temperature and drought stress,and their direct expression levels increased from 0 h to 48 h during low temperature stress,and reached the highest at 48 h.As exposure to the drought stress,the expression level under severe drought stress was 1.29-1.56 times higher than that in the control.The expression level of PmSUS1 gradually increased and then decreased under methyl jasmonate treatment,while the opposite pattern was investigateed from that of PmSUS2.4.Genetic transformation of PmSUS1 and PmSUS2 genesThe overexpression vectors of PmSUS1 and PmSUS2 were constructed.The two genes were transformed into tobacco and Arabidopsis by Agrobacterium-mediated leaf disc and inflorescence infection.After resistance screening and molecular detection,we successfully identified eight transgenic tobacco lines of PmSUS1 and PmSUS2.Four T₁ generation lines of transgenic Arabidopsis thaliana with PmSUS1 and PmSUS2 were also identified.The over-expressed PmSUS1 and PmSUS2 tobacco clones,i.e.line 6and line 2,respectively,were obtained after q RT-PCR detection.5.Effects of transgenic PmSUS1 and PmSUS2 on sugar content of tobaccoThe sugar content of the three lines with the highest expression levels in PmSUS1and PmSUS2 transgenic tobacco and wild-type tobacco were determined.The sucrose and soluble sugar contents of PmSUS1 transgenic tobacco were lower than those of wild-type tobacco.In contrast,both sucrose and soluble sugar contents of PmSUS2transgenic tobacco were greater than those of wild type.6.Transgenic PmSUS2 gene improved stress resistance of Arabidopsis thaliana.The results showed that the activities of antioxidant enzymes,sucrose content and soluble sugar content of transgenic Arabidopsis thaliana were increased under low temperature and drought stress,and the content of malondialdehyde（MDA）was lower than that of wild type.It is speculated that the overexpression of PmSUS2 gene improves the stress resistance of Arabidopsis thaliana.The results of this study laid the foundation for further research on the function of sucrose synthase in masson pine.