The Function Analysis Of GPPS Gene And Its Promoter From Pinus Massoniana

Pinus massoniana is a commodity material and a good raw material for the fiber industry in our country.It growes fast and its wood quality is fine.It is also the main tree species of oleoresin tapping.Turpentine and rosin can be extracted from pine oleoresin,which are mainly composed of terpenoids and have a specific odor,both of which are important raw materials for the chemical industry.Oleoresin also has the function of assistant the biological stress.This dissertation mainly conducts preliminary research on the function of GPPS gene and its promoter in the synthesis pathway of terpenoid,and aims to help for the selection and breeding of fine varieties and molecular breedingof P.massoniana.P.massoniana was used as the test material,its total RNA was extracted,and the ORF of P.massoniana GPPS gene was obtained by homologous cloning technique,and the bioinformatics analysis was performed on it.Through real-time fluorescence quantification,the expression of PmGPPS gene in different tissues of P.massoniana was analyzed.The expression level of PmGPPS gene was analyzed with the seedlings of P.massoniana treated with PEG 6000 osmotic stress,mechanical damage,H₂O₂,Me JA,ETH and SA,respectively.The PmGPPS gene promoter fragment of P.massoniana was cloned,the core elements contained in it were predicted,the recombinant plasmids of the promoter fragments were constructed,and transient expression analysis was performed in tobacco leaves.Prokaryotic expression analysis of PmGPPS gene was performed,and the protein was purified.In addition,the PmGPPS gene was transferred to Arabidopsis thaliana to analyze the chlorophyll content,enzyme activity,and expression of genes related to terpenoid synthesis.The results are as follows:（1）The ORF of PmGPPS gene is 1164 bp in length and encodes 387 aa amino acids.The analysis of physical and chemical properties showed that the molecular formula of PmGPPS protein is C₁₈₅₀H₃₀₀₁N₅₀₇O₅₇₂S₁₉,the molecular weight is 42.11 k Da,the theoretical isoelectric point（p I）is 6.08,and the total hydrophilic mean is-0.069.The protein is a hydrophilic protein.The protein instability coefficient（II）is 41.20,which is an unstable protein.According to the online software prediction results,it is preliminarily speculated that the PmGPPS protein is a chloroplast localization protein and does not contain a signal peptide sequence and has no transmembrane structure.The predicted result of the conserved domains indicate that the PmGPPS protein belongs to the member of isoprenoid superfamily.（2）The PmGPPS gene of P.massoniana has the highest expression in young leaf,followed by mature leaf.Under osmotic stress,mechanical damage and SA treatment,PmGPPS gene expression reached the highest level at 3 h;Under the H₂O₂treatment,the expression level rose to the highest at 24 h;the expression level under the ETH treatment reached the highest at 12 h;Under Me JA treatment,PmGPPS gene expression reached the highest value at 6 h.（3）The length of the PmGPPS gene promoter fragment is 1690 bp.The results of bioinformatics analysis showed that the PmGPPS promoter contained many core elements,including many hormone response elements,cis-acting elements involved in light response,defense and stress response,and defensive elements such as the MYB binding site involved in drought-induced,as well as some elements with unknown functions.The transient expression results showed that each deleted fragment can initiate the expression of GUS gene.Under different hormone treatments,the p BI121-PmGPPS-570 bp-GUS fragment had the darkest leaf color under Me JA treatment;The leaf color of p BI121-PmGPPS-1130 bp-GUS fragment is relatively dark under Me JA and GA treatments;The p BI121-PmGPPS-1690 bp-GUS fragment was relatively dark in leaf color under IAA treatment.（4）Prokaryotic expression induced a protein with a size of about 42.1 k Da.After purification,a target protein with the correct size and a single band was obtained.（5）The chlorophyll content of the Arabidopsis thaliana transformed PmGPPS gene was higher than that of the wild-type and the enzyme activity was also generally increased,up to52.74 IU/L,which was about 1.28 times that of wild-type.In the case of overexpression of PmGPPS,the expression of DXR and HDR decreased slightly,and the expression of HMGR did not change significantly.The expression levels of MK,PSY and TPS all increased,especially the TPS gene expression level increased the most,up to more than 40 times the wild-type expression level.This dissertation initially found that PmGPPS gene can respond to some abiotic stress and hormone treatments,and overexpression can positively regulate the expression of some genes on the MVA and MEP pathways to promote the synthesis of more terpenoids.