| Eucommia ulmoides Oliver is a unique Tertiary relict tree species in China,belonging to the Eucommia family and genus.It is a traditional and precious Chinese medicinal herb with anti-tumor,antibacterial,antioxidant,anti-inflammatory,and bidirectional blood pressure regulation effects.The stem bark,leaves,and pericarp cells of E.ulmoides contain rubber with the dual properties of rubber and plastic,known as E.ulmoides gum,which can be used in medical,transportation,communications,national defense,construction,and other fields.However,the biosynthetic mechanism of E.ulmoides gum has not yet been fully studied.In this study,the protein eluted from E.ulmoides rubber particles in the early stage of the laboratory was sequenced,and a sequence containing the peptide AKEEGYDVTK was selected based on the comparative analysis of the E.ulmoides whole genome annotation database established in the early stage of the laboratory.The full length sequence of the Eu TIL1 gene was obtained using RACE cloning technology,and the function of the gene was analyzed.The following research results were obtained:1.Cloning,structure,and protein prediction analysis of Eu TIL1 geneThe Eu TIL1 gene c DNA was cloned using RACE technology with a total length of 917 bp,containing an open reading frame sequence of 567 bp,a 5 ’non coding region(5’UTR)of 103 bp,a3’ non coding region(3’UTR)of 227 bp,and a Ploy(A)tail of 20 bp,encoding 188 amino acid residues of the protein.To stabilize the hydrophilic protein,the protein has no transmembrane domain,no signal peptide,15 phosphorylation sites,2 glycosylation sites,and Lipocalin-2 domain,belonging to the temperature induced lipocalin protein superfamily.There is a short hydrophobic proline rich motif(HPR)located at the 91-98 amino acids,which can interact with the cell membrane and bind to it.Phylogenetic analysis shows that the protein encoded by the Eu TIL1 gene belongs to the temperature induced temperature induced lipocalin protein and has the closest genetic relationship with Brassica napus and Arabidopsis.2.Eu TIL1 gene promoter region analysisUsing the complete genome annotation database of E.ulmoides established in the laboratory,the 2000 bp upstream sequence of E.ulmoides temperature induced lipocalin protein gene Eu TIL1 was compared and retrieved.After analysis on the Plant CARE online website,it was found that in addition to the core elements such as TATA-box and CAAT-box,there are also elements such as ACE that participate in light response;MYC binding sites involved in drought induced responses;LTR elements related to low-temperature response;A component CGTCA-motif participating in the methyl jasmonate response;Abscisic acid response element and other response elements.In order to understand whether the expression of Eu TIL1 gene is induced by abscisic acid,methyl jasmonic acid,drought and low temperature,the true leaves of E.ulmoides seedlings at 5-leaf stage were sprayed with 200 μ mol of ABA solution,and 100 μ mol of Me JA solution until water droplets drop from the page.The results showed that after spraying ABA and Me JA solution for 6 hours,the expression of Eu TIL1 gene in E.ulmoides.leaves decreased,and the expression began to increase at12 hours,reaching the maximum at 24 hours,significantly higher than 0 hours(P<0.05),4.13 and1.07 times higher than 0 hours,respectively.During the treatment of E.ulmoides seedlings with 20%PEG and low temperature for 0-24 hours,the expression level of Eu TIL1 gene showed a gradually increasing trend,reaching its maximum value at 24 hours and significantly higher than 0 hours(P<0.05),indicating that the expression of Eu TIL1 gene is regulated by ABA,Me JA,PEG,and low temperature.3.Analysis of temporal and spatial expression of Eu TIL1 geneIn this study,the Eu TIL1 gene was expressed in different parts of E.ulmoides,indicating that the Eu TIL1 gene is involved in the growth and development process of E.ulmoides and plays an important role.From April to September,the expression level of Eu TIL1 gene in the stem bark and leaves of both female and male plants showed an initial upward trend,both significantly higher than in April,and reached its maximum value in September.Similar to the expression pattern of Eu SRPP1 gene,which was previously studied in our laboratory,it is speculated that Eu TIL1 gene is related to the glue synthesis process.4.Genetic transformation of E.ulmoides with Eu TIL1 geneAgrobacterium tumefaciens containing p SH737-35S-Eu TIL1 overexpression vector was used to infect the hypocotyl of E.ulmoides 38 E.ulmoides resistant buds were detected and obtained.Three E.ulmoides buds with different expression levels were selected.The expression levels of associated genes in the E.ulmoides rubber biosynthesis pathway were examined using real-time PCR.It was found that the expression levels of Eu SRPP1,Eu SRPP4,and Eu FPS1 genes in overexpressed E.ulmoides were significantly higher than those in WT plants,4.65,9.45,and 32.26 times higher than those in WT plants,respectively.Therefore,it is speculated that E.ulmoides temperature induced lipocalin proteins play an important biological role in rubber synthesis.5.Genetic transformation of Eu TIL1 gene in tobacco to improve cold resistanceWhen treated for 24 hours at 4 °C(16 hours of light and 8 hours of darkness)in a climatecontrolled environment with the Eu TIL1 transgenic tobacco plants,empty vector plants,and WT plants.The third leaf of WT and transgenic vector plants began to wilt from top to bottom,while the degree of leaf wilting of transgenic Eu TIL1 tobacco plants was relatively light.During cold treatment,the activities of SOD,POD,and CAT in transgenic tobacco plants with Eu TIL1 gene first increased and then decreased.Compared with wild type and empty vector plants,the protective enzyme activity of transgenic plants significantly increased and the content of malondialdehyde(MDA)significantly decreased after 12 hours of cold treatment(P<0.05).The soluble sugar content of Eu TIL1 transgenic tobacco plants increased with the increase of low-temperature treatment time,reaching the maximum value at 24 h,significantly higher than WT(P<0.05),1.69 and 1.58 times higher than WT and EV.When treated at 4 ℃ for 0 h,the expression levels of Nt DREB2 and Nt DREB4 in Eu TIL1 transgenic tobacco plants were 2.25 and 7.59 times higher than those in WT,and 2.27 and 7.66 times higher than those in empty vector plants.There was no significant difference in the expression levels of Nt DREB1 in TP plants compared to WT and EV.After tobacco plants were treated at 4 ℃ for 6 hours,the expression of Nt DREB1,Nt DREB2,Nt DREB4,and Nt COR15 a genes in the leaves of Eu TIL1 transgenic tobacco plants was upregulated,significantly higher than that of wild type and empty vector plants(P<0.05).It is suggested that low temperature induction can increase the sensitivity of the cold response gene Nt DREB1 to low temperature,thereby increasing the expression level;Overexpression of Eu TIL1 gene can increase the expression of Nt DREB2,4,and Nt COR15 a genes in transgenic plants,further improving the activity of protective enzymes,thereby improving the ability of tobacco to scavenge active oxygen species,and enhancing tobacco’s tolerance to low temperature. |
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