Establishment Of Genetic Transformation System In Eucalyptus And Preliminary Function Identification Of EgrBRI1 Gene

Eucalyptus is the generic name of Eucalyptus L Herit..Eucalyptus plantation is widely planted all over the world,which has brought huge economic benefits to human society and is an important afforestation and paper-making tree species.The aim of this study was to optimize the in vitro regeneration system of Eucalyptus urophylla×tereticornis YL02 clone,constructing a complete genetic transformation system and obtaining stable transgenic plants.And BRI1 gene of Eucalyptus grandis was cloned successfully and its function was preliminarily analyzed.The results are as follows:1)Through the selection of explants,hormone types and concentration,the upper and middle stem segments of tissue-cultured seedlings were selected and placed on modified MS medium containing TDZ 0.025 mg/L and IBA 0.1 mg/L,and the differentiation rate of adventitious buds was the highest.2)Using Agrobacterium tumefaciens GV3101 strain containing PBI121 plasmid,choosing well-growing YL02 rooting seedlings,cutting middle and upper stem segments without pre-culture,putting the cut stem segments into the suspended bacterial solution for 30 minutes,then putting the stem segments into the improved MS medium after the end,putting a filter paper sterilized by high pressure on the co-culture medium,the dark treatment can not exceed 48 hours,and then transferring.In the screening medium,the screening medium was changed every two weeks.After the adventitious buds were differentiated from the screening medium,they were transferred to the Eucalyptus proliferation medium with antibiotics.When the seedlings were strong enough to transfer to the rooting medium with antibiotics,the DNA of the positive plants was extracted for PCR detection(using GUS primers and NPTII primers),and GUS staining was carried out to prove that the positive plants with GUS gene were successfully obtained.3)Brassinosteroids(BRs)are a kind of important plant hormones,which play an important role in plant growth and development.Therefore,BRI1(brassinosteroid insensitive 1),a receptor protein of BR signal,was selected as the research object in this study.The BRI1 gene of Eucalyptus grandis was cloned successfully,with a total length of 3893 bp and encoding 1197 amino acids.Bioinformatics analysis and subcellular localization prediction showed that the protein was stable,highly conservative,with multiple leucine repeats,and played a physiological role on the cell membrane.The analysis of BRI1 gene expression in different tissues showed that BRI1 gene was most expressed in young leaves.The least expression was found in mature leaves,xylem,phloem and root.The expression of BRI1 in Eucalyptus grandis treated with Me JA for 1 h was 593 times higher than that of the control.The expression of BRI1 in Eucalyptus grandis treated with BR for 1 h was slightly lower than that of the control.The expression of BRI1 in Eucalyptus grandis treated with BR for 1 h was the highest at 168 h,which was 2.9 times higher than that of the control.External application of salicylic acid had no significant effect on the expression of BRI1 gene in Eucalyptus grandis.At the same time,real-time fluorescence quantitative PCR also analyzed the BRI1 gene expression pattern of Eucalyptus grandis under salt stress and cold stress.It was found that the BRI1 expression level of Eucalyptus grandis increased significantly after 6 hours of salt treatment,which was 2.3 times higher than that of the control.The BRI1 expression of Eucalyptus grandis treated with cold stress for 48 hours was 1.6 times higher than that of the control.These results indicate that Egr BRI1 gene can be specifically expressed in response to stress and exogenous hormone treatment,and participates in the regulation mechanism of biological and abiotic stress response.These results indicate that Egr BRI1 gene plays an important role in the growth and development of Eucalyptus.