Study On Direct Transformation Of Eucommia Ulmoides Oliver

Eucommia ulmoides Oliver belongs to the Eucommia genus of the Eucommia family.It is a high-quality Chinese medicinal material and gum-producing plant.It iswidely distributed in my country and is a unique economic forest tree species in mycountry.Studies have shown that Eucommia ulmoides is rich in medicinal compounds,and Eucommia ulmoides gum has excellent properties in terms of insulation,durability,plasticity,and flexibility,so it is one of the high-quality natural rubberresources with great development prospects.At present,in the research of Eucommia ulmoides gene function,it is usuallynecessaryto carry out genetic transformation of the target gene in Eucommia ulmoides to achievethe purpose of analyzing its gene function.However,Eucommia ulmoides is a woodyplant,and its tissue culture method has problems such as poor callus induction and differentiation,difficulty in rooting of resistant buds,low survival rate of transgenicplants,and long transformation cycle,which lead to such researches as Eucommia.Relatively lagging behind.The non-tissue culture genetic transformation method has the advantages of simple operation,low cost,short time,and can effectively avoid thelengthy and cumbersome tissue culture process.It is of great significance for the studyof Eucommia ulmoides gene function.At present,there are few researches on the nontissue culture genetic transformation technology of Eucommia u Lmoides.In this study,paraffin section technology was used to determine the best injection site,and Agrobacterium injection was used to carry out non-tissue culture genetic transformation of Eucommia ulmoides seedlings.The effect of different bacterial concentration on plant survival was explored.GUS histochemical staining,PCR identification,and sequencing determination method to identify transformed plants,in order to establish a non-tissue culture genetic transformation system of Eucommia u Lmoides.The results of the study are as follows:1 Determination of the best site of injury to Eucommia seedlingsIn this study,Eucommia ulmoides seedlings germinated for 2-4 days were selected,and the first true leaf between the two cotyledons was removed as the transformation recipient.A 1m L medical syringe was used to inject the stem apex of the eucommiaseedlings,each treatment was performed three times repeatedly process100 Eucommia ulmoides seedlings each time.After culturing in the dark at 28°C for 3days,move thetransformed plants to an artificial climate room（photoperiod 16h/8h,light intensity 3500lx,humidity 70%）,and manage the plants normally.Through the paraffin section technique,with the Eucommia ulmoides seedlings without injection treatment as acontrol,the longitudinal section of the paraffin section of the eucommia seedlings stem tip was observed.The results showed that the cells at the injection site were strong cell division ability,so it was suitable for the introduction and integration of the target gene.Therefore,this area was determined to be the best injection site for Eucommia ulmoides seedlings.2 Study on the survival rate of Eucommia ulmoides seedlings with different bacterial concentrationIn this study,8 bacterial liquid concentration gradients were designed to infect Eucommia ulmoides seedlings,and the survival rateof Eucommia ulmoides seedlings after 15 days of transformation was calculated and analyzed.The results showed that different bacterial concentration had different effects on the survival rate of Eucommia ulmoides seedlings,and the difference was significant.When the OD₆₀₀ value was 0.8,the survival rate was the highest,which was 77.67%.As the concentration of the bacterial solution increased,the survival rate of Eucommia ulmoides seedlings decreased.When the OD₆₀₀ value is 1.8,the survival rate is the lowest,53.00%;the SPSS software was used to analyze the correlation between different Agrobacterium concentrations and the plant survival rate,and it was found that the Agrobacterium concentration was highly negatively correlated with the plantsurvival rate,and the Pearson correlation coefficient was-0.626,the results showed that the concentration of bacterial fluid had an important effect on the survival rate of Eucommia ulmoides seedlings.3 Study on GUS positive rate of Eucommia ulmoides seedlings with different bacterial concentrationThe seedlings of Eucommia ulmoides transformed for 1 month were selected,and the third new leaf that grew from bottom to top was subjected to GUS histochemical staining,and 75%ethanol was used for decolorization.The results showed that the leaves of the successfully transformed Eucommia ulmoides were blue.The positive rate of GUS was analyzed statistically.The results showed that when the OD₆₀₀ value was1.8,the positive rate of GUS was the highest,which was 11.00%,followed by 0.8 and2.0,which were 10.00%and 9.67%,respectively.The SPSS software was used to analyze the correlation between different Agrobacterium concentrations and the positive rate of GUS in plant,and it was found The concentration of Agrobacterium was negatively correlated with the positive rate of GUS in plant,and the Pearson correlation coefficient was-0.128,the results showed that the bacterial concentration had no significant effect on the positive rate of GUS.4 PCR detection and sequencing identification of transformed plants of Eucommia ulmoidesThe transformed plants of Eucommia ulmoides that were positive for GUS staining were amplified by PCR with specific primers specific to GUS and 35S-Eu SRPP3,and detected by 1.2%agarose gel electrophoresis.The results showed that wild-typeEucommia ulmoides had no amplified bands.Some Eucommia ulmoides transformed plants had GUS（304bp）and 35S-Eu SRPP3（768bp）specific primer amplified bands,which were the same as the plasmid positive control bands.The 35S-Eu SRPP3specific PCR product was recovered and sequenced.According to the forward and reverse sequencing results of the transgenic plants and the target nucleotide sequence were compared and analyzed,it was found that the similarity between the forward and reverse sequencing results and the target sequence was 99%,which further proved the purpose the genes have been integrated into the Eucommia ulmoides genome.By GUS histochemical staining and PCR detection,a total of 11 transgenic Eucommia ulmoides plants have been obtained.When the OD₆₀₀ value is 0.8,the conversion rate is 3%;when the OD₆₀₀ value is 0.6,the conversion rate is 0.67%.5 Relative expression of target genesThe transgenic plants identified by PCR were taken,RNA was extracted and reversed to synthesise c DNA for fluorescence quantitative PCR detection.The results showed that the target genes in transgenic Eucommia ulmoides were overexpressed compared with those in photographs.The relative expression level of the target gene in TP4 was the highest,which was 10.5 times that of the control group.The relative expression level of target gene in TP7 was 7 times that in control group,and the relative expression level of target gene in TP5 was 3.8 times that in control group.