Study On The Method Of Cell Model To Evaluate Cytokine Activity By Using Cis-elements

Cytokinins is one of the classic plant hormones,and many scholars have confirmed that it play an important role in all aspects of plant growth and development,and have the characteristics of micro-efficient,which has gradually been developed as plant growth regulators widely used in agricultural production.However,due to the uncertainty of its bioactivity,improper use of dose will seriously affect the benefits of agricultural production.The traditional hormone detection method son-focused on quantitative detection,the evaluation of its bioactivity method is less research.The content of cytokinin in plants is related to many metabolic regulation,and ARR1 is a transcription factor in the transmission of cytokinin signals,which can respond quickly to the signaling of cytokines.In this study,the protoplast as a detection model of plant hormone bioactivity cell level,successfully constructed a cell model to detect cytokinin bioactivity.Based on ARR1's preferred binding site(5'-AAGATTT-3')drives the expression of the GUS gene,the specificity of the response to cytokines is analyzed with GUS activity.Further exploration of optimal treatment time for hormones,the most responsive cell divider species and the working range of hormone concentrations,the biological activity of the sample chloroquine was detected through the working interval.First,construct two fusion expression vectors,ARR1AT::GUS and 35S::GUS,and use Agrobacterium transformation to transfect Arabidopsis plants respectively.Transgenic plants were obtained through resistance plate screening and PCR identification.The homozygous screening of positive plants was carried out by double fluorescence quantitative method,and homozygous plants with good growth and development were obtained,and it was found that the higher the copy number,the more difficult it is to obtain homozygous plants.Using homozygous Arabidopsis plant leaves as materials,the mesophyll protoplasts qualified in terms of activity and number were prepared by enzymatic hydrolysis,and the survival time of protoplasts was explored.It was found that the protoplast activity was relatively stable from 0 to8 h.Then,mesophyll protoplasts were used as the detection model for the level of phytohormone active cells,and the expression level of GUS protein was induced to evaluate the activity level of hormones.The specificity of ARR1AT::GUS response to cytokinin was explored.Induced expression of mitogen;according to the survival time of protoplasts,the optimal hormone treatment time was explored,and the results showed that the best hormone treatment time was 6 h;this study responded to different structures of cytokinin response and hormone concentration The interval was explored,and it was found that ARR1AT::GUS responded differently to cytokinins of different structures,but the overall trend remained the same.Among them,the purine cytokinins with isoprene side chains,t Z and 2-IP,had the highest activity.The side chain is aromatic 6-BA,followed by non-adenine TDZ with the least activity.The linear interval was further explored with t Z with the largest response intensity,and it was found that when the hormone concentration was 10^-8?10^-3mg/m L,GUS activity increased with the increase of concentration.Further determining the repeatability of the linear interval,it was found that the results of different batches were different,but the trend lines were similar,and R² was greater than 0.99.Finally,the linear interval was used as the standard curve to determine the activity of forchlorfenuron in the sample.The results showed that the activity of forchlorfenuron per ml was 0.0195,and the relative standard error of three times was 2.71%,indicating that the measurement method has good repeatability and has certain feasibility.The determination of hormone activity injects new vitality.