Cloning Of Arabidopsis Resistance Gene RIN4 And RPM1 And Functional Verification In Soybean

As an important oil crop in China,soybean is widely used in various fields of social life.However,in recent years,frequent outbreaks of uncontrolled soy disease and uncontrolled spread have become an important cause of soybean yield and quality.In particular,losses caused by soybean gray spot disease are particularly serious.The use of traditional breeding methods to breed disease-resistant gene varieties is an effective way to solve the soybean gray spot disease.but there are problems such as genetic limitations and resistance to degradation.Transgenic technology can effectively solve such problems and become a new way to cultivate new varieties of soybean disease resistance.The study found that the resistance genes RIN4 and RPM1 derived from Arabidopsis thaliana can cause allergic reactions in plants through a series of signal transduction and to stimulate the non-specialized disease resistance of plants,that is,systemic acquired resistance,and causing the infection of the pathogen and the surrounding tissue cells programmed cell death so that the pathogen does not spread to healthy tissues and organs,and achieve the purpose of disease resistance,it has important application value.Among them,the RIN4 gene showed negative regulation on gray leaf spot resistance,and the RPM1 gene showed positive regulation on gray leaf spot resistance.In this experiment,Arabidopsis genome is used as a template to obtain a DNA fragment with a sequence length of 458 bp?RIN4 gene?and 2592 bp?RPM1 gene?by PCR amplification,and is ligated into the pMD18T cloning vector.Because of RIN4 gene can be detected in soybean,RPM1 gene can not be detected in soybean.Therefore,using the seamless cloning technology,the plant interfering expression vector pCAMBIA-3301-35s-RIN4-nos and the plant overexpression vector pCAMBIA-3301-35s-RPM1-nos containing the selection marker Bar were constructed using pCAMBIA-3301 as the basic vector.To identify the function of RIN4and RPM1 resistance genes.The constructed recombinant plasmid is transferred into soybean receptor variety JN28 by Agrobacterium-mediated method.Through the preliminary detection by PCR,the obtained positive plant seeds are added.The T₁ generation transgenic plants are subjected to routine PCR,Southern blotting,fluorescence quantitative PCR and indoor disease resistance identification.and the resistance of soybean plants to hrpZpsta resistance gene is compared to identify the resistance of the two target genes in soybean.The test results are as follows:1.Through the PCR detection of the target gene RIN4 and RPM1 and the comparison of the homology of the sequencing results,it has been shown that the two resistant genes have been successfully cloned,and plant interference expression vectors pCAMBIA-3301-35s-RIN4-nos and plant overexpression vector pCAMBIA-3301-35s-RPM1-nos containing the oxaphosphine-selective marker are constructed.2.Detection obtained by PCR containing the resistance gene RIN4 positive T₀ generation plants 3,T₁-generation 11 have positive plants,and containing the resistance gene RPM1positive T₀ generation plants 5,T₁-generation 14 have positive plants.3.The promoter 35s,terminator Nos,target band,and selection marker Bar of the target gene of interest are detected in the T₁ transformant lines,indicating that the target gene is stably inherited.4.The results of Southern hybridization of T₁ transgenic plants show that the gene for RIN4interfering expression vector and the gene for RPM1 overexpression vector are all integrated into the soybean genome with a single copy,and the integration sites were different.5.Fluorescent quantitative PCR detection of transgenic plants with southern hybridization signals,and the results show that the gene for RPM1 overexpression vector is expressed in roots,stems and leaves of the plants,of which the highest expression levels in leaves and the lower expression levels in roots and stems.The gene for RIN4 interfering expression vector inhibited the expression of soybean endogenous genes,resulting in a decrease in the expression of roots,stems,and leaves.Among them,the amount of expression in leaves decreased the most,and the expression in roots and stems decreased less.6.The foliar spray method is used to identify the resistance of the transgenic plants to soybean grey spot.The results show that:?1?Increase resistance to RPM1 overexpression vector gene plants compare to control plants,and the resistance increase from the susceptible to the middle resistance level.Through the correlation analysis of different tissue sites and gray spot disease index,the results show that the correlation coefficient is-0.92096,showing a significant positive correlation.That is,the higher the expression level of the target gene in the leaves,the stronger the resistance of the plant and the lower the disease index.?2?Compare with the control plants,the resistance to RIN4 interference expression vector gene plants is enhanced.And resistance increase from the sensation to the middle level.The correlation analysis between different tissue sites and gray spot disease index show that the correlation coefficient is0.844375,showing a significant negative correlation.That is,the lower the expression level of the target gene in the leaves,the stronger the disease resistance of the plants and the lower the disease index.