| Soybean[Glycine max(L.)Merr.]originated in China and has been cultivated in my country for more than 5,000 years.It is a protein-rich and cheap food crop and oil crop in legumes.Soybean mosaic virus(SMV)is widely present in soybean producing areas around the world,and the virus disease caused by it can cause serious reduction in soybean production and deterioration of quality.Cultivating disease-resistant soybean varieties is an extremely effective way to prevent and control SMV diseases.Due to the complexity and diversity of the division of SMV strains,and the resistance of soybeans to SMV is strainspecific,it is difficult to quickly cultivate it using traditional breeding methods.Soybean varieties with broad-spectrum resistance to SMV were developed.The development of genetically modified breeding methods provides new possibilities for the cultivation of soybean mosaic virus-resistant soybean varieties,which also plays a key role in the effective control of SMV.Soybean eukaryotic translation initiation factor 4E(eukaryotic translation initiation factor 4E)is a recessive disease resistance gene ubiquitous in eukaryotic organisms,and it is also an essential element for many RNA viruses to complete the infection process.By constructing RNAi vectors,our laboratory introduced the interference fragments of eIF4E into the genome of the recipient material Tianlong No.1 soybean,and specifically silenced the eIF4E gene to block the combination of eIF4E and SMV elements,thereby achieving the goal of resisting SMV.Predecessors conducted broad-spectrum disease resistance identification on the T3 and T4 generations of the constructed transgenic material eIF4EiTL1,indicating that the T3 and T4 generation materials have broad-spectrum disease resistance to multiple viruses of the potato Y virus genus,but not all have reached High disease resistance(HR)level.In order to select highly disease-resistant transgenic families and individual plants in the offspring,the T6 generation and T7 generation were further identified for disease resistance and the families with excellent disease resistance and agronomic traits in the T7 generation were selected.By PCR detection,Southern hybridization detection,Western hybridization detection,Elisa kit detection,PAT/bar test strip detection,herbicide smearing and flanking sequence analysis methods to detect the existence of the target gene to ensure disease resistance in the offspring family and it can be inherited stably.The PCR test results show that the target gene in the T5-T7 generation transgenic materials can be inherited stably;the Southern test results show that the DNA of the T5-T7 generation eIF4Ei-TL1 plant hybridized with the Bar probe has bands and the size is in line with expectations,indicating in three generation materials Bar gene can inherited stably;Western and Elisa kit test results show that the transgenic materials have BAR protein expression in roots,stems,leaves,flowers,pods,and seeds,and the highest expression in roots is 149.17ng/g,followed by leaves,the minimum expression in seeds is 5.07ng/g;PAT/bar test strip test shows that the T5-T7 generation eIF4Ei-TL1 plants all have control lines and test lines,while the recipient plants have only control lines,indicating that the Bar gene was successfully expressed protein;the results of herbicide application experiments show that the target gene is stably present in the genome of T5 and T6 soybeans;the flanking sequence analysis experiment showed that the insertion position of the foreign T-DNA of the transgenic eIF4Ei gene was located at the 4906903 site of chromosome 11 of the genome,and the insertion direction was forward single copy insertion.Based on the above results,it is speculated that the target gene has been successfully inserted into the soybean genome and inherited stably.The T6 generation transgenic materials were inoculated with SMV strains SC3,SC7,SC 15,SC 1 8 of the potato Y virus genus and recombinant SMV virus(SMV-R),bean common mosaic virus(BCMV),watermelon mosaic virus(WMV),Bean pod mottle virus(BPMV)was identified,and the results showed that all plants of the recipient control variety Tianlong No.1 were moderately disease-resistant(MR),while highly disease-resistant(HR)plants of the transgenic material accounted for 95.36%of the identified plants,and the rest were moderate disease resistance.In the identification of the four SMV strains and BCMV and WMV inoculation of materials from 15 families of the T7 generation,the recipient control variety Tianlong No.1 was moderately disease-resistant(MR),while transgenic materials were highly disease-resistant(HR)plants accounted for 97.14%of the identified plants,the rest were moderate disease resistance.The leaves of 15 days and 30 days after the inoculation of SMV were sampled for DAS-ELISA detection,it was found that the amount of virus accumulation in the transgenic material was significantly lower than that of the recipient control.The identification of resistance to bean pod mottle virus(BPMV)showed that both the recipient material and the genetically modified material were susceptible(S).Through the identification of disease resistance of the T7 generation materials,8 transgenic families with high disease resistance(HR)levels and 180 transgenic plants were finally obtained,a total of 408 transgenic plants.In this study,the leaf shape,flower color,growth habit,pod setting habit,plant type,pod splitting,growth days before harvest of Tianlong No.1(control)and T7 generation transgenic materials inoculated with PBS agronomic traits were investigated and analyzed.The plant height and bottom pod height,number of branches,number of main stem nodes,number of pods per plant,number of seeds per plant,100-seed weight and seed weight per plant were investigated and analyzed after harvest.It was found that there was a certain difference of plant height between the control and transgenic plants.There are no significant differences in other traits.Comparison of the agronomic traits among the 15 transgenic families inoculated with the four SMV strains SC3,SC7,SC15,SC18 and the other two potato Y virus genus viruses BCMV and WMV,found that the agronomy among the transgenic families inoculated with different viruses were different.Comparison of agronomic traitsbetween the T7 generation transgenic families inoculated with the virus and Tianlong No.1 inoculated with PBS showed that the Tianlong No.1 had a decrease in plant height,a decrease in the number of pods per plant and the number of seeds per plant,100 seed weight and seed weight per plant reduce.Nutritional analysis showed that the protein content and oil content of the T7 transgenic material seeds were stable at 43.28%and 21.20%,respectively,there was no significant difference compared with the recipient material,indicating that the transgene had no significant effect on the protein and fat content of soybean. |
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