Study On The Resistant To Fungal Disease Of EuDIR1 In Transgenic Tomato

Tomatoes（Solanum Lycopersicum Mill.）are annual or perennial herbs,It is one of the most susceptible vegetable crops in the world.DIRs proteins are involved in lignin and lignin biosynthesis.Studies have shown that,EuDIR1 protein can inhibit the growth of Fusarium oxysporum.Besides,Phytophthora infestans,H₂O₂and NO,Can induce the up-regulation of potato St DIR1 gene."Exogenous gene clearance"technology（Gene-Deletor）uses organ-specific or inducible promoters,recombinant enzymes and fusion recognition sites to construct gene expression elements,An attempt was made to remove all exogenous a in transgenic plants from pollen,seeds and fruits automatically after completing their functions.To obtain resistant tomato plants and tomato fruit without exogenous genes,Using genetic engineering,The expression vector of Eucommia ulmoides EuDIR1 gene with Gene-Deletor system was constructed,Genetic transformation of Micro-Tom tomatoes,Effects of EuDIR1expression on phenotype,Erysiphales,Fusarium oxysporum resistance and lignin content of transgenic tomato plants,The deletion efficiency of exogenous genes in fruit was counted.The results were as follows:1.Genetic transformation and relative expression of EuDIR1 genes in tomatoApplication of Prophase Carrier p GM626-Act1,in Laboratory to construct the Gene-Deletor system of fruit specific E8 promoter-initiated FLP gene,Bar genes as screening genes,GUS genes as reporter genes,Act1 plant expression vectors that drive the EuDIR1 gene of Eucommia ulmoides.Agrobacterium mediated tomato genetic transformation,After herbicide basta resistance screening,to GUS histochemical staining and PCR amplification of target fragments,Finally,30transgenic tomato plants were obtained.Fluorescence quantitative PCR of transgenic tomato plants.The results showed that the relative expression of EuDIR1 genes was the lowest in the TP9 of transgenic tomato lines,1.16 times the EV of the vector plants.And the relative expression was the highest in TP1,3.52 times of plant EV;The relative expression of EuDIR1 genes in plant TP2,TP4,TP5 and TP12 is also high,2.36 times,3.23 times,2.24 times and2.18 times of plant EV,respectively.EuDIR1 genes have been integrated into the tomato genome and transcribed normally,and subsequent experiments can be carried out.2.Effects of EuDIR1 gene expression on tomato phenotypePhenotypic observation of 13 transgenic tomato plants after 30d of transplanting.The difference was not significant.Indicating that EuDIR1 gene expression had no significant effect on tomato plant height,leaf size and number.3.Effects of EuDIR1 gene expression on resistance to Botrytis cinerea,Erysiphales,Fusarium oxysporumInoculation method of isolated leaves was used to inoculate the pathogen of Botrytis cinerea.After 7 d of inoculation,the Botrytis cinerea spots of wild-type and transfer empty vector plants leaves had spread to other uninfected leaves,while the ttransgenic tomato Botrytis cinerea spots had just begun to spread.The results showed that the mean diameter of leaf spot was 2.415±0.13cm,2.496±0.08cm,and that of transgenic tomato leaves was 1.539±0.10cm,36.3%lower than that of wild type and38.3%lower than that of transvacuum type,the difference reached a very significant level（P<0.01）.Indicating that the expression of eucommia ulmoides EuDIR1 gene can improve the resistance of tomato to Botrytis cinerea.Inoculation of Erysiphales bacteria was also carried out in vitro.After 7d of inoculation with Erysiphales,the Erysiphales spots in wild-type and transfer empty vector plants leaves were significantly increased,and there were spots on the uninfected leaflets,while the Erysiphales spots in transgenic tomato leaves were small,and no spots appeared on the uninfected leaflets.The mean diameter of leaf spot after7d of docking bacteria was 1.819±0.11cm,1.777±0.07cm,and 1.314±0.06cm,27.8%lower than that of wild type and 26.1%lower than that of empty type,the difference reached a very significant level（P<0.01）.Eucommia ulmoides EuDIR1 gene expression can also improve the resistance of tomato to Erysiphales.Millet seed inoculation of Fusarium oxysporum.The results showed that the average plant height of wild-type and transfer empty vector plantses was 6.93±0.55cm,6.72±0.41cm,and the average root length was 6.09±0.59cm,6.29±0.46cm,respectively.The average plant height of transgenic tomatoes was 7.38±0.49cm,which was 6.5%and 9.8%higher than that of control,respectively,and the difference was significant（0.01<P<0.05）.The average root length of transgenic tomato was6.86±0.48cm,which was 12.6%and 9.1%higher than that of the control,and the difference was significant（0.01<P<0.05）.The average aboveground biomass of wild-type and no-load tomatoes was 129±6mg,131±4mg,respectively,and the underground biomass was 6.74±0.7mg,6.51±0.9mg,and the average aboveground biomass of transgenic tomato was 142±4mg,which was 10.1%and 8.4%higher than that of control,and the difference was significant（0.01<P<0.05）.The average underground biomass of transgenic tomatoes was 7.81±0.5mg,which was 15.9%and19.9%higher than that of the control.The extremely significant difference（P<0.01）.Indicated that under the stress of Fusarium oxysporum,the incidence degree of transgenic tomatoes was lower than that of the empty-loaded and wild-type transgenic tomatoes,that is,the expression of EuDIR1 gene could significantly enhance the resistance of tomato to Fusarium oxysporum.4.Effects of EuDIR1 gene expression on lignin content in tomatoDetermination of lignin content in leaves and stems of transgenic tomato,transgenic tomato,A total of 1.87±0.06,1.76±0.05（OD280/g,FW）were found in the leaves of wild-type and transfer empty vector plants,2.09±0.11（OD280/g,FW）of lignin in transgenic tomato leaves,11.7%and 18.8%higher than the control,Significant differences;2.31±0.06,2.25±0.04（OD280/g,FW）of lignin in wild-type and transloaded tomato stems,2.63±0.13（OD280/g,FW）in transgenic tomato stems,13.9%and 16.9%higher than the control,the difference was significant（0.01<P<0.05）.Conclusion EuDIR1 gene can increase the lignin content of tomato,thicken the cell wall of tomato tissue and enhance the defense against pathogenic bacteria.5.Effect of EuDIR1 gene on development of tomato cell wallThe results showed that the cell wall of the cells near the xylem duct of transgenic leaves was thicker than that of the transfer empty tomato.6.Deletion efficiency of exogenous gene from tomato EuDIR1 geneUsing target gene EuDIR1 and GUS gene design primers to PCR and verify the transgenic and empty carrier tomato fruit,it was found that 11 of the 30 transgenic tomato fruits were deleted.The deletion rate was 36.67%and 13 of the 30 transfer empty tomato fruits were deleted,and the deletion rate was 43.33%,the extremely significant difference（P<0.01）.