Cloning And Resistance Effect Of Tomato LncRNA39042 And PI100

Tomato(Solanum lycopersicum)is native to South America and is widely cultivated in China due to its nutrient-rich and special flavor.Late blight caused by Phytophthora infestans is the most serious disease occurring on tomato.So it is of great importance to control this disease at the molecular level.The synthesis and accumulation of self-protecting compounds in plants can directly affect the effect of induced proteins in pathogens.These resistance proteins include pathogenesis related protein(PR),which are regulated by lncRNA(long non-coding RNA)when expressed.LncRNA is a linear RNA fragment of more than 200 nt in length and doesn't have the ability to encode proteins.At present,it has been revealed that it is related to plant diseases,but the lncRNA related to the function of PR-6 protease inhibitor(PI)in tomato has not been reported.Therefore,in order to study the scientific issues above,specific processes and experimental results as follows:Firstly,this study screened the late blight-associated SLPI100(S.lycopersicum Protease Inhibitor 100)and lncRNA39042 on its antisense strand,both of which existed on tomato chromosome 10 and contained in response to the initiation of biotic stress.The subunits were cloned from the tomato leaves with lncRNA39042 and SLPI100 gene fragments,which were310 bp and 285 bp in length,SLPI100 had a half-life of 30 h,an average hydrophobicity of0.189,a theoretical isoelectric point of 9.38,and a relative molecular weight of 10.52 kD,a hydrophobic protein,the gene had a PI functional domain and belonged to the potato protease inhibitor I family.Secondly,lncRNA39042 and SLPI100 had the highest expression in tomato leaves and roots respectively,and it was speculated that they play important roles in the process of resistance to soil-borne diseases and leaf diseases.Under the Phytophthora infestans infection,the relative expression of both increased first and then decreased,indicated that it could respond well to the infection of Phytophthora infestans;then the cloning vector and overexpression vector of lncRNA39042 and SLPI100 gene were successfully obtained;After detecting the sgRNA activity in vitro,the CRISPR-Cas9 double-target vector of lncRNA39042 was constructed.Finally,the Agrobacterium tumefaciens engineering strain was obtained,the tomatohomologous and tobacco heterologous transient expression system were established respectively.Compared with the control group which was transformed pBI121 plasmid,in the tomato leaves showed a disease-resistant phenotype and PR expression were significantly increased which was overexpressed lncRNA39042 and SLPI100 genes,a similar trend was shown in tobacco;while lncRNA39042 knocked out tomato leaves showed with disease susceptible,PR significantly down-regulated.It was indicated that lncRNA39042 and SLPI100 can play a role in the interaction between tomato and P.infestans.In conclusion,this study explored that lncRNA39042 and SLPI100 gene can improve the resistance of plants to pathogens,through the combination of bioinformatics analysis and biological experiments,which can not only lay a foundation for the study of the mechanism between plants and pathogens,it can also provide a basis for the study of disease resistance of other plants at the same time.