A Preliminary Study On The Mechanism Of Upstream Open Reading Frames And Screening Of Phytophthora Toxicity Effectors

Upstream open reading frame is a type of regulatory element widely present in the transcripts of eukaryotes.It is a small open reading frame in the 5’ untranslated region of m RNA,the start codon of which is located upstream of the start codon in the coding region and has an important influence on the translation of the downstream coding region.In agricultural production,crops are infected by fungi,bacteria,viruses and other pathogens,causing a variety of diseases,resulting in reduced yields,low quality,human and livestock poisoning.Breeding disease-resistant varieties is the most economical and environmentally effective measure to prevent and control crop diseases.Transgenic breeding is a major breakthrough in plant disease resistance breeding.It improves the disease resistance of crops by transferring genes from other organisms into crops.While gaining resistance,it often has an adverse effect on the growth and development of plants.In order to solve this problem,a new uORF-mediated transgenic breeding strategy is proposed in this study,which uses the regulatory effect of uORF to balance the resistance and growth of transgenic plants.In addition,Phytophthora is a type of pathogen that is more harmful to many crops.In the process of interaction between plants and Phytophthora,the effector secreted by Phytophthora is an important weapon for plant disease.In this study,the effector of Phytophthora was used as the starting point and the interaction system of Arabidopsis thaliana and Pseudomonas syringae pv.Tomato strain DC3000 was used to carry out the study.The specific research contents are as follows:Establishment of a system to study uORF function and preliminary exploration of ACD11-uORFs function: In this study,In this study,the uORFs of an important Arabidopsis immune system regulatory factor accelerated cell death 11 was selected as the research object.ACD11-uORFs,green fluorescent protein,luciferase were constructed on the p SUPER vector to form a modified vector uORFs-GFP.The modified vector is expressed in tobacco.The green fluorescence of GFP and the biofluorescence emitted by the oxidation of LUC catalytic substrate are observed by the in vivo imager and detected by western blot.The target band with the same size as the target protein indicates that the recombinant vector uORFs-GFP can be expressed normally.The system established in this study to study the function of uORF is successful.Using the established system,the seven mutants obtained from uORFs-GFP were studied and it was found that different combinations of uORFs have different inhibitory effects on downstream GFP translation and uORFs acted as regulatory elements to regulate GFP expression at the m RNA translation level.Exploration of uORFs in transgenic disease-resistant breeding: Different combinations of uORFs were expressed in series with the disease resistance gene Lec RK4.1.It was found that the expression level of Lec RK4.1 in different combinations was different,causing different tobacco cell deaths.All of them have resistance to Phytophthora capsici,but the resistance intensity was different.Using different combinations of uORFs and the susceptible gene BZR1 to express in tandem,tobacco can obtain different resistance intensity.Using the interaction system of Arabidopsis and DC3000 to screen the effectors of Phytophthora: Using the screening system established in the early stage of the laboratory,two screening schemes are used to screen the effectors of Phytophthora.The first is to transfer the effectors into the DC3000 mutant strain D36 E for phenotypic screening.Nine effectors of Phytophthora capsici and four effectors of Phytophthora sojae were screened.Compared with the control,the inoculated leaves of Arabidopsis thaliana showed yellow phenomenon after transforming Ps CRN63 into D36 E and the others have no obvious phenotype.The second screening scheme is to transfer the effector into the DC3000 strain and screen the effectors that caused the change in the number of DC3000.Five effectors are screened.Compared with the control,there is no significant difference in the amount of bacteria.