Cloning And Construction Of Vector On Resistant Gene StWRKY11 Induced By Sodium Silicate In Potato By Rhizoctonia Solani

Rhizoctonia solani Kühn is one of the most important potato diseases in Inner Mongolia.At present,with the promotion of environmental friendly agricultural production and the reduction of the use of chemical pesticides,the role of mineral nutrients in the regulation of plant disease resistance has been gradually paid attention to.Among them silicon and silicate have been proved to induce plant disease resistance.Our previous study confirmed that sodium silicate can induce potato resistance to nevus in indoor and outdoor experiments.We would like to further explore its resistance mechanism from the perspective of molecular biology.In this study,through transcriptome data analysis,mportant genes related to potato resistance to nevus were screened out,and an important regulatory gene StWRKY11 was selected for study.First,StWRKY11 gene was cloned from potato variety Atlantic,and its nucleotide sequence was analyzed,which confirmed that StWRKY11 was a WRKY transcriptionfactor.Then,thegeneoverexpressionvector p CAMBIA1302-StWRKY11-GV3101 was constructed to explore the optimal regeneration system of potato variety Atlantic,and the genetic transformation system of Agrobacterium mediated StWRKY11 gene expression vector was studied.1.Extraction of total RNA,from potato to amplified the gene and cloned it to construct recombinant carriers.Structure prediction and analysis were conducted through bioinformatics-related software.Results show that the StWRKY11 gene with a reading frame of 1005bp was cloned from the potato Atlantic,encoding 334 amino acids.Expression protein molecular formula C₃₀₁₃H₅₀₂₃N₁₀₀₅O₁₂₆₀S₁₉₉,contains a typical WRKYGQK conserved domain,and zinc finger structure CX₅CX₂₃HXH,belong to the second class II d subfamily.Expressed as hydrophobic stability proteins,No transmembrane region and signal peptide domain,the secondary structural elements are a-spiral,extended chain,β-folding and irregular winding,with the highest,61.68%and 29 phosphorylation sites,possibly localized within the nucleus.Upstream of the promoter has shun-regulatory elements related to resistance to stress response and shun-regulatory regulatory elements for growth,development and hormone response.The gene is closely related to the potato StWRKY5 gene,with a homology of 95%.2.The regeneration system of stem segments and leaves of virus-free potato seedlings was established.Callus and adventitious buds could be produced from stem and leaves on the medium with different concentrations of 6-BA,NAA and GA₃.The optimal medium for callus induction was MS+0.25 mg/L 6-BA+0.4 mg/L NAA and MS+5 mg/L 6-BA+0.3 mg/L NAA,respectively.The induction rates were 96.67%and 77.04%,respectively.The callus growth was light green and rice grain.The optimal medium for callus adventitious bud differentiation was with MS+5.0 mg/L6-BA+0.1 mg/L NAA+10.0 mg/L GA₃,the adventitious bud differentiation rate reached more than 40%,and the buds were more and stronger.3.The recombinant clone vector was used as a template,and the plant overexpression vector p CAMBIA1302-StWRKY11 was successfully constructed by in-fusion method,and the recombinant plasmid was successfully transferred into Agrobacterium tumefaciens strain GV3101.Stem segments and leaves were used as explants to construct the potato genetic transformation system.PCR results showed that Agrobacterium-mediated gene StWRKY11 could be successfully transferred into the Atlantic potato varieties.