Study On Anti-stress And Mechanism Of Panax Ginseng PR Family Proteins—defensin And Lipid Transfer Protein

Objective:Taking the plant immune response as the starting point,screening important anti-stress genes of ginseng,clarifying its biological activity and exploring its mechanism,so as to lay scientific and technological support for the prevention and control of ginseng diseases and the development of biological pesticides.Methods:Taking the roots,stems,and leaves of five-year-olds exhibiting leaf,flowering,green fruit,red fruit,and late fruit as the research objects,the modified Trizol method was used to extract the total RNA of ginseng,and the Illumina/Solexa sequencing platform and paired ends were used.Sequencing methods are used to sequence the transcriptome of ginseng to construct a transcriptome database of different tissues of ginseng;combine spatio-temporal expression analysis with gene expression feature analysis to screen ginseng stress resistance candidate genes from the database;use bioinformatics analysis methods to analyze defensins and lipids The structure prediction analysis of mass transfer protein;Arabidopsis thaliana is used as a model organism,and Agrobacterium-mediated methods are used to screen transdefensin and lipid transfer protein gene-positive plants;to prevent root rot,rust,high salt,drought,etc.Stress resistance is used as an indicator to study the biological activities of two genes;by detecting the key signals in plant resistance signal transduction:salicylic acid,jasmonic acid,chlorophyll,electrolyte rate,malondialdehyde,proline,SOD,etc.And the PR expression of genes related to stress resistance,and explore the biological mechanism of the two genes.Results:1.Established 15 ginseng transcriptome databases with different growth periods and different growth parts,and through spatio-temporal analysis,18genes related to stress resistance were screened,including 11 members of the family and 7 members of other families.It was further correlated with ginseng disease,and two candidate genes were screened out:defensin and lipid transfer protein.2.The structural analysis results of the protein showed that the total molecular weight of the defensin protein is 8927.33,the total number of amino acids is 80,and the theoretical isoelectric point is 8.36;all amino acid residues are located outside the biofilm;it has a typical structure of the defensin family,1 alpha helix and 3 A?-sheet;it has relatively high sequence homology with the defensins of Panax notoginseng,ragweed,and carrot,confirming that it belongs to the defensin family.The total molecular weight of lipid transfer protein is 12091.19,the total number of amino acids is 120,and the theoretical isoelectric point is 8.46;the protein has a transmembrane structure;the typical structure of lipid transfer protein includes 4alpha helices;the sequence homology is relatively compared with tea tree and kiwi fruit high.3.Successfully constructed defensin and lipid transfer protein plant expression vectors p CAMBIA1303-DEF,p CAMBIA1303-LTP;selected Arabidopsis homozygous strains D₁,D₂,D₃,D₄and translipid transfer that were trans-defensin proteins Protein homozygous lines L1 and L2 of Arabidopsis thaliana.4.Investigate the defensive effects of transgenic Arabidopsis against Fusarium oxysporum,rust fungi,high salt,and drought stress.The results show that high expression of defensin genes can significantly improve the resistance of Arabidopsis to root rot,but it is The effect of rust is weak;it can significantly increase the germination rate of Arabidopsis seeds under salt stress and drought stress,but it fails to resist the decline in survival rate caused by salt stress and drought stress.High expression of translipid transfer protein gene can improve the resistance of Arabidopsis to root rot and rust;it can significantly increase the germination rate of Arabidopsis seeds under salt stress and drought stress,and can resist salt stress and drought stress The resulting decrease in survival rate.5.Arabidopsis thaliana transfected with defensin genes can increase the salicylic acid content and reduce the jasmonic acid content under Fusarium oxysporum;at the same time,it can increase the salicylic acid synthase key gene PAL and salicylic acid activation in the salicylic acid signaling pathway Marker genes PR1,PR2 and PR5;inhibit the expression of key genes AOS and COR-I of jasmonate synthase.To achieve.In conclusion,the effect of defensins on the stress of Fusarium oxysporum depends on the salicylic acid signaling pathway and inhibits the jasmonic acid signaling pathway.6.Transgenic Arabidopsis thaliana with defensin gene can inhibit the increase of malondialdehyde content under salt stress,thereby reducing the damage of plant cell membrane caused by high salt.It can increase the SOD activity under drought stress and inhibit the increase of malondialdehyde content,thereby reducing the toxic effect of reactive oxygen species and slowing down the damage of plant cell membranes caused by water loss.7.Transformation of lipid transfer protein gene into Arabidopsis can increase chlorophyll synthesis and reduce malondialdehyde content under salt stress,thereby reducing plant cell membrane damage caused by high salt.It can increase proline synthesis,increase SOD activity,and reduce malondialdehyde content under drought stress,indicating that the high expression of lipid transfer protein protects the osmotic potential balance between cytoplasm and vacuoles under osmotic stress,and reduces the toxicity of reactive oxygen species.Function and slow down the damage of plant cell membrane caused by water loss.Conclusion:1.Establishing the ginseng transcriptome database of different growth stages and different growth parts and analyzing the spatio-temporal expression of genes is a feas ible way to screen ginseng stress resistance candidate genes.2.During the growth of ginseng,ginseng can specifically and highly express anti-stress genes represented by the PR family through the activation of its own immune response system.Among them,defensins and lipid transfer protein ginseng diseases are more closely related.3.Ginseng defensins and lipid transfer proteins have typical structures of their families.4.Ginseng defensin and lipid transfer protein have different biological activities.High expression of defensin genes can significantly improve the resistance of Arabidopsis to root rot,but has a weak effect on rust;high expression of translipid transfer protein genes can have significant resistance to root rot and rust.improve.Highly expressed lipid transfer protein genes are more resistant to abiotic stress than highly expressed defensin genes.5.The disease resistance of transgenic Arabidopsis to root rot bacteria is achieved by activating the salicylic acid signaling pathway and inhibiting the jasmonic acid signaling pathway.6.Under abiotic stress conditions,both genes can increase SOD activity and reduce malondialdehyde content,but lipid transfer protein can also increase the synthesis of chlorophyll and proline.