Cloning And Disease Resistance Analysis Of Soybean Disease Resistance Gene GmLNDR

The frequent occurrence of Phytophthora root rot seriously affects the production of soybean.The study found:(1)The disease-resistant protein containing the NBS-LRR domain can act as a receptor to recognize the elicitor and stimulate the defense response in the plant;(2)This kind of protein has important application value in the process of plant cell death and development;(3)Studies in Arabidopsis have found that the resistance of the protein to disease is positively regulated.In the early stage of this study,the differential expression of the Gm LNDR(大豆含LRR and NB-ARC结构域抗病基因),Arabidopsis thaliana LRR and NB-ARC domains-containing disease resistance protein)was obtained by sequencing the transcriptome of the soybean"Jinong 18"mutant"M18"seedling stage,and further explore the specific mechanism and biological function of this gene in soybean disease resistance.First,the homologous PCR technology(同源PCR技术,Homologous PCR)was used to clone the Gm LNDR gene from the"M18"seedling leaf genome;Furthermore,the plant overexpression vector p3301-Gm LNDR and plant RNAi interference expression vector p3301-RNAi-Gm LNDR were constructed respectively by the technology of the seamless cloning(无缝克隆技术,Seamless cloning);Transform the vector plasmid to the receptor"JN18"by the method of Agrobacterium-mediated and the method of pollen tube pathway;Under the environment of inoculation with Phytophthora sojae,to explore the expression of this gene in the roots,the stems and leaves of"JN18"at the seedling stage by the method of hypocotyl infection;Finally,the transgenic offspring were tested,and the disease resistance to be identified for verifying the function of the gene.The main findings are as follows:1.The analysis of the nucleic acid sequence structure of the target gene shows:the gene is a stable spatial conformation based on theα-helical structure,with a size of 501bp,a hydrophilic protein;It is closely related to the known Arabidopsis AT3G14460 gene(拟南芥含LRR and NB-ARC结构域抗病基因,AT3G14460);The gene was cloned by homologousPCR technology,consequently the cloning vector PMD-18T-Gm LNDR was constructed successfully.2.The Construction structures of plant expression vector show that: based on plant expression vector p CAMBIA3301,it is by seamless cloning technology to construct plant overexpression vector p3301-Gm LNDR and plant interference expression vector p3301-RNAi-Gm LNDR.3.The result of genetic transformation shows: it is by Agrobacterium-mediated method and pollen tube pathway,The achieved plant overexpression vector p3301-Gm LNDR and plant RNAi interference expression vector p3301-RNAi-Gm LNDR were transferred to soybean receptor "JN18".4.The PCR test results of the transformed strains showed that: there appeared the specific bands respectively consistent with the known sequence of promoter 35 S,terminator Nos,and screening marker Bar,which initially proved that the target gene Gm LNDR was successfully transferred into soybean receptor Jinong 18.(1)Agrobacterium-mediated method was used to obtain 7 plants of TO generation transgenic Gm LNDR overexpression vector gene,and 9 plants of TO generation transgenic Gm LNDR interference expression vector gene;(2)the method of Agrobacterium-mediated was used to obtain 4 T1 generation Gm LNDR overexpression vector gene plants and 4 T1 generation Gm LNDR interference vector gene plants;the pollen tube pathway was used to obtain 2 T1 generation Gm LNDR overexpression vector gene plants and 3T1 generation Gm LNDR interference vector gene plants;(3)the method of Agrobacterium-mediated was used to obtain 3 T2 generation Gm LNDR overexpression vector gene plants and 3 T2 generation Gm LNDR interference vector gene plants;the pollen tube pathway was used to obtain 3 T2 generation Gm LNDR overexpression vector gene plants and 3T2 generation Gm LNDR interference vector gene plants;5.The probe was prepared with the screening marker Bar.The Southern test results of T2 transgenic plants showed that: the trans-Gm LNDR overexpression vector and the trans-Gm LNDR interference expression vector,were successfully integrated into the soybean receptor genome,and the integration sites were different.6.Fluorescence quantitative PCR results of T2 generation transgenic plants showed that:The expression level of Gm LNDR overexpression vector gene in soybean roots,stems and leaves is higher than that of receptor CK,and the expression level of Gm LNDR interference expression vector gene in soybean roots,stems and leaves is lower than receptor CK;In both types of expression,the expression level of the leaf is the highest,followed by the root and the stem at least;This shows that transgenic Gm LNDR overexpression vector gene promotes theexpression of soybean endogenous genes,while transfection of Gm LNDR interference expression vector gene suppresses the expression of soybean endogenous genes.7.The identification and test results of Phytophthora root rot resistance in transgenic offspring showed that: the mortality rate of plants infected with Phytophthora infestation by the expression vector was 22.22%,which was a level of resistance;The mortality rate of the receptor ‘JN18’ is 48.14%,which belongs to the level of intermediate resistance;The mortality rate of Phytophthora inoculated with the expression vector plants was 70.37%,which was susceptible.This indicates that the expression level of this gene is directly related to the resistance of soybean plants to Phytophthora infestans.8.The results of disease resistance comparison showed that :the mortality rate of Phytophthora root rot inoculated with Gm LNDR overexpression vector was 22.22%,while the mortality rate of Phytophthora root rot inoculated with hrp Zpsta overexpression vector were11.11% and 18.51%,the former gene is only lower than the latter gene with broad-spectrum disease resistance,which may be related to the function of Gm LNDR gene.