Integrated top-down and bottom-up proteomics mass spectrometry for the characterization of endogenous ribosomal protein heterogeneity

Ribosomes are abundant,large RNA-protein complexes that are the sites of all protein synthesis in cells.Defects in ribosomal proteins(RPs),including proteoforms arising from genetic variations,alternative splicing of RNA transcripts,post-translational modifications and alterations of protein expression level,have been linked to a diverse range of diseases,including cancer and aging.Comprehensive characterization of ribosomal proteoforms is challenging but important for the discovery of potential disease biomarkers or protein targets.In the present work,using E.coli 70S RPs as an example,we first developed a top-down proteomics approach on a Waters Synapt G2 Si mass spectrometry(MS)system,and then applied it to the HeLa 80S ribosome.The results were complemented by a bottom-up approach.In total,50 out of 55 RPs were identified using the top-down approach.Among these,more than 30 RPs were found to have their N-terminal methionine removed.Additional modifications such as methylation,acetylation,and hydroxylation were also observed,and the modification sites were identified by bottomup MS.In a HeLa 80S ribosomal sample,we identified 98 ribosomal proteoforms,among which multiple truncated 80S ribosomal proteoforms were observed,the type of information which is often overlooked by bottom-up experiments.Although their relevance to diseases is not yet known,the integration of topdown and bottom-up proteomics approaches paves the way for the discovery of proteoform-specific disease biomarkers or targets.

Effects of electroacupuncture on the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of rats with vascular dementia

This study investigated the mechanism underlying electroacupuncture therapy for vascular dementia through electroacupuncture at the acupoints of Baihui （DU20）, Dazhui （DU14）, and bilateral Shenshu （BL23） in a rat model of vascular dementia produced by bilateral middle cerebral artery occlusion. Morris water maze test showed that electroacupuncture improved the learning ability of vascular dementia rats. Western blot assay revealed that the expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in vascular dementia rats was significantly increased after electroacupuncture, compared with the model group that was not treated with acupuncture. The average escape latency was also shortened after electroacupuncture, and escape strategies in the spatial probe test improved from edge and random searches, to linear and trending swim pathways. The experimental findings indicate that electroacupuncture improves learning and memory ability by up-regulating expression of p70 ribosomal protein S6 kinase and ribosomal protein S6 in the hippocampus of vascular dementia rats.

Molecular Cloning and Bioinformatical Analysis of a cDNA Encoding Mitochondrial 50S Ribosomal Protein L21 from Hevea brasiliensis Muell. Arg.

[Objective] ＂Tapping panel dryness （TPD）＂, a syndrome known as tapping incision blocked partly or entirely during latex exploiting, has become the most important factor causing great losses for rubber production. Aiming to elucidate the molecular mechanism of tapping panel dryness occurrence, this study carried out molecular cloning and bioinformatical analysis of a mRPL21 cDNA sequence, a gene associated with TPD. [Method] In a preliminary study, an expressed sequence tag （EST） encoding a deduced protein homologous to mitochondrial 50S ribosomal protein L21 （mRPL21）, which showed to be down-regulated in the latex of TPD-affected rubber trees, was isolated by suppression subtractive hybridization （SSH）. After ESTs assembling and RT-PCR validation, an 853 bp cDNA sequence with an open reading frame （ORF） was cloned, which was named as HbmRPL21 under GenBank accession number of HM230670. [Result] Bioinformatical analysis suggests that HbmRPL21 encodes a deduced polypeptide of 271 amino acids with a theoretical molecular weight （Mw） of 30.52 kDa and isolectric point （pI） of 8.40, and HbmRPL21 is a mitochondrion-targeted protein with a conserved domain of Ribosomal_L21p involving translation. Homology analysis reveals high amino acid sequence identity of mRPL21 from plants, while diversity of that between plant and animal kingdom. [Conclusion] This study laid the basis for further revealing the biological functions of mRPL21 in TPD-affected rubber trees.

18β-glycyrrhetinic acid regulates mitochondrial ribosomal protein L35-associated apoptosis signaling pathways to inhibit proliferation of gastric carcinoma cells

BACKGROUND Gastric carcinoma(GC)is a common gastrointestinal malignancy worldwide.Based on the cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the mechanism by which 18β-glycyrrhetinic acid(18β-GRA)regulates mitochondrial ribosomal protein L35(MRPL35)related signal proteins to inhibit the proliferation of GC cells.METHODS Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823.The apoptosis and cell cycle were assessed by flow cytometry.Cell invasion and migration were evaluated by Transwell assay,and cell scratch test was used to detect cell migration.Furthermore,a tumor model was established by hypodermic injection of 2.5×106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect MRPL35 expression in the engrafted tumors in mice.We used the term tandem mass tag(TMT)labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins(DEPs)extracted from GC cells and control cells after 18β-GRA intervention.A detailed bioinformatics analysis of these DEPs was performed,including Gene Ontology annotation and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis,and so on.Moreover,STRING database(https://string-db.org/)was used to predict proteinprotein interaction(PPI)relationships and Western blot was used to detect the expression of proteins of interest in GC cells.RESULTS The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose-and timedependent manner.It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase.The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose,and the migration and invasiveness of GC cells were inhibited.The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice,and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice.Using TMT technology,609 DEPs,among which 335 were up-regulated and 274 were down-regulated,were identified in 18β-GRA intervention compared with control.We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways,such as cellular processes,biological regulation,and TP53 signaling pathway.Notably,after the drug intervention,MRPL35 expression was significantly down-regulated(P=0.000247),TP53 expression was up-regulated(P=0.02676),and BCL2L1 was down-regulated(P=0.01699).Combined with the Retrieval of Interacting Genes/Proteins database,we analyzed the relationship between MRPL35,TP53,and BCL2L1 signaling proteins,and we found that COPS5,BAX,and BAD proteins can form a PPI network with MRPL35,TP53,and BCL2L1.Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells,MRPL35,TP53,and BCL2L1 showed dose-dependent up/down-regulation,and the expression of COPS5,BAX,and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION 18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35,COPS5,TP53,BCL2L1,BAX,and BAD.

Characterization and analysis of ribosomal proteins in two marine calanoid copepods

Copepods are among the most abundant and successful metazoans in the marine ecosystem. However, genomic resources related to fundamental cellular processes are still limited in this particular group of crustaceans. Ribosomal proteins are the building blocks of ribosomes, the primary site for protein synthesis. In this study, we characterized and analyzed the c DNAs of cytoplasmic ribosomal proteins(c RPs) of two calanoid copepods, P seudodiaptomus poplesia and A cartia pacifi ca. We obtained 79 c RP c DNAs from P. poplesia and 67 from A. pacifi ca by c DNA library construction/sequencing and rapid amplifi cation of c DNA ends. Analysis of the nucleic acid composition showed that the copepod c RP-encoding genes had higher GC content in the protein-coding regions(CDSs) than in the untranslated regions(UTRs), and single nucleotide repeats(>3 repeats) were common, with "A" repeats being the most frequent, especially in the CDSs. The 3′-UTRs of the c RP genes were signifi cantly longer than the 5′-UTRs. Codon usage analysis showed that the third positions of the codons were dominated by C or G. The deduced amino acid sequences of the c RPs contained high proportions of positively charged residues and had high p I values. This is the fi rst report of a complete set of c RP-encoding genes from copepods. Our results shed light on the characteristics of c RPs in copepods, and provide fundamental data for further studies of protein synthesis in copepods. The copepod c RP information revealed in this study indicates that additional comparisons and analysis should be performed on different taxonomic categories such as orders and families.

Antibodies against ribosomal protein S29(RPS29)fused with glutathione's transferase specially react with native RPS29 in mouse and human cells

The ribosomal protein S29 also known as RPS29, is not only a component of the 40S subunit of ribosome, but also involved in embryonic development, oncogenesis and other pathologic conditions. However, rare commercial antibody against RPS29 restricts the discovery of precise physiological and pathological function of this protein. In this study, the whole RPS29 gene was inserted into plasmid pGEX-6p-1 to express glutathione＇s transferase （GST） fusion proteins in Escherichia eoli （E. coli） strain BL21. High yields of soluble recombinant proteins were obtained. Mice were immunized with the recombinant RPS29 protein. The serum from the immunized mice could specially react with purified recombinant RPS29 proteins and native RPS29 proteins in CCE cells by western blotting, immunofluorescence staining and flow cytometric analysis. Further more the polyclonal antibodies also reacted specifically with human cell strain ECV304, which showed typical cytoplasmatic fluorescence. The polyclonal antibodies we prepared would be an available tool for studying the roles of RPS29 in embryonic development and human diseases.

Knockdown of 60S ribosomal protein L14‑2 reveals their potential regulatory roles to enhance drought and salt tolerance in cotton

Background:Cotton is a valuable economic crop and the main significant source of natural fiber for textile industries globally.The effects of drought and salt stress pose a challenge to strong fiber and large-scale production due to the ever-changing climatic conditions.However,plants have evolved a number of survival strategies,among them is the induction of various stress-responsive genes such as the ribosomal protein large(RPL)gene.The RPL gene families encode critical proteins,which alleviate the effects of drought and salt stress in plants.In this study,comprehensive and functional analysis of the cotton RPL genes was carried out under drought and salt stresses.Results:Based on the genome-wide evaluation,26,8,and 5 proteins containing the RPL14B domain were identified in Gossypium hirsutum,G.raimondii,and G.arboreum,respectively.Furthermore,through bioinformatics analysis,key cis-regulatory elements related to RPL14B genes were discovered.The Myb binding sites(MBS),abscisic acid-responsive element(ABRE),CAAT-box,TATA box,TGACG-motif,and CGTCA-motif responsive to methyl jasmonate,as well as the TCA-motif responsive to salicylic acid,were identified.Expression analysis revealed a key gene,Gh_D01G0234(RPL14B),with significantly higher induction levels was further evaluated through a reverse genetic approach.The knockdown of Gh_D01G0234(RPL14B)significantly affected the performance of cotton seedlings under drought/salt stress conditions,as evidenced by a substantial reduction in various morphological and physiological traits.Moreover,the level of the antioxidant enzyme was significantly reduced in VIGS-plants,while oxidant enzyme levels increased significantly,as demonstrated by the higher malondialdehyde concentration level.Conclusion:The results revealed the potential role of the RPL14B gene in promoting the induction of antioxidant enzymes,which are key in oxidizing the various oxidants.The key pathways need to be investigated and even as we exploit these genes in the developing of more stress-resilient cotton germplasms.

Development of a competitive array for discriminative determination of amphenicols in egg based on ribosomal protein L16

Objective:Amphenicols(chloramphenicol,thiamphenicol and forfenicol)can cause aplastic anaemia and other severe side effects to consumers;therefore,it is necessary to inspect their residues in foods of animal origin.However,there has been no report on the use of amphenicols receptor for the determination of their residues,and none of the previously reported immunoassays for amphenicols can differentiate the specifc species.Materials and Methods:In this study,the ribosomal protein L16 of Escherichia coli was frst expressed,and its intermolecular interaction mechanisms with the three amphenicols was studied using the molecular docking technique.The protein was then combined with three enzymelabelled conjugates to develop a direct competitive array on microplate for determination of the three drugs in egg.Results:Due to the use of principal component analysis to analyse the data,this method could discriminate the three drugs in the range 0.1–10 ng/mL,and the limits of detection for the three drugs were in the range of 0.0002–0.0009 ng/mL.The analysis results for the unknown egg samples were consistent with a liquid chromatography–tandem mass spectrometry method,and the method performances were superior to the previous immunoassays for amphenicols.Conclusion:This is the frst paper reporting the use of ribosomal protein L16 to develop a competitive array for discriminative determination of amphenicols in food samples.

Roles of ribosomal proteins in hematologic disorders and cancers:a review

Ribosomes are important organelles for synthesizing proteins in cells.They are composed of ribosomal RNA and more than 80 ribosomal proteins.It is well known that an essential function of ribosomal proteins is to participate in protein translation.In addition,ribosomal proteins also perform extra-ribosomal functions,such as participating in DNA replication,transcription,and damage repair,regulating cell growth,proliferation,apoptosis,and transformation.In recent years,studies have shown that alterations in ribosomal protein synthesis or function can lead to various hematologic diseases,including Diamond-Blackfan anemia,5q-syndrome,Shwachman-Diamond syndrome,and other blood system diseases.Moreover,abnormal expressions of specific ribosomal protein genes have been reported in many malignant tumors.In this review,we elaborated on the changes in ribosomal proteins in hepatocellular carcinoma and colorectal,prostate,gastric,esophageal,and other cancers and discussed the relationship between ribosomal proteins and the occurrence of hematologic disorders and cancers.

Hmo1:A versatile member of the high mobility group box family of chromosomal architecture proteins

Eukaryotic chromatin consisting of nucleosomes connected by linker DNA is organized into higher order structures,which is facilitated by linker histone H1.Formation of chromatin compacts and protects the genome,but also hinders DNA transactions.Cells have evolved mechanisms to modify/remodel chromatin resulting in chromatin states suitable for genome functions.The high mobility group box(HMGB)proteins are non-histone chromatin architectural factors characterized by one or more HMGB motifs that bind DNA in a sequence nonspecific fashion.They play a major role in chromatin dynamics.The Saccharomyces cerevisiae(yeast hereafter)HMGB protein Hmo1 contains two HMGB motifs.However,unlike a canonical HMGB protein that has an acidic C-terminus,Hmo1 ends with a lysine rich,basic,C-terminus,resembling linker histone H1.Hmo1 exhibits characteristics of both HMGB proteins and linker histones in its multiple functions.For instance,Hmo1 promotes transcription by RNA polymerases I and II like canonical HMGB proteins but makes chromatin more compact/stable like linker histones.Recent studies have demonstrated that Hmo1 destabilizes/disrupts nucleosome similarly as other HMGB proteins in vitro and acts to maintain a common topological architecture of genes in yeast genome.This minireview reviews the functions of Hmo1 and the underlying mechanisms,highlighting recent discoveries.

Ribosomal proteins:functions beyond the ribosome

Although ribosomal proteins are known for playing an essential role in ribosome assembly and protein translation,their ribosomeindependent functions have also been greatly appreciated.Over the past decade,more than a dozen of ribosomal proteins have been found to activate the tumor suppressor p53 pathway in response to ribosomal stress.In addition,these ribosomal proteins are involved in various physiological and pathological processes.This review is composed to overview the current understanding of how ribosomal stress provokes the accumulation of ribosome-free ribosomal proteins,as well as the ribosome-independent functions of ribosomal proteins in tumorigenesis,immune signaling,and development.Wealso propose the potential of applying these pieces of knowledge to the development of ribosomal stress-based cancer therapeutics.

The role of ribosomal proteins in the regulation of cell proliferation, tumorigenesis, and genomic integrity

Ribosomal proteins （RPs）, the essential components of the ribosome, are a family of RNA-binding proteins, which play prime roles in ribosome biogenesis and protein translation. Recent studies revealed that RPs have additional extra-ribosomal func- tions, independent of protein biosynthesis, in regulation of diverse cellular processes. Here, we review recent advances in our understanding of how RPs regulate apoptosis, cell cycle arrest, cell proliferation, neoplastic transformation, cell migration and invasion, and tumorigenesis through both MDM2/p53-dependent and p53-independent mechanisms. We also discuss the roles of RPs in the maintenance of genome integrity via modulating DNA damage response and repair. We further discuss mutations or deletions at the somatic or gennline levels of some RPs in human cancers as well as in patients of Diamond-Blackfan ane- mia and 5q- syndrome with high susceptibility to cancer development. Moreover, we discuss the potential clinical application, based upon abnormal levels of RPs, in biomarker development for early diagnosis and/or prognosis of certain human cancers. Finally, we discuss the pressing issues in the field as future perspectives for better understanding the roles of RPs in human cancers to eventually benefit human health.

Effects of Yiqi Huoxue Recipe （益气活血方） and Coxsackie Virus B Type 3 on the Expression of Ribosomal Protein S20 in Rat Cardiac Myocytes

Objective： To study the effects of Yiqi Huoxue Recipe （益气活血方） and Coxsackie B virus type 3 （CVB3） on the expression of ribosomal protein S20 in rat cardiac myocytes, to explore the pathogenesis of myocarditis induced by CVB3 and the mechanism of Yiqi Huoxue Recipe on gene level, and to further investigate whether Yiqi Huoxue Recipe is an effective prescription for CVB3 myocarditis. Methods： A modified suppression subtractive hybridization （SSH） was used to isolate differentially expressed genes between the CVB3 infection group and the treatment group with Yiqi Huoxue Recipe. The results were further verified by fluorescence RT-PCR. Results： The results of SSH showed that the gene expression of ribosomal protein S20 in the treatment group was higher than that in the CVB3 infection group （P〈0.05）, which agreed with the results of fluorescent RT-PCR. Conclusion： Down-regulation of ribosomal protein S20 mRNA expression might be one of the mechanisms in CVB3 myocarditis, and Yiqi Huoxue Recipe could achieve the treatment of viral myocarditis by regulating the expression of ribosomal protein S20.

cDNA sequence analysis of ribosomal protein S13 gene in Plutella xylostella （Lepidoptera： Plutellidae）

Ribosomal protein S 13 gene has been cloned and analyzed in many organisms,but there are few documents relating to insects. In this communication, the full-length cDNA sequence of ribosomal protein S 13 gene in the diamondback moth, Plutella xylostella(Lepidoptera: Plutellidae), was determined by using PCR amplification technique. The features of the ribosomal protein S 13 gene sequence were analyzed and the deduced amino acids sequence was compared with those from other insects. The results of multi-alignment of the amino acid sequences between the diamondback moth and other insect species revealed that this gene sequence is highly conserved in insects. Based on maximum likelihood method, a phylogenetic tree was constructed from 10 different species using PHYLIP software. It showed that nematode is one separate lineage and the five insect speciesbe long to another lineage, whereas those species higher than insects form the third one. The pattern of this phylogenetic tree evidently represented the evolution of different species.

Molecular characterization and m RNA expression of ribosomal protein L8 in Rana nigromaculata during development and under exposure to hormones

Like Xenopus laeuis, some species of the Rang genus are also used to study endocrine disrupting chemicals （EDCs）. Although ribosomal protein L8 （rp18） is the most-used reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction in Rang, its suitability as the reference gene has never been validated in any species of the Rana genus. We characterized rp18 cDNA in Rana nigromaculata, a promising native species in East Asia for assaying endocrine disrupting effects. We found that the rp18 cDNA consisted of 919 bp and encoded 257 amino acids, exhibiting high identities of amino acid sequence with known rp18 in other Rana species. Then, we examined the stability of mRNA expression during development. Compared with elongation factor 1 alpha 1, another common housekeeping gene, neither stage-specific nor tissue-specific expression of the rp18 gene was found in all tissues examined （brain, liver, intestine, tail, testis and ovary） during R. nigromaculata development. Finally, we investigated rp18 expression under exposure to hormones. No change in rp18 mRNA expression was found under exposure to thyroid hormone （T4） and estrogen （estradiol）, whereas expression of the corresponding biomarker genes was induced. Our results show that rp18 is an appropriate reference gene for analyzing gene expression by quantitative reverse transcription polymerase chain reaction for assaying EDCs using R. nigromaculata, and might also provide support for using rp18 as a reference gene in other Rang species due to the high conservation of rp18 among the Rana genus.

Partial genomic organization of ribosomal protein S7 gene from malaria vector Anopheles stephensi

In this study, we describe the partial genomic organization of ribosomal protein S7 gene isolated from the mosquito Anopheles stephensi. Initially a 558 bp partial cDNA sequence was amplified as precursor mRNA sequence containing 223 bp long intron. 5＇ and 3＇ end sequences were recovered using end specific rapid amplification of cDNA ends （RACE） polymerase chain reaction. The full-length cDNA sequence was 914 nucleotide long with an open reading frame capable of encoding 192 amino acid long protein with calculated molecular mass of 22174 Da and a pI point of 9.94. Protein homology search revealed 〉75% identity to other insect＇s S7 ribosomal proteins. Analysis of sequence alignment revealed several highly conserved domains, one of which is related to nuclear localization signal （NLS） region of human rpS7. Interestingly, intron nucleotide sequence comparison with A. gambiae showed a lesser degree of conservation as compared to coding and untranslated regions. Like this, early studies on the genomic organization and cDNA/ Expressed sequence tag analysis （EST） could help in genome annotation ofA. stephensi, and would be likely to be sequenced in the future.

A novel full-length gene of human ribosomal protein L14.22 related to human glioma

Background This study was undertaken to obtain differentially expressed genes related to human glioma by cDNA microarray and the characterization of a novel full-length gene. Methods Total RNA was extracted from human glioma and normal brain tissues, and mRNA was used as a probe. The results of hybridization procedure were scanned with the computer system. The gene named 507E08 clone was subsequently analyzed by northern blot, bioinformatic approach, and protein expression. Results Fifteen differentially expressed genes were obtained from human glioma by hybridization and scanning for four times. Northern blot analysis confirmed that the 507E08 clone was low expressed in human brain tissue and over expressed in human glioma tissues. The analysis of BLASTn and BLASTx showed that the 507E08 clone was a novel full-length gene, which codes 203 amino acid of protein and is called human ribosomal protein 14.22 gene. The nucleotide sequence had been submitted to the GenBankTM with the accession number of AF329277. After expression in E.coli., protein yielded a major band of apparent molecular mass 22 kDa on an SDS-PAGE gel. Conclusions cDNA microarray technology can be successfully used to identify differentially expressed genes. The novel full-length gene of human ribosomal protein 14.22 may be correlated with the development of human glioma.

RIBOSOMAL PROTEIN S12 GENE MUTATION AFFECTING THE EXPRESSION OF BACTERIOPHAGE LAMBDA N GENE

Previous results in this laboratory indicated that in ribosomal protein S12 strepto-mycin-dependent mutants of Bacillus subtilis, the burst size of bacteriophage φ105 was decreased, and the protein synthesis was inhibited, while the DNA and

Ribosome Inactivating Proteins from Plants Inhibiting Viruses

Many plants contain ribosome inactivating proteins (RIPs) with N-glycosidase activity, which depurinate large ribosomal RNA and arrest protein synthesis. RIPs so far tested inhibit replication of mRNA as well as DNA viruses and these proteins, isolated from plants, are found to be effective against a broad range of viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV) and herpes simplex virus (HSV). Most of the research work related to RIPs has been focused on antiviral activity against HIV; however, the exact mechanism of antiviral activity is still not clear. The mechanism of antiviral activity was thought to follow inactivation of the host cell ribosome, leading to inhibition of viral protein translation and host cell death. Enzymatic activity of RIPs is not limited to depurination of the large rRNA, in addition they can depurinate viral DNA as well as RNA. Recently, Phase I/II clinical trials have demonstrated the potential use of RIPs for treating patients with HIV disease. The aim of this review is to focus on various RIPs from plants associated with anti-HIV activity.

Isolation of a genomic DNA for Jatropha curcas ribosome inactivating protein and its tobacco transformation

Genomic DNA for Jatropha curcas ribosome inactivating protein （JRIP） was cloned from total DNA of its leaves by polymerase chain reaction （PCR）. The no intron character was confirmed. The plant expression vector pBI121-JRIP was constructed by inserting the JRIP gene into pBI121 plasmid. The recombinant Agrobacterium EHA105 strain harboring pBI121-JRIP was constructed by conducting pBI121-JRIP to strain EHA 105. PCR and Southern blotting were carried out, and the results proved that the JRIP gene was integrated into tobacco genome. It might provide a new material for disease resistance tobacco species breeding.