Interstitial tissue-specific gene expression in mouse testis by intra-tunica albuguineal injection of recombinant baculovirus

这研究的目的是用修改 recombinant baculovirus 在鼠标睾丸为空隙的织物特定的蛋白质表示建立一个基因交货系统。格林荧光灯表示 recombinant baculovirus (GFP-baculovirus ) 的蛋白质(GFP ) ，在哪个昆虫房间特定的多面体倡导者被 cytomegalovirus (CMV ) 代替 IE 倡导者，习惯于 transfect 在 vitro 的阴囊的房间，并且为睾丸的空隙的织物的 intra-tunica albuguineal 注射。GFP 表示被荧光显微镜学在冻结的睾丸节监视。在阴囊的纸巾的 GFP 的表示被反向的抄写聚合酶链反应(RT-PCR ) 也估计，并且蛋白质表示被西方的污点估计。在 vitro 的阴囊的房间被修改 recombinant GFP-baculovirus 高效地感染。进老鼠睾丸的 GFP-baculovirus 的 Intra-tunica albuguineal 注射在空隙的纸巾导致了 GFP 表示的高水平。RT-PCR 分析清楚地在睾丸显示出 GFP 基因表示，特别地空隙的纸巾。编码了 recombinant 老鼠的修改 baculovirus 的 Intra-tunica albuguineal 注射像胰岛素的生长因素绑定蛋白质(IGFBP )-5 导致了睾丸和精液的 IGFBP-5 的增加。在结论，我们在阴囊的房间在 vivo 为基因表示开发了一个有效交货系统，特别地用修改 recombinant baculovirus 的 intra-tunica albuguineal 注射的空隙的织物的房间。这个方法将为在睾丸的外部生精的小管的阴囊的房间要求基因交货和蛋白质表示的申请是特别地相关的。

Construction of recombinant industrial Saccharomyces cerevisiae strain with bglS gene insertion into PEP4 locus by homologous recombination

The bglS gene encoding endo-l,3-1,4-β-glucanase from Bacillus subtilis was cloned and sequenced in this study. The bglS expression cassette, including PGK1 promoter, bglS gene fused to the signal sequence of the yeast mating pheromone α-factor (MFα1S), and ADH1 terminator with G418-resistance as the selected marker, was constructed. Then one of the PEP4 allele of Saccharomyces cerevisiae WZ65 strain was replaced by bglS expression cassette using chromosomal integration of polymerase chain reaction (PCR)-mediated homologous recombination, and the bglS gene was expressed simultaneously. The recombinant strain S. cerevisiae (SC-βG) was preliminarily screened by the clearing hydrolysis zone formed after the barley β-glucan was hydrolyzed in the plate and no proteinase A (PrA) activity was measured in fermenting liquor. The results of PCR analysis of genome DNA showed that one of the PEP4 allele had been replaced and bglS gene had been inserted into the locus of PEP4 gene in recombinant strains. Different endo-l,3-1,4-β-glucanase assay methods showed that the recombinant strain SC-βG had high endo-l,3-1,4-β-glucanase expression level with the maximum of 69.3 U/(h·ml) after 60 h of incubation. Meanwhile, the Congo Red method was suitable for the determination of endo-l,3-1,4-β-glucanase activity during the actual brewing process. The current research implies that the constructed yeast strain could be utilized to improve the industrial brewing property of beer.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

Induction of Chondrogenesis of Adipose-derived Stem Cells by Novel Recombinant TGF-β3 Fusion Protein

A new type of TGF-β3 fusion protein with targeted therapy function was constructed,and its feasibility and target specificity of inducing chondrogenesis were investigated by transfecting LAP-MMP-mTGF-β3 gene into adipose-derived stem cells (ADSCs).The recombinant pIRESEGFP-MMP was constructed by inserting the sense and antisense DNA of encoding the amino acid of the synthetic MMP enzyme cutting site into the eukaryotic expression vector pIRES-EGFP.LAP and mTGF-β3 fragments were obtained by using RT-PCR and inserted into the upstream and downstream of MMP from pIRES-EGFP-MMP respectively,and the recombinant plasmid of pIRES-EGFPLAP-MMP-mTGF-β3 was constructed,which was transferred to ADSCs.The ADSCs were cultured and divided in three groups:experimental group (MMP group),negative control group (no MMP) and non-transfection group.The morphological changes were observed microscopically,and the expression of proteoglycan and type Ⅱ collagen (ColⅡ) was detected by using Alcian blue staining and immunohistochemistry staining at 7th,14th and 21st day after culture.The recombinant plasmid of pIRES-EGFP-LAP-MMP-mTGF-β3 was correctly constructed by methods of enzyme cutting and sequencing analysis.The mTGF-β3 fusion protein was successfully expressed after transfection,and in the presence of the MMP,active protein mTGF-β3 was generated,which significantly promoted differentiation of ADSCs into chondrocytes and the expression of cartilage matrix.The novel fusion protein LAP-MMP-mTGF-β3 can targetedly induce differentiation of ADSCs into chondrocytes,which would open up prospects for target therapy of cartilage damage repair in future.

Construction and expression of SET gene and siRNA recombinant adenovirus vectors

Objective:To construct SET gene recombinant adenovirus vector and SET gene small interfering RNA(SiRNA) recombinant adenovirus vector for over-expression or knock-down of SET levels. Methods:The cDNA sequence of SET was cloned by reverse transcriptive polymerase chain reaction(RT-PCR) and the SET gene fragment was subcloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-SET.The shuttle plasmid pAdtrack-SET was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant Ad-CMV-SET and the recombinant Ad-CMV-SET was packaged and amplified in the AD293 cells.The expression of SET in AD293 cells was detected by Western blot.In addition,we constructed SET gene SiRNA recombinant adenovirus vector(Ad-H1-SiRNA/SET) and its efficacy of knockdown of SET protein was detected in infected GC-2spd(ts) cells by Western blot. Results:The recombinant adenovirus vectors,both SET gene recombinant adenovirus vector Ad-CMV-SET and SET gene SiRNA recombinant adenovirus vector Ad-H1-SiRNA/SET,were proven to be constructed successfully by the evidence of endonulease digestion and sequencing.AD293 cells infected with either recombinant adenovirus vector of Ad-CMV-SET or Ad-H1-SiRNA/SET were observed to express GFP.The expression of SET protein was up-regulated significantly in AD293 cells infected with SET gene recombinant adenovirus vector.On the contrast, SET protein was significantly down-regulated in the GC-2spd(ts) cells infected with Ad-H1-SiRNA/SET (P<0.05) and the knockdown efficiency was approximately 50%-70%. Conclusion:The recombinant adenovirus vector Ad-CMV-SET and Ad-H1-SiRNA/SET were successfully constructed and effectively expressed in germ cells and somatic cells.It provides an experimental tool for further study of SET gene in the physiological and pathophysiological mechanism of reproduction-related diseases.

Construction of the recombinant adenovirus vectors of CALB_2 gene and small interfering RNA,and application in testicular Leydig cells

Objective:To construct the recombinant adenovirus vectors of calretinin(CALB_2) gene and small interfering RNA(siRNA),for over-expression or knock-down of CALB_2,as the basis of functional investigation of CALB_2 in testicular Leydig cells. Methods:The cDNA sequence of CALB_2 was cloned by the reverse transcriptive polymerase chain reaction (RT-PCR).A CALB_2 gene fragment was sub-cloned into adenovirus shuttle plasmid pAdTrack-CMV to construct the shuttle plasmid pAdTrack-CALB_2.Then it was transformed into BJ5183 cells with the adenoviral backbone pAdEasy-1 to obtain the homologous recombinant AdCMV-CALB_2.The recombinant AdCMV-CALB_2 was further packaged and amplificated in AD293 cells.The expression of CALB_2 protein in AD293 cells was detected by Western blotting.CALB_2 protein was over-expressed in mouse Leydig cell line(MLTC-1 cells) by the constructed AdCMV-CALB_2. CALB_2 gene siRNA recombinant adenovirus vector(Ad-H1-siRNA/CALB_2 was also constructed simultaneously. Its efficacy was detected in AD293 cells by Western blotting. Results:The CALB_2 gene recombinant adenovirus vector AdCMV-CALB_2 and the CALB_2 gene siRNA recombinant adenovirus vector Ad-H1-siRNA/CALB_2 were constructed successfully by endonulease digestion and sequencing. AD293 cells infected with AdCMV-CALB_2 or Ad-H1-SiRNA/CALB_2 significantly expressed GFP protein. The expression of CALB_2 protein was significantly up-regulated in AD293 cells infected with AdCMV-CALB_2 plasmids, while the expression of CALB_2 protein was down-regulated by 60%in the CALB_2 cells infected with Ad-H1-SiRNA/CALB_2. MLTC-1 cells did not markedly express CALB_2 protein,while MLTC-1 cells infected with AdCMV-CALB_2 expressed CALB_2 protein at a high level. Conclusions:The recombinant adenovirus vectors of AdCMV-CALB_2 and Ad-H1-SiRNA/CALB_2 were successfully constructed.Both vectors effectively expressed in AD293.CALB_2 protein was over-expressed in the cultured MLTC-1 cells by AdCMV-CALB_2.These vectors of CALB_2 gene and Leydig cell line are useful tools for investigating the testicular function.

Intratracheal administration of recombinant adenovirus containing IL-18 gene in treatment of experimental metastatic lung carcinoma

Objective: To study the treatment of experimental metastatic lung carcinoma by intratracheal injection of IL-l8 gene recombinant adenovirus. Methods: (1)The mouse IL-18 mRNA was detected by RT-PCR, and the concentration of IL-18 and associated cytokines in lung lavages and blood were determined by ELISA at different time points after intratracheal injection of IL-18 recombinant adenovirus. (2)The lung metastasis nodes, mouse survival periods and survival rates were evaluated. NK activity and CTL activity were determined by 51Cr 4 h release method. Results: (1) IL-18 mRNA was detectable in lung tissue 6 h after intratracheal use of IL-18 recombinant adenovirus. and the concentration of IL-18 in lung lavage was higher than that in peripheral blood. Neither IL-18 mRNA nor IL-18 was detectable in control group. (2) Intratracheal use of IL-18 recombinant adenovirus resulted in increased CTL and NK activity, longer survival time and higher survival rates compared with the control group, showing significant therapeutic effect on expermental lung metastasis. Conclusion: Intratracheal use of adenovirus vector containing IL- 18 gene has therapeutic effect on the lung metastasis, denoting that gene therapy of lung diseases could be applied through airway directly with recombinant adenovirus.

Cloning and expression of human arresten gene and effect of its recombinant protein on endothelial cell proliferation

To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.

Construction of the recombinant expression vector for CD80-IgG fusion gene and its expression in Chinese hamster ovary cells

To construct the recombinant expression vector for CD80-IgG fusion gene and to express it functionally in Chinese hamster ovary cells in order to be used as an effective method to eliminate the immune escape of leukemic cells, the cDNA encoding the signal and extracellular domains of murine CD80 was generated by PCR amplification from plasmid pcDNA/B7 containing the full length cDNA of murine CD80 and those of murine IgG1, in which the Fc fragment was obtained through RT-PCR amplification from murine spleen cells. These two cDNAs were then cloned in tandem into eukaryotic expression vector pcDNA3.0 and the resultant recombinant plasmid pcDNA/CD80-IgG was then transfected to Chinese hamster ovary cells with liposome transfection reagent. The cell clones constitutively expressing CD80-IgG fusion protein were obtained by G418 screening. Western blotting and dot ELISA assay were used to detect the expression of the fusion protein in the supernatants of these cells. Meanwhile, the fusion protein expressed was then purified with affinity chromatography, and its biological activity was demonstrated by flow cytometry, MTT colorimetry and ELISA assay. The experimental results showed that these two inserts were successfully cloned into plasmid pcDNA3.0, and the highly purified fusion protein was obtained. This fusion protein was proved to be able to upregulate the density of CD80 on leukemic cells, deliberately promote the proliferative reactions of mouse allogenic lymphocytes and increase the killing activity against WEHI-3 cells from 49.7% up to 84.6%. In addition, this fusion protein could also enhance the IL-2 secretion from allogenic lymphocytes activated by tumor-specific antigens. It is concluded that the recombinant vector constructed can be functionally expressed in the mammalian cells, thus providing a solid foundation for the further investigation on the mechanism to eliminate the immune escape of leukemic cells in vivo.

Construction of HBV-specific ribozyme and its recombinant with HDV and their cleavage activity in vitro

AIM To construct the recombinant of HDVcDNA and HBV-specific ribozyme gene byrecombinant PCR in order to use HDV as atransporting vector carrying HBV-specificribozyme into liver cells for inhibiting thereplication of HBV.METHODS We separately cloned the ribozyme（RZ）gene and recombinant DVRZ（comprisingHDV cDNA and HBV-specific ribozyme gene）intothe downstream of T7 promoter of pTAdv-Tvector and studied the in vitro cleavage activityof their transcripts（rRZ,rDVRZ）on target RNA（rBVCF）from in vitro transcription of HBV Cgene fragment（BVCF）.RESULTS Both the simple（rRZ）and therecombinant ribozyme rDVRZ could efficientlycatalyze the cleavage of target RNA（rBVCF）under different temperatures（37℃,42℃ and55℃）and Mg2+concentrations（10 mmol/L,15 mmol/L and 20 mmol/L）and their catalyticactivity tended to increase as the temperaturewas rising.But the activity of rRZ was evidentlyhigher than that of rDVRZ.CONCLUSION The recombinant of HDV cDNAand ribozyme gene had the potential of beingfurther explored and used in gene therapy of HBVinfection.

Secretory Expression of Recombinant Diphtheria ToxinMutants in B. Subtilis

Three diphtheria toxin (DT) mutants CRM-197, DT-del (148) and DT-El48S-K516A-F530A were cloned in B- Subtilis plasmid PSM604 under the subtilisin signal sequence. The expression was effective in both SMS300 and SMS118, but higher yield of 7. 1 mg/L was observed in SMS300 compared with 2. 1 mg/L in SMS118. Western blot showed that the recombinant protein could be effectively secreted into the culture medium as a 58 ku peptide, and could be de-graded into two peptides of 37ku and 21ku.

Identification of reference genes provides functional insights into meiotic recombination suppressors in Gerbera hybrida

Gerbera Hybrida is one of the important cut flowers across the world.The novel traits are the primarily market requirements and the breeding targets,mainly determined by the degree of genetic variation after hybridization.However,meiotic recombination is highly conserved in most eukaryotes which suppressed the crossover formation and limited the genetic diversity.Recently,several meiotic recombination suppressors have been identified and characterized in plants,whereas it remains elusive in G.hybrida.In order to characterize the expression patterns of these suppressors in G.hybrida,20 candidate reference genes were identified from the transcriptome datasets of G.hybrida,and their expression stabilities during plant development were evaluated by geNorm,NormFinder and BestKeeper.Although the most stable reference genes were variable in different softwares,comprehensive ranking revealed that PGK2 was the most stable reference gene and GAPDH was the most unstable one.The expression patterns of FANCM,FIGL1,RECQ4,RM1,and FLIP further validated that PGK2 was suitable for normalization of gene expression.Our study identified a reliable reference gene for gene expression during meiotic recombination,and provided functional insights into meiotic recombination suppressors in G.hybrida.

Targeting of human aFGF gene into silkworm,Bombyx mori L. through homologous recombination

The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL 1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.

Bioassay of Estrogenic Activity of Effluent and Influent in a Farm Wastewater Treatment Plant Using an in vitro Recombinant Assay with Yeast Cells

Objective Environmental estrogens at an elevated concentration are known to produce adverse effects on human and animal life.However,the majority of researches have been focused on industrial discharges,while the impact of livestock wastes as a source of endocrine disrupters in aquatic environments has been rarely elucidated.In order to investigate the contribution of environmental estrogens from livestock,the estrogenic activity in water samples from a farm wastewater treatment plant was analyzed by a recombinant yeast screening method.Methods The extracts prepared from 15 selected water samples from the farm wastewater treatment plant,among which 6 samples were from pre-treatment section(influents) and 9 from post-treatment section(effluents),were analyzed for estrogenic activity by cellar bioassay.Yeast cells transfected with the expression plasmid of human estrogen receptor and the Lac Z reporter plasmid encoding β-galactossidase,were used to measure the estrogen-like compounds in the farm wastewater treatment plant.Results The wastewater samples from influents showed a higher estrogenic potency than the effluent samples showing a low induction of β-galactossidase relative to solvent control condition.By comparison with a standard curve for 17β-estradiol(E2),estrogenic potency in water samples from the influents was calculated as E2-equivalent and ranged from 0.1 to 150 pM E2-equivalent.The estrogenic potency in water samples from the effluents was significantly lower than that in the influents,and 7 water samples had less detectable limit in the total of 9 samples.Conclusion Yeast bioassay of estrogenic activity in most of the samples from the farm wastewater after disposal by traditional sewage treatment showed negative results.

Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

Rat calcineurin (CaN) A α isoform (Ppp3ca) cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rat, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EGFP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment (3.97 kb) obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno-Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect (CPE) appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A α (Ppp3ca) cDNA as well as a report gene—enhancer green fluorescent protein gene was successfully constructed in this experiment.

Packaging and Functional Identification of Recombinant Adeno-associated Virus Encoding cdc2-siRNA

Cyclin dependent kinases (cdks) play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus (rAAV) encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA (rAAV-EGFP-U6-cdc2-siRNA) was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

Construction and Expression of Recombinant Plasmid pCD-rbFGF in Osteoblasts

Summary: To construct basic fibroblast growth factor (bFGF) eukaryotic expression vector and to evaluate the possibility of bFGF gene therapy in orthopedic disease, the pCD-rbFGF recombinant plasmid was constructed by cloning rat basic fibroblast growth factor (bFGF) cDNA into an eukaryotic expression vector, pcDNA 3. Rat osteoblasts were transfected with pCD-rbFGF plasmid by lopofectin mediated gene transfer, the transient expression was detected by streptavidin-biotin-enzyme complex (SABC) method. It was observed that the expression of rat bFGF gene was detected 72 h after transfected distinctly. Basic fibroblast growth factor gene therapy is a method of potential for a wide array of orthopedic diseases.

A VNTR ELEMENT ASSOCIATED WITH STEROID SULFATAES GENE DELETIONS STIMULATES RECOMBINATIONIN CULTURED CELLS

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Identification of QTLs for Blast, Bacterial Blight, and Planthopper Resistance Using SNP-Based Linkage Maps from Two Recombinant Inbred Rice Lines

Rice is the most significant global food security. Several biotic factors limit rice production, breeding biotic-resistant rice has, therefore, become an increasingly important goal. Two elite rice lines, IR71033-121-15 (IR71033) and IR57514-PMI-5-B-1-2 (IR57514), provide potential genes for biotic stress resistance traits. In this study, genotyping by sequencing (GBS) for single nucleotide polymorphism (SNP)-based linkage map construction was used to detect quantitative trait loci (QTLs) for blast (BL), bacterial blight (BB), whitebacked planthopper (WBPH), and brown planthopper (BPH) resistance. IR71033 was derived from Oryza minuta and carried BL, BB, WBPH, and BPH resistance QTLs. IR57514 is a well-adapted rainfed lowland line that carries BL and BB resistance QTLs. Two sets of recombinant inbred line (RIL) populations derived from crosses of KDML105 × IR71033 and KDML105 × IR57514 were used to dissect the genetic basis of disease and insect pest resistance. The RIL populations were evaluated for BL, BB, WBPH, and BPH resistance from 2016 to 2018 at four rice research centers in Thailand. From these, we identified a large number of SNPs through GBS and constructed high-resolution linkage maps. By combining phenotypic evaluation with the GBS data, a total of 24 QTLs on four chromosomes were detected that confered pest resistance and explained 7.3% - 61.4% of the phenotypic variance. These findings should facilitate identifying novel resistance genes and applying marker-assisted selection for resistance to the four major rice pests investigated here. These strategies will improve the resilience and reliability of rice varieties adapted to the low-yielding environment of rainfed lowland areas worldwide.