Polymorphism of Coding Sequences of IGF1R Gene in Baise Horses and Thoroughbred

[ Objective]The aim was to study polymorphism of mat-peptide sequence of IGF1 R gene in Baise horses and thoroughbred, which would af- ford reference for the further studies on the dwarf mechanism and molecular breeding in horses. [Method] A total of 57 blood samples of each breed were collected and genomic DNA was extracted by the standard phenol -chloroform method. Five DNA pools of each breed were constituted and polymorphism sites were identified by sequencing PCR products. Frequencies of genotypes and alleles at these sites of each breed were checked by PCR-RFLP. [Result] Four polymorphism sites were identified in exon 2, 5 and 16, including mutations of T406C, T179 627C, G212 077A and G2.12 110A. No difference was found in the frequency of T179 627C between the Baise horses and thoroughbred. The mutation （3212 077A was only found in the thorou- ghbred, and the mutations, T406C and G212 110A, were only checked out in the Baise horses. [ Conclusion] Whether these mutations are associated with horse growth needs further studies.

An atypical NLR gene confers bacterial wilt susceptibility in Arabidopsis

Quantitative disease resistance(QDR)remains the most prevalent form of plant resistance in crop fields and wild habitats.Genome-wide association studies(GWAS)have proved to be successful in deciphering the quantitative genetic basis of complex traits such as QDR.To unravel the genetics of QDR to the devastating worldwide bacterial pathogen Ralstonia solanacearum,we performed a GWAS by challenging a highly polymorphic local mapping population of Arabidopsis thaliana with four R.solanacearum type III effector(T3E)mutants,identified as key pathogenicity determinants after a first screen on an A.thaliana core collection of 25 accessions.Although most quantitative trait loci(QTLs)were highly specific to the identity of the T3E mutant(ripAC,ripAG,ripAQ,and ripU),we finely mapped a common QTL located on a cluster of nucleotide-binding domain and leucine-rich repeat(NLR)genes that exhibited structural variation.We functionally validated one of these NLRs as a susceptibility factor in response to R.solanacearum,named it Bacterial Wilt Susceptibility 1(BWS1),and cloned two alleles that conferred contrasting levels of QDR.Further characterization indicated that expression of BWS1 leads to suppression of immunity triggered by different R.solanacearum effectors.In addition,we showed a direct interaction between BWS1 and RipAC T3E,and BWS1 and SUPPRESSOR OF G2 ALLELE OF skp1(SGT1b),the latter interaction being suppressed by RipAC.Together,our results highlight a putative role for BWS1 as a quantitative susceptibility factor directly targeted by the T3E RipAC,mediating negative regulation of the SGT1-dependent immune response.

(R)-(+)-长叶薄荷酮对耐药结核分枝杆菌抑制作用及其生物被膜调控基因的影响

目的 研究(R)-(+)-长叶薄荷酮对耐药结核分枝杆菌体外抑制作用及其生物被膜调控基因Rv0024、Rv2872、Ra1362的影响。方法 通过聚合酶链反应(Polymerase chain reaction, PCR)筛选含生物被膜调控基因Rv0024、Rv2872、Ra1362的耐药结核分枝杆菌,采用刃天青显色法和液体培养计数法测定(R)-(+)-长叶薄荷酮对生物被膜介导的耐药结核分枝杆菌的最小抑菌浓度(Minimal inhibit concentration, MIC)和最低杀菌浓度(Minimum bactericidal concentration, MBC),应用反转录-聚合酶链式反应(Reverse transcription-polymerase chain reaction, RT-PCR)检测(R)-(+)-长叶薄荷酮对耐药结核分枝杆菌生物被膜调控基因Rv0024、Rv2872、Ra1362的影响。结果 在3×10^(7) CFU/mL和3×10^(6) CFU/mL菌液浓度下,刃天青显色法测得(R)-(+)-长叶薄荷酮对耐药结核分枝杆菌的MIC和MBC分别为14.79、29.59 mg/mL;液体培养计数法测得(R)-(+)-长叶薄荷酮对耐药结核分枝杆菌的MIC_(90)和MBC分别为19.89、24.62 mg/mL。RT-PCR法结果表明长叶薄荷酮可抑制Rv0024、Ra1362的表达,促进Rv2872的表达(P<0.05)。结论 (R)-(+)-长叶薄荷酮通过调控耐药结核分枝杆菌生物被膜主要基因Rv0024、Rv2872、Ra1362的表达,达到抑制耐药结核分枝杆菌的生长的作用。

Genetic polymorphisms of MC2R gene associated with responsiveness to adrenocorticotropic hormone therapy in infantile spasms

Background Infantile spasms is a severe epileptic encephalopathy, which is refractory to conventional antiepileptic drugs. Adrenocorticotropic hormone （ACTH） has been the major therapy for infantile spasms; however, ACTH therapy is ineffective for some patients. The variations in the receptor genes can contribute to antiepileptic drug resistance. This study was to elucidate the possible associations between the variations of the MC2R gene and ACTH responsiveness in patients with infantile spasms. Methods We screened for variations in the promoter and coding region of the MC2R gene in 91 Chinese patients with infantile spasms and 94 controls, using PCR and a direct sequencing method. The frequencies of the genotypes, alleles and reconstructed haplotypes were analyzed in the cases and controls. The association between ACTH responsiveness and genetic variations of the MC2R gene was also assessed. Results Four single nucleotide polymorphisms （SNPs） were identified in the MC2R promoter, one of which was a novel specimen at position-2 from the transcription start site ATT, -2T〉C. Three SNPs （rs1893220, rs2186944 and -2T〉C） showed a significant difference between the cases and controls （P 〈0.05 for all）. The frequency of the common TCCT haplotype carrying four-SNP major alleles was significantly lower in the cases （39%） than in the controls （60%） （P=-0.00003）. The homozygous carriers of the TCCT haplotype had a much lower relative risk than the non-carriers （RR=O.42, 95%C/ 0.26-0.70, P=-0.0001）. ACTH responsiveness was strongly associated with the TCCT haplotype （P=-0.000082）. Compared with non-carriers of the TCCT haplotype, the homozygous and heterozygous carriers were more responsive to ACTH therapy （P=0.0002; P=-0.0003, respectively）. Conclusions Our results indicated that the TCCT haplotype in the MC2R promoter is strongly associated with the responsiveness of the ACTH therapy performed on patients with infantile spasms. The polymorphisms of the MC2R promoter might be one important factor that influences the efficacy of ACTH therapy on infantile spasms.

Xa7, a new executor R gene that confers durable and broad-spectrum resistance to bacterial blight disease in rice

Bacterial blight(BB)is a globally devastating rice disease caused by Xanthomonas oryzae pv.oryzae(Xoo).The use of disease resistance(R)genes in rice breeding is an effective and economical strategy for the control of this disease.Nevertheless,a majority of R genes lack durable resistance for long-term use under global warming conditions.Here,we report the isolation of a novel executor R gene,Xa7,that confers extremely durable,broad-spectrum,and heat-tolerant resistance to Xoo.The expression of Xa7 was induced by incompatible Xoo strains that secreted the transcription activator-like effector(TALE)AvrXa7 or PthXo3,which recognized effector binding elements(EBEs)in the Xa7 promoter.Furthermore,Xa7 induction was faster and stronger under high temperatures.Overexpression of Xa7 or co-transformation of Xa7 with avrXa7 triggered a hypersensitive response in plants.Constitutive expression of Xa7 activated a defense response in the absence of Xoo but inhibited the growth of transgenic rice plants.In addition,analysis of over 3000 rice varieties showed that the Xa7 locuswas found primarily in the indica and aus subgroups.A variation consisting of an 11-bp insertion and a base substitution(G to T)was found in EBEAvrXa7 in the tested varieties,resulting in a loss of Xa7 BB resistance.Through a decade of effort,we have identified an important BB resistance gene and characterized its distinctive interaction with Xoo strains;these findings will greatly facilitate research on the molecular mechanism of Xa7-mediated resistance and promote the use of this valuable gene in breeding.

Cloning and Characterization of Full Length cDNA of a CC-NBS-LRR Resistance Gene in Sweetpotato

Conserved domain such as nucleotide binding site （NBS） was found in several cloned plant disease resistance genes. Based on the NBS domain, resistance gene analogues （RGAs） have been isolated. A full-length cDNA, SPR1 was obtained by rapid amplification of cDNA ends （RACE） method. Sequence analysis indicated that the length of SPR1 was 3 066 bp, including a complete open reading frame of 2 667 bp encoding SPR1 protein of 888 amino acids. Compared with known NBS-LRR genes, it presented relatively high amino acid sequence identity. The polypeptide has a typical structure of nonT1R-NBS-LRR genes, with NB-ARC, CC, and LRR domains. The SPR1-related sequences belonged to multicopy gene family in sweetpotato genome according to the result of Southern blotting. Semi-quantitative RT-PCR analysis showed SPR1 expressed in all tested tissues. The cloning of putative resistance gene from sweetpotato provides a basis for studying the structure and function of sweetpotato disease-resistance relating genes and disease resistant genetic breeding in sweetpotato. The gene has been submitted to the GenBank database, and the accession number is EF428453.

Improving Rice Blast Resistance by Mining Broad-Spectrum Resistance Genes at Pik Locus

Magnaporthe oryzae is known for its genetic diversity and pathogenic variability,leading to rapid breakdown of resistance in rice.Incorporating multiple broad-spectrum blast resistance genes into rice cultivars would extend disease resistance longevity.Effective resistance breeding in rice therefore requires continual enrichment of the reservoir of resistance genes and alleles.We conducted a large-scale screen of rice blast resistance in about 2000 rice accessions.Among them,247 accessions showed at least medium resistance to the natural infection of rice blast and 7 novel Pik alleles were identified from them.Variations in gene sequences were then correlated with the phenotypic trait to enable the identification of favorable alleles.Among the seven novel Pik alleles,the resistant rate of Pik-R0/ME/7017 donors was greater than 80%,and the disease score was less than 3.Through molecular marker-assisted backcross breeding,we successfully transferred the three Pik alleles,Pik-R0/ME/7017,into an elite cultivated line Kongyu 131 to obtain BC_(3)F_(2)lines,which showed enhanced resistance to rice blast compared with the recurrent parent.Assessment of these near-isogenic lines in the greenhouse using 31 isolates of M.oryzae from Heilongjiang Province of China revealed that the resistant levels of the BC_(3)F_(2)lines with Pik-R0/ME/7017 were significantly higher than those of the established cloned resistance genes Pik-m and Pi1.Exploring such alleles will enrich our gene library for resistance to rice blast.

Complement Gene Mutation and Ehlers-Danlos Syndrome

Background: Dental complications of Ehlers-Danlos syndrome (EDS) include periodontitis with gum fragility and inflammation, enamel hypoplasia with frequent caries, high palate with dental crowding, TMJ instability, sutural dehiscence or scarring, and insensitivity to anesthetics. Objective: Determine if EDS dental complications always define a specific type and genetic cause or if they can arise as a general consequence of altered inflammatory response in EDS. Method: We compared findings of a 58-year-old female with complement component 1R (C1R) gene mutation (c.1553A > T, p.Asp518Val) found by whole exome sequencing to 43 patients with C1R gene mutations ascertained because of periodontal disease and to 710 EDS patients conventially ascertained because of joint and skin laxity. Result: Female patients ascertained as periodontal EDS showed the expected higher frequency of periodontitis (96% versus 14%) but had similar frequencies of hypermobility (81% versus 90%) and some skin findings (84% versus 92% with skin fragility) as the general group and our female patient who shared their C1R gene change. Her oromandibular bone loss rather than gum disease may reflect the more carboxy-terminal position of her C1R gene mutation compared to those in the patients identified as periodontal EDS. Conclusion: While mutation of the C1R gene may predict more frequent periodontal, skin, and vascular complications, focus on an articulo-autonomic dysplasia process that includes mast-cell activation and altered inflammatory response rather than extreme EDS types will help dentists and other subspecialists identify all EDS patients and anticipate their frequent oral manifestations.

An association study of protein tyrosine phosphatase receptor type R gene polymorphism and the resting-state functional magnetic resonance imaging in major depressive disorder

Objective To explore the influence of a polymorphism of protein tyrosine phosphatase receptor type R(PTPRR)gene rs1513105 on abnormal brain activities in resting-state patients with major depressive disorder(MDD)using the gene-imaging technology.Methods 54MDD and 43 gender-,age-,and education-matched con-

Phylogenetic Diversity of Microorganisms from Chemocline of the Meromictic Soda Lake Doroninskoe(Zabaikalie,Russia)

1 Introduction Many soda and salt lakes are characterized by the formation of the meromictic conditions under which a part of the water column is not involved in the annual process of mixing(Mac Intyre,Melack,1982).This creates an

Effect of adeno-associated virus-mediated transfer of low density lipoprotein receptor gene on treatment of hypercholesterolemia

Low density lipoprotein receptor (LDL\|R) plays an important role in removing LDL from plasma and preventing producing atherosclerosis. To test hepatic reconstitution of LDL\|R expression is sufficient for metabolic correction of subjects with hypercholesterolemia; a recombinant adeno\|associated virus (rAAV) carrying the human LDL receptor cDNA (AAV/LDL\|R) was constructed. Using adenosome, a complex of adenoviral capsid protein and liposome, to transfer the rAAV into livers of rabbits with experimental hypercholesterolemia could elicit the production of LDL\|R and lower plasma cholesterol level. These results demonstrate that AAV\|mediated transfer of LDL\|R gene may provide an alternative means to gene therapy of hypercholesterolemia.

Development and mapping of SSR markers linked to resistance-gene homologue clusters in common bean

Common bean is an important but often a disease-susceptible legume crop of temperate,subtropical and tropical regions worldwide. The crop is affected by bacterial, fungal and viral pathogens. The strategy of resistance-gene homologue(RGH) cloning has proven to be an efficient tool for identifying markers and R(resistance) genes associated with resistances to diseases. Microsatellite or SSR markers can be identified by physical association with RGH clones on large-insert DNA clones such as bacterial artificial chromosomes(BACs). Our objectives in this work were to identify RGH-SSR in a BAC library from the Andean genotype G19833 and to test and map any polymorphic markers to identify associations with known positions of disease resistance genes. We developed a set of specific probes designed for clades of common bean RGH genes and then identified positive BAC clones and developed microsatellites from BACs having SSR loci in their end sequences. A total of 629 new RGH-SSRs were identified and named BMr(bean microsatellite RGH-associated markers). A subset of these markers was screened for detecting polymorphism in the genetic mapping population DOR364 × G19833. A genetic map was constructed with a total of 264 markers,among which were 80 RGH loci anchored to single-copy RFLP and SSR markers. Clusters of RGH-SSRs were observed on most of the linkage groups of common bean and in positions associated with R-genes and QTL. The use of these new markers to select for disease resistance is discussed.

多发性骨髓瘤中p53基因表达与R-ISS分期及预后的相关性分析

目的 研究多发性骨髓瘤患者p53基因表达情况与修订的国际分期系统(R-ISS)及预后的相关性。方法 回顾性纳入2018年10月至2021年12月阜阳市人民医院收治的86例多发性骨髓瘤患者为研究对象。采用荧光原位杂交技术检测患者p53基因表达情况,并收集所有患者临床一般资料,包括性别、年龄、体重指数、免疫球蛋白类型及R-ISS分期,分析多发性骨髓瘤患者p53基因表达情况及其与临床特征的关系。所有患者均接受标准化疗方案治疗,评估治疗疗效,并分析p53基因表达情况与临床疗效的关系。所有患者均接受1年随访,观察患者生存情况,比较p53基因阳性及缺失组与p53基因阴性组患者1年存活率。结果 86例多发性骨髓瘤患者中,p53基因阳性及缺失组患者7例,占8.14%,p53基因阴性组患者79例,占91.86%。p53基因阳性及缺失组和p53基因阴性组患者性别构成比、年龄、体重指数、免疫球蛋白类型比较,差异均无统计学意义(P>0.05);p53基因阳性及缺失组患者中R-ISS分期为Ⅲ期的患者占比为71.43%,显著高于p53基因阴性组(21.52%),差异有统计学意义(P<0.05)。经标准化疗方案治疗后,86例多发性骨髓瘤患者中,完全缓解13例,部分缓解38例,疾病稳定24例,疾病进展11例。有效组患者共51例,无效组患者共35例。无效组患者中p53基因阳性及缺失患者占比高于有效组患者,但差异无统计学意义(P>0.05)。入组患者均获得有效随访,p53基因阳性及缺失组患者1年累计生存率为71.43%(5/7);p53基因阴性组患者1年累计生存率为84.81%(67/79)。两组患者1年累计生存率比较,差异无统计学意义(P>0.05)。结论 p53基因表达情况影响多发性骨髓瘤的发生、发展,并与R-ISS分期密切相关,伴有p53基因阳性及缺失的患者的化疗疗效及预后可能较差,临床需对多发性骨髓瘤细胞中p53基因异常的患者进一步探寻新型治疗方法,延长患者生存期。

Cloning and Expression Analysis of <i>TTG</i>1 Gene Related to <i>Rosa rugosa</i>Trichomes Formation

The TTG1 transcription factor plays an important role in the formation of plant trichomes. Based on the R. rugosa transcriptome data, this study cloned a R. rugosa TTG1 gene, named RrTTG1, and carried out bioinformatics analysis and fluorescence quantitative analysis to explore the relationship between TTG1 gene and R. rugosa trichomes formation, in order to lay a good foundation to cultivate a thornless plant in the family Rosaceae. In this experiment, six hybrid cultivars of R. rugosa “Zizhi”, R. rugosa “Xizi”, R. rugosa “Tang fen”, R. rugosa “Hun chun”, R. rugosa “Zi long wo chi” and R. rugosa “Tian e huang” were used as experimental materials, and the cDNA full length of this gene was obtained by RT-PCR and RACE, and the full length of the cDNA was 1348 bp. After bioinformatics analysis, it is predicted that its molecular formula is C1723H2661N465O529S12, the molecular weight is 38.71 KB, and the isoelectric point is 5.00. Its instability index is 54.30, which belongs to unstable protein;and its hydrophilic amino acid distribution is relatively uniform, and the amount is larger than hydrophobic amino acid, which belongs to hydrophilic protein. Phylogenetic tree was constructed for the TTG1 gene. Evolutionary analysis indicated that RrTTG1 is closely related to the TTG1 protein of Rosaceae family, and has a close relationship with other families. The expression analysis showed that the expression of RrTTG1 protein was negatively correlated with the trichome content of R. rugosa stems and leaves. The expression levels of the three spiny varieties of R. rugosa “Hun chun”, R. rugosa “Xizi” and R. rugosa “Zi long wo chi” were lower, and the expressions of the three less thorn varieties of R. rugosa “Zizhi”, R. rugosa “Tian e huang” and R. rugosa “Tang fen” were higher. According to the above results, it was speculated that RrTTG1 is involved in the synthesis of R. rugosa trichomes and belongs to the negative regulation mechanism.

Cloning and Expression Level Analysis of Melanocyte-stimulating Hormone Receptor 1 Gene(MC1R) in Alpacas with Different Coat Color

Specific primers for the MC1R gene of alpacas(GenBank EU1358800) were designed to amplify the cDNA sequence using RT-PCR to seek variation in the sequence and explore the relationship between the expression level of MC1R gene and alpaca coat color.The MC1R gene from white alpaca was cloned successfully and sequence analysis verified that the MC1R gene,encoding 317 amino acids,was 1081 bp in length.Compared with the existing sequence in GenBank,sequence identity was 99.9%and 7 mutations were found.Primers,designed from the sequence obtained,were used to assess the relative expression of MC1R in alpacas of different coat color using QRT-PCR and SPSS 13.0 software.Relative expression of MC1R in the skin of brown alpacas was 4.32 times higher than that in white alpacas after normalization with GAPDH(P【0.01),indicating that MC1R expression may be related to coat color of alpacas.

pGenesil-1-EGFP-shRNA/survivin真核表达载体构建及其表达验证

目的构建用survivin启动子驱动增强绿色荧光蛋白(EGFP)的shRNA真核表达载体pGenesil-1-EGFP-shRNA/survivin并验证其在宫颈癌Caski细胞中的表达。方法用有活性的survivin启动子取代pGenesil-1-EGFP-shRNA/U6载体中的U6启动子从而获得新的shRNA真核表达载体pGenesil-1-EGFP-shRNA/survivin;将pGenesil-1-EGFP-shBNA/survivin转染到宫颈癌Caski细胞及正常人脐静脉内皮HuVEC细胞,观察EGFP在两种细胞中的表达变化。结果成功构建pGenesil-1-EGFP-shRNA/survivin载体,分别转染Caski及HuVEC细胞,在Caski细胞中观察到较少的绿色荧光蛋白表达,而在HuVEC细胞中观察到较多的绿色荧光蛋白表达。结论survivin启动子能驱动shRNA在宫颈癌Caski细胞中特异性表达,而在正常HuVEC细胞中不表达。

Detection of the FecX^R Mutation of BMP15 Gene in Sheep and Goats

PCR-SSCP was used to detect mutations of bone morphogenetic protein 15(BMP15) gene in both high prolificacy(Small Tail Han sheep,Hu sheep,Jining Grey goat and Boer goat) and low prolificacy breeds(Dorset sheep,Texel sheep,Inner Mongolia Cashmere goat and Angora goat).Both the nucleotide sequences and the amino acid sequences were compared in amplification fragments of both Small Tail Han sheep and Jining Grey goat.The results indicated that none of the four sheep and the four goat breeds carried the same FecXR mutation of the BMP15 gene as do Rasa Aragonesa sheep.The nucleotide sequence of Small Tail Han sheep was completely identical with that of the sheep BMP15 sequence(GenBank AF236079,NM001114767).Three base substitutions(T529G,C530G and T576C) and two amino acid changes(V155G and S171P) were found in Jining Grey goat compared with Small Tail Han sheep.The FecXR mutation of the BMP15 gene had no significant effect on high prolificacy of Small Tail Han sheep, Hu sheep,Jining Grey goat and Boer goat.

Management of the Case of a Young Female Patient with Multiple Malignancies and Germline R24P CDKN2A Gene Mutation

The case of a young female patient with metachronous primary melanomas, advanced breast and pancreatic cancers is reported. The 5 different tumors diagnosed within six years, were managed with curative intent. Genetic analysis revealed the mutation of the R24P CDKN2A gene in a heterozygote form in both the patient and her father. Careful tertiary prevention during the follow-up of the patient is needed.

乳酸片球菌R-4细菌素PA-1原核表达及其理化特性

为实现原核表达产出细菌素并检测其理化特性,作者将乳酸片球菌R-4细菌素pedA基因进行扩增回收,与pMD19-T载体连接后转入E.coli DH5α感受态细胞进行克隆。提取克隆后的pedA基因与表达载体pET-32a(+)连接,形成重组质粒pET-32a-pedA并转入E.coli BL21(DE3)感受态细胞,经异丙基硫代半乳糖苷诱导,乳酸片球菌R-4细菌素PA-1在大肠杆菌细胞进行表达。表达蛋白质经Ni-NTA柱纯化后,以金黄色葡萄球菌为指示菌检测其理化特性。结果表明,在E.coli BL21(DE3)细胞中成功表达相对分子质量为26000的乳酸片球菌R-4细菌素PA-1并完成纯化。纯化后的乳酸片球菌R-4细菌素PA-1在40~121℃作用20 min、在pH 2~12、紫外线照射0~10 h、过氧化氢酶作用2 h后,其抑菌范围分别为14.7~15.6 mm、14.0~16.5 mm、15.1~15.8 mm和14.9 mm,而分别经胃蛋白酶和胰蛋白酶作用2 h均失去抑菌作用。这表明乳酸片球菌R-4细菌素PA-1对高温、强酸强碱、紫外线和过氧化氢酶均具有较好的稳定性,而胃蛋白酶和胰蛋白酶会使其失活。

结球甘蓝NBS-LRR类R基因同源序列的分离

根据大多数抗病基因编码蛋白质的核苷酸结合区(NBS)和富含亮氨酸重复序列(LRR)保守区域的特点,设计PCR特异简并引物,从抗TuMV的结球甘蓝材料84075中,扩增出513 bp的DNA片段,经克隆、测序后,得到4个含有NBS-LRR保守区域的R基因同源序列,分别命名为:Bor1、Bor2、Bor3和Bor4,同源性比较分析表明,它们与已克隆的抗病基因或抗病基因片段有不同程度的同源性。以Bor1为探针,对84075进行Southern blot和RFLP分析的结果表明,Bor1以多拷贝形式存在。