Prognostic significance of X-ray cross-complementing gene 1 expression in gastric cancer

Objective： The aim of this study is to identify the prognostic significance of X-ray cross-complementing gene 1 （XRCCI） in patients with gastric cancer undergoing surgery and platinum-based adjuvant chemotherapy. Methods： Immunohistochemistry （IHC） was used to evaluate XRCCI protein expression profiles on surgical specimens of 612 gastric cancer patients. The relationship between XRCC1 expression and existing prognostic factors, platinum-based adjuvant chemotherapy, disease-free survival （DFS） and overall survival （OS） were analyzed. Results： Among 612 patients staged II/III in our study, 182 （29.74%） were evaluated as XRCC1 IHC positive. XRCC1 expression was not significantly related to OS （P=0.347） or DFS （P=0.297）. Compared with surgery only, platinum-based adjuvant chemotherapy significantly improved the OS （P=0.031）. And the patients with negative XRCC1 expression benefited more from platinum-based adjuvant chemotherapy （P=0.049）. Multivariate analysis demonstrated that tumor size, T category, N category, vascular or nerve invasion and platinum-based chemotherapy were good prognostic factors for OS （P〈0.05）. Though XRCCI plays an important role in DNA repair pathways, no significant relationship is found in XRCCI expression and OS among gastric cancer in our study. Conclusions： XRCC1 might be an alternative prognostic marker for the patients of gastric cancer after radical resection. The patients with negative XRCC1 expression can benefit more from platinum-based adjuvant chemotherapy.

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.

ERCC1、K-ras、TP-73在替雷利珠单抗联合TP化疗方案治疗非小细胞肺癌中的评估价值

目的研究探讨核苷酸切除修复交叉互补基因1(ERCC1)、Kirsten-Rous肉瘤病毒蛋白(K-ras)、肿瘤蛋白P73(TP73)在替雷利珠单抗结合紫杉醇+顺铂(TP)化疗方案治疗NSCLC中的评估价值。方法选取2020年1月至2021年12月本院收治的126例NSCLC肺癌患者为研究对象,按随机抽签法分为对照组、观察组,各63例。对照组以TP化疗方案治疗,观察组增加替雷利珠单抗治疗。评估组间临床疗效、肿瘤标记蛋白、免疫指标、生存周期、不良反应。结果观察组患者的客观缓解率为69.84%(44/63)高于对照组患者为52.38%(33/63),观察组疾病控制率为82.54%(52/63),高于对照组患者为66.67%(42/63)(P<0.05)。化疗1周期、化疗3周期、化疗6周期时,观察组ERCC1、K-ras、TP-73水平均低于对照组(P<0.05)。治疗后观察组免疫功能补体C3、补体C4、CD40细胞低于对照组,NK细胞高于对照组(P<0.05)。观察组患者的TTP、PFS、总生存期均高于对照组(P<0.05)。观察组不良反应发生率为19.05%(12/63),对照组为12.70%(8/63),组间比较差异无统计学意义(P>0.05)。结论替雷利珠单抗联合TP化疗方案治疗肺癌有良好的治疗效果,能够改善患者免疫功能,延长患者生存周期,治疗安全性较好,且ERCC1、K-ras、TP-73水平变化可反映替雷利珠单抗联合TP化疗方案在肺癌治疗中的效果,在综合疗效评估中有较高的应用价值。

补体蛋白调控靶点在糖尿病视网膜病变中的作用及临床研究进展

补体系统是一个具有精密调控机制的蛋白质反应系统,具有介导炎症、调节免疫应答、溶解细胞和清除免疫复合物等功能。糖尿病视网膜病变(DR)是糖尿病常见和严重的眼部并发症,是眼科常见的不可逆性致盲性眼病之一,且其发病机制错综复杂,包括缺氧、氧化应激、炎症反应、多元醇代谢通路的异常等。近年来有越来越多的证据表明,免疫系统的失调和炎症是DR发病的重要因素,且多种补体蛋白在炎症调节、血管新生等关键过程中发挥着重要作用。因此,本综述的中心主旨在于研究补体系统及相关调节蛋白在DR中的作用,目的是阐明多种补体蛋白与DR发生发展的紧密联系,为我们防治DR提供重要参考和新的思路。同时本文也对补体系统靶向药物的临床研究进一步阐述。

SPP1、DEC1、C1QTNF6蛋白与口腔鳞状细胞癌患者临床病理指标及预后的关系

目的:探讨口腔鳞状细胞癌患者血清重组人分泌型磷蛋白1(SPP1)、软骨分化的表达基因1(DEC1)和补体C1q/肿瘤坏死因子相关蛋白6(C1QTNF6)的表达水平与其临床病理指标及预后的关系。方法:免疫组织化学染色法和电化学发光免疫分析法检测88例口腔鳞状细胞癌患者癌组织和血清中SPP1、DEC1和C1QTNF6蛋白的表达水平;Pearson相关性分析和Kaplan-Meier生存分析法分析患者血清SPP1、DEC1和C1QTNF6蛋白表达水平与其临床病理指标的相关性和对患者预后的影响。多因素Cox回归法分析影响口腔鳞状细胞癌患者预后的危险因素。结果:免疫组织化学染色结果显示口腔鳞状细胞癌患者癌组织中SPP1、DEC1和C1QTNF6蛋白的阳性表达率较癌旁组织分别增加了1.94、2.98和2.35倍(P<0.05或P<0.01);电化学发光免疫分析法结果显示口腔鳞状细胞癌患者血清SPP1、DEC1和C1QTNF6蛋白表达水平较正常人分别上调了8.61、6.20和4.03倍(P<0.05或P<0.01);Pearson相关性分析结果显示患者血清SPP1、DEC1和C1QTNF6蛋白表达水平与患者发生多灶嗜神经侵犯、肿瘤浸润程度、淋巴结转移、较高的TNM分期呈正相关(P<0.05);Kaplan-Meier生存分析结果显示,血清SPP1、DEC1和C1QTNF6蛋白高表达水平的口腔鳞状细胞癌患者总生存率低;多因素Cox回归分析结果表明多灶嗜神经侵犯、肿瘤高浸润度、淋巴结转移和高的TNM分期是影响预后的危险因素(P<0.05)。结论:口腔鳞状细胞癌患者癌组织和血清中SPP1、DEC1和C1QTNF6蛋白表达水平升高,且与患者发生多灶嗜神经侵犯、肿瘤浸润和淋巴结转移、较高的TNM分期呈正相关,而与患者预后呈负相关。

ERCC1 mRNA和X线修复交叉互补组1基因多态性联合检测在局部晚期鼻咽癌患者放化疗中的应用价值

目的 探讨核苷酸切除修复交叉互补组1(ERCC1)m RNA和X线修复交叉互补组1(XRCC1)基因多态性联合检测在局部晚期鼻咽癌患者放化疗中的应用价值。方法 选取2020年1月至2021年1月在江西省上饶市人民医院接受放化疗的41例局部晚期鼻咽癌患者,采用定量反转录聚合酶链反应检测外周血中ERCC1 mRNA的表达水平,采用限制性片段长度多态性聚合酶链反应检测XRCC1基因型(Arg194Trp、Arg280His、Arg399Gln),探讨ERCC1 mRNA和XRCC1多态性与局部晚期鼻咽癌患者放化疗效果、肿瘤复发及药物不良反应(ADR)的关系,并采用logistic回归分析局部晚期鼻咽癌患者ADR的影响因素。结果 完全缓解和部分缓解患者的ERCC1 mRNA及XRCC1多态性与疾病稳定和疾病进展患者比较差异无统计学意义(P>0.05)。肿瘤复发患者的ERCC1 mRNA及XRCC1多态性与非复发患者比较差异无统计学意义(P>0.05)。ADR患者XRCC1 Arg194Trp位点携带AG基因型、ERCC1 mRNA高表达的频率均高于非ADR患者,差异有统计学意义(P<0.05)。多因素logistic回归分析显示,XRCC1 Arg194Trp AG基因型(OR=1.876)、ERCC1mRNA高表达(OR=1.109)是局部晚期鼻咽癌患者放化疗期间发生ADR的影响因素(P<0.05)。结论 与单一检测相比,ERCC1 mRNA和XRCC1多态性联合检测预测局部晚期鼻咽癌患者放化疗期间ADR的价值更高,值得临床应用。

XRCC1 genetic polymorphism Arg399Gln and gastric cancer risk:A meta-analysis

AIM:TO evaluate the association between X-ray cross- complementing gene 1 (XRCC1) genetic polymorphism Arg399GIn and gastric cancer risk by means of meta- analysis. METHODS:We searched PubMed and NCBI up to June 1,2008.A total of 16 clinical trials and reports were identified,but only 8 trials qualified under our selection criteria.Statistical analysis was performed with the software program Review Manage,version 4.2.8. RESULTS:Of the 8 case-control studies selected for this meta-analysis,a total of 1334 gastric cancer cases and 2194 controls were included.For Arg399Gln,the Gln/Gln genotype carriers did not have a decreased cancer risk compared with those individuals with the Arg/Arg genotype (OR=0.92,95% CI,0.71-1.19; P=0.51).Similarly,no associations were found in the recessive and dominant modeling (Gln/Gln vs Arg/Gln +Arg/Arg:OR=0.96;95% CI,0.77-1.19;P=0.70 and Gln/Gln+Arg/Gln vs Arg/Arg:OR=0.90,95% CI,0.77-1.05;P=0.18). CONCLUSION:No association is found between the XRCC1 polymorphism Arg399Gln and gastric cancer risk.

DNA repair gene XRCC1 polymorphisms and susceptibility to childhood acute lymphoblastic leukemia: a meta-analysis

Objective： To estimate the relationship between genetic polymorphisms of X-ray repair cross- complementing group 1 （XRCC1） and the susceptibility to childhood acute lymphoblastic leukemia （ALL）. Methods： Relevant case-control studies were enrolled in the meta-analysis. We applied Rev Man 4.2 software to pool raw data and test studies＇ heterogeneity and to calculate the incorporated odds ratio （OR） and 95% confidence interval （95% CI）. Results： Our data showed that the OR for the Gln allele of the Arg399Gln polymorphism, compared with the Arg allele, was 1.35 （95% CI, 1.16-1.57; P〈0.0001） for childhood ALL patients. Similarly, the homozygous genotype Gln/Gln and heterozygous genotype Arg/Gln both significantly increased the risk of childhood ALL compared with the wild genotype Arg/Arg （OR =1.58; 95% CI, 1.13-2.21; P=0.008; OR =1.51; 95% CI, 1.21-1.87; P=0.0002）. The dominant model of Arg399Gln was associated with childhood ALL risk （OR =1.54; 95% CI, 1.25-1.89; P〈0.0001）. The ethnic subgroup analysis demonstrated that the Gln allele in all five ethnic groups was prone to be a risk factor for childhood ALL just with different degrees of correlation while Arg194Trp SNP showed a protective or risk factor or irrelevant thing in different races. Conclusions： XRCC1 399 polymorphism may increase the risk of childhood ALL. Different ethnic groups with some gene polymorphism have different disease risks.

Age-related macular degeneration treatment in the era of molecular medicine

Age-related macular degeneration(AMD) is the leading cause of irreversible blindness in the developed world. The quality of life of both patients and families is impacted by this prevalent disease. Previously, macular degeneration had no known effective treatment. Today, vitamins for non-exudative AMD and intravitreal injection of medications for its exudative form are primary forms of current treatment. Modern advances in molecular science give rise to new possibilities of disease management. In the year 2003 the sequencing of the entire human genome was completed. Since that time, genes such as complement factor H, high-temperature requirement factor A1, and age-relateed maculopathy susceptibility 2 have been discovered and associated with a higher risk of AMD. A patient's genetic make-up may dictate the effectiveness of current or future therapeutic options. In addition, utilizing genetic data and incorporating it into new treatments(such as viral vectors) may lead to longer-lasting(or permanent) VEGF blockade and specific targeting of complement related genes. There have also been considerable advances in stem cell directed treatment of AMD. Retinal pigment epithelial(RPE) cells can be derived from human embryonic stem cells, induced pluripotent stem cells, or adult human RPE stem cells. Utilizing animal models of RPE and retinal degeneration, stem cell-derived RPE cells have been successfully implanted into the subretinal space. They have been injected as a cell mass or as a pre-prepared monolayer on a thin membrane. Visual recovery has been demonstrated in a retinal dystrophic rat model. Preliminary data on 2 human subjects also demonstrates possible early visual benefit from transplantation of stem cell-derived RPE. As more data is published, and as differentiation and implantation techniques are optimized, the stabilization and possible improvement of vision in individuals with non-exudative macular becomes a real possibility. We conclude that the technologic advances that continue to unfold in both genetic and stem cell research offer optimism in the future treatment of AMD.

Correlation between X-ray cross-complementing group 1 polymorphisms and the onset risk of glioma A meta-analysis

OBJECTIVE： To evaluate the association of X-ray cross-complementing group 1 （XRCC1） Arg399GIn, Arg194Trp and Arg280His polymorphisms with the risk of glioma. DATA SOURCES： A systematic literature search of papers published from January 2000 to August 2012 in PubMed, Embase, China National Knowledge Infrastructure database, and Wanfang da- tabase was performed. The key words used were ＂glioma＂, ＂polymorphism＂, and ＂XRCC1 or X-ray repair cross-complementing group 1＂. References cited in the retrieved articles were screened manually to identify additional eligible studies. STUDY SELECTION： Studies were identified according to the following inclusion criteria： case-control design was based on unrelated individuals; and genotype frequency was available to estimate an odds ratio （OR） and 95% confidence interval （CI）. Meta-analysis was performed for the selected studies after strict screening. Dominant and recessive genetic models were used and the relationship between homozygous mutant genotype frequencies and mutant gene frequency and glioma incidence was investigated. We chose the fixed or random effect model according to the heterogeneity to calculate OR and 95%CI, and sensitivity analyses were conducted. Publication bias was examined using the inverted funnel plot and the Egger＇s test using Stata 12.0 software. MAIN OUTCOME MEASURES： Association of XRCC1 Arg399GIn, Arg194Trp, and Arg280His polymorphisms with the risk of glioma, and subgroup analyses were performed according to differ- ent ethnicities of the subjects.RESULTS： Twelve articles were included in the meta-analysis. Eleven of the articles were concerned with the Arg399GIn polymorphism and glioma onset risk. Significantly increased glioma risks were found only in the dominant model （Gin/Gin ＋ GIn/Arg versus Arg/Arg： OR = 1.26, 95%CI= 1.03-1.54, P = 0.02）. In the subgroup analysis by ethnicity, significantly increased risk was found in Asian subjects in the recessive （OR = 1.46, 95%CI= 1.04-2.45, P = 0.03） and dominant models （OR = 1.40, 95%CI= 1.10-1.78, P = 0.007）, and homozygote contrast （OR = 1.69, 95%CI= 1.17-2.45, P = 0.005）, but not in Caucasian sub- jects. For association of the Arg194Trp （eight studies） and Arg280His （four studies） polymorphisms with glioma risk, the meta-analysis did not reveal a significant effect in the allele contrast, the recessive genetic model, the dominant genetic model, or homozygote contrast. CONCLUSION： The XRCC1 Arg399GIn polymorphism may be a biomarker of glioma susceptibility, espe- cially in Asian populations. The Arg194Trp and Arg280His polymorphisms were not associated with overall glioma risk.

Cell cycle and complement inhibitors may be specific for treatment of spinal cord injury in aged and young mice: transcriptomic analyses

Previous studies have reported age-specific pathological and functional outcomes in young and aged patients suffering spinal cord injury,but the mechanisms remain poorly understood. In this study, we examined mice with spinal cord injury. Gene expression profiles from the Gene Expression Omnibus database （accession number GSE93561） were used, including spinal cord samples from 3 young injured mice （2–3-months old, induced by Impactor at Th9 level） and 3 control mice （2–3-months old, no treatment）, as well as 2 aged injured mice （15–18-months old, induced by Impactor at Th9 level） and 2 control mice （15–18-months old, no treatment）. Differentially expressed genes （DEGs） in spinal cord tissue from injured and control mice were identified using the Linear Models for Microarray data method,with a threshold of adjusted P 〈 0.05 and ｜logFC（fold change）｜ 〉 1.5. Protein–protein interaction networks were constructed using data from the STRING database, followed by module analysis by Cytoscape software to screen crucial genes. Kyoto encyclopedia of genes and genomes pathway and Gene Ontology enrichment analyses were performed to investigate the underlying functions of DEGs using Database for Annotation, Visualization and Integrated Discovery. Consequently, 1,604 and 1,153 DEGs were identified between injured and normal control mice in spinal cord tissue of aged and young mice, respectively. Furthermore, a Venn diagram showed that 960 DEGs were shared among aged and young mice, while 644 and 193 DEGs were specific to aged and young mice, respectively. Functional enrichment indicates that shared DEGs are involved in osteoclast differentiation, extracellular matrix–receptor interaction, nuclear factor-kappa B signaling pathway, and focal adhesion. Unique genes for aged and young injured groups were involved in the cell cycle （upregulation of PLK1） and complement （upregulation of C3） activation, respectively. These findings were confirmed by functional analysis of genes in modules （common, 4; aged, 2; young, 1） screened from protein–protein interaction networks. Accordingly, cell cycle and complement inhibitors may be specific treatments for spinal cord injury in aged and young mice, respectively.

玉米番茄红素环化酶基因的克隆、表达及功能分析

从玉米中克隆获得β-环化酶(lycopene β-cyclase,LCYb)和ε-环化酶(lycopene ε-cyclase,LCYe)基因,对编码产物进行生物学信息分析,通过体外大肠杆菌表达系统,借助于颜色互补及产物分析实验,探究了玉米LCYb和LCYe的催化功能特性。序列分析结果显示,玉米LCYb和LCYe的cDNA全长分别1 470 bp和1 611 bp,与高粱和小米物种的同源性均在90%以上。通过与谷胱甘肽巯基转移酶标签融合表达,成功纯化出LCYe和LCYb蛋白。颜色互补实验及高效液相产物分析结果表明,玉米LCYb具有β-环催化活性,可将番茄红素两端环化形成β-胡萝卜素,同时也具有微量的ε-环活性,可以通过中间体γ-胡萝卜素生成α-胡萝卜素;而玉米LCYe具有双ε-环活性可以同时催化番茄红素两端形成ε-胡萝卜素。本研究揭示了玉米番茄红素环化酶的催化特性,为探究玉米类胡萝卜素分子调控机制奠定了基础。

羊草ZIP家族成员LcZIP10的功能

羊草(Leymus chinensis)作为内蒙古草原的建群物种,是优质牧草资源,但目前对其矿质营养调控机理研究仍少有报道。植物ZIP家族被认为是植物从土壤吸收锌铁离子的主要功能蛋白,主要负责吸收和转运植物必需微量元素。本研究从羊草转录组数据库中筛选获得的LcZIP10,为拟南芥ZIP家族AtZIP10的同源基因,预测其具有锌铁转运功能。结果表明,LcZIP10在根叶中均有表达,且在叶中表达量显著高于根,同时高铁条件下会诱导LcZIP10表达,且该基因编码蛋白定位于液泡膜。随后,酵母功能互补实验证明,LcZIP10能够恢复铁敏感酵母突变体(Δfet3/Δfet4)的生长,说明具有Fe^(2+)转运功能。以上结果对日后开发和利用微量元素强化农作物品质具有一定的参考价值。

PARPi通过抑制XRCC1的表达增加食管鳞癌放射治疗敏感性

探讨PARP抑制剂(PARPi)对XRCC1的表达以及对食管鳞癌(ESCC)放射治疗敏感性的影响。收集接受直线加速器辐照治疗的ESCC患者组织标本,免疫组化法检测其中XRCC1、PARP-1表达,观察其表达对ESCC患者放疗近期疗效的影响。ECA109细胞经AZD2281(PARP抑制剂)处理后接受加速器辐照,检测PARPi的放疗增敏比(SER)。利用RT-PCR实验检测AZD2281联合辐照后XRCC1mRNA转录情况,探讨PARPi对辐照后ECA109细胞XRCC1mRNA转录的影响。结果发现,XRCC1阳性者放疗的客观有效率(ORR)低于阴性者(38.1%vs.88.9%,p=0.017);PARP-1阳性者放疗的ORR低于阴性者(36.8%vs.81.8%,p=0.026)。AZD2281的浓度为3μmol/L时,联合组的SER=1.744。AZD2281可增强ECA109细胞的辐射损伤作用。辐射后48 h XRCC1mRNA相对表达量明显上升;联合PARPi可抑制辐射诱导的XRCC1mRNA表达上调。本组结果显示,XRCC1、PARP-1高表达者放疗近期疗效较差,放疗可诱导XRCC1基因转录,AZD2281能有效抑制辐射诱导的XRCC1 mRNA表达上调,其可能的机制是PARPi抑制DNA-PKcs进而下调XRCC1表达。该结果提示PARPi可能通过抑制XRCC1表达、减少DNA损伤修复,从而增加ESCC放疗敏感性。

XRCC6基因多态性与术后化疗胃癌患者预后的关系

目的探讨X-射线修复交叉互补蛋白6(XRCC6)基因多态性与术后化疗胃癌患者预后的关系。方法选取99例术后行奥沙利铂+替吉奥化疗方案治疗的胃癌患者作为研究对象,检测其入院时外周血XRCC6基因rs2267437位点基因型。随访患者化疗36个月后的生存情况,将其分为存活组56例和死亡组43例。比较两组患者的临床病理资料和XRCC6基因基因型的分布频率,并采用COX回归模型分析XRCC6基因多态性与术后化疗胃癌患者预后的关系。结果死亡组肿瘤直径>5 cm、肿瘤临床分期为Ⅲ期、发生淋巴结转移、存在远处转移的患者比例更高(均P<0.05)。两组患者的XRCC6基因的基因型分布频率差异有统计学意义(P<0.05),其中死亡组患者的XRCC6基因CG基因型、GG基因型频率低于存活组。COX回归分析结果显示,校正相关临床病理特征后,携带XRCC6基因CG/GG基因型的术后化疗胃癌患者的预后更好(P<0.05)。结论XRCC6基因多态性与术后化疗的胃癌患者的预后密切相关,携带CG/GG基因型的患者预后更好。

L-乳酸脱氢酶基因克隆及功能分析

构建了一株产D ,L_乳酸的乳杆菌 (Lactobacillussp .)MD_1的基因文库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的EscherichiacoliFMJ14 4作为宿主 ,通过互补筛选分离克隆到乳酸脱氢酶基因 (ldhL)。核酸序列分析表明 ,该基因以ATG为起始密码子编码 316个氨基酸残基组成的蛋白质 ,预测的分子量为 33 84kD ;5′端存在典型的启动子结构 ,3′端的终止子是不依赖于 ρ因子的转录终止子。ldhL编码的蛋白质有 3个保守区域 ,其中Gly13～Asp5 0保守区域是NADH的结合位点 ,Asp73～Ile10 0和Asn12 3～Arg15 4保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低 ,核苷酸序列相似性最高仅为 6 4 1% ,氨基酸序列相似性最高仅为 6 8 9% 。

格尔德霉素生物合成基因功能的验证

格尔德霉素(Geldanamycin,Gdm)作为热休克蛋白90的特异性抑制剂,是非常有前景的抗肿瘤和抗病毒的药物,我们已从吸水链霉菌17997(Streptomyces hygroscopicus17997)的基因文库中获得了Gdm大部分生物合成基因。为了研究主要基因的功能,选择了聚酮合酶基因(Polyketide synthase gene,pks)的第六模块、单加氧酶基因(Mono-oxygenase gene,gdmM)和氨甲酰基转移酶基因(Carbamoyltransferase gene,gdmN)3个基因作为靶点分别进行基因阻断,获得了基因同源双交换的阻断变株△pks、△gdmM和△gdmN。经HPLC检测证实这些基因的阻断变株均不产生Gdm,基因回复实验排除了基因阻断所可能造成的极性效应对其它基因表达的影响,说明所克隆的pks、gdmM和gdmN基因确实是Gdm生物合成所必需的基因。

副溶血弧菌基因回补实验方法的建立与应用

目的建立以pBAD33质粒为载体的副溶血弧菌(Vibrio parahaemolyticus,VP)基因回补实验平台。方法 PCR扩增aphA和opaR的整个ORF区序列,并将其直接克隆入pBAD33质粒中,构建重组质粒。分别将重组质粒转入到ΔopaR和ΔaphA中(分别代表opaR和aphA的突变株),以构建出相应的回补株C-ΔaphA和C-ΔopaR。分别在aphA和opaR基因内设计特异性引物,采用RT-PCR方法,验证在相应的回补突变株中aphA和opaR是否转录。利用引物延伸实验研究野生株(WT)、ΔopaR、ΔaphA、C-ΔaphA和C-ΔopaR中mfpA(aphA负调控,opaR正调控其表达)的相对RNA水平。结果 aphA和opaR在相应的回补株中发生转录;且mRNA水平与野生株一致。结论成功建立了以pBAD33质粒为载体的VP基因回补方法,并得到应用。

DUF784基因在花粉管导向中的功能分析

花粉管导向是高等植物完成双受精过程的重要环节,是受多重信号调控的复杂过程.最近的研究揭示,配子体阶段花粉管导向的诱导信号分子是一类具多态性的富含半胱氨酸的防卫素类似蛋白,如来自玉米的ZmEA1和蓝猪耳草中的LUREs在吸引花粉管进入珠孔起重要作用.但是拟南芥及其它植物中此类信号未知.转录组学分析表明,一组DUF784基因可能在花粉管导向中起到重要作用.通过RNAi技术降低一组DUF784基因的表达,分析发现在RNAi转基因植株中,出现胚珠败育现象,花粉管导向出现异常,一部分花粉管不能进入珠孔.另外,用MYB98基因的启动子携带1个DUF基因的编码区,然后转化ccg突变体,发现ccg转基因株系中胚胎败育率下降,即DUF基因能部分互补ccg突变体的表型;从这两方面证实了DUF784基因在花粉管定向导入过程中的作用.

黑腹果蝇黑条体突变型的基因定位研究

黑腹果蝇的体色突变类型常见的有黄体（ｙｅｌｌｏｗ；ｙ）、黑体（ｂｌａｃｋ，ｂ）和黑檀体（ｅｂｏｎｙ，ｅ），分别位于Ｘ染色体，第二染色体和第三染色体上。黑条体突变型是本实验室１９９１年９月从野外采集的黑腹果蝇野生型单雌系后代中发现的自发突变品系，为了探明黑条体突变型是原有黑体突变类型的再现还是新的突变，采用常规杂交方法和互补实验技术对黑腹果蝇黑条体突变型的基因定位进行了探讨。互补测验的结果表明，黑条体与黑檀体杂交的子一代为反式排列的杂合体无互补，表现为突变型，子二代中，由于交换而产生重组类型的顺式排列的杂合体表现为野生型。因此确定黑条体突变基因（ｂｓｒ）与黑檀体突变基因（ｅ）是等位的，位于第三染色体的９３Ｄ２区，但分别位于不同的位点上，属于同一顺反子的新的点突变。同时对于各体色基因间的相互作用及遗传传递方式的进行了讨论。