长根菇与鸡從菌属间不对称融合后代特性研究

以长根菇和鸡菌为亲本进行属间不对称融合研究 ,从原生质体再生菌株中得到能够双核化的单核无锁状联合的菌株 2 0株。栽培结果显示 ,这类菌株不能形成子实体 ,且菌株间菌丝的生长差异显著。但在特定的培养条件下 ,单核菌株均可双核化形成双核菌株 ,双核菌株具有出菇能力 ,菌株间子实体颜色、菌柄着生方式及菌丝生长速度等与亲本有明显的差异     

长根菇与鸡菌属间不对称融合后代特性研究

以长根菇和鸡Zong菌为亲本进行属间不对称融合研究，从原生质体再生菌株中得到能够双核化的单核无锁状联合的菌株20株。栽培结果显示，这类菌株不能形成子实体，且菌株间菌丝的生长差异显著。但在特定的培养条件下，单核菌株均可双核化形成双核菌株，双核菌株具有出菇能力，菌株间子实体颜色，菌柄着生方式及菌丝生长速度等与亲本有明显的差异。     

糙皮侧耳HMG-box转录因子分析与候选基因克隆表达

在糙皮侧耳(Pleurotus ostreatus)的全基因组中鉴定出14个高速泳动蛋白(high-mobility group box,HMG-box)的编码基因(PoHMG1～PoHMG14)，对其进行特征、结构与系统进化分析，发现所有的HMGbox转录因子蛋白的三级结构高度相似，相对分子质量为6893.79～60397.29，蛋白PoHMG6、PoHMG11、PoHMG12与PoHMG13之间的亲缘关系较近。基于糙皮侧耳在菌丝、原基、幼嫩子实体和成熟子实体时期的转录组数据，对筛选到的转录因子基因PoHMG11进行克隆分析并探究其功能及表达特性，发现该基因全长序列1 517 bp，编码489个氨基酸，含有一个HMG-box结构域。通过大肠杆菌原核表达载体体外诱导表达出相对分子质量为5.5×104的蛋白，该重组蛋白在1 mol·L^-1 IPTG、25℃诱导表达6 h时表达量最高。利用实时荧光定量PCR(qRT-PCR)分析糙皮侧耳在菌丝、原基、幼嫩子实体和成熟子实体阶段PoHMG11基因的相对表达量，发现该基因在成熟糙皮侧耳子实体中的表达量比其他生长发育时期显著...     

金针菇寡肽转运蛋白编码基因fvopt1与fvopt2的鉴定与表达分析

基于金针菇(Flammulina velutipes)的基因组和转录组数据,通过本地BLAST方法,获得2个寡肽转运蛋白编码基因,命名为fvopt1和fvopt2,并对其结构、编码蛋白的基本性质、系统进化与表达模式进行分析。结果表明:fvopt1和fvopt2分别含有13和18个外显子,编码蛋白FVOPT1与FVOPT2各含774和772个氨基酸,相对分子质量为86120.25与86192.24,等电点为7.46与7.92,且均无信号肽。保守结构域分析与系统进化分析结果表明,FVOPT1与FVOPT2为寡肽转运蛋白家族成员。实时荧光定量PCR结果表明:在菌丝阶段,fvopt1在无氮培养基中的表达量高于其在含氮培养基中的表达量;在含氮培养基中,fvopt1在整个子实体发育阶段(原基、菌柄和菌盖)中表达量上调,fvopt2只在原基中高表达,推测fvopt1和fvopt2可能与子实体和原基的发育有关。     

农杆菌介导法转化金针菇不同受体的效率比较

以金针菇(Flammulina velutipes)单核体菌株Dan3和双核体菌株G1的菌丝碎片以及原生质体分别为转化受体,根癌农杆菌(Agrobacterium tumefaciens)LBA4404为侵染菌株,潮霉素为筛选标记,绿色荧光蛋白编码基因(egfp)为外源基因,进行农杆菌介导转化金针菇。结果显示:金针菇双核体菌株G1的菌丝碎片作为受体时转化效率最高,为40.31%;单核体菌株Dan3的原生质体作为受体时转化效率最低,为21.87%。     

金针菇单核体DAN3和M与其杂交双核体G1菌丝阶段基因表达差异的转录组初步分析

以金针菇(Flammulinavelutipes)单核菌株DAN3的基因组为参考,完成单核体DAN3和M及其杂交双核体G1在菌丝阶段的转录组测序与数据分析,比较两个样本间的差异基因,并对差异基因进行GO功能分析和KEGG pathway分析.结果表明:两个样本中共有显著性差异表达的基因86个,其中,在G1中呈上调、下调表达的基因数分别为41、45个,有2个基因在G1中特异表达.GO功能分析结果表明,86个差异基因中有40个基因比对上了GO功能注释,其中18个基因在G1中呈上调表达;单一生物过程和催化活性为显著性富集的功能,相关基因在G1中呈上调表达.KEGG pathway分析结果表明,22个差异基因被定位到17条Pathway,其中10个基因在G1中呈上调表达,包括赖氨酸代谢途径对应基因,3_M和G1菌丝样品中赖氨酸含量分别为1.70×103 ng/mg和1.06×103 ng/mg,说明G1中上调的基因可能与之降解相关;DNA复制是显著性富集的代谢途径,相关基因在G1中呈下调表达.     

转Pofst3糙皮侧耳菌株农艺性状和胞外酶活性分析

通过袋栽实验比较野生型、Pofst3过表达型和Pofst3反义沉默型糙皮侧耳（Pleurotus ostreatus）菌株（分别记为Ⅰ、Ⅱ、Ⅲ）的菌丝生长速度、原基数量、子实体数量、菌柄直径、菌盖直径等农艺性状;测定其漆酶、木聚糖酶、纤维素酶活性。结果表明：Ⅰ、Ⅱ、Ⅲ菌丝生长速度差异不显著；与Ⅰ相比，Ⅲ的原基和子实体数量多，菌柄直径大，菌盖直径小，原基和子实体中Pofst3的相对表达量低；与Ⅰ、Ⅱ相比，Ⅲ的漆酶、木聚糖酶及纤维素酶活性较高；Pofst3对糙皮侧耳发育的影响可能与其胞外酶活性相关。     

金针菇假定蛋白E3泛素连接酶基因的鉴定与表达分析

以金针菇基因组与转录组数据为基础,通过本地BLAST方法鉴定金针菇假定蛋白E3泛素连接酶基因,命名为GENE5116,并对其结构、编码蛋白的基本性质与表达模式进行了分析,以期了解其在金针菇子实体生长发育过程中的作用。结果表明:该基因有9个外显子,8个内含子,基因总长为1998 bp,共编码438个氨基酸残基,相对分子质量为48987.36,等电点8.83;系统进化分析结果表明该基因与真菌E3泛素连接酶具有高度同源性,与陶兰柱担菌(Cylindrobasidium torrendii)亲缘关系最近;实时荧光定量PCR结果表明该基因在菌柄的表达量最高且菌柄上部基因表达量显著高于下部,在菌丝的表达量最低,所以推测该基因可能与子实体菌柄伸长有关。     

斑玉蕈β-葡萄糖苷酶基因的序列分析及皂苷对基因表达的影响

通过转录组测序获得了斑玉蕈(Hypsizygus marmoreus)SIEF3133菌株的β-葡萄糖苷酶基因的cDNA序列(命名为bgl1),采用荧光定量PCR检测bgl1的相对表达量。当PDB中加入0.05g/L的皂苷溶液,菌丝培养4~6d时,bgl1相对表达量降低了1/2左右(相比对照),8d开始升高,第10天为对照组的1.8倍;向工厂化生产用的栽培瓶(接种培养85d后搔菌处理)中添加0.05g/L的皂苷溶液15mL,可缩短子实体采收时间约3d(与对照组相比),在此过程中bgl1相对表达量在第5天时最低,20d时超过对照组,5d时为对照组的1.66倍。     

构建香菇转化的同源筛选标记的研究

通过比对分析多种导致真菌内源萎锈灵抗性的琥珀酸脱氢酶B亚基(SdhB)氨基酸序列的突变位点,将香菇(Lentinula edodes)SdhB氨基酸序列第243位的组氨酸替换为亮氨酸,构建了香菇萎锈灵抗性同源筛选标记载体,以农杆菌介导转化小米粒培养的香菇菌丝。转化子经5次传代后,PCR扩增验证插入片段,并观察绿色荧光蛋白(EGFP)的表达情况。结果显示:香菇单核体和双核体菌株经转化后,均能筛选到具有萎锈灵抗性的转化子,单核体菌株的转化率为34%,显著高于双核体菌株(4%);单核体转化子中的同源筛选标记及报告基因稳定性较好。本研究构建的萎锈灵抗性同源筛选标记可有效地在香菇遗传转化体系中应用。     

Interaction between hHBrk1 and WAVE1 protein

AIM: To identify the binding sites between hHBrk1 and WAVE1 protein.METHODS: PCR was applied to clone the WAVE1 gene and its truncated domains from human fetal brain library. The gene fragments were sub-cloned into eukaryotic expression vector. Co-immunoprecipitation assay was used to identify the binding sites between hHBrk1 and WAVE1.RESULTS: The sequences of WAVE1 and the truncated domains were successfully amplified and sub-cloned into pCMV-HA vector. An HA tag was added to the fused protein at N-terminal. The interaction between hHBrk1 and the Scar homology domain(SHD) of WAVE1 was identified by co-immunoprecipitation. We also observed that WAVE1 bound to the heptad repeat(HR) domain of hHBrk1, and the site-directed mutations at HR domain abolished the interaction between hHBrk1 and WAVE1.CONCLUSION: hHBrk1 may play an important role in migration of tumor cells through binding to SHD of WAVE1 protein.     

梨‘中矮1号’LUE1基因的克隆及功能分析

以梨优良矮化砧木‘中矮1号’新梢嫩茎为试材,根据白梨(Pyrus bretschneideri Rehd.)基因组预测的相关基因序列信息设计特异引物,克隆获得1个Katanin蛋白家族成员基因。序列分析显示该基因编码区全长1 566 bp,编码521个氨基酸,预测蛋白分子量约为57.2 kD,等电点为8.11,与其他物种的Katanin P60家族氨基酸序列具有78.01%~98.66%的相似性,将该基因命名为PcLUE1。实时荧光定量RT-PCR(qRT-PCR)分析显示,生长慢的矮化品种新梢快速生长时期PcLUE1的表达量显著高于生长快的乔化品种。石蜡切片显示,矮化品种与半矮化及乔化品种间茎组织细胞的排列方式和细胞大小存在较大差异。过表达PcLUE1的烟草植株与对照相比,株高降低,叶片变小,颜色变深,与‘中矮1号’中PcLUE1高表达效应极为相似,说明PcLUE1可能与‘中矮1号’的矮化有一定的关系。     

Relationship between IL-1 gene polymorphism and severity of rheumatoid arthritis and secretion of IL-1

AIM: To determine the influence of interleukin-1α(IL-1α) and β(IL-1β) gene polymorphisms on rheumatoid arthritis(RA) disease severity and secretion of IL-1β. METHODS: The study included 136 RA patients and 102 healthy controls. PCR-RFLP was used to detect site mutation at IL-1 gene. Meanwhile the IL-1β was also measured in the supernatant of the cultured and stimulated peripheral blood mononuclear cells(PBMC). RESULTS: No difference in the allele frequencies or genotypes of the IL-1α gene polymorphisms was found between the controls and RA patients.IL-1β allele 2 was overrepresented in patients with erosive RA but not in nonerosive patients. The patients with IL-1β allele 2 had a higher swollen joint index, higher tender joint index and erythrocyte sedimentation rate than those without IL-1β allele 2.The IL-1β in supernatant of stimulated PBMC from patients with IL-1β allele 2 had a higher level than that from those without allele 2. CONCLUSION: IL-1 gene polymorphisms may influence the occurrence of RA. Detection of IL-1β allele 2 have a potential prognostic value in RA.     

桃PpSnRK1β1和PpSnRK1β2在番茄中超表达对光合碳代谢的影响

从毛桃[Prunus persica(Linn.)Batsch.]叶片中克隆了PpSnRK1蛋白激酶β亚基编码基因PpSnRK1β1和PpSnRK1β2,其c DNA全长分别为720 bp和867 bp。q RT-PCR组织表达分析表明,PpSnRK1β1在‘鲁星’桃叶片中的表达量最高,PpSnRK1β2在其果实中表达量最高,两者在其花中的表达量均较低。通过农杆菌介导转化番茄,获得超表达PpSnRK1β1的番茄株系T21、T23和超表达PpSnRK1β2的株系T91、T92。研究发现,与野生型(WT)相比,超表达株系T23和T91叶片的Sn RK1蛋白激酶活性分别增加9.84%和53.32%。T91叶片净光合速率比WT提高17.02%;叶片可溶性糖含量比WT增加34.00%。T91和T92叶片淀粉含量分别约是WT的2倍。T23、T91和T92果实维生素C含量分别比WT下降19.21%、38.49%和39.76%。因此说明PpSnRK1β1和PpSnRK1β2参与了光合作用过程,调控碳代谢及次生代谢过程。     

Expression of SREBP-1 and SREBP-2 in kidney of type 1 diabetic rats

AIM:To investigate the expression of sterol regulatory element binding protein-1 (SREBP-1) and sterol regulatory element binding protein-2 (SREBP-2) in the kidney of type 1 diabetic rats.METHODS:The triglyceride (TG) and cholesterol content in the kidney of experimental rats were measured by the assay kits and oil red O staining.Furthermore, the expressions of SREBP-1 and SREBP-2 protein were detected by the methods of Western blotting and immunohistochemistry.The analysis of SREBP-1 mRNA was performed by in situ hybridization.RESULTS:Renal triglyceride content was markedly higher in diabetic rats than that in normal rats and the result of oil red O showed that lipid deposited in the renal tubular epithelium.Immunohistochemistry and Western blotting presented similar results that SREBP-1 protein was up-regulated in renal tubular epithelium in diabetic rats.On the other hand, SREBP-2 protein didnt show difference between diabetic rats and normal control rats.In situ hybridization confirmed the increasing of SREBP-1 mRNA in renal tubular epithelium in diabetic rats.CONCLUSION:Above results suggest that altered SREBP-1 may play an important role in the pathogenesis of renal lipid accumulation in type 1 diabetic rats.     

Effect of ginsenoside Rg1 and Rh1 onthe anti-tumor activity of dendritic cell

AIM: To study the effect of ginsenoside Rg1 and Rh1 on the anti-tumor activity of dendritic cells (DC). METHODS: Effect of Rg1 and Rh1 on the production of IL-12 p40 protein was detected by ELISA, and the IL-12 p40 mRNA level of DC was monitored by RT-PCR. Anti-tumor activity of DC-LPAK was determined by neutral red staining assay. RESULTS: The results of ELISA showed that Rg1 and Rh1 significantly enhanced the production of IL-12 p40 of DC. Rg1 at 1 mg/L and Rh1 at 100 mg/L upregulated the IL-12 p40 mRNA level. Rg1 and Rh1 enhanced the anti-tumor ability of DC, induced lymphokine and PHA activated killer (DC- LPAK) on human papillate tumor cell line. Each dose of Rg1 can obviously accelerate the cytotoxity to L929 at the E∶T ratio of 5∶1(P<0.05,0.01), while only Rh1 10 mg/L enhanced the cytotoxity ability of DC-LPAK (P<0.05). CONCLUSION: Rg1 and Rh1 enhanced the production of IL-12 p40. This effect may be mediated by the increase in the mRNA level. As a result, Rg1 and Rh1 promote the ability of DC to stimulate the cytotoxitic acticity of DC-LPAK.