PCR-based INDEL markers co-dominant between Oryza sativa,japonica cultivars and closely-related wild Oryza species

Wild relatives genetically close to cultivars are precious genetic resources for plant breeding. Oryza rufipogon, O. barthii, O. glumaepatula, O. meridionalis and O. longistaminata are such wild species, and are also categorized as AA genome species based on their structural similarities. Chromosome segment substitution lines (CSSLs) are a powerful resource in breeding and genetics, and numerous rice CSSLs have been produced. This study aimed to develop DNA markers for evaluation of CSSLs directly by PCR and subsequent gel electrophoresis. We confirmed that up to 155 of 188 markers developed for detection of japonica-indica INDELs could also detect INDELs between rice cultivars and wild AA-species accessions. Percentages of applicable markers were higher in O. rufipogon accessions (61.7 to 85.6%), and lower in accessions of other four AA species (39.8 to 51.4%). These markers were distributed throughout the rice chromosomes, and will be useful for genotyping of CSSLs and other genetic resources derived from crosses between rice cultivars and closely related wild species.     

Deletion of the LMP-1 gene integrated in the nasopharyngeal carcinoma cell line SUNE-1

AIM: To study the EBV LMP-1 gene integrated in the chromosome of poorly differentiated nasopharyngeal carcinoma cell line SUNE-1. METHODS: The LMP-1 gene of SUNE-1 was detected with PCR; Deletion of LMP-1 was examined by restriction endonuclease analysis and PCR. The deletion was precisely localized by DNA sequencing. RESULTS: The LMP-1 gene integrated in the chromosome of SUNE-1 could be deleted or non-deleted. The two introns of LMP-1 gene were shown being lost in SUNE-1 cell line. CONCLUSION: Deletion of intron 1 and intron 2 happen in some of the LMP-1 gene integrated in the chromosome of SUNE-1.     

西瓜专化型尖孢镰刀菌FonSIX6缺失突变体的构建

病原菌和植物相互作用时分泌效应蛋白（effectors）抑制植物的防卫反应。枯萎病是由尖孢镰刀菌引起的真菌病害,尖孢镰刀菌和寄主相互作用时分泌几个特定的富含半胱氨酸的小分子量蛋白（15.8～29.9 kD）进入木质部启动致病力,称为SIX（secreted in xylem）蛋白,其中,SIX6蛋白是一个效应蛋白。西瓜枯萎病是由西瓜专化型尖孢镰刀菌（Fusarium oxysporum f. sp.Niveurn,Fon）引起的真菌病害。为了明确FonSIX6在侵染西瓜时的作用,克隆了该基因的上下游序列,以潮霉素为筛选标记构建了该基因的缺失突变体载体pDH\|SIX6;将构建好的基因缺失载体转入农杆菌AGL\|1中,通过农杆菌介导的遗传转化和转化子的筛选获得了FonSIX6基因的缺失突变体。     

布鲁氏菌外膜蛋白BP26细胞毒性作用的研究

【目的】布鲁氏菌外膜蛋白BP26是布鲁氏菌的重要毒力因子,本研究采用bp26基因缺失株和BP26蛋白分别与胚胎滋养层细胞(HPT-8)作用,探讨布鲁氏菌bp26基因的生物学功能。【方法】采用Ni柱亲和层析法纯化BP26蛋白,overlap技术构建布鲁氏菌bp26基因缺失株,用BP26蛋白和bp26基因缺失株分别侵染HPT-8细胞,观察细胞形态变化,ELISA检测上清中的细胞因子。【结果】 从布鲁氏菌疫苗株M5-90克隆出bp26基因并在大肠杆菌E.coli BL21(DE3)中成功表达,获得纯化的BP26蛋白,经SDS-PAGE验证正确,Western-Blot鉴定具有免疫原性。将目的片段bp26基因的上下臂插入到自杀载体pGEM-7zf中,电转至布鲁氏菌疫苗株M5-90感受态细胞中,成功筛选出了具有遗传稳定性的bp26基因缺失株。用BP26蛋白与bp26基因缺失株分别侵染HPT-8层细胞,BP26蛋白使细胞变形且贴壁不牢,缺失株导致细胞大量脱落溶解,ELISA检测蛋白侵染HPT-8细胞诱导产生的细胞因子IL-6、TNF-α和LDH均高于PBS对照组,差异极显著（P＜0.01）, 而细胞因子IL-10的分泌下降。缺失株侵染HPT-8细胞诱导产生的细胞因子IL-6、TNF-α均高于M5-90对照组,LDH和IL-10均低于M5-90对照组,差异显著（P＜0.05）。【结论】成功获得了布鲁氏菌BP26蛋白和M5-90Δbp26缺失株,BP26蛋白可以引起HPT-8细胞的炎性反应,有细胞毒性作用,bp26基因缺失株对HPT-8细胞有损伤作用,bp26基因在布鲁氏菌侵染宿主细胞过程中起重要作用。     

牛羊朊蛋白基因（PRNP）多态性与抗病性的研究进展

 朊蛋白（PRNP）是近年来造成人和部分哺乳动物传染性海绵状脑病(TSE)的主要根源,该基因的多态性显著影响了人和动物对TSE的易感性或抗病性。本文分析了朊蛋白基因及其编码蛋白的结构与功能;简要介绍了绵羊基因编码区突变与致病性的关系;系统分析了牛科动物启动子区域内23bp的插入/缺失、第一内含子区域内12bp的插入/缺失及其与疯牛病（BSE）抗病性的作用机制;全面总结了全球已经报道的牛科动物12和23bp插入/缺失的等位基因与单倍体频率,评价了其发病的可能性。该研究将为牛的分子育种提供指导。     

高分子量谷蛋白单亚基缺失对软质小麦宁麦9号加工品质的影响

基因敲除是研究高分子量谷蛋白(HMW-GS)亚基功能的重要方法。本研究以软质小麦宁麦9号野生型及其单亚基缺失系为材料,探讨了HMW-GS缺失对籽粒品质性状、谷蛋白组分含量和加工品质的影响。在29份参试品系中,野生型有3个穗系,Glu-A1x、Glu-B1x、Glu-B1y、Glu-D1x和Glu-D1y缺失型分别有5、7、5、5和4份。野生型与缺失型,以及缺失型之间的蛋白质含量、湿面筋含量、籽粒硬度和溶剂保持力无显著差异。缺失型的谷蛋白/醇溶蛋白、高分量谷蛋白/低分子量谷蛋白含量比值低于野生型,其中Glu-B1x和Glu-D1x缺失型的比值显著低于野生型(P<0.05)。缺失型的揉面仪峰值时间和8 min带宽变异范围分别为1.38~1.64 min和3.38%~3.98%,显著低于野生型的2.00 min和4.57% (P<0.05),以Glu-B1x和Glu-D1x缺失型表现最低。与野生型相比,缺失型的糖酥饼干直径均有增加,其中Glu-B1x、Glu-B1y和Glu-D1y缺失型饼干直径的增加达显著水平(P<0.05),而缺失型之间的差异不显著。在宁麦9号背景下,高分子量麦谷蛋白单亚基缺失弱化了面筋强度,改善了糖酥饼干加工品质,亚基敲除可能是进一步提高软质小麦加工品质的有效途径。     

中国部分地区对虾白斑综合征病毒胶原蛋白基因缺失变异分析

为了解我国主要对虾养殖区的白斑综合征病毒(WSSV)胶原蛋白基因(wsv001)蛋白结构变异情况,以2015年13个省市发病地区采集的57份WSSV阳性样品为模板,用特异引物对目的片段进行扩增,连接转化扩增片段并进行测序分析。结果显示,57份WSSV阳性样品中,扩增出现条带的样品共有18份。氨基酸序列比对结果显示,扩增的wsv001片段所编码氨基酸出现三类变异,包括18个氨基酸小片段缺失,121个氨基酸大片段缺失及氨基酸插入,此外,还有8个氨基酸存在突变。结果表明,我国不同地区wsv001基因及其编码氨基酸存在明显缺失变异。对wsv001蛋白结构缺失变异的分析,为进一步对其蛋白功能和WSSV毒株毒力及WSSV对环境适应能力的研究提供参考。     

测定重复序列的一种有效方法——定向删除法

基因组中存在大量的重复序列,它们具有调节基因表达和染色体的生理代谢等重要的生物功能。重复序列由于其结构的特殊性,不但鸟枪法基因组测序不能有效地测定它们的序列,而且采用引物步移法等一般的定向测序方法也不能对其准确测定。本研究利用核酸外切酶Ⅲ定向删除技术,结合菌液电泳等快速筛选方法,对黄蜂蜘蛛三个高度重复序列丝蛋白基因MaSp2、CySp1和CySp2进行了准确的测定。研究结果表明核酸外切酶Ⅲ定向删除法是一种测定高度重复序列的有效方法,利用普通的琼脂糖电泳技术,该方法可以单向地有效测定重复序列的长度可达10kb,同时我们还发现合适内切酶的选择,定向删除亚克隆的筛选与排序以及序列的准确拼接是该方法有效而准确测定高度重复序列的关键。     

Deletion of TTTTA in 5′UTR of goat MSTN gene and its distribution in different population groups and genetic effect on bodyweight at different ages

A total of 436 goat samples from twenty-six populations were scanned for single nucleotide polymorphisms (SNPs) in 5′UTR of goat MSTN gene using sequencing and the polymerase chain reaction technique combined with restriction enzyme fragment length polymorphism (PCR-RFLP). The deletion of TTTTA found in 5′UTR of goat MSTN gene was conserved in different species and might be a unique mutation in goats. The Foreign Group and the Boer Goat backcrossed offspring had the highest and smallest genetic diversity of this mutation respectively, and also with the smallest and highest heterozygosity deficiency respectively. The genetic diversity of this mutation for the South Group of Chinese indigenous goat breeds was higher than that of the North Group, and the South Group had the smaller heterozygosity deficiency than the North Group. The estimated marginal means based on the modified population marginal mean and pairwise comparison between significant pairs showed that there was a trend that genotype AA had a significant effect on body weight and size from birth to four-month old (P<0.05 or P<0.01). It could be inferred that the deletion of TTTTA in 5′UTR of goat MSTN gene had a significant effect on body weight and size.     

牛种布鲁氏菌Ⅳ型分泌系统对巨噬细胞内质网应激和细胞凋亡的影响

旨在研究牛种布鲁氏菌Ⅳ型分泌系统(T4SS)对巨噬细胞内质网应激和细胞凋亡的影响,深入探究布鲁氏菌的分子致病机制.本研究以牛种布鲁氏菌疫苗株A19及其T4SS启动子缺失株A19ΔVirB侵染小鼠巨噬细胞,分别在侵染的6、12、24 h收集细胞RNA和总蛋白,通过qRT-PCR以及Western blot检测内质网应激标...