一款老人助力起身智能马桶研究

研究一款能在现有旧式马桶基础上,通过加装助力起身智能马桶盖,实现助力起身和智能冲洗的结合,简化操作动作,适应老人使用的设备。     

Identification of Pathogenicity Defective Mutants from a T-DNA Insertion Mutant Library of Verticillium dahliae

[Objective] Our research aimed to identify pathogenicity defective mutants from a T-DNA insertion mutant library of Verticillium dahliae strain Vd991 and to analyze pathogenicity-related genes. [Method] In total, 294 T-DNA insertion mutants of V. dahliae were tested for their virulence using a cotton infection assay. Southern blot assays were performed to identify the T-DNA insertion copy number of each pathogenicity defective mutant. DNA sequences flanking the T-DNA insertional sites of each mutant were analyzed by high-efficiency thermal asymmetric interlaced PCR. [Result] Based on the virulence assay, the disease indices of cotton plants inoculated with each mutant decreased very significantly in comparison with the index of those inoculated with Vd991. The Southern blot assay revealed that only one mutant contained two T-DNA insertions, while the remaining eight mutants harbored a single T-DNA insert. An analysis of biological characteristics found that the growth and conidial production of these mutants were impaired by the T-DNA insertions compared with the wild type Vd991. The T-DNAs' insertion position and distribution in each mutant were identified by comparison with genome sequences of strain VdLs.17. Furthermore, the pathogenicity-related genes were cloned from strain Vd991. [Conclusion] The screening and identification of T-DNA insertion mutants is an effective method to identify the pathogenicity-related genes of V. dahliae on a genome-wide scale. This laid a foundation for the further breeding of disease-resistant cotton varieties and will promote the study of the pathogenic molecular mechanisms of V. dahliae.     

基于甘蔗热带种(LA-purple)全基因组序列的SSR开发及特征分析

现代甘蔗栽培品种(2n=100~130)是由甘蔗热带种(2n=80)与割手密(2n=40~128)种间杂交而来,形成异源多倍体、非整倍体作物,使得甘蔗栽培品种中80%~90%的染色体来源于热带种。开发热带种基因组SSR分子标记,有助于甘蔗遗传多样性分析、分子标记辅助选择、遗传图谱的构建等。本研究基于热带种LA-purple的全基因组测序数据的255 398个预测基因序列(累计总长为1 029 222 285 bp),利用Perl程序与生物信息学软件结合,发掘SSR位点,获得了153 150个SSR位点,平均每1.67个基因有1个SSR位点,其中二、三核苷酸重复基序分别为39 556个和50 072个,占总SSR位点数的58.5%。在二核苷酸重复基序中,TA/AT所占比例最高,占41.4%,CG/GC所占比例最低,占4.6%;在三核苷酸碱基重复基序中,TGT/ACA所占比例最高,为15.6%。在TA/AT重复类型中选取100个基序重复次数在60~90之间的SSR位点,进行引物设计与合成,在12个甘蔗属材料中进行PCR扩增分析,从中筛选出52对具有多态性SSR引物,其中有27对引物在研究的2个甘蔗栽培品种间表现为多态。这些基因组SSR标记的开发,不仅可以用于甘蔗栽培品种DNA指纹图谱分析,而且为甘蔗属不同种的遗传图谱构建、遗传多样性分析和重要性状的遗传机制解析奠定基础,为甘蔗分子育种研究提供重要支撑。     

The integration and insertion site of GbVe1 gene in the genome of transgenic cotton (Gossypium hirsutum)

[Objective] The aim of this study was to obtain the flanking sequences of T-DNA in the transgenic cotton containing a GbVe1 over-expression cassette. [Method] The T-DNA insertion copy number in the transgenic GbVe1 cotton was analyzed by southern blot. Flanking sequences of the transgenic lines with putative single T-DNA insertion copy were obtained using high-efficiency Thermal asymmetric interlaced polymerase chain reaction (hiTAIL-PCR). The T-DNA insertion sites were further confirmed by PCR with specific primers. [Result] RB-flanking sequences (119-1 018 bp) and LB-flanking sequences (243-516 bp) were obtained from three transgenic lines with low copy number of T-DNA insertion. The AT content was more than 63% in these flanking sequences. A same single insertion site in the intron of Gohir.D01G157600.1 was found in the two transgenic lines 7/100826-152 and 12/100826-393, while two separated insertion sites, one also in the intron of Gohir.-D01G157600.1 and the other in the intergenic region of A12 chromosome, were found in the transgenic line 1/w-ch14. A deletion of 21 bp was found in the insertion site in the intron of Gohir.D01G157600.1. The T-DNA insertion in the intron of Gohir.D01G157600.1 was further confirmed by the specific PCR. [Conclusion] The flanking sequences of T-DNA in the transgenic GbVe1 cotton were obtained and the specific transformation event in the intron of Gohir.D01G157600.1 was further confirmed by PCR.     

烟草疫霉SSR分子标记的开发与初步应用

为开发烟草疫霉的SSR分子标记,利用MISA软件搜索烟草疫霉基因组序列中的SSR位点,共发现1311个SSR位点,优势SSR位点为二核苷酸和三核苷酸,分别占总SSR位点的56.45%和39.36%;根据分析到的SSR位点使用Primer 5.0软件设计48对SSR引物,以7株烟草疫霉的DNA为模板对这些引物进行筛选,共获得扩增条带清晰且具有多态性的SSR引物20对,然后使用其中的6对SSR对32株烟草疫霉进行UPGMA聚类分析,遗传相似系数在0.60~1.00之间,在相似系数0.70水平上,可将其划分为3个类群,类群Ⅰ包含29个菌株,类群Ⅱ包含1个菌株,类群Ⅲ包含2个菌株,显示出较低的遗传分化水平。这些SSR引物的开发将为研究我国烟草疫霉的遗传多样性、群体遗传结构以及遗传图谱构建等奠定基础。     

河北省玉米小斑病菌优势生理小种鉴定及ITS序列分析

2007~2016年在河北各地采集玉米小斑病标样,对分离出的356株玉米小斑病菌菌株进行生理小种鉴定。结果表明,在鉴定的年度范围内,玉米小斑病菌T小种、C小种、S生理型、O小种的分离频率在年度间存有差异,O小种的平均分离频率94.94%,是河北省玉米小斑病菌的优势小种;O小种对主栽品种郑单958和自交系C103的致病力呈下降趋势,在C103上致病力下降幅度小于郑单958。对河北省石家庄、保定、衡水、沧州邯郸、邢台采集的45株菌株进行ITS序列分析构建UPGMA进化树,分析表明,河北省内玉米小斑病菌株的遗传进化与地域有一定关系,差异不明显。     

转基因甘蔗BtG-2的T-DNA侧翼序列分析及其转化事件特异性检测

转基因甘蔗BtG-2是利用农杆菌介导法把Cry1Ac-2A-gna融合抗虫基因导入‘新台糖22号’的转基因甘蔗株系,具有良好的抗虫特性和农艺性状。为了明确转基因甘蔗BtG-2的分子特征及其检测方法,推进其生物安全性评价工作,以BtG-2的T2代为研究材料,利用Southern杂交检测外源基因在转基因甘蔗基因组内的拷贝数;利用染色体步移技术分离外源基因在甘蔗基因组中插入位点的侧翼序列,并建立了该转化体高效灵敏的特异性PCR检测方法。结果表明：Southern杂交检测证明外源T-DNA以单拷贝方式插入BtG-2株系;经过3次的热不对称巢式PCR扩增,获得外源基因T-DNA左边侧翼序列984 bp和右边侧翼序列705 bp;以这2个序列和相应的T-DNA的左右端序列分别设计3对检测引物对,建立了BtG-2株系的转化事件特异性PCR检测方法,扩增效率最高的引物对LS011/LA451和RS160/RA588分别扩增到440 bp和428 bp的特异片段。其中T-DNA左侧设计的LS011/LA451检测引物对扩增的灵敏度高、特异性强,能够在甘蔗BtG-2基因组DNA相对含量为0.1%的模板中检测出转基因目的成分,相当于9个单倍体基因组拷贝数。本研究完成了转基因株系BtG-2的分子特征及其转化事件特异性检测,为该转基因甘蔗及其衍生产品的检测和身份识别提供技术依据。     

蛙皮素样肽家族及其受体氨基酸序列信息学比较分析

【目的】研究反刍动物蛙皮素样肽家族多肽及其受体的保守性。为不同动物,特别是反刍动物这2种蛙皮素样多肽及其受体蛋白抗原设计和活性多肽筛选等研究提供参考。【方法】采用生物信息分析学的方法,通过UniProt数据库比较牛的蛙皮素样肽家族胃泌素释放肽(Gastrin-releasing peptide, GRP)和神经介素B(Neuromedin B, NMB)2种多肽及其特异性受体GRP-R和NMB-R与其他动物氨基酸序列的差异性。【结果】13种动物成熟GRP多肽C-端的8个氨基酸序列完全相同,牛GRP氨基酸序列与绵羊、猪和豚鼠最高,分别为88.7%、77.8%和77.8%,与其他动物相似性低于74.1%。10种动物成熟NMB多肽C-端10个氨基酸序列完全相同。牛GRP-R与狗、马和猪等相似性最高,分别为96.1%、94.3%和94.0%,牛NMB-R与猪、人和马相似性最高,分别为92.3%、91.5%和91.0%;牛GRP-R与NMB-R相似性仅为62.2%。【结论】GRP和NMB的C-端氨基酸序列在不同动物之间均具有高度的保守性,而N-端为变化区域,且GRP-R和NMB-R氨基酸序列在动物之间保守性较高。     

林麝肺源ST1249型铜绿假单胞菌的分离鉴定及其全基因组序列分析

【目的】对分离自林麝化脓肺脏的1株疑似铜绿假单胞菌(PA)进行鉴定,并分析其致病和耐药机制,为林麝PA感染化脓性疾病的防控奠定基础。【方法】将病原菌分离纯化后,依次进行生化试验、16S rRNA序列分析、药敏试验和小鼠致病性试验,并通过全基因组测序,对分离菌株进行群体进化和物种分型分析以及基因功能注释。【结果】分离自林麝化脓肺脏的1株疑似铜绿假单胞菌经鉴定与PA相符,命名为FMDP001。药敏试验显示其对阿莫西林、头孢曲松、氨曲南、多粘菌素B和林可霉素耐药;对小鼠半数致死量为9.4×10~5 CFU/mL。全基因组序列分析显示该菌基因组大小为6 955 100 bp,序列类型为ST1249,与B136-33株同源性最高,且两菌株基因组平均核苷酸一致性(ANI)值达98.93%;全基因组中共有357个序列编码FMDP001与致病性相关的基因,根据功能分为黏附、铁摄取、胞外毒性蛋白和调控系统;84个序列编码耐药基因,其中多药耐药外排泵为主要成分。【结论】从林麝化脓肺脏中分离到1株致病性较强的PA,并获得ST1249型林麝源PA的全基因组序列,序列显示该菌携带大量药物外排泵及生物膜形成相关基因,决定其具有多重耐药特性,哌拉西林等可作为该类型PA感染的临床用药。