An efficient method for constructing a random insertional mutant library for forward genetics in Nannochloropsis oceanica

Insertional mutation,phenotypic evaluation,and mutated gene cloning are widely used to clone genes from scratch.Exogenous genes can be integrated into the genome during non-homologous end joining(NHEJ)of the double-strand breaks of DNA,causing insertional mutation.The random insertional mutant library constructed using this method has become a method of forward genetics for gene cloning.However,the establishment of a random insertional mutant library requires a high transformation efficiency of exogenous genes.Many microalgal species show a low transformation efficiency,making constructing random insertional mutant libraries difficult.In this study,we established a highly efficient transformation method for constructing a random insertional mutant library of Nannochloropsis oceanica,and tentatively tried to isolate its genes to prove the feasibility of the method.A gene that may control the growth rate and cell size was identified.This method will facilitate the genetic studies of N.oceanica,which should also be a reference for other microalgal species.

The relationship among amyloid-βdeposition,sphingomyelin level,and the expression and function of P-glycoprotein in Alzheimer’s disease pathological process

In Alzheimer’s disease,the transporter P-glycoprotein is responsible for the clearance of amyloid-βin the brain.Amyloid-βcorrelates with the sphingomyelin metabolism,and sphingomyelin participates in the regulation of P-glycoprotein.The amyloid cascade hypothesis describes amyloid-βas the central cause of Alzheimer’s disease neuropathology.Better understanding of the change of P-glycoprotein and sphingomyelin along with amyloid-βand their potential association in the pathological process of Alzheimer’s disease is critical.Herein,we found that the expression of P-glycoprotein in APP/PS1 mice tended to increase with age and was significantly higher at 9 and 12 months of age than that in wild-type mice at comparable age.The functionality of P-glycoprotein of APP/PS1 mice did not change with age but was significantly lower than that of wild-type mice at 12 months of age.Decreased sphingomyelin levels,increased ceramide levels,and the increased expression and activity of neutral sphingomyelinase 1 were observed in APP/PS1 mice at 9 and 12 months of age compared with the levels in wild-type mice.Similar results were observed in the Alzheimer’s disease mouse model induced by intracerebroventricular injection of amyloid-β1-42 and human cerebral microvascular endothelial cells treated with amyloid-β1-42.In human cerebral microvascular endothelial cells,neutral sphingomyelinase 1 inhibitor interfered with the changes of sphingomyelin metabolism and P-glycoprotein expression and functionality caused by amyloid-β1-42 treatment.Neutral sphingomyelinase 1 regulated the expression and functionality of P-glycoprotein and the levels of sphingomyelin and ceramide.Together,these findings indicate that neutral sphingomyelinase 1 regulates the expression and function of P-glycoprotein via the sphingomyelin/ceramide pathway.These studies may serve as new pursuits for the development of anti-Alzheimer’s disease drugs.

Variations in chlorophyll content, stomatal conductance, and photosynthesis in Setaria EMS mutants

Chlorophyll (Chl) content,especially Chl b content,and stomatal conductance (G_s) are the key factors affecting the net photosynthetic rate (P_n).Setaria italica,a diploid C_4 panicoid species with a simple genome and high transformation efficiency,has been widely accepted as a model in photosynthesis and drought-tolerance research.The current study characterized Chl content,G_s,and P_n of 48 Setaria mutants induced by ethyl methanesulfonate.A total of 24,34,and 35 mutants had significant variations in Chl content,G_s,and P_n,respectively.Correlation analysis showed a positive correlation between increased G_s and increased P_n,and a weak correlation between decreased Chl b content and decreased P_n was also found.Remarkably,two mutants behaved with significantly decreased Chl b content but increased P_n compared to Yugu 1.Seven mutants behaved with significantly decreased G_s but did not decrease P_(n )compared to Yugu 1.The current study thus identified various genetic lines,further exploration of which would be beneficial to elucidate the relationship between Chl content,G_s,and P_n and the mechanism underlying why C_4 species are efficient at photosynthesis and water saving.

Construction of EMS-Induced Peanut Mutant Libraries and Identification of Pod-Related Traits Mutant Lines

Peanut(Arachis hypogaea L.)is an oil and economic crop of vital importance,and peanut pod is the key organ influencing the yield and processing quality.Hence,the Pod-related traits(PRTs)are considered as important agronomic traits in peanut breeding.To broaden the variability of PRTs in current peanut germplasms,three elite peanut cultivars were used to construct Ethyl methane sulfonate(EMS)-induced mutant libraries in this study.The optimal EMS treatment conditions for the three peanut varieties were determined.It was found that the median lethal dose(LD50)of EMS treatment varied greatly among different genotypes.Finally,the EMS-induced peanut mutant libraries were constructed and a total of 124 mutant lines for PRTs were identified and evaluated.Furthermore,“M-8070”,one of the mutant lines for pod constriction,was re-sequenced via high-throughput sequencing technology.The genome-wide variations between“M-8070”and its wild parent“Fuhua 8”(FH 8)were detected.2994 EMS-induced single nucleotide polymorphisms(SNPs)and 1188 insertion-deletions(InDels)between“M-8070”and its wild parent were identified.The predominant SNP mutation type was C/G to T/A transitions,while the predominant InDel mutation type was“1-bp”.We analyzed the distribution of identified mutations and annotated their functions.Most of the mutations(91.68%of the SNPs and 77.69%of the InDels)were located in the intergenic region.72 SNPs were identified in the exonic region,leading to 27 synonymous,43 nonsynonymous and 2 stop-gain variation for gene structure.13 Indels were identified in the exonic region,leading to 4 frame-shift,8 non-frame-shift and 1 stop-gain variations of genes.These mutations may lead to the phenotypic variation of“M-8070”.Our study provided valuable resources for peanut improvement and functional genomic research.

Effect of Cyclooxygenase Inhibition on P-Glycoprotein Expression and Phenytoin Level in Brain Tissue of Pilocarpine Induced Epilepsy in Rats

Background: Increased brain P-glycoprotein (P-gp) expression may play important role in resistance to antiseizure drugs. The present work aimed to overcome the drug resistance that develop due to overexpression of P-gp with subsequent increase in brain phenytoin level in epileptic rats, using either non-selective (indomethacin) or selective (celecoxib) cyclooxygenase inhibitors. Methods: Fifty-six adult male albino rats were randomly divided into seven groups. Epilepsy was induced using the lithium pilocarpine model. Rats received indomethacin (2.5 mg/kg) or celecoxib (20 mg/kg), either alone or combined with phenytoin (50 mg/kg). Seizures were evaluated using Racine score. Motor coordination was assessed using open field and rotarod tests. Phenytoin brain level was measured using High Performance Liquid Chromatography (HPLC), glutamate expression was measured using Enzyme Linked Immunosorbent Assay (ELISA), ATP Binding Cassette Subfamily B Member 1 (ABCB1) gene expression was assessed using Real Time-Polymerase Chain Reaction (RT-PCR), and immunohistochemical analysis was done for P-gp expression. Results: Phenytoin combination with either indomethacin or celecoxib had improved the Racine score, motor coordination on rotarod apparatus, and open field test results. Also, phenytoin combination with either indomethacin or celecoxib decreased brain glutamate level, ABCB1 gene and P-gp expression, and increased brain phenytoin level compared to treatment with phenytoin alone. This indicated that both P-gp inhibitors indomethacin and celecoxib, increased the level of phenytoin that reached the brain of rats. However, brain uptake of phenytoin was significantly enhanced using celecoxib rather than indomethacin (CI 95%, 17.092: 32.808, P-value Conclusion: Cyclooxygenase inhibition using either celecoxib or indomethacin resulted in downregulation of P-gp expression, with subsequent increase in brain phenytoin level in epileptic rats.

Transcriptome Analysis of Molecular Mechanisms Underlying Phenotypic Variation in Phaseolus vulgaris Mutant‘nts’

The phenotype of a common bean plant is often closely related to its yield,and the yield of plants with reduced height or poor stem development during growth is low.Mutants serve as an essential gene resource for common bean breeding genetic research.Although model plants and crops are studied to comprehend the molecular mechanisms and genetic basis of plant phenotypes,the molecular mechanism of phenotypic variation in common beans remains underexplored.We here used the mutant‘nts’as material for transcriptome sequencing analysis.This mutant was obtained through 60Co-γirradiation from the common bean variety‘A18’.Differentially expressed genes were mainly enriched in GO functional entries such as cell wall organization,auxin response and transcription factor activity.Metabolic pathways significantly enriched in KEGG analysis included plant hormone signal transduction pathways,phenylpropanoid biosynthesis pathways,and fructose and mannose metabolic pathways.AUX1(Phvul.001G241500),the gene responsible for auxin transport,may be the key gene for auxin content inhibition.In the plant hormone signal transduction pathway,AUX1 expression was downregulated and auxin transport across the membrane was blocked,resulting in stunted growth of the mutant‘nts’.The results provide important clues for revealing the molecular mechanism of‘nts’phenotype regulation in bean mutants and offer basic materials for breeding beneficial phenotypes of bean varieties.

Mapping and Functional Analysis of LE Gene in a Lethal Etiolated Rice Mutant at Seedling Stage

An EMS(ethy methanesulfonate)-induced lethal etiolated(le)mutant obtained from the rice variety Zhongjian 100 was characterized by lethal etiolated phenotypes,with significantly reduced levels of chlorophyll a,chlorophyll b,total chlorophyll,and carotenoids.Additionally,the mutant displayed a significantly decreased number of chloroplast grana,along with irregular and less-stacked grana lamellae.The le mutant showed markedly diminished root length,root surface area,and root volume compared with the wild type.It also exhibited significantly lower catalase activity and total protein content,while peroxidase activity was significantly higher.Using the map-based cloning method,we successfully mapped the LE gene to a 48-kb interval between markers RM16107 and RM16110 on rice chromosome 3.A mutation(from T to C)was identified at nucleotide position 692 bp of LOC_Os03g59640(ChlD),resulting in a change from leucine to proline.By crossing HM133(a pale green mutant with a single-base substitution of A for G in exon 10 of ChlD subunit)with a heterozygous line of le(LEle),we obtained two plant lines heterozygous at both the LE and HM133 loci.Among 15 transgenic plants,3 complementation lines displayed normal leaf color with significantly higher total chlorophyll,chlorophyll a,and chlorophyll b contents.The mutation in le led to a lethal etiolated phenotype,which has not been observed in other ChlD mutants.The mutation in the AAA+domain of ChlD disrupted the interaction of ChlDle with ChlI as demonstrated by a yeast two-hybrid assay,leading to the loss of ChlD function and hindering chlorophyll synthesis and chloroplast development.Consequently,this disruption is responsible for the lethal etiolated phenotype in the mutant.

靶向敲除棉花GhAGL16高效sgRNA的筛选

【目的】AGL16是棉花MADS-box基因家族中一个重要的转录负调控因子,在抵御干旱和盐胁迫过程中发挥着重要作用。利用病毒介导的基因编辑技术(virus-induced gene editing,VIGE)筛选靶向敲除棉花GhAGL16的sgRNAs,并验证这些sgRNAs的特异性,为定向创制棉花agl16突变体奠定重要基础。【方法】根据在棉花YZ-1中A亚组和D亚组上实际克隆GhAGL16的基因组序列,预测了3个能靶向敲除GhAGL16的sgRNAs;基于棉花叶皱缩病毒(cotton leaf crumple virus,CLCrV)介导的VIGE系统,构建3个CLCrV-AtU6-26::GhAGL16-sgRNAs表达载体;利用qPCR检测Cas9超表达(Cas9 over-expression,Cas9-OE)植株中Cas9的表达量,确定Cas9是否稳定遗传表达;将3个CLCrV-AtU6-26::GhAGL16-sgRNAs表达载体分别转化Cas9-OE棉花子叶,并通过PCR/RE方法检测3个靶点的突变情况;利用生物信息学方法分析3个GhAGL16-sgRNAs的二级结构;对突变植株进行Hi-TOM高通量测序,明确基因编辑效率。同时,对转化GhAGL16-sgRNA2的突变植株进行脱靶率鉴定,检测基因编辑的特异性。【结果】成功构建了3个能同时靶向敲除GhAGL16-A亚组和GhAGL16-D亚组序列的sgRNAs。对不同Cas9-OE植株中Cas9表达量检测结果表明,Cas9在不同Cas9-OE棉株中稳定表达。用3个CLCrV-AtU6-26::GhAGL16-sgRNAs表达载体转化Cas9-OE棉花子叶,PCR/RE突变检测结果表明,GhAGL16-sgRNA2可有效用于GhAGL16的靶向敲除,在棉花A亚组和D亚组靶位点上出现了不同碱基缺失的突变类型,而GhAGL16-sgRNA1和GhAGL16-sgRNA3是2个无效sgRNA。3个GhAGL16-sgRNAs的二级结构分析结果表明,GhAGL16-sgRNA1和GhAGL16-sgRNA3可能存在引导序列容易与其他序列发生配对并且不易解链的现象,干扰了引导序列对目标位点的识别,导致sgRNA无效。进一步量化GhAGL16-sgRNA2对GhAGL16的编辑效率,对每一株转化CLCrV-AtU6-26::GhAGL16-sgRNA2的Cas9-OE植株突变检测结果表明,在转化的9株Cas9-OE植株中有6株发生了突变,突变效率为66.67%。此外,Hi-TOM高通量测序结果表明,GhAGL16-sgRNA2对GhAGL16的编辑效率为13.69%—54.42%。脱靶率鉴定结果表明,预测的4个潜在脱靶位点都没有发现脱靶现象,说明GhAGL16-sgRNA2不仅具有高效的基因编辑效率,而且具有专一的基因编辑特异性。【结论】利用CLCrV介导的VIGE系统转化Cas9-OE棉花筛选获得1个能高效敲除GhAGL16的sgRNA,为定向创制棉花agl16突变体提供了理想的sgRNA。

玉米矮秆突变体20F421的表型鉴定及遗传分析

矮秆资源是农作物矮化育种的物质基础,发掘矮秆基因资源对培育矮秆新品种具有重要作用。为了明确^(252)CF裂变快中子辐射诱变玉米自交系KWS49筛选得到的矮秆突变体20F421的遗传特性和矮化机理,以20F421为材料分别与玉米自交系PH6WC、B73、Mo17及KWS49杂交构建F_(1)和F_(2)分离群体,分析矮秆性状的遗传模式,并以(20F421/B73)F_(2)为定位群体,采用混池转录组测序(bulked segregant RNA-seq,BSR-seq)方法初步定位突变基因。结果表明,与KWS49相比,20F421的植株高度为95.2 cm,降低47.89%;穗位高度为23.9 cm,降低64.54%;茎秆节间长度显著缩短、叶片较直立密生,自交结实良好。遗传分析表明,F2分离群体野生型(高秆)与突变型(矮秆)植株性状分离比例符合3∶1,表明该突变体受单个核隐性基因控制;BSR-seq结果将突变基因定位在1号染色体177~255 Mb之间。通过与B73参考基因组进行比对发现,该区间内含有矮秆基因Br2,将20F421与br2突变体杂交进行等位性检测,F_(1)和F_(2)的株高均没有发生性状分离,表现为突变体20F421和br2表型,推测20F421的矮秆突变基因为矮秆基因Br2的等位突变。研究结果为进一步精细定位、克隆突变基因奠定基础,也为解析玉米矮化机理和培育矮秆玉米新品种提供重要基因资源和理论支撑。

抗枯萎病宝岛蕉早花突变体的筛选与鉴定

【目的】筛选鉴定抗枯萎病香蕉品种宝岛蕉的早花突变体,为进一步利用突变体株系选育适应性更广的香蕉新品种提供参考依据。【方法】利用多代组织培养繁育结合田间种植对比试验,依据生育期短、抗病性强、株高矮、株产高和遗传稳定筛选目标,从宝岛蕉早花突变后代中筛选优良株系,以宝岛蕉为对照,按照香蕉种质资源描述规范对优良突变株系的形态特征和生物学特性等进行观察鉴定,测定分析植株球茎生长点部位碳、氮营养累积以及成花相关激素吲哚乙酸(IAA)、玉米素(ZR)、赤霉素(GA)和脱落酸(ABA)含量差异,通过ISSR分子标记检测突变体及其野生型之间的遗传变异和亲缘关系。以宝岛蕉和主栽品种(桂蕉6号及巴西蕉)为对照,利用伤根浸菌法进行苗期抗病性鉴定,并在广西和海南开展区域种植试验及田间抗病性鉴定。【结果】筛选获得优良突变株系0523(简称0523),其新植蕉平均株高280.0 cm,假茎基围80.5 cm、中围60.0 cm,较宝岛蕉分别减小14.0%、5.8%和11.8%,新抽总叶片数26~29片;果实营养品质和单株产量与宝岛蕉无显著差异(P>0.05,下同)。0523球茎生长点碳氮比较宝岛蕉显著提高19.0%(P<0.05,下同),GA和IAA含量显著降低24.8%和22.6%,ABA和ZR含量与宝岛蕉相比略有增加,差异不显著。抗病性苗期鉴定结果显示0523为中抗且偏强,抗性水平与宝岛蕉相比略有提高,桂蕉6号为高感。突变株系0523与宝岛蕉的多态性比率为8.8%,遗传相似系数为0.95,二者存在差异。0523在广西和海南各试验点第1、2造蕉平均发病率分别为4.5%和3.5%,较主栽品种降低87.8%和94.1%,各试验点发病率均显著低于主栽品种。0523第1、2造蕉的生育期较宝岛蕉显著缩短,与主栽品种无显著差异。【结论】突变株系0523具有抗枯萎病、早熟、矮化、适应性广等优良性状,可用来培育大面积推广的香蕉新品种,或作为亲本育种材料应用于香蕉育种研究。

一株拟南芥宽叶形突变体atscamp的分离鉴定

叶片是主要的光合作用器官,选育利于光合作用的叶片形态已成为重要的育种目标。atscamp是从拟南芥(Arabidopsis thaliana)突变体库(约6000株系)中筛选获得的1株叶片宽大突变体。Tail-PCR分析该突变体为AT1G11180位点的插入,该基因位点编码1个分泌载体膜蛋白(SCAMP)。RT-PCR检测显示,该基因转录表达水平基本为零。进一步研究发现,该突变体叶片的宽度和叶面积极显著大于野生型植株(P<0.01),但是冠幅基本保持不变;同时atscamp突变体叶绿素含量增加,叶绿素最大荧光、PSⅡ潜在光化学效率显著增加(P<0.05);相应地,突变体植株蒸腾系数(Tr)、净光合速率(Pn)和叶片水分利用效率(WUE)显著增加(P<0.05)。拟南芥AT1G11180基因的时空特异性表达分析显示,该基因仅在叶片中高表达,在其他器官中表达量很低;且随着植物发育成熟,该基因表达量逐渐增加。研究结果表明AtSCAMP基因在叶形发育中发挥着重要作用。

大白菜蜡质缺失突变体YW71的遗传及序列变异分析

以大白菜蜡质突变体YW71为对象,研究其亮绿无蜡粉性状的遗传规律和调控基因。通过遗传分析,表明YW71中的亮绿无蜡粉性状由隐性单基因控制。通过等位基因互补实验证明YW71的亮绿性状是BrWAX2等位突变引起的。序列分析表明,在YW71中BrWAX2基因在第3个外显子末端发生了39 bp的缺失,进而引起转录水平的可变剪切和翻译水平的提前终止。表达模式分析表明,BrWAX2基因在YW71茎和叶中表达量显著下降。此外,研究针对39 bp的变异开发并验证了共显性标记BrWAX2-In Del1。研究结果丰富了白菜类蔬菜蜡质突变遗传资源,将为亮绿无蜡粉品种的分子育种提供理论指导和技术支持。

基于变异的正则表达式反例测试串生成算法

正则表达式在计算机科学的许多领域具有广泛应用.然而,由于正则表达式语法比较复杂,并且允许使用大量元字符,导致开发人员在定义和使用时容易出错.测试是保证正则表达式语义正确性的实用和有效手段,常用的方法是根据被测表达式生成一些字符串,并检查它们是否符合预期.现有的测试数据生成大多只关注正例串,而研究表明,实际开发中存在的错误大部分在于定义的语言比预期语言小,这类错误只能通过反例串才能发现.研究基于变异的正则表达式反例测试串生成.首先通过变异向被测表达式中注入缺陷得到一组变异体,然后在被测表达式所定义语言的补集中选取反例字符串揭示相应变异体所模拟的错误.为了能够模拟复杂缺陷类型,以及避免出现变异体特化而无法获得反例串的问题,引入二阶变异机制.同时采取冗余变异体消除、变异算子选择等优化技术对变异体进行约简,从而控制最终生成的测试集规模.实验结果表明,与已有工具相比,所提算法生成的反例测试串规模适中,并且具有较强的揭示错误能力.

苜蓿抗寒突变体生理生化及性状指标分析

为选育在黑龙江省可越冬的理想紫花苜蓿品种。以‘龙牧806’零磁空间诱变处理后二代群体中获得的lm1609、lm1625、lm1704三个抗寒突变体为实验材料,通过测定发芽指标、越冬率、产量特性指标及根系生理生化指标,进行抗寒性能鉴定。3个突变体发芽指数与对照差异不显著;发芽势、简化活力指数lm1609显著高于lm1625、lm1704,分别为82.05%、469.82(P<0.05);3个突变体的越冬率都显著低于对照组;突变体和对照6月6日起始株高差异不显著,随着植株生长,7月6日和8月6日lm1609、lm1704显著高于lm1625和对照;突变体lm1609、lm1625、lm1704三者鲜草产量差异不显著,但显著高于对照(P<0.05);三个苜蓿突变体植株根系过氧化物酶、超氧化物歧化酶均显著高于对照,并且突变体lm1609 POD、SOD最高,分别达到304.85 OD470/(min·g)、208.41 U/g(P<0.05)。lm1609突变体具有较高的抗寒特性及产量特性,是为理想的抗寒突变体材料。

玉米籽粒皱缩突变体sh2019的分子标记初步定位

玉米籽粒作为光合产物的重要贮藏器官,其发育直接影响百粒重和产量。本研究以玉米籽粒皱缩突变体sh2019、野生型玉米自交系Mo17及两者的杂交F_(2)代为材料,进行表型分析、遗传分析和分子标记初步定位。结果表明,突变体sh2019籽粒干瘪,种皮皱缩,百粒重和出苗率均比野生型显著下降,分别下降43.55%和69.56%。遗传分析发现突变体sh2019的籽粒皱缩表型受1对隐性核基因控制。利用F_(2)分离群体借助集团分离分析法进行的分子标记定位结果表明,sh2019基因被定位于玉米3号染色体上约30.18 Mb的物理距离内。本研究结果可为开展sh2019基因的精细定位及分子标记辅助育种奠定基础。

陆地棉自然早落叶突变系的激素含量分析

为研究陆地棉自然早落叶性状的激素表达特征,本研究采用自然早落叶突变系落叶棉501为试验材料,以叶片功能期长的近等基因系N065、陆地棉品系N002作为对照1与对照2,采用酶联免疫法(ELISA)定量测定不同组织的脱落酸(ABA)、赤霉素(GA3)、玉米素(ZR)和生长素(IAA)含量。从种胚激素含量看,落叶棉501的ABA含量极显著高于对照,分别是对照1与对照2的2.86倍和2.39倍;其GA3含量极显著低于对照,仅为对照1、对照2的21.4%、23.6%;其ZR含量也显著低于对照,分别为对照1、对照2的70.7%、60.4%;但其IAA含量与对照无显著差异。从叶片激素含量看,落叶棉501的ABA含量极显著高于对照,分别是对照1与对照2的2.65倍和5.63倍;其GA3含量则极显著低于对照,分别为对照1与对照2的21.8%、31.8%;其ZR含量与对照无显著差异;IAA的含量则显著低于对照,分别为对照1、对照2的21.7%和31.6%。综合分析,陆地棉早落叶性状与种胚、叶片中的ABA及GA3含量存在明显关系,可根据陆地棉顶端小真叶或种胚中的ABA、GA3含量,初步预判其是否具有早落叶特性。

局灶性脑缺血大鼠脑内mdr1／P-glycoprotein表达的变化

目的观察大鼠局灶性脑缺血损害后脑内mdr1／P—glycoprotein的表达变化。方法大鼠大脑中动脉栓塞法(MCAO法)制作局灶性脑缺血模型,脑切片免疫组织化学染色检测mdr-1／P—glycoprotein在脑内的表达部位及表达时程的变化。结果 mdr-1／P—glycoprootein在缺血侧皮层和纹状体的血管内皮细胞表达增多,并出现在同侧损伤部位的神经元,其在损伤后2h开始出现,6h达到高峰,之后开始下降,到24h不能被检测到。结论大脑中动脉阻塞后可以诱导缺血损伤侧的血管内皮细胞P—glycoprotein过量表达,而同侧的神经元短暂表达P-glyco- protein．

无乳链球菌nrdD基因缺失株的构建及其对小鼠的致病性检测

为研究nrdD基因对无乳链球菌毒力的影响,本研究参照GenBank中无乳链球菌GD201008-001(登录号:NC_018646)的nrdD全基因序列设计引物,采用融合PCR的方法将nrdD基因上、下游同源臂连接到一起,构建nrdD基因上、下游同源臂融合片段,连接至温敏型自杀质粒pSET4s。PCR克隆含有启动子序列的nrdD基因,连接至链球菌-大肠杆菌穿梭质粒pSET2。分别构建nrdD基因的缺失株ΔnrdD及互补株CΔnrdD。经PCR和基因测序,证明nrdD基因的缺失株及互补株构建成功。通过对ΔnrdD和CΔnrdD的形态学变化的观察,生长速率、小鼠血清中增殖能力、小鼠组织载量的测定以及小鼠攻毒试验的分析,评价nrdD基因对无乳链球菌毒力的影响。结果表明:与野生株GD201008-001相比,ΔnrdD在细菌染色形态上链长明显增加,但不影响荚膜的形成;在相同培养条件下,ΔnrdD在与GD201008-001生长速率无明显差异;在小鼠血清中增殖试验中,ΔnrdD在血清中的增殖能力较野生株明显减缓;在小鼠感染14 h后,ΔnrdD在其血液、脾脏及脑部的载量明显低于野生株,而CΔnrdD在各组织中的载量与野生株相比差异不显著;小鼠的攻毒试验显示,ΔnrdD注射组小鼠存活时间较野生株与互补株延长,差异显著。研究证明,nrdD基因与无乳链球菌毒力密切相关,本试验结果也为深入研究无乳链球菌nrdD基因的功能奠定了基础。

一种绿豆柱头外露突变体的转录组分析

柱头外露作为提高作物异交率、制种纯度和降低制种成本的优良性状,在杂交制种中得到了广泛的利用。绿豆是一种闭花授粉的作物,被报道的柱头外露突变体很少。通过对冀绿7号的化学诱变,发现了1个柱头外露突变体se2,为明确该突变体柱头外露的分子机制,对该突变体及其野生型冀绿7号即将开放的花蕾进行了转录组测序(RNA-seq)分析。根据差异倍数|log2(Fold Change)|≥1,P≤0.05的标准筛选,在se2中共得到572个差异表达基因(differentially expressed genes,DEGs),其中262个DEGs上调,310个DEGs下调。在基因本体(gene ontology,GO)数据库中,差异表达基因显著富集到代谢和生物合成等生物过程,定位在质外体和细胞壁、细胞膜等区域,与结合、氧化还原等分子功能有关。在京都基因与基因组百科全书(kyoto encyclopedia of genes and genome,KEGG)数据库中,差异表达基因显著富集在植物激素信号传导、次生代谢物生物合成等通路。功能注释发现许多有关细胞壁合成和代谢、细胞分裂和细胞扩张、植物激素相关的基因,因此推测se2突变体中龙骨瓣的细胞分裂、细胞扩张以及植物激素信号传导过程受到影响,从而导致了柱头外露。本研究为今后探究绿豆柱头外露的分子机制以及该性状在绿豆杂种优势中的利用奠定了基础。

核偏最小二乘法及其在P-glycoprotein抑制剂设计中应用

介绍了一个新的基于优化推导出的核偏最小二乘(kernel partial least squares,K-PLS)的算法原理和实现步骤,并且给出了利用K-PLS法构建P-糖蛋白(P-glycoprotein,P-gp)黄酮类抑制剂的定量构效关系(QSAR)模型.利用该方法结合几个简单的分子拓扑参数,构建了具有高准确率的预测模型.该模型将有助于P-gp黄酮类抑制剂的虚拟筛选和理性设计.结果证明K-PLS是一个十分稳定可靠的方法,将会在化学计量学领域得到较好的应用和推广.