Study on introduction of XIOsPR10 gene into rice and the functional analysis of XIOsPR10

PR10蛋白的表达与植物系统获得性抗性反应（SAR）有着紧密的联系，在很多植物对病原菌的防御反应中PR10类蛋白都具有转录表达活性，许多研究表明PR10类蛋白在植物防御反应中具有重要的作用。为了更好地研究水稻XIOsPR10基因的功能，及其在水稻抗病反应中的的作用，我们构建了相应的植物过量表达载体以及RNAi表达载体，并通过农杆菌介导的方法将这两个载体分别转入水稻中，希望通过对比能够更好地了解XIOsPR10基因的功能；同时通过原核表达得到了融合了His标签的目的蛋白，希望以此研究其活性，也为进一步研究水稻XIOsPR10基因功能奠定了基础。 首先，我们根据得到的XIOsPR10基因cDNA...It has been regarded that there are closed relationship between PR10s and systemic acquired resistance (SAR). And PR10s always shows its transcriptional activity when plant response to pathogen infection. Many studies showed that PR10s played an important role in plant defense response. In order to study the function of rice XIOsPR10 gene and its effects in plant resist disease reaction more clear...学位：理学硕士院系专业：生命科学学院生物化学与生物技术系_细胞生物学学号：20042606

Preliminary Study on Regulation of XIOsPR10 Gene Expression in Rice

为探索水稻Pr10蛋白在水稻抗细菌性条斑病中的作用,构建了XIOSPr10基因的植物过量表达载体P1301-XIOSPr10及rnAI表达载体PdS1301-XIOSPr10,通过农杆菌介导分别转化水稻愈伤组织,获得了相应的再生植株。经guS检测和PCr分析,证实XIOSPr10基因以及rnAI片段分别整合到水稻再生植株基因组中;半定量rT-PCr分析显示,过量表达植株中XIOSPr10基因的表达量高于对照,而rnAI转基因植株中XIO-SPr10基因的表达被抑制。In order to study the function of rice XIOsPR10 gene which is related to the plant disease resistant reaction,we constructed plant over expression and RNAi expression vector and integrated into the genome of rice via Agrobacterium tumefaciens EHA105,respectively.The transgenic cultivars were identified by PCR and GUS gene expression detection.The analysis result showed that the transcription level of XIOsPR10 of over-expression transgenic rice was higher than that of control.In construct,XIOsPR10 gene expression was blocked in RNAi transgenic rice.科技部863专题(2007AA10Z132);国家重大科技专项(2008ZX08001-001;2009ZX08009-045B);教育部重点项目(01102

Expression Analysis of a Stress Repressed Gene OsDSR4 from DUF966 Family and Generation of OsDSR4- overexpressing Transgenic Rice(Oryza sativa L. ssp. japonica)

OSdSr4基因是duf966基因家族中的一个未知功能基因,目前其生物学功能尚不清楚。本研究生物信息学分析显示,OSdSr4基因CdnA全长2 167 bP,包含一个1 149 bP的开放阅读框(Orf),编码382个氨基酸,推测的蛋白中包含一个高度保守的duf966结构域;表达模式分析表明,OSdSr4主要在水稻(OryzA SATIVA l.SSP.JAPOnICA)的茎节间和叶片中表达,干旱、高盐和低温等非生物胁迫明显抑制了OSd Sr4的表达,而脱落酸(AbSCISIC ACId,AbA)则显著诱导了它的表达;利用重叠延伸PCr方法成功克隆了OSdSr4,并将其转化进水稻中,获得了32株超表达转基因植株。分子鉴定结果表明,该基因已被整合进水稻基因组中,并在部分转基因植株中实现了超量表达。本实验为进一步开展OSdSr4基因的生物学功能研究提供了基础资料。OsDSR4 is a gene of unkown function in DUF966 gene family,and the function of DUF966 family genes have not been reported until now.In this study,the bioinformatic analysis showed that the cDNA of OsDSR4 had 2 167 bp containing an open reading frame(ORF) of 1 149 bp,and it encoded a putative protein of 372 amino acids with a highly conserved DUF966 domain.The gene expression profile analysis indicated that OsDSR4 was expressed mainly in internode and leaf blade of rice(Oryza sativa L.),and it was repressed markedly by drought,salt and cold stresses,and induced significantly by abscisic acid(ABA).OsDSR4 was cloned using overlap extension PCR,and the fusion construct containing OsDSR4 was introduced into rice(Oryza sativa L.ssp.japonica) by Agrobacterium- mediated transformation method.Thirty- two OsDSR4- overexpressing transgenic plants were obtained and identified by PCR and qRT-PCR,which was demonstated that OsDSR4 had been integrated into rice genome and was overexpressed in some positive transgenic plants.These results establish the foundation for further study of the precise function of OsDSR4.国家重点基础研究发展计划(973)前期研究专项(No.2012CB126312

Genetic Analysis and Cloning of the Rice Dwarf Mutant d-ss

通过γ射线诱变,从粳稻品种9522的M2代中筛选出一株矮秆水稻(OryzA SATIVA l.)突变体,定名d-SS.d-SS突变体表现为叶色深绿、短宽的叶片、以及小而圆的籽粒.以d-SS突变体与籼稻品种龙特普杂交的f2代群体为基因定位群体,利用IndEl分子标记将d-SS突变位点定位在5号染色体上的IndEl标记zz5-6和zz1343之间,物理距离为412kb.最终通过图位克隆的方法获得了此基因,测序结果表明此基因在编码区发生了两处缺失突变.Rice height character is one of the most important agronomic traits of rice(Oryza sativa L.).Generally,the dwarf varieties with proper plant height have a greater harvest and higher lodging resistance.Identifying new useful dwarf mutant and understanding its regulating mechanism is an important subject for practice rice breeding.In this study,the d-ss,a mutant of Oryza sativa L.spp.japonicacv.9522,was mutagenized by irradiation with 60 Coγ-ray.The d-ss mutant plants display short stem,dark green leaves,compact panicles,and short,round grains.To map the d-ss locus,an F2population generated by crossing between d-ss(japonica)mutant and Longtepu(indica)was analyzed.The d-ss locus was mapped to rice chromosome 5,between the two InDel markers,ZZ5-6and ZZ1343.The region was delimited to about 412kb.At last,the d-ss gene was cloned by map-based cloning.The analysis of sequencing indicated two deletions happened in translated regions of d-ss gene.国家重点基础研究发展计划(973)项目(2012CB126312

Establishment of H5N1 Real-time Fluorescence Quantitative PCR Detection Method Using Virus-like Particles as the Positive Control

合适的标准品对实时荧光定量PCr(QPCr)检测高致病性H5n1禽流感病毒(AVAIn InfluEnzA VIruS,AIV)十分重要.本研究将H5n1AIV HA基因的部分序列插入到能够表达MS2噬菌体病毒样颗粒(VIruS-lIkE PArTIClE,VlP)dnA序列的表达载体上,诱导表达后得到了包裹有H5n1AIV HA基因rnA片段的VlP.该VlP能够耐受核酶的消化,形态与MS2噬菌体病毒颗粒形态相同.利用表达的VlP作为阳性标准品及设计的特异性荧光探针、淬灭链,使用优化的QPCr反应体系,得到QPCr检测H5n1亚型AIV的阳性对照标准曲线.研究结果为高致病性H5n1亚型AIV的准确定量检测提供了基础.Appropriate standards are very important in detecting highly pathogenic H5N1 subtype avain influenza virus(AIV)by real-time fluorescent quantitative PCR(qPCR).In this study,part of HA gene sequence of the H5N1 AIV was inserted into the expression vector with the expression of MS2 bacteriophage virus-like particles(VLP).After that,the derivative HA VLP products were obtained,containing the RNA of parts of HA gene sequences by isopropyl β-D-1-thiogalactopyranoside induced expression.The HA-VLP was able to tolerate nuclear enzyme digestion,of which morphological structure was the same as VLP.Using the expressed HA-VLP as a positive control,the specific fluorescent probes and quenching were designed,and the positive control standard curve of qPCR detection of H5N1 subtype AIV was described through the optimum qPCR reaction system.The results laid a basic means for the quantitative detection of the highly pathogenic H5N1 subtype AIV.福建省自然科学基金(2010J01240); 厦门市科技计划项目(3502Z20103007

Prokaryotic Expression and RNase Activity Analysis of Rice XIOsPR10

Pr10(病程相关蛋白10)类蛋白与植物的抵御外来病害及系统获得性抗性(SAr)有着紧密联系,而且许多Pr10类蛋白都具有rnASE活性,并通过这种活性抵抗外源病害。根据获得的XIOSPr10基因,将其构建到PET28A中,通过原核表达及磁珠纯化获得目的蛋白,通过rnA消化实验证实XIOSPr10重组蛋白具有rnASE活性,进一步揭示了XIOSPr10蛋白的功能。It has been regarded that PR10s played an important role in systemic acquired resistance(SAR).Furthermore,many PR10 proteins have been reported to exhibit RNase activity and predicted to be responsible for its antibiotic activity.So the Prokaryotic Expression Vector pET28a-XIOsPR10 was constructed and transferred to BL21(DE3).The recombinant protein XIOsPR10 was obtained by Prokaryotic expression and purification of magnetic microbeads,and showed the RNase activity.国家“863”计划(2007AA10Z132);国家科技重大专项(2008ZX08001-001、2009ZX08009-045B);教育部科技研究重点项目(01102)资助项

Over-expression of PC-1 Gene Increases Survival Ability of C4-2B Prostate Cancer Cell Line

建立稳定表达外源PC-1基因的人前列腺癌骨转移C4-2B细胞模型,初步探讨PC-1基因表达对前列腺癌发展的影响.通过脂质体介导的方法,将融合PC-1基因的真核表达载体pcDNA3·1-PC-1稳定转染C4-2B细胞,Western印迹和RT-PCR技术,分别从蛋白水平和RNA水平确定外源PC-1基因表达.MTT和软琼脂集落形成能力等一系列方法,研究PC-1基因的功能,RT-PCR和实时定量PCR检测前列腺癌发生发展相关基因表达的变化.结果表明,PC-1基因的高表达能够诱导雄激素受体(AR)调控基因和一系列重要的信号通路成员基因PSA、PSMA、NKX3·1、Jagged1、EphA3、SGEF和NOTCH3等表达发生变化.实验结果初步证明,PC-1基因表达在晚期前列腺癌中,以及在雄激素非依赖的转变中可以发挥作用,PC-1基因表达可调控一些重要信号通路.对PC-1基因功能深入研究将有可能为发现新的前列腺癌的诊断治疗分子靶标提供线索.To investigate the potential role of PC-1 gene over-expression on prostate cancer progression, a metastasis and androgen independent prostate cancer cell line C4-2B was used, cell clones constitutively expressing PC-1, and their mock-transfected counterparts were constructed. The expression of ectopic PC-1 was analyzed by Western blot and RT-PCR assay. MTT and colony anchor-independent growth in soft agar were performed to evaluate the effects of PC-1 gene expression on prostate cancer progression. RT-PCR and real-time PCR were used to analyse the expression of differential genes between C4-2B-PC-1 and C4-2B-neo cell lines. Our results showed that stable expression PC-1 induced transcription change of some genes, including PSA,PSMA,NKX3.1,Jagged1, EphA3, SGEF and NOTCH3, which were the androgen receptor, regulated genes and important signal transduction pathway molecules. These observations indicated that PC-1 over-expression might play important role in advanced prostate cancer progression, promote prostate cancer cell androgen independent growth, and may regulate multiple signal transduction pathways. Further study of PC-1 gene function may provide clue to discover new molecular targets for prostate cancer diagnosis and treatment.国家自然科学基金资助(No.30070296)~

Genetic Analysis and Mapping of Rice(Oryza sativa.L) sd-sl Mutant

chenlg @xmu. edu. cn[中文文摘]新的矮秆基因的发掘、研究和利用对水稻育种和植物生长发育机制研究有重要的作用.用60Coγ射线辐照粳稻9522,获得一个能稳定遗传的突变体.该突变体表型为株高较野生型矮,叶片短而微卷.将该突变体与籼稻广陆矮杂交,F2代呈3∶1分离,说明该突变体受隐性单基因控制.通过InDel分子标记对F2代分离群体进行遗传定位,将该基因定位于第6染色体InDel标记OS604附近.随后又发展了多对有多态性的InDel分子标记,将该基因座位精细定位在InDel标记XL6-6和XL6-1之间,AP003490和AP005619上,两个引物之间的物理距离为118 kb.本研究为该克隆基因及其作用机理的探究奠定了基础.[英文文摘]It is the bright road of rice breeding to study about dwarf mutant . On the other hand ,dwarf mutant s in rice are crucial for elucidating regulating mechanisms for plant growth and development . In the present study ,the j aponica cultivar 9522 was muta-genized by 60Coγ-Ray and then a rice dwarf mutant s d2sl was found which was dwarf and had smallish ,olled leaves. Genetic analysis indicated that the phenotype was cont rolled by a single recessive gene. To map sd-sl locus ,a F2 population was const ructed f rom the cross between the s d2sl ( j a ponica) and guangluai ( indica) . Firstly ,the s d2sl locus was roughly mapped by InDel marker ,OS604 ,in rice chromosome 6. And then several polymorphic markers were developed for further fine2mapping. At last the sd-sl locus was mapped between the two InDel markers ,XL626 and XL621. The region was delimited to about 118 kb. These result s provide a basis for molecular cloning and functional analysis of sd-sl .科技部“功能基因组与生物芯片”之“水稻功能基因组”子课题(2002AA2Z1002)资