水稻淀粉分支酶基因5′上游区缺失对基因表达的影响

为研究水稻淀粉分支酶基因 (sbe1) 5′上游调控区中存在的顺式作用元件 ,我们将水稻sbe1基因翻译起始点 (ATG) 5′上游区 1.2kb(- 10 96～ 74bp)片段经过不同限制性内切酶消化及外切核酸酶ExoIII部分消化 ,得到 4个 5′端缺失的片段。将这些缺失片段分别与 gus基因编码区连接 ,构建成融合质粒 ,经土壤农杆菌 (Agrobacterium)介导引入水稻 ,定量测定转基因水稻植株未成熟种子中的 gus酶活力。结果表明 ,- 5 16～ 6 4bp的sbe1启动子片段可以驱动gus基因的高表达 ,其它 3个启动子片段 (- 10 96～ 74bp ,- 2 95～ 74bp ,- 146～ 6 4bp)驱动 gus基因表达的能力较低。推测在sbe1基因 5′上游区 - 5 16～ - 2 95bp片段中可能存在能使 gus基因高表达的增强元件     

Expression Enhancement of a Rice Polyubiquitin Gene Promoter

An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold. Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes.     

籼稻蜡质基因5＇上游区与GUS基因编码区融合基因的构建和在水稻中的瞬间表达

为了鉴定水稻蜡质基因５＇上游区的顺式作用因子与研究它们在组织专一性表达中的作用,我们对籼稻品种２３２的蜡质基因５＇上游－１１５至－２１２０的区域进行了顺序测定,并将此基因的５＇上游区同报告基因ＧＵＳ构建成融合基因,用基因枪粒子轰击的方法将此融合基因导入水稻未成熟种子的幼胚和糊粉层细胞中,瞬间表达检测的结果表明,我们构建的融合基因中蜡质基因５＇上游区的长度已足以使ＧＵＳ基因在上述组织中表达。     

番茄rbcS3A启动子控制的GUS融合基因在转基因水稻中的表达

为研究不同启动子用于转基因水稻,克隆了番茄Rubisco小亚基rbcS3A基因的5′上游调控区,构建了由rbcS3A启动子引导的GUS嵌合基因,并经农杆菌介导导入到水稻中。对转基因水稻植株中GUS活性的定性与定量测定结果表明,rbcS3A启动子可驱动GUS报告基因在转基因水稻植株茎和叶组织中高效表达,而在根和种子等器官中不表达或表达活性极弱,表现出一定的组织特异性。在转基因水稻中,番茄rbcS3A启动子驱动外源基因的表达不受光诱导。     

Homologous recombination-mediated knock-in targeting of the MET1a gene for a maintenance DNA methyltransferase reproducibly reveals dosage-dependent spatiotemporal gene expression in rice

Although homologous recombination-promoted knock-in targeting to monitor the expression of a gene by fusing a reporter gene with its promoter is routine practice in mice, gene targeting to modify endogenous genes in flowering plants remains in its infancy. In the knock-in targeting, the junction sequence between a reporter gene and an endogenous target promoter can be designed properly, and transgenic plants carrying an identical and desired knock-in allele can be repeatedly obtained. By employing a reproducible gene-targeting procedure with positive–negative selection in rice, we were able to obtain fertile transgenic knock-in plants with the promoterless GUS reporter gene encoding β-glucuronidase fused with the endogenous promoter of MET1a , one of two rice MET1 genes encoding a maintenance DNA methyltransferase. All of the primary (T₀) transgenic knock-in plants obtained were found to carry only one copy of GUS , with the anticipated structure in the heterozygous condition, and no ectopic events associated with gene targeting could be detected. We showed the reproducible, dosage-dependent and spatiotemporal expression of GUS in the selfed progenies of independently isolated knock-in targeted plants. The results in knock-in targeted plants contrast sharply with the results in transgenic plants with the MET1a promoter -fused GUS reporter gene integrated randomly in the genome: clear interindividual variation of GUS expression was observed among independently obtained plants bearing the randomly integrated transgenes. As our homologous recombination-mediated gene-targeting strategy with positive–negative selection is, in principle, applicable to modify any endogenous gene, knock-in targeting would facilitate basic and applied plant research.     

水稻OsGSTL1启动子的分离和表达分析

从水稻基因组文库中筛选得到一个水稻GST基因,命名为OsGSTL1.半定量RT-PCR分析表明OsGSTL1基因的表达不受绿磺隆、乙烯利、脱落酸、水杨酸和茉莉酸甲酯的诱导,因此该基因可能与植物抗逆性无关.为了研究OsGSTL1启动子在植物体内的表达特性,将OsGSTL1起始位点5＇端上游不同长度的调控序列与报告基因GUS融合,并在洋葱表皮瞬间表达和拟南芥中稳定表达.研究表明：在洋葱表皮细胞中,160bp及更长的上游调控序列均能启动GUS基因的表达;而在转基因拟南芥中,含有2155 bp的上游序列的PGZ2.1：：GUS具有时空表达的特性,在转基因的早期幼苗中GUS基因在子叶中特异性表达,但在根中没有表达;而在幼苗生长的后期,根、茎、叶中都有少量的表达.但包含1 224 bp的上游序列的PGZ1.2：：GUS却表现为组成型表达的特性.由此推测,OsGSTL1启动子启动的基因表达可能与幼苗的营养代谢相关;而OsGSTL1启动子的时空表达相关元件可能位于OsGSTL1翻译起始位点5＇端上游-2155 bp至-1224 bp范围内.     

骨肉瘤中RASSF1A基因的甲基化状态研究

目的：分析骨肉瘤组织中RASSF1A基因甲基化状况。方法：运用甲基化特异性PCR（MSP）分别检测44例骨肉瘤组织及相应的癌旁组织中RASSF1A基因启动子甲基化状态并分析其临床病理意义。结果：骨肉瘤组织中RASSF1A基因异常甲基化率（61.4%）显著高于癌旁正常骨组织中RASSF1A基因的异常甲基化率（20.5%）,二者之间差异具有统计学意义（P〈0.05）。RASSF1A基因异常甲基化导致组织中RASSF1A基因mRNA和蛋白表达水平均显著降低。另外,RASSF1A基因异常甲基化和肿瘤组织分化程度及全身有无转移情况有相关性（P值分别为0.022和0.016）,而与患者年龄、性别、肿瘤位置及大小等临床特征无关（P值分别为0.6944,0.977,0.786和0.831）。结论：RASSF1A基因启动子高甲基化可能是导致其在骨肉瘤中表达水平降低的分子机制之一,有望成为骨肉瘤早期辅助诊断的一个重要分子标志物。     

在转基因水稻植株中蜡质基因第1内含子对-基因表达影响的分析

为阐明水稻Wx基因第1内含子在整体植株的胚乳发育阶段是否确有增强基因表达的功能,以及弄清高和中、低直链淀粉含量的水稻品种Wx基因第1内含子1 126个碱基之间有差异的16个碱基中哪几个碱基影响了该内含子的正常剪接从而降低了基因的表达水平,我们分别用高直链淀粉含量品种的Wx基因翻译起始密码子ATG上游3.1和2.1 kb片段与GUS基因编码区融合构建成嵌合质粒,并在此基础上,(1)去除嵌合质粒中Wx基因的第1内含子;(2)将嵌合质粒Wx基因的第1内含子中(3.1 kb)与中、低直链淀粉含量的水稻品种Wx基因第1内含子有差异的6个碱基以中、低直链淀粉含量的水稻品种的碱基替换.将上述改造过的几种质粒分别转化粳稻品种中花11,测定转化植株未成熟种子胚乳中的GUS活性.结果表明第1内含子的缺失或此内含子的5′端剪接点上的碱基G以T替换均造成GUS活性的急剧下降,说明第l内含子在植株体内的确有增强基因表达的功能,而且在中、低直链淀粉含量的水稻品种中Wx基因第1内含子5′端剪接点上自然存在的G→T突变是造成这些品种中该内含子剪接不正常、从而使Wx基因表达水平和直链淀粉含量下降的主要原因.     

橡胶树白粉菌(HO-73)启动子WY172不同长度片段的克隆及表达活性分析

旨在克隆橡胶树白粉菌启动子WY172及其上游2K序列上4个不同长度缺失片段,以分析启动子各片段的表达活性。基于实验室前期研究基础,以WY172上游2K序列作为研究对象进行渐变缺失突变,得到4个不同长度的可能具有启动子活性的片段,结合WY172,选用pBI121载体作为骨架,分别替换GUS基因前的CaMV35S启动子,并分别构建重组表达载体,通过ATMT法转化农杆菌;利用GUS染色法和酶活性检测,分析WY172启动子及不同长度片段的酶活性。分别构建了pBI121-WY172、pBI121-WY172Q、pBI121-WY172Q1、pBI121-WY172Q2、pBI121-WY172Q3共5个重组的植物表达载体,所有植物表达载体烟草瞬时表达GUS染色均有蓝色出现,且蓝色程度均强于阳性对照CaMV35S启动子,其中pBI121-WY172Q3的GUS染色相对最深;GUS酶活性测定结果显示所有缺失突变片段都具有调控基因表达的启动子活性,且启动活性均强于CaMV35S启动子,WY172Q3调控GUS基因表达的活性最高。因此我们判断WY172及其上游2K序列上4个不同长度缺失片段均具有启动子活性,其中以WY172Q3启动子片段的表达活性最强。