5′UTR序列对DR5GUS基因瞬时表达的影响

利用生长素响应原件(DR5)构建了DR5::GUS报告基因表达载体,并在载体构建时,对GUS的5′UTR序列进行2种设计:一种是利用载体GUS基因原有的5′UTR构建成pBI121-DR5::GUS;另一种采用植物增强表达的常用原件烟草花叶病毒(TMV)的5′端序列(Ω′)替换GUS基因原有的5′UTR序列,构建成pBI121-DR5(Ω′)::GUS。将2种载体转化根癌农杆菌后,采用烟草叶片注射浸染的瞬时表达检测法对2个载体的植物表达效果进行检测,2种载体分别转化烟草叶片2 d后,对浸染的叶盘进行GUS染色分析,pBI121-DR5::GUS的转化叶片有明显的GUS反应,而pBI121-DR5(Ω′)::GUS的转化叶片则无GUS反应。分离注射浸染部位叶盘RNA后,对GUS特异引物进行RT-PCR分析,2种转化的基因都已有效转录出RNA,但前者能翻译出相应的Ω-葡萄糖苷酸酶,后者则不能完成翻译过程,说明Ω′序列的引入并未有效提高GUS基因的表达,反而抑制了mRNA的翻译。     

棉花GhCCR4基因表达载体构建及GUS基因的瞬时表达

研究以GUS基因为报告基因,构建CCR4基因的瞬时表达载体,首先酶切带有CCR4基因的 pGEM-T-CCR4载体,获得CCR4基因目的片段,然后将其克隆到瞬时表达载体pGUS/NIa中,获得由CaMV35S启动子调控目的基因的pGUS-CCR4融合表达载体,使目的基因能够和GUS基因同时表达.采用基因枪法将pGUS-CCR4转化到洋葱表皮细胞中,暗培养24 h,经GUS组织化学染色,检测到多个洋葱细胞呈现蓝色,表明构建的瞬时表达载体可在植物细胞中高效表达.为进一步在棉花中研究GhCCR4基因的功能奠定了实验基础.     

棉花GhCAD6基因表达载体构建及GUS基因的瞬时表达

[目的]以GUS基因为报告基因,构建GhCAD6基因的瞬时表达载体.[方法]采用PCR方法和基因枪法.[结果]用PCR法,以pGEM-T-CAD6质粒为模板,获得GhCAD6基因目的片段.然后将其克隆到瞬时表达载体pRTL2-GUS/NIa中,获得由CaMV35S启动子调控目的基因的pGUS-CAD6融合表达载体,使目的基因能够和GUS基因同时表达.采用基因枪法将pGUS-CAD6转化到洋葱表皮细胞中,暗培养24 h,经GUS组织化学染色,检测到多个洋葱细胞呈现蓝色.[结论]构建的瞬时表达载体可在植物细胞中高效表达,为进一步研究棉花GhCAD6基因的功能奠定了实验基础.     

观赏海棠'火焰'果皮中可变剪接基因分析

[目的]研究观赏海棠'火焰'果皮中可变剪接基因.[方法]利用已有苹果属观赏海棠品种'火焰'果皮转录组测序数据,分析可变剪接类型和数量,并对发生可变剪接的基因进行KEGG分析,研究观赏海棠可变剪接基因与其发育过程的关系.[结果]在观赏海棠果皮发育过程中,主要发生的可变剪接是第一个外显子可变剪接和最后一个外显子可变剪接两种类型.可变剪接在果皮发育前期发生较多,同时KEGG通路分析表明可变剪接主要发生在嘌呤代谢、氧化磷酸化、糖酵解/糖异生等通路过程中.[结论]可变剪接在观赏海棠果皮的整个发育过程中普遍存在.观赏海棠果皮发育过程中共有的可变剪接基因主要与磷酸化和糖代谢有关.     

真核生物pre-mRNA剪接的研究进展

Pre-mRNA剪接,即把内含子去除并把外显子序列连接成为成熟的mRNA,是基因表达与调控的重要环节之一。本文在概述真核基因mRNA剪接反应机制的基础上,主要讨论pre-mRNA的剪接机器,剪接体如何催化pre-mRNA剪接反应和pre-mRNA选择性剪接等方面的研究进展。     

拟南芥 AtNHX5基因启动子的克隆和组织定位分析

为了探明拟南芥内膜反向转运体 AtNHX5基因启动子（proNHX5）功能及基因的组织表达模式,从基因组中克隆 AtNHX5基因开放阅读框（ORF）上游侧翼调控区1 807 bp序列,发现该启动子具有典型启动子一般特征,不仅具有TATA-box、CAAT-box等核心元件,还含有与光响应、激素响应、逆境诱导响应和分生组织表达调控元件。构建 AtNHX5基因启动子与GUS的融合表达载体,通过农杆菌花序浸染法转化野生型拟南芥获得转基因植株。利用组织染色法鉴定转基因拟南芥的GUS表达模式,发现在子叶、下胚轴和花中有显著的GUS酶活性,成熟叶片和根中只有局部检测到GUS表达,在未成熟果荚中只有在果荚顶端和基部存在GUS酶活性,说明 AtNHX5基因启动子与GUS的融合表达载体成功构建且正常启动GUS基因表达,同时也表明, AtNHX5基因主要在这些部位表达,且表达具有时空特异性。     

棉花蔗糖合成酶3基因第1个内含子在基因表达中的作用

为了研究棉花SUS3基因编码区的第1个内含子在基因表达调控中的作用,分离该内含子并将其插入到GUS基因编码区中构建载体Susfig::121,将载体Susfig::121通过农杆菌介导转入拟南芥.对转基因拟南芥进行GUS活性分析,发现该内含子对GUS基因在拟南芥中的表达具有负调控作用,并且特异抑制报告基因在花粉中表达....     

崂山奶山羊不同泌乳期乳腺组织可变剪接的鉴定与分析

本研究对崂山奶山羊泌乳初期、高峰期和末期乳腺组织RNA-Seq数据进行可变剪接分析,共鉴定到151 503个可变剪接事件,对应17 627个可变剪接基因,6 323个差异剪接基因,显著富集到细胞器、蛋白质结合、mRNA加工和RNA剪接等相关GO条目,主要参与催产素信号通路、钙信号通路、内质网中的蛋白质加工等,与乳腺组织生理活性密切相关的KEGG通路,筛选出PTEN作为参与乳腺发育及泌乳相关生理过程中调控网络的核心基因。研究结果表明,可变剪接在奶山羊不同泌乳期的乳腺组织具有发育阶段特异性,特有可变剪接基因与乳腺组织生理活性特点密切相关,在调控奶山羊乳腺组织发育及泌乳生理中发挥重要作用。本研究可为探究可变剪接机制调控奶山羊乳腺发育与泌乳性状的分子机理提供理论依据。     

农杆菌介导银杏遗传转化体系的建立及Gb-DXR基因表达载体的构建

[目的]为提高农杆菌介导的银杏遗传转化效率提供参考。[方法]以成熟的银杏种子胚为外植体,在无激素MS培养基上预培养48h后,采用3种农杆菌介导将GUS基因导入银杏胚中,经过组织化学染色法检测到GUS瞬时表达活性,对影响GUS基因表达的因素进行初步分析;并构建了银杏内酯前体合成途径上1-脱氧-D-木酮糖-5-磷酸还原异构酶（DXR）的表达载体。[结果]该研究得到较合适的遗传转化方案,即用银杏胚作为外植体,用携带pCAMBIA1304＋的EHA105农杆菌进行侵染,共培养3d,进行GUS染色,结果显示转化后GUS阳性率较高。[结论]该研究为进一步的银杏转基因研究工作奠定了基础。     

烟草PR3b转录后剪切元件NRSE1与GUS融合表达后的可变剪切

【目的】烟草(Nicotiana tabacum L.)碱性几丁质酶基因PR3b在低烟碱突变体(nic1和nic2)中存在转录后mRNA可变剪切现象,但其可变剪切的发生机制仍不清楚。将PR3b的可变剪切元件NRSE1(nicotinesynthesis related splicing element 1)与GUS融合表达,分析NRSE1元件的独立可变剪切特性,以揭示其作用机制。【方法】利用PCR扩增方法获得PR3b cDNA序列中的NRSE1元件片段,并利用基因重组技术构建了烟草PR3b可变剪切元件NRSE1与GUS的融合表达载体。将融合表达载体导入农杆菌LBA4404后,通过农杆菌介导的叶盘转化法培育了表达NRSE1与GUS融合子的低烟碱突变体nic1和nic2及野生型烟草转基因植株;通过RT-PCR检测及GUS染色鉴定出阳性植株后,利用RT-PCR分析NRSE1与GUS融合表达后在低烟碱突变体和野生型烟草中的可变剪切特性;对转基因植株的幼苗进行乙烯(ET)和茉莉酸(JA)处理,通过GUS染色方法分析ET和JA处理对转基因植株中GUS活性的影响,并通过RT-PCR方法分析ET和JA处理对转基因植株中NRSE1与GUS融合子的可变剪切特性影响,以及对转基因植株中NRSE1与GUS融合子表达水平的影响。【结果】通过RT-PCR检测及GUS染色鉴定出表达NRSE1元件与GUS融合子的低烟碱突变体和野生型烟草转基因植株;RT-PCR检测及测序分析证明,NRSE1元件与GUS融合表达后仍能在低烟碱突变体发生高水平的可变剪切,剪切修饰区段的序列变化与烟草中PR3b的mRNA可变剪切修饰一致;利用ET和JA处理转基因植株进行的GUS染色表明,ET和JA处理对转基因植株的GUS活性有不同程度的影响;但利用ET和JA处理转基因植株进行的RT-PCR分析表明,ET和JA处理不改变NRSE1元件原有的诱导剪切特性,也不影响转基因植株中NRSE1元件与GUS融合子的表达水平。【结论】PR3b的可变剪切元件NRSE1与GUS在烟草中融合表达后,仍能在低烟碱突变体nic1和nic2中发生高水平的可变剪切;NRSE1在烟草中的可变剪切不依赖PR3b的其他mRNA区段,是烟草PR3b发生可变剪切的独立元件;ET和JA处理对NRSE1元件与GUS融合表达植株的GUS活性具有一定影响,可能存在翻译水平的调控作用。     

内含子的识别和选择性剪切

真核生物内含子的一个显著特征是在很多生物中其5'和3'剪切位点的基本序列都具有相对很高的保守性,内含子从mRNA前体转录产物中的去除和伴随的外显子的连接称作mRNA前体的剪切,它是构成真核基因表达和基因调控水平的一个重要方面。这个过程由许多具有有限序列和特殊空间结构的顺式作用元件控制,由被称为剪切体的核糖核蛋白复合体来执行。以内含子的识别和由于识别造成的选择性剪切进行了综述,试图去理解造成选择性剪切的分子机理。     

可用于植物转化的RNAi载体构建方法

利用PCR方法扩增杨树Poptr;CYCD1;1基因的一段内含子序列,并克隆到pROKⅡ载体中,以构建可用于植物特定基因表达干扰的RNAi载体。为了验证载体的效果,构建pCAMBIA-1303载体转化烟草,经检测,转基因烟草能过量表达外源GUS与GFP融合蛋白。把GUS及GFP片段序列连接于RNAi载体,并导入pCAMBIA-1303载体转化烟草。试验结果表明,GUS及GFP干扰序列的导入均能大大降低外源GUS与GFP融合基因的表达。结果说明构建的载体能有效干扰特定基因的表达。     

烟草Bex inhibitor-1基因植物沉默表达载体的构建及对农杆菌的转化

试验首先对质粒pGSA1285进行改造,将pFGC5941质粒的查尔酮合酶的Intron片段取代pGSA1285的GUS片段,构建含有内含子并具有卡那抗性的pGSA2285植物沉默表达载体。同时设计引物、PCR扩增、在BI-1基因两端各加上两个酶切位点,将BI-1基因分别反向、正向插入改造后的质粒pGSA2285Intron片段两端的多克隆位点中,构建得到烟草BI-1基因沉默载体pGSA4285。此沉默质粒经PCR鉴定、限制性酶切分析以及回复动员试验证明已正确构建并成功转入农杆菌,为获得含有BI-1基因沉默烟草植株及其在植物程序性死亡中调控作用的研究奠定试验基础。     

烟草PR3b转录后剪切元件NRSE1与GUS融合表达后的可变剪切

【目的】烟草（Nicotiana tabacum L.）碱性几丁质酶基因PR3b在低烟碱突变体（nic1和nic2）中存在转录后mRNA可变剪切现象,但其可变剪切的发生机制仍不清楚。将PR3b的可变剪切元件NRSE1（nicotine- synthesis related splicing element 1）与GUS融合表达,分析NRSE1元件的独立可变剪切特性,以揭示其作用机制。【方法】利用PCR扩增方法获得PR3b cDNA序列中的NRSE1元件片段,并利用基因重组技术构建了烟草PR3b可变剪切元件NRSE1与GUS的融合表达载体。将融合表达载体导入农杆菌LBA4404后,通过农杆菌介导的叶盘转化法培育了表达NRSE1与GUS融合子的低烟碱突变体nic1和nic2及野生型烟草转基因植株;通过RT-PCR检测及GUS染色鉴定出阳性植株后,利用RT-PCR分析NRSE1与GUS融合表达后在低烟碱突变体和野生型烟草中的可变剪切特性;对转基因植株的幼苗进行乙烯（ET）和茉莉酸（JA）处理,通过GUS染色方法分析ET和JA处理对转基因植株中GUS活性的影响,并通过RT-PCR方法分析ET和JA处理对转基因植株中NRSE1与GUS融合子的可变剪切特性影响,以及对转基因植株中NRSE1与GUS融合子表达水平的影响。【结果】通过RT-PCR检测及GUS染色鉴定出表达NRSE1元件与GUS融合子的低烟碱突变体和野生型烟草转基因植株;RT-PCR检测及测序分析证明,NRSE1元件与GUS融合表达后仍能在低烟碱突变体发生高水平的可变剪切,剪切修饰区段的序列变化与烟草中PR3b的mRNA可变剪切修饰一致;利用ET和JA处理转基因植株进行的GUS染色表明,ET和JA处理对转基因植株的GUS活性有不同程度的影响;但利用ET和JA处理转基因植株进行的RT-PCR分析表明,ET和JA处理不改变NRSE1元件原有的诱导剪切特性,也不影响转基因植株中NRSE1元件与GUS融合子的表达水平。【结论】PR3b的可变剪切元件NRSE1与GUS在烟草中融合表达后,仍能在低烟碱突变体nic1和nic2中发生高水平的可变剪切;NRSE1在烟草中的可变剪切不依赖PR3b的其他mRNA区段,是烟草PR3b发生可变剪切的独立元件;ET和JA处理对NRSE1元件与GUS融合表达植株的GUS活性具有一定影响,可能存在翻译水平的调控作用。     

Regulated splicing of the Drosophila P transposable element third intron in vitro: somatic repression

In eukaryotic cells alternative splicing of messenger RNA precursors (pre-mRNA's) is a means of regulating gene expression. Although a number of the components that participate in regulating some alternative splicing events have been identified by molecular genetic procedures, the elucidation of the biochemical mechanisms governing alternative splicing requires in vitro reaction systems. The tissue specificity of P element transposition in Drosophila depends on the germline restriction of pre-mRNA splicing of the P element third intron (IVS3). Drosophila P element IVS3 pre-mRNA substrates were spliced accurately in vitro in heterologous human cell extracts but not in Drosophila somatic cell splicing extracts. Components in Drosophila somatic cell extracts that specifically inhibited IVS3 splicing in vitro were detected by a complementation assay. Biochemical assays for Drosophila RNA binding proteins were then used to detect a 97-kilodalton protein that interacts specifically with 5' exon sequences previously implicated in the control of IVS3 splicing in vivo. Inhibition of IVS3 splicing in vitro could be correlated with binding of the 97-kD protein to 5' exon sequences, suggesting that one aspect of IVS3 tissue-specific splicing involves somatic repression by specific RNA-protein interactions.     

Using the Phosphomannose Isomerase (PMI) Gene from Saccharomyces cerevisiae for Selection in Rice Transformation

The phosphomannose isomerase (PMI) gene from Saccharomyces cerevisiae acted as selectable marker and mannose acted as selective agent for the production of transgenic plants of rice (Oryza sativa L.) via Agrobacterium-mediated transformation. The concentration of mannose during the selection was stepwise increased, 5 g L-1 mannose combined with 15 g L-1 sucrose and 500 mg L-1 cefotaxime was used in the initial selection stage, then the concentration of mannose was increased to 11 g L-1, the highest transformation rate was 20.0%. The integration of PMI gene was confirmed by PCR, and the result of RT-PCR assay proved that the intron of PMI gene can be excised correctly during RNA splicing. β- Glucuronidase (GUS) activity analysis confirmed the expression of GUS gene. All those means the PMI gene from yeast can be used as a selectable marker in rice transformation.     

Construction of Tobacco Bax lnhibitor-1 ihpRNA Gene Silencing Vector and Transformation into Agrobacterium tumefaciens EHA105

The plasmid pGSA1285 was first modified by substituting its GUS sequence with the Chalcone synthase intron fragment from vector pFGC5941 to get the plant silencing expression vector that contained Kanamycin resistance site and was named as pGSA2285. Using PCR-based amplification, two different restriction sites at both ends of tobacco Bax inhibitor-1 (NtBI-1) gene were created, respectively, which made the construction of ihpRNA gene silencing vector more efficiently. Then, NtBI-1 genes were inserted into Multiple Cloning Site (MCS) of pGSA2285 respectively to form Bax inhibitor-1 ihpRNA gene silencing vector, named as pGSA4285, containing sense and anti-sense BI-1 sequence which was spliced by chalcone synthase intron. Combined PCR identification and enzyme restriction analyses, the results showed that Bax inhibitor-1 ihpRNA gene silencing vector had been constructed and transferred into Agrobacterium tumefaciens EHA105 successfully, which laid a foundation for the further study on the function of BI-1 in plant PCD regulation.     

Construction of Tobacco Bax Inhibitor-1 ihpRNA Gene Silencing Vector and Transformation into Agrobacterium tumefaciens EHA105

The plasmid pGSA1285 was first modified by substituting its GUS sequence with the Chalcone synthase intron fragment from vector pFGC5941 to get the plant silencing expression vector that contained Kanamycin resistance site and was named as pGSA2285. Using PCR-based amplification, two different restriction sites at both ends of tobacco Bax inhibitor-1 (NtBI-1) gene were created, respectively, which made the construction of ihpRNA gene silencing vector more efficiently. Then, NtBI-1 genes were inserted into M...     

RNA secondary structure repression of a muscle-specific exon in HeLa cell nuclear extracts

The chicken beta-tropomyosin pre-messenger RNA (pre-mRNA) is spliced in a tissue-specific manner to yield messenger RNA's (mRNA's) coding for different isoforms of this protein. Exons 6A and 6B are spliced in a mutually exclusive manner; exon 6B was included in skeletal muscle, whereas exon 6A was preferred in all other tissues. The distal portion of the intron upstream of exon 6B was shown to form stable double-stranded regions with part of the intron downstream of exon 6B and with sequences in exon 6B. This structure repressed splicing of exon 6B to exon 7 in a HeLa cell extract. Derepression of splicing occurred on disruption of this structure and repression followed when the structure was re-formed, even if the structure was formed between two different RNA molecules. Repression leads to inhibition of formation of spliceosomes. Disrupting either of the two double-stranded regions could lead to derepression, whereas re-forming the helices by suppressor mutations reestablished repression. These results support a simple model of tissue-specific splicing in this region of the pre-mRNA.