tritordeum花粉特异性表达的遗传分析

为明确转基因tritordeum中被外源uidA基因标记的启动子的调控特异性,对筛选到的一株标记材料进行了两代uidA基因的遗传表达分析,结果表明,后代材料基因组中都含有uidA基因,没有发生分离,且都只在花粉中检测到GUS活性,在其他组织没有检测到GUS活性.进一步RT-PCR分析显示uidA基因在根和叶中没有发生转录,说明该株材料为花粉特异性启动子被uidA基因标记的阳性纯合体,实验证明该特异性能稳定遗传.     

拟南芥ACOS5基因启动子的克隆和表达载体构建

花药组织特异性启动子在花药发育的分子遗传学中有重要的研究价值.采用PCR方法克隆了1kb长度的ACOS5基因启动子,并将其连入含有GFP基因的拟南芥表达载体p1300中.电激转化农杆菌GV3101后,通过农杆菌介导转化拟南芥,获得转基因植株.显微观察显示,转基因植物花药只在绒毡层中有荧光表达.这说明ACOS5基因启动子可以在拟南芥花药中驱动外源GFP基因的特异性表达.     

一个水稻花发育特异性启动子的克隆与活性分析

用PCR方法从水稻中克隆了EXPANSIN家族基因OsEXPB1的启动子,利用PLACE软件在线分析其上游2.7kb的序列,发现存在2种与花粉特异性表达有关的顺式作用元件.将该启动子与GUS报告基因融合,构建重组表达载体并转移到水稻中,对水稻各组织进行GUS染色.结果显示:水稻幼嫩组织中均有GUS染色信号;进入生殖生长期后,在营养组织中均没有检测到GUS的表达;只在花器官中检测到GUS信号,在花发育晚期,GUS信号则主要出现在花粉粒中.荧光实时定量PCR进一步验证了GUS染色的结果.综上所述,在水稻成熟植株中,OsEXPB1启动子是一个花发育特异性启动子,可为利用水稻生殖生长期的花特异性表达启动子来进行转基因实验提供备选.     

Gus基因在佛手叶片中的瞬间表达

采用基因枪法对佛手(Citrus medica L.var.sarcodactylis(Noot)Swingle)嫩叶和老叶叶盘进行了Gus基因转化和瞬时表达研究,分析了轰击距离、叶片组织特性对转化效果的影响.结果显示,在7.584 5×106Pa轰击压力下,采用6 cm轰击靶距转化嫩叶可获得比较高的Gus基因瞬间表达频率;老叶的转化效果比嫩叶差;增加轰击次数并不一定能提高瞬间表达频率.     

植物维管组织特异性定位启动子研究进展

组织特异性基因的启动子对该基因的组织特异性表达起重要作用 .本文综述了植物维管组织特异性定位表达启动子的研究进展 .研究表明 ,在植物维管组织特异性定位表达启动子中 ,顺式调控元件与反式作用因子协同作用 ,调控基因的表达 .     

拟南芥器官大小调控基因AtKIX8启动子的克隆和表达分析

为研究拟南芥器官大小调控基因AtKIX8的表达模式,以期进一步研究其功能机制,克隆了该基因启动子序列,获取了启动子-GUS转基因报告植株.利用GUS组织化学染色检测了该启动子作用位置,利用荧光定量PCR分析了启动子对外源激素及冷胁迫的响应.结果表明该启动子诱导GUS基因在幼苗茎尖分生区,成株茎、叶主脉中特异性表达,生长素和低温处理显著提高GUS基因的表达量.说明拟南芥AtKIX8基因启动子具备组织特异性表达模式,且能受到外源生长素和低温的诱导.     

ChIFN-γ基因植物表达载体的构建与瞬时表达

 为了使鸡干扰素-γ（ChIFN-γ）基因在油菜中获得高效表达,优化了油菜偏爱的密码子,在ChIFN-γ的5′端添加Kozark序列,3′端增加内质网滞留信号肽SEKDEL.应用同尾酶将人工合成的ChIFN-γ,Napin特异性启动子及信号肽与NOS终止子克隆到质粒pSH中构建成植物表达载体pSH-NGN.经生菜瞬时表达,ELISA检测表明ChIFN-γ蛋白获得有效表达.研究结果为进一步遗传转化油菜奠定了基础,同时也对构建基因的正确表达提供了一种新的快速鉴定方法.     

拟南芥NCED3基因在水稻中的过量表达可以提高水稻的干旱胁迫耐受性

9-顺-环氧类胡萝卜素双加氧酶(NCED)是高等植物脱落酸(ABA)生物合成途径中的关键酶,其催化的裂解反应直接生成ABA的前体物质.本文应用拟南芥rd29A诱导型启动子和35S组成型启动子成功地将AtNCED3基因在水稻中过量表达,通过耐旱性筛选实验证明转基因水稻对干旱胁迫的耐受性有了显著提高,并且该优良性状在T2代中得到稳定遗传.进一步的分析表明,过量表达AtNCED3可以促进转基因水稻种子的休眠;在含rd29A诱导型启动子的转基因水稻中检测到ABA下游基因OsBZ8的表达;推测 AtNCED3在水稻中的过量表达可能会提高水稻中内源 ABA的水平,从而提高植株的耐旱性.     

烟草维管束发育相关基因Nvas启动子顺式作用元件鉴定

为研究烟草维管束发育相关基因Nvas启动子顺式作用元件,将克隆得到的Nvas启动子ATG+1~-463区域分为10个不同长度的片段与GFP报告基因融合构建了植物表达载体并完成了5'端缺失分析.利用根癌农杆菌介导转化W38型烟草叶盘,得到了含有不同长度Nvas启动子片段的转基因烟草植株.体视显微镜下观察到含Nvas启动子-216~-463区域的转基因烟草均有绿色荧光蛋白的表达,表达部位集中在维管束组织.结果表明Nvas启动子能使外源基因在维管束组织中特异表达.该启动子活性高于Ca MV35S,在ATG+1~-216区域存在核心启动子元件,在-337~-379区域存在能增强启动子活性的元件.     

杂交鹅掌楸LhARF2基因启动子的克隆及瞬时表达分析

生长素响应因子(Auxin response factor, ARF)参与调控植物生长发育的多个途径。本研究利用Tail-PCR技术从杂交鹅掌楸中克隆LhARF2基因5′端上游2 637 bp序列,即pLhARF2启动子,并利用生物信息学软件PlantCARE分析其包含的调控元件,发现其含有启动子必需元件TATA-box和CAAT-box,以及压力响应、激素响应、光信号转导和代谢循环过程元件。构建pLhARF2-pCAMBIA1300:GUS植物表达载体并瞬时浸染本氏烟草叶片,利用GUS组织化学染色法鉴定烟草叶片中瞬时表达活性,发现在烟草叶片中有蓝色斑块,但颜色较浅。实验结果推测杂交鹅掌楸pLhARF2启动子可以调控GUS基因活性,为下一步研究启动子遗传转化的组织表达活性奠定了基础。     

Activity of the truncated promoter of pollen-specific G9 gene of cotton and the transgenic recessive nuclei-sterile tobacco

A 557 bp fragment from the translation initiation site of the G9 gene expressed in maturing pollens of cotton was isolated from genomic DNA of upland cotton (Gossypium hirsutum L.) cv. “Zhongkang 17”, and two expression vectors for plant transformation were constructed via fusing this fragment with β-glucuronidase gene (Gus) and cytotoxin gene Barnase. The promoter activity of this fragment was demonstrated via transient expression of Gus gene in cotton and by the integrated expression of Barnase gene in tobacco. This promoter can initiate the expression of exogenous gene specifically and efficiently in plant pollen. The transgenic tobacco plant containing G9-Barnase fusion gene showed the characteristics of recessive nuclei-sterility.     

Influence of DNA methylation on transgene expression

DNA methylation plays an important role in gene expression in eukaryote. But DNA methylation of transgene usually leads to target gene silencing in plant genetic engineering. In this research, reporter gene b-glu- curonidase (GUS) gene ( uidA ) was introduced into tobaccos via Agrobacterium-mediated transformation method, and the foreign uidA gene became inactive in some transgenic tobaccos. No mRNA of uidA was detected in these plants by Northern blotting analysis, and DNA methylation of promoter region was found. The results indicated that gene silencing might be caused by DNA methylation of promoter.     

Isolation of a pollen specific promoter in tritordeum

The promoter is a cis-acting element in regulating gene expression. A promoterless plasmid containing UidA gene was transformed into tritordeum by barmbadment. Histochemical analysis of various tissues in transgenic tritordeum was carried to examine tissue-specific expression of GUS(beta-glucuronidase) activity. The pollen-specific promoter was trapped and identified successfully in a transformant line. PCR(polymerase chain reaction) method was used to isolate this pollen-specific promoter. By sequencing and analyzing the amplified fragment from PCR, a part of UidA gene and a flanking sequence were obtained. Some essential elements of plant promoters were found in the sequence. To determine the function of it, the cloned fragment was fused with UidA gene, then cloned and transformed into Triticum durum. The transgenic plant transformed by this vector showed GUS expression only in pollen. Therefore a pollen-specific promoter was isolated successfully.     

Effect of 5'-deletion of Arabi-dopsis profilin2 promoter on its vascular specific expression

Based on the published sequence of profilin2 promoter of Arabidopsis thaliana, a full-length promoter (1667 bp) was amplified by PCR. The 5' -end deletion fragments with length of 1380, 1153, 969 and 597 bp were then fused with gus (uidA.) gene respectively. Constructed plant expression vectors were individually transferred into Kalan-choe laciniata and transgenic plants regenerated. GUS his-tochemical assay confirmed that the full-length promoter Pfn1.7 was vascular-specific. Deletion assays showed that profilin2 promoter could be divided into three parts. Deletion of fragment 1 ( -1667--1380 bp) resulted in constitutive expression, suggesting that element(s) responsible for vascular-specific expression might exist in this region. Fragment 2 located at -1153 - -597 bp strongly inhibited gus gene expression. Fragment 3 ( -597 - -1 bp) is considered as a basic domain of profilin2.     

Effect of 5′-deletion ofArabidopsis profilin2 promoter on its vascular specific expression

Based on the published sequence of profilin2 promoter ofArabidopsis thaliana, a full-length promoter (1667 bp) was amplified by PCR. The 5′-end deletion fragments with length of 1380, 1153, 969 and 597 bp were then fused withgus (uidA) gene respectively. Constructed plant expression vectors were individually transferred intoKalanchoe laciniata and transgenic plants regenerated. GUS histochemical assay confirmed that the full-length promoter Pfn1.7 was vascular-specific. Deletion assays showed that profilin2 promoter could be divided into three parts. Deletion of fragment 1 (−1667—−1380 bp) resulted in constitutive expression, suggesting that element(s) responsible for vascular-specific expression might exist in this region. Fragment 2 located at −1153—−597 bp strongly inhibitedgus gene expression. Fragment 3 (−597—−1 bp) is considered as a basic domain of profilin2.     

Cre-lox重组系统介导转基因烟草中外源基因删除的研究

对Cre在转基因个体中介导的重组效率进行了研究。构建了含有Cre 基因(p35S-Cre)和 GUS 基因侧翼含同向loxP 位点的(loxP-p35S-GUS-loxP)两种植物表达载体。以共转化的技术将两种基因元件同时转化烟草得到转基因植株,根据对共转化植株GUS 基因的活性分析、分子检测、PCR检测及对重组后扩增DNA片段进行序列分析表明：Cre-loxP 重组系统在转基因烟草中能精确高效地介导转基因的删除,但也存在部分植株不能完全删除的现象。     

Isolation of a strong matrix attachment region (MAR) and identification of its function in vitro and in vivo

Inclusion of MARs in transgene cassettes enhances their expression and reduces position-effect variations in the transgenic host. Four new MARs (TM2, TM3, AM1 and AM2) were isolated from tobacco and Arabidopsis by PCR method. The nuclei isolated from suspension- cultured cells of rice were used to prepare nuclear matrix. With a characterized MAR (TM1) as a positive control, the Matrix-MAR interactions were tested by an in vitro binding assay to identify the DNA sequences as MARs and their binding strength to nuclear matrix in vitro was compared. The results showed that TM2 and TM3 had stronger binding strength than TM1. To determine the functions of the four new MARs in vivo, binary vectors pBI121 carrying a uidA GUS reporter gene were modified with direct repeat MARs inserted on both sides of the reporter gene cassette and were transferred into tobaccos via Agrobacterium-mediated transformation procedure. Quantitative GUS assays of the transgenic tobaccos showed that when flanking a GUS reporter gene TM1, TM2, TM3 and AM1 increased uidA GUS gene expression level approximately 1.5-fold, 5-fold, 1.35-fold, 1.3-fold respectively and AM2 has no effect on gene expression. TM2 was found to be a strong MAR that could effectively increase gene expression level and could be used as an effective enhancing element to construct high efficient expression vectors. In this note the relations among the sequence features, binding strength in vitro and function in vivo of the five MARs were analyzed, and the potential significance of TM2 in plant genetic engineering was dis- cussed.     

Identification of a bi-directional promoter from Tomato yellow leaf curl China virus

A bi-directional promoter of Tomato yellow leaf curl China virus (TYLCCNV) was obtained with the total DNA from TYLCCNV isolate Y10 infected tobacco leaves as a template. Plant expression vectors were constructed by fusing the amplified DNA fragment with the gus gene and nopaline terminator in different orientations. The vectors containing promoter fragments were transferred into leaf cells and plant stems of Nicotiana benthamiana by Agrobacterium-mediated method. Transient expression results showed that both the complementary and virion-sense promoters could drive the gus gene to express, and the GUS activity of the complementary-sense promoter was stronger than that of the virion-sense. Co-expression of the vector containing βC1 gene of TYLCCNV DNAβ with the vector containing a bi-directional promoter revealed that the βC1 protein has no impact on expression of either the virion- or the complementary-sense promoter.