转基因大麦中gfp基因的染色体位置及其表达

通过对大麦小孢子进行基因枪轰击获得4株转绿色荧光蛋白基因(gfp)的植株(A、C、D、E),以gfp基因为探针进行荧光原位杂交(FISH)研究转化植株中转基因插入位置和基因表达。4个株系在染色体7L(5HL)的不同位置都有一个插入点,而E株系在染色体5S(7HS)还有第2个插入点。所有的转基因T0代植株都是半合子并在T1、T2代发生分离。D株系GFP未表达,但FISH和PCR分析表明gfp基因已成功插入其染色体。各株系在根尖和花粉中的GFP表达水平不同：C株系在花粉表达强而在根尖表达中等;A株系在花粉中等表达而在根尖表达较淡;E株系则在根尖高表达,花粉中等表达。A和C株系在根尖和花粉的GFP分离都表现单位点特性,而E株系的根尖分离表现重叠作用(15：1)特征,但在花粉中表达GFP的频率低。PCR结果和3个分离株系的根尖表达结果一致。D和E株系的GFP表达不正常可能和加基因插入位置或基因的结构有关。     

启动子cbh1的改造与里氏木霉通用质粒载体的构建

里氏木霉具有优异的表达及分泌异源蛋白的能力。本研究为了提高里氏木霉在工业生产中异源蛋白表达产量,对cbh1启动子中四处葡萄糖反馈抑制位点进行突变,并且成功构建两株定点插入型表达质粒pG与pH。在后续的报告基因红色荧光蛋白DsRed表达过程中,pG比pH表现出更优异的表达能力,并且异源蛋白基因被定点插入了cbh1基因位点而对该基因进行了敲除。pG质粒具有工业应用前景。     

非洲爪蛙Klf4基因新转录本的克隆及功能研究

[目的]克隆非洲爪蛙Klf4基因的新转录本,并研究其在早期胚胎发育中的功能。[方法]提取胚胎总RNA,反转录构建c DNA文库。从Xenbase数据库中获得Klf4的基因组序列,分析序列发现新的ATG位点,设计引物、PCR扩增,将扩增的目的片段插入p CS2+,插入位点为EcoRⅠ和XhoⅠ。经双酶切和测序鉴定,发现载体中插入的目的片段为编码483个氨基酸的Klf4的新转录本。体外转录获得mRNA,显微注射入胚胎,进行表型分析以及原位杂交检测。[结果]成功克隆出了Klf4基因的新转录本;过表达该转录本,表型分析发现胚胎发育异常,原位杂交检测发现Xag2和Dkk1的表达上升。[结论]成功克隆编码483个氨基酸的Klf4基因的新转录本,过表达该转录本胚胎发育异常,Xag2和Dkk1的表达被激活。     

显花植物自体和异体花粉识别的分子机理研究(英文)

显花植物的受精涉及许多识别过程;其中第一个是雌性生殖组织心皮对花粉的识别。自交不亲和性（Ｓｅｌｆ－ｉｎｃｏｍｐａｔｉｂｉｌｉｔｙ,ＳＩ）是一种广泛分布于显花植物的种内生殖障碍。在多数自交不亲和的植物中,ＳＩ的遗传控制比较简单,受控于一个由复等位基因构成的单一位点,称为Ｓ位点。在以茄科、玄参科和蔷薇科为代表的配子体自交不亲和植物中,Ｓ位点编码一类核酸酶,即Ｓ核酸酶（Ｆｉｇ．１）,控制ＳＩ在花柱中的表达,但是与花粉自交不亲和性的表达无关。后者可能由与Ｓ核酸酶不同的基因控制,这种基因常被称为花粉Ｓ基因。它是目前了解显花植物花粉识别生化和分子机理的关键。近来;通过对影响花粉ＳＩ表达突变体的分子遗传分析提出了一个花粉Ｓ基因产物如何与Ｓ核酸酶相互作用完成自体和异体花粉识别过程的模型（Ｆｉｇ．２）。另外,描述了两个在金鱼草中克隆花粉Ｓ基因的方法,即Ｓ位点选择性的转座子标记和图位克隆。     

显花植物自体和异体花粉识别的分子机理研究

显花植物的受精涉及许多识别过程,其中和线个是雌性生殖组织心皮对花粉的识别。自交不亲和性（Self-incompatibility,SI)是一种广泛分布于显花植物的种内生殖障碍。在多数自交不亲和的植物中,SI的遗传控制比较简单,受控于一个由复等位基因构成的单一位点,称为S位点。在以茄科、玄参科和蔷薇科为代表的配子体自交不亲和植物中,S位编码一类核酸酶,即S核酸酶（Fig.1),控制SI在花柱中的表达,但是与花粉自交不亲和性的表达无关。后者可能由与S核酸酶不同的基因控制,这种基因常被称为花粉S基因。它是目前了解显花植物花粉识别生化和分子机理的关键。近来,通过对影响花粉SI表达突变体的前了解 花植物花粉识别生化和分子机理的关键。近来,通过对影响花粉SI表达突变体的分子遗传分析提出了一个花粉S基因产物如何与S核酸酶相互作用完成自体和异体花粉识别过程的模型（Fig.2)。另外,描述了两个在金鱼草中克隆花粉S基因的方法,即S位点选择性的转座子标记和图位克隆。     

青花菜花粉发育基因MF21的克隆及表达特征分析

从青花菜自交系‘WN12-95B’中克隆得到花粉发育基因MF21。序列生物信息学分析表明,该基因全长468 bp,编码151个氨基酸,相对分子量为16.70 kD,等电点为8.56,为疏水性蛋白。青花菜MF21蛋白的信号肽长度为22个氨基酸残基。且该蛋白含有两个蛋白激酶C磷酸化位点,4个N端豆蔻酰化位点和一个酪氨酸磷酸化位点。不规则卷曲和β-转角是其二级结构主要构成成分。分子进化表明,MF21基因与同属的埃塞俄比亚芥进化关系最近。通过荧光定量PCR对青花菜MF21基因的时空表达特性进行分析,结果表明该基因在保持系花蕾中表达较高,具有明显的组织特异性。在Ogu不育系及其保持系不同发育阶段的花蕾中MF21相对表达量差异明显。     

矮牵牛PMADS9基因启动子的克隆及分析

矮牵牛PMADS9基因是MADS-box基因AGL15亚家族的成员。该亚家族基因可能具有调控开花时间、抑制花器官衰老脱落和促进体胚形成等功能。本文应用YADE和hiTAIL-PCR等方法,克隆了PMADS9基因5′端翻译起始位点上游1853bp的启动子区域序列(FJ798977);RACE分析发现该基因至少有4个转录起始位点,2个位于编码区第一外显子内。启动子调控元件分析显示,PMADS9启动子富集花粉和种子发育过程中特异表达元件和与环境应答相关的元件;AGL15同源基因启动子存在非常保守的RY-repeat元件,启动子的保守性与物种的遗传距离不一致;推测PMADS9启动子翻译起始位点上游200～400bp和800～1000bp区域具重要功能。     

转座子挽救法对转座子突变菌株中插入位点的定位分析

为寻找谷氨酸棒杆菌转座子插入突变菌株中的转座子插入位点,采用了转座子挽救法对转座子及其插入位点附近的序列进行分离,并测定插入位点相邻DNA序列,获得了三个转座子插入位点DNA序列,其中一个是柠檬酸合成酶基因,另两个为目前未知基因,暂命名为orfA和orfB。该方法简便易行,是分析转座子插入位点的理想方法。     

基于启动子捕获系统的水稻基因的分离

为了分离水稻的基因及其启动子,该实验室构建了T-DNA(GUS)结构的水稻启动子捕获系统,对其中编号为113#、T-DNA单拷贝插入、GUS报告基因为组成型表达的阳性捕获系进行了进一步分析。潮霉素筛选结合GUS组织化学染色获得了T-DNA插入的纯合株(113#-22和113#-26);Inverse法分离得到T-DNA插入位点水稻基因组DNA旁邻序列,测序和BLAST结果表明,T-DNA反方向插在水稻基因组4号染色体预测基因的内含子中;扩增T-DNA插入位点上游2kb左右DNA片段,构建启动子分析质粒转化水稻‘中花11’胚性愈伤组织,获得转基因植株,GUS组织化学染色模式与113#阳性株系一致。结果表明,该预测基因及其启动子是利用启动子捕获系统所捕获到的候选基因。     

木糖异构酶基因xylA的克隆及其表达载体的构建

木糖异构酶基因xylA是一种正向选择标记基因,在植物基因工程中使用该标记可以获得安全的转基因植物.构建了以xylA基因为选择标记的植物表达载体.从大肠杆菌Top10中扩增出xylA基因,插入到质粒pCAMBIA2301的Xho Ⅰ位点,通过酶切和PCR检测插入片段的正确性,得到载体pCAMBIA2301-xylA,将pBI121载体上的‘35S-GUS-Nos'表达框插入到pCAMBIA2301-xylA的EcoR Ⅰ和Hind Ⅲ位点.得到中间载体pCAMBIA2301-xylA-GUS,用Sac Ⅰ和Sma Ⅰ切下克隆载体上的CBF1基因替代pCAMBIA2301-xylA-GUS中的GUS片段,用电转化法将获得的表达载体转化到农杆菌中,为将来获得安全的转基因抗寒植株奠定基础.     

Chromosomal translocations are a common phenomenon in Arabidopsis thaliana T-DNA insertion lines

Ordered collections of Arabidopsis thaliana lines containing mapped T-DNA insertions have become an important resource for plant scientists performing genetic studies. Previous reports have indicated that T-DNA insertion lines can have chromosomal translocations associated with the T-DNA insertion site, but the prevalence of these rearrangements has not been well documented. To determine the frequency with which translocations are present in a widely-used collection of T-DNA insertion lines, we analyzed 64 independent lines from the Salk T-DNA mutant collection. Chromosomal translocations were detected in 12 of the 64 lines surveyed (19%). Two assays were used to screen the T-DNA lines for translocations: pollen viability and genome-wide genetic mapping. Although the measurement of pollen viability is an indirect screen for the presence of a translocation, all 11 of the T-DNA lines showing an abnormal pollen phenotype were found to contain a translocation when analyzed using genetic mapping. A normal pollen phenotype does not, however, guarantee the absence of a translocation. We observed one T-DNA line with normal pollen that nevertheless had a translocation based on genetic mapping results. One additional phenomenon that we observed through our genetic mapping experiments was that the T-DNA junctions on the 5'- and 3'-sides of a targeted gene can genetically separate from each other in some cases. Two of the lines in our survey displayed this 'T-DNA borders separate' phenomenon. Experimental procedures for efficiently screening T-DNA lines for the presence of chromosomal abnormalities are presented and discussed.     

Pollen tube tip growth depends on plasma membrane polarization mediated by tobacco PLC3 activity and endocytic membrane recycling

Phosphatidyl inositol 4,5-bisphosphate (PI 4,5-P2) accumulates in a Rac/Rop-dependent manner in the pollen tube tip plasma membrane, where it may control actin organization and membrane traffic. PI 4,5-P2 is hydrolyzed by phospholipase C (PLC) activity to the signaling molecules inositol 1,4,5-trisphosphate and diacyl glycerol (DAG). To investigate PLC activity during tip growth, we cloned Nt PLC3, specifically expressed in tobacco (Nicotiana tabacum) pollen tubes. Recombinant Nt PLC3 displayed Ca2+-dependent PI 4,5-P2-hydrolyzing activity sensitive to U-73122 and to mutations in the active site. Nt PLC3 overexpression, but not that of inactive mutants, inhibited pollen tube growth. Yellow fluorescent protein (YFP) fused to Nt PLC3, or to its EF and C2 domains, accumulated laterally at the pollen tube tip plasma membrane in a pattern complementary to the distribution of PI 4,5-P2. The DAG marker Cys1:YFP displayed a similar intracellular localization as PI 4,5-P2. Blocking endocytic membrane recycling affected the intracellular distribution of DAG but not of PI 4,5-P2. U-73122 at low micromolar concentrations inhibited and partially depolarized pollen tube growth, caused PI 4,5-P2 spreading at the apex, and abolished DAG membrane accumulation. We show that Nt PLC3 is targeted by its EF and C2 domains to the plasma membrane laterally at the pollen tube tip and that it maintains, together with endocytic membrane recycling, an apical domain enriched in PI 4,5-P2 and DAG required for polar cell growth.     

Phosphoinositides regulate clathrin-dependent endocytosis at the tip of pollen tubes in Arabidopsis and tobacco

Using the tip-growing pollen tube of Arabidopsis thaliana and Nicotiana tabacum as a model to investigate endocytosis mechanisms, we show that phosphatidylinositol-4-phosphate 5-kinase 6 (PIP5K6) regulates clathrin-dependent endocytosis in pollen tubes. Green fluorescent protein-tagged PIP5K6 was preferentially localized to the subapical plasma membrane (PM) in pollen tubes where it apparently converts phosphatidylinositol 4-phosphate (PI4P) to phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)]. RNA interference-induced suppression of PIP5K6 expression impaired tip growth and inhibited clathrin-dependent endocytosis in pollen tubes. By contrast, PIP5K6 overexpression induced massive aggregation of the PM in pollen tube tips. This PM abnormality was apparently due to excessive clathrin-dependent membrane invagination because this defect was suppressed by the expression of a dominant-negative mutant of clathrin heavy chain. These results support a role for PI(4,5)P(2) in promoting early stages of clathrin-dependent endocytosis (i.e., membrane invagination). Interestingly, the PIP5K6 overexpression-induced PM abnormality was partially suppressed not only by the overexpression of PLC2, which breaks down PI(4,5)P(2), but also by that of PI4Kβ1, which increases the pool of PI4P. Based on these observations, we propose that a proper balance between PI4P and PI(4,5)P(2) is required for clathrin-dependent endocytosis in the tip of pollen tubes.     

Agrobacterium-Mediated Transfer of the GUS Gene into Pollen of Petunia

Transfer of the GUS gene into pollen has been studied in co-cultures of Agrobacterium and Petunia pollen. The Agrobacterium strain used contains a GUS gene between the two borders of the T-DNA. Uptake and integration of this GUS gene could be shown using two different restriction systems. First, by appropriate cleavage within the T-DNA the GUS gene could be isolated intact from Agrobacterium-treated pollen. Second, using enzymes with cleave the T-DNA only once, integration of this T-DNA into individual pollen genomes could be shown. The fragments obtained could not be obtained from Agrobacterium alone. The positive Southern blots were reprobed with vir probes, but all were negative. Also following plating, no Agrobacteria could be detected from our pollen DNA preparations. Therefore, the signals obtained were not due to contaminating bacteria. Due to a high endogenous GUS activity of Petunia pollen the expression of the transferred gene could not be studied. The data demonstrate the uptake and integration of T-DNA into pollen and are closing a gap in the line of evidence for the functioning of an indirect Agrobacterium — mediated gene transfer. Besides this, it should be stressed that only this indirect pollen system leads to success.     

Arabidopsis glucan synthase-like 10 functions in male gametogenesis

Callose or beta-1,3-glucan performs multiple functions during male and female gametophyte development. Callose is synthesized by 12 members of the glucan synthase-like (GSL) gene family in Arabidopsis thaliana. To elucidate the biological roles of Arabidopsis GSL family members during sexual development, we initiated a reverse genetic approach with T-DNA insertional mutagenesis lines. We screened T-DNA insertion lines for all members of the GSL gene family and detected homozygous mutant seedlings for all members except GSL10. Three independent alleles in GSL10, gsl10-1, gsl10-3 and gsl10-4 showed distorted segregation (1:1:0) of T-DNA inserts rather than Mendelian segregation (1:2:1). By genetic analysis through reciprocal cross, we determined that gsl10 pollen could not be transmitted to descendent. The mutant pollen of GSL10/gsl10 plants at tetrad and microspore stages were not different from that of wild type, suggesting that GSL10 is not essential for normal microspore growth. Analysis of GSL10/gsl10 hemizygous pollen during development revealed abnormal function in asymmetric microspore division. gsl10 mutant microspores failed to enter into mitosis. Unlike the previously described functions of GSL1, GSL2 and GSL5, GSL10 involves an independent process of pollen development at the mitotic division stage.     

Type B Phosphatidylinositol-4-Phosphate 5-Kinases Mediate Arabidopsis and Nicotiana tabacum Pollen Tube Growth by Regulating Apical Pectin Secretion

Phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P₂] occurs in the apical plasma membrane of growing pollen tubes. Because enzymes responsible for PtdIns(4,5)P₂ production at that location are uncharacterized, functions of PtdIns(4,5)P₂ in pollen tube tip growth are unresolved. Two candidate genes encoding pollen-expressed Arabidopsis thaliana phosphatidylinositol-4-phosphate 5-kinases (PI4P 5-kinases) of Arabidopsis subfamily B were identified (PIP5K4 and PIP5K5), and their recombinant proteins were characterized as being PI4P 5-kinases. Pollen of T-DNA insertion lines deficient in both PIP5K4 and PIP5K5 exhibited reduced pollen germination and defects in pollen tube elongation. Fluorescence-tagged PIP5K4 and PIP5K5 localized to an apical plasma membrane microdomain in Arabidopsis and tobacco (Nicotiana tabacum) pollen tubes, and overexpression of either PIP5K4 or PIP5K5 triggered multiple tip branching events. Further studies using the tobacco system revealed that overexpression caused massive apical pectin deposition accompanied by plasma membrane invaginations. By contrast, callose deposition and cytoskeletal structures were unaltered in the overexpressors. Morphological effects depended on PtdIns(4,5)P₂ production, as an inactive enzyme variant did not produce any effects. The data indicate that excessive PtdIns(4,5)P₂ production by type B PI4P 5-kinases disturbs the balance of membrane trafficking and apical pectin deposition. Polar tip growth of pollen tubes may thus be modulated by PtdIns(4,5)P₂ via regulatory effects on membrane trafficking and/or apical pectin deposition.     

Isolation and Characterization of a Rice Cysteine Protease Gene, OsCP1, Using T-DNA Gene-Trap System

The T-DNA gene-trap system has been efficiently used to elucidate gene functions in plants. We report here a functional analysis of a cysteine protease gene, OsCP1, isolated from a pool of T-DNA insertional rice. GUS assay with the T-DNA tagged line indicated that the OsCP1 promoter was highly active in the rice anther. Sequence analysis revealed that the deduced amino acid sequence of OsCP1 was homologous to those of papain family cysteine proteases containing the highly conserved interspersed amino acid motif, ERFNIN. This result suggested that the gene encodes a cysteine protease in rice. We also identified a suppressed mutant from T2 progeny of the T-DNA tagged line. The mutant showed a significant defect in pollen development. Taken together, the results demonstrated that OsCP1 is a cysteine protease gene that might play an important role in pollen development.     

Petunia phospholipase c1 is involved in pollen tube growth

Although pollen tube growth is essential for plant fertilization and reproductive success, the regulators of the actin-related growth machinery and the cytosolic Ca2+ gradient thought to determine how these cells elongate remain poorly defined. Phospholipases, their substrates, and their phospholipid turnover products have been proposed as such regulators; however, the relevant phospholipase(s) have not been characterized. Therefore, we cloned cDNA for a pollen-expressed phosphatidylinositol 4,5-bisphosphate (PtdInsP2)-cleaving phospholipase C (PLC) from Petunia inflata, named Pet PLC1. Expressing a catalytically inactive form of Pet PLC1 in pollen tubes caused expansion of the apical Ca2+ gradient, disruption of the organization of the actin cytoskeleton, and delocalization of growth at the tube tip. These phenotypes were suppressed by depolymerizing actin with low concentrations of latrunculin B, suggesting that a critical site of action of Pet PLC1 is in regulating actin structure at the growing tip. A green fluorescent protein (GFP) fusion to Pet PLC1 caused enrichment in regions of the apical plasma membrane not undergoing rapid expansion, whereas a GFP fusion to the PtdInsP2 binding domain of mammalian PLC delta1 caused enrichment in apical regions depleted in PLC. Thus, Pet PLC1 appears to be involved in the machinery that restricts growth to the very apex of the elongating pollen tube, likely through its regulatory action on PtdInsP2 distribution within the cell.     

<Emphasis Type="Italic">pgd1</Emphasis>, an <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> deletion mutant,is defective in pollen germination

An Arabidopsis deletion mutant was fortuitously identified from the alpha population of T-DNA insertional mutants generated at the University of Wisconsin Arabidopsis Knockout Facility. Segregation and reciprocal crosses indicated that the mutant was a gametophytic pollen sterile mutant. Pollen carrying the mutation has the unusual phenotype that it is viable, but cannot germinate. Thus, the mutant was named pollen germination defective mutant 1 (pgd1), based on the pollen phenotype. Flanking sequences of the T-DNA insertion in the pgd1 mutant were identified by thermal asymmetric interlaced (TAIL) PCR. Sequencing of bands from TAIL PCR revealed that the T-DNA was linked to the gene XLG1, At2g23460, at its downstream end, while directly upstream of the T-DNA was a region between At2g22830 and At2g22840, which was 65 genes upstream of XLG1. Southern blotting and genomic PCR confirmed that the 65 genes plus part of XLG1 were deleted in the pgd1 mutant. A 9,177 bp genomic sequence containing the XLG1 gene and upstream and downstream intergenic regions could not rescue the pgd1 pollen phenotype. One or more genes from the deleted region were presumably responsible for the pollen germination defect observed in the pgd1 mutant. Because relatively few mutations have been identified that affect pollen germination independent of any effect on pollen viability, this mutant line provides a new tool for identification of genes specifically involved in this phase of the reproductive cycle.