拟南芥光形态突变体的筛选与基因鉴定

以哥伦比亚(Columbia)野生型拟南芥(Arabidopsis thaliana)为实验材料,用含有激活标记双元质粒pCB260的农杆菌浸花进行转化,构建拟南芥T-DNA插入突变体库.通过突变体的筛选和表型分析,获得了两株光形态突变体,子叶下胚轴伸长的光抑制效应减弱.通过TAIL-PCR(thermal asymmetric interlaced-PCR)技术,成功扩增出突变植株T-DNA插入位点侧翼序列,经NCBI序列比对,T-DNA分别插在CRY1第一和第三外显子部位.突变体的表型分析及PCR鉴定结果表明,T-DNA插入CRY1并影响到突变植株的光形态建成.     

烟曲霉突变体库的构建及伊曲康唑耐药菌的筛选

目的构建烟曲霉突变体库并筛选耐药突变子,初步探讨烟曲霉的耐药机制。方法利用根瘤农杆菌T-DNA介导的插入突变构建烟曲霉突变体库,用伊曲康唑对突变体库进行筛选,挑取烟曲霉耐药突变株。结果利用根瘤农杆菌T-DNA插入突变,本研究获得了1805株突变子,诊断PCR显示突变子基因组DNA上均含有潮霉素标记,说明T-DNA均插入到了宿主的基因组上。通过筛选,本研究成功获得了16株烟曲霉伊曲康唑耐药菌,其中有两株对两性霉素B表现为不同程度的耐药性。结论通过构建烟曲霉突变体库以及耐药突变子筛选获得了16株烟曲霉耐药菌,为进一步研究烟曲霉的耐药机制奠定了基础。     

拟南芥光周期开花突变体的筛选和基因的鉴定

以双子叶模式植物拟南芥（Arabidopsis thaliana）突变体crylcry2为实验材料,用舍有激活标记质粒DSK1015的农杆菌浸花进行转化,构建了拟南芥T-DNA插入突变体库．通过筛选和观察分析,获得了一些开花时间比crylcry2明显延迟或明显提早的突变体．采用IPCR（inverse PCR）和TAIL-PCR（thermal asymmetric interlaced PCR）等方法,鉴定了这些突变体T-DNA插入位点的基因组旁邻序列,并采用半定量RT-PCR对插入位点两侧基因的mRNA水平进行了分析,初步鉴定了与开花相关的候选基因,为进一步研究其功能,深入研究隐花素调节光周期开花的作用机制奠定了基础．     

拟南芥与大豆疫霉菌的非寄主互作及一个感病突变体的遗传分析

非寄主抗病性是一种普遍的自然现象, 该文通过建立拟南芥-大豆疫霉菌(Arabidopsis thaliana-Phytophthora sojae)非寄主互作系统, 筛选对大豆疫霉菌感病的拟南芥突变体, 为研究植物对卵菌的非寄主抗病性遗传机制奠定基础。以大豆疫霉菌游动孢子接种拟南芥T-DNA插入突变体离体叶片, 从代表12 000个独立转化株系的40 000株T₃代T-DNA插入拟南芥突变体中获得一系列对大豆疫霉菌感病的突变体。其中突变体581-51感病性状表现稳定, 离体叶片接菌后3天内出现明显的水渍状病斑, 4–5天后产生大量卵孢子和/或孢子囊。细胞学观察发现有典型的吸器形成。Southern杂交和遗传分析结果表明, 581-51突变体含有4个T-DNA插入事件, 其感病性状可能由隐性单基因控制。     

植物功能基因组研究中的基因敲除技术

综述了T-DNA插入突变和转座子插入突变等基因敲除技术的原理及其在植物功能基因组研究中的应用,并简要介绍了向国际有关机构索要或购买拟南芥插入突变体的方法.     

拟南芥复制因子C亚基1在胚胎发生中起重要作用

复制因子C包含1个大亚基和4个小亚基,在DNA复制、损伤修复和细胞增殖中起重要作用,拟南芥复制因子C亚基1(AtRFC1)是人类复制因子C大亚基p140的同源蛋白。在对3个复制因子C亚基1的T-DNA插入突变株系rfc1-1、rfc1-2和rfc1-3的检验中,证实插入位点分别位于第16、19号外显子和启动子区域。T-DNA在外显子中的插入突变引起胚胎发育异常并导致胚胎和种子败育。将野生型拟南芥复制因子C亚基1基因转化到突变株系rfc1-1和rfc1-2后恢复了突变株的野生型表型,证明胚胎发生异常表型是由拟南芥复制因子C亚基1基因突变所引起的,AtRFC1在拟南芥胚胎发生中起重要作用。     

ST273是拟南芥绒毡层和小孢子发育的必需基因

绒毡层在拟南芥花药花粉发育过程中具有重要作用,包括分泌降解胼胝质的胼胝质酶、为花粉壁的形成提供原料以及为小孢子发育提供营养物质.本文通过对拟南芥雄性不育突变体st273的分析,研究了ST273基因在花药花粉发育过程中的功能.st273是通过T-DNA插入诱变野生型拟南芥得到的一株突变体,遗传分析表明st273是单隐性核基因控制的.利用图位克隆的方法对不育基因ST273进行了定位,结果表明ST273基因与拟南芥第三条染色体上分子标记CIW11连锁.生物信息学分析发现该分子标记附近有一个调控花粉发育的基因TDF1.测序分析结果表明在st273突变体中,TDF1基因第三个外显子上459位的碱基发生了由G459变成了A459的单碱基变化,导致ST273基因该位点提前终止突变.等位分析结果表明st273与tdf1是等位突变体.st273突变体营养生长期发育正常,但生殖生长发育出现异常.亚历山大染色结果显示st273突变体花药中没有花粉.组织切片观察结果表明,突变体花药绒毡层异常肥大且空泡化,四分体不能正常释放小孢子,最终无法形成花粉.这些结果揭示了ST273蛋白质参与调控了绒毡层和小孢子发育过程.     

球孢白僵菌T-DNA突变体库的构建及高温和高渗敏感型突变体的筛选

摘要：【目的】筛选对高温和高渗等逆境胁迫敏感的球孢白僵菌T-DNA随机插入突变体,并克隆相关基因,为研究杀虫真菌适应逆境的分子机理奠定基础。【方法】利用逆境胁迫的方法从球孢白僵菌的T-DNA随机插入突变库中筛选对高温和高渗敏感的突变体,进而利用YADE (Y-shaped adaptor dependent extension)技术克隆相关基因。【结果】筛选得到5个对高温和高渗敏感的突变体。其中2个（212和2550）对高温敏感,在32oC下生长完全受抑;其它3个突变体对高渗胁迫敏感。突变体212的分生孢     

拟南芥温度诱导脂质运载蛋白TIL1参与雌配子体发育

雌配子体的正常发育是种子形成的前提条件之一,拟南芥温度诱导的脂质运载蛋白编码基因TIL1突变使胚珠败育,结实率下降明显。基因表达分析表明T-DNA插入使得TIL1基因敲除,突变体TIL1基因功能缺失;互交实验、Alexander染色、花粉离体培养和胚珠透明实验结果表明till-1突变体雄配子体发育正常、雌配子体胚囊发育有缺陷;通过遗传互补实验证明外源克隆的TIL1基因能恢复突变体的败育表型,并确定了TIL1基因主要在胚珠的胚囊中表达。实验结果表明TIL1基因参与了植物雌配子体发育这一重要的生理过程。     

一种产黄青霉突变体库的简单快速构建方法

产黄青霉(Pennicillium chrysogenum)是重要的工业丝状真菌,为更好地提高青霉素产量,了解青霉素合成的调控途径及相关基因的功能,突变体库的构建是一种有效途径。本研究以PCR扩增得到的T-DNA片段为外源DNA,通过PEG介导转化至产黄青霉原生质体中,成功地将含有外源博来霉素抗性基因(ble)和绿色荧光蛋白基因(gfp)的T-DNA插入到产黄青霉基因组中,构建了产黄青霉突变体库,并实现了ble基因和gfp基因在产黄青霉中的表达。该方法不依赖于农杆菌介导,无需构建二元表达载体,只需一步PCR即可实现外源基因的制备,简单快速,也为研究其它真菌基因功能和突变体库构建提供参考。     

Five gametophytic mutations affecting pollen development and pollen tube growth in Arabidopsis thaliana

Mutant analysis represents one of the most reliable approaches to identifying genes involved in plant development. The screening of the Versailles collection of Arabidopsis thaliana T-DNA insertion transformants has allowed us to isolate different mutations affecting male gametophytic functions and viability. Among several mutated lines, five have been extensively studied at the genetic, molecular, and cytological levels. For each mutant, several generations of selfing and outcrossing have been carried out, leading to the conclusion that all these mutations are tagged and affect only the male gametophyte. However, only one out of the five mutations is completely penetrant. A variable number of T-DNA copies has integrated in the mutant lines, although all segregate at one mutated locus. Two mutants could be defined as "early mutants": the mutated genes are presumably expressed during pollen grain maturation and their alteration leads to the production of nonfunctional pollen grains. Two other mutants could be defined as "late mutant" since their pollen is able to germinate but pollen tube growth is highly disturbed. Screening for segregation ratio distortions followed by thorough genetic analysis proved to be a powerful tool for identifying gametophytic mutations of all phases of pollen development.     

EMBRYONIC FACTOR 1 encodes an AMP deaminase and is essential for the zygote to embryo transition in Arabidopsis

Fusion of the egg and the sperm cells in plants produces a zygote that develops into an embryo. Screening of ethyl methanesulfonate-mutagenized populations of Arabidopsis led to the identification of EMBRYONIC FACTOR 1 (FAC1), a locus that gives a zygote-lethal phenotype when mutated. The FAC1 gene was identified by positional cloning and confirmed by a genetic complementation test against a T-DNA insertion allele. It encodes an AMP deaminase (AMPD) that is known in human and yeast to convert AMP to IMP to maintain the energy potential. Expression of FAC1 in a yeast AMPD mutant after removal of its N-terminal putative transmembrane domain complemented the mutant phenotype, suggesting a functional conservancy but a structural divergence through evolution. Although a low level of FAC1 expression was observed in all organs tested, using a reporter construct we observed a significantly increased FAC1 expression in the zygote, early embryo and endosperm. Furthermore, during somatic embryogenesis, a high level of FAC1 expression was observed in developing embryos including putative embryogenic cells. FAC1, therefore, represents one of the earliest expressed genes known in plants. It may act through AMP depletion to provide sufficient energy for the zygote to proceed through development.     

EMBRYONIC FACTOR 31 encodes a tyrosyl-tRNA synthetase that is essential for seed development

Aminoacyl-tRNA synthetases (AARSs) involve the process of catalyzing the ligation of specific amino acids to their cognate tRNAs. Here we identified an Arabidopsis mutant embryonic factor 31 (fac31), its embryos arrested at development from one cell to globular stage. The FAC31 gene was identified by positional cloning and confirmed by a genetic complementation test with two independent T-DNA insertion lines and transgenic rescue with full-length genomic DNA. FAC31 encodes a Tyrosyl-tRNA synthetase and localize to mitochondria and cytoplasm. Fac31 mutants contain a point mutation from CAA to a stop codon TAA which may lead to a truncated protein. The phenotype of fac31 mutants are very similar to the T-DNA insertion lines Salk_016722 and Salk_045570 displayed smaller embryo sac contains only less number of endosperm nucleolus. Genetic analysis showed that the FAC31 gene had no parental effects through the transmission of mutated FAC31 gene by gametes. FAC31 is a high-conserved protein among animals and plants. RT-PCR analysis and promoter-GUS expression showed that it is expressed in nearly all tissues tested, strongly expressed in meristem of seedlings, the primordium of lateral root, young inflorescences, mature pollen, germinated pollen tubes and embryo sacs before heart stage. Our findings suggest that FAC31 is essential for the seed development through regulation the expanding of embryo sac and proliferation of endosperm nucleolus.     

激活标签法构建拟南芥突变体库及其表型分析

以拟南芥(ArabMopsis thaliana)野生生态型(Columbia)植株为实验材料,以含有激活标记双元质粒pCB260的农杆菌进行转化,并以抗除草剂Basta为筛选标记,构建了拟南芥激活标签突变体库,所用pCB260双元质粒含有两个Ds位点、一个GFP标记基因与一个抗basta标记基因,可以方便高效地筛选转基因植物.目前经初步筛选获得了约10 000个独立转化株系(T1代),其中约50个株系具有明显的表型变化,包括花期改变、株型变异、叶形特异、育性降低、花发育异常、种子颜色变浅等.运用TAIL-PCR技术,成功获得了其中10个表型特异株系的T-DNA侧翼序列,分别分布于拟南芥基因组的5条染色体上.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Genetic and molecular characterization of embryonic mutants identified following seed transformation in Arabidopsis

Over 5000 transgenic families of Arabidopsis thaliana produced following seed transformation with Agrobacterium tumefaciens were screened for embryonic lethals, defectives, and pattern mutants. One hundred and seventy-eight mutants with a wide range of developmental abnormalities were identified. Forty-one mutants appear from genetic studies to be tagged (36% of the 115 mutants examined in detail). Mapping with visible markers demonstrated that mutant genes were randomly distributed throughout the genome. Seven mutant families appeared to contain chromosomal translocations because the mutant genes exhibited linkage to visible markers on two different chromosomes. Chromosomal rearrangements may therefore be widespread following seed transformation. DNA gel blot hybridizations with 34 tagged mutants and three T-DNA probes revealed a wide range of insertion patterns. Models of T-DNA structure at each mutant locus were constructed to facilitate gene isolation. The value of such models was demonstrated by using plasmid rescue to clone flanking plant DNA from four tagged mutants. Further analysis of genes isolated from these insertional mutants should help to elucidate the relationship between gene function and plant embryogenesis.     

Arabidopsis AtVPS15 plays essential roles in pollen germination possibly by interacting with AtVPS34

VPS 15 protein is a component of the phosphatidylinositol 3-kinase complex which plays a pivotal role in the development of yeast and mammalian cells.The knowledge about the function of its homologue in plants remains limited.Here we report that AtVPS15, a homologue of yeast VPS15p in Arabidopsis,plays an essential role in pollen germination.Homozygous T-DNA insertion mutants of AtVPS15 could not be obtained from the progenies of self-pollinated heterozygous mutants.Reciprocal crosses between atvpslS mutants and wild-type Arabidopsis revealed that the T-DNA insertion was not able to be transmitted by male gametophytes.DAPI staining, Alexander’s stain and scanning electron microscopic analysis showed that atvpsl5 heterozygous plants produced pollen grains that were morphologically indistinguishable from wild-type pollen,whereas in vitro germination experiments revealed that germination of the pollen grains was defective.GUS staining analysis of transgenic plants expressing the GUS reporter gene driven by the AtVPS15 promoter showed that AtVPSI5 was mainly expressed in pollen grains.Finally,DUALmembrane yeast two-hybrid analysis demonstrated that AtVPS15 might interact directly with AtVPS34.These results suggest that AtVPS15 is very important for pollen germination,possibly through modulation of the activity of PI3-kinase.