激发标签技术在植物功能基因组研究中的应用

获得突变体是研究植物功能基因组的有效方法。与传统的T-DNA插入功能缺失突变相比, 建立在功能获得突变基础之上的激发标签技术具有独特的优势, 主要表现为可以获得功能冗余基因的显性突变体并方便地克隆相关基因。文章对激发标签技术的原理, 及其在拟南芥、水稻等植物功能基因组研究中的进展进行了综述, 并介绍了激发标签技术在植物抗逆、抗病和生长发育机理研究方面的新进展。文章最后探讨了激发标签技术的发展前景。     

TILLING在水稻育种中的应用前景

TILLING(Targeting Induced Local Lesions in Genomes)是功能基因组研究中应用的一种反向遗传学技术。它能高通量低成本地在EMS诱变群体中鉴定出发生在特定基因上的点突变。在其基础上发展出的EcoTILLING技术则可发现种质资源中的SNP位点及小插入或缺失多态性位点。水稻是非常重要的粮食作物, 也是已经完成了全基因组序列测定,有丰富的生物信息学资源可以利用的基因组研究模式植物。水稻的分子标记辅助育种将在育种中扮演越来越重要的角色。在这样的背景下,本文从基于特定基因的种质资源鉴定、EMS诱变育种、及水稻功能标记开发等方面论述了其在水稻育种中的应用前景。     

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.     

Ac/Ds标签系统与水稻功能基因组学

自2002年水稻基因组测序完成后,水稻功能基因组学研究正在成为水稻研究的重要内容.构建突变体库是研究功能基因组学的一条重要而有效的途径.利用外源的Ac/Ds (Activator/Dissociation)标签系统是构建插入突变体库较为理想的方法,经过多年发展完善,其在水稻中已有广泛的应用,但仍面临着一些需要解决的实际问题.文章对Ac/Ds标签系统的转座行为及其构建突变体库的问题和优点进行了综述,总结了近年来Ac/Ds标签系统在水稻中的研究进展,分析了利用Ac/Ds标签系统进行功能基因组学研究所面临的挑战.     

快速筛选拟南芥受精和早期胚胎发生相关基因的方法

阐明拟南芥受精和早期胚胎发生过程对理解被子植物生殖发育有着重要的指导意义,而利用正向遗传学方法研究拟南芥突变体的表型及其分子机理是探究植物基因功能最常用的一种方法。基于常规的插入突变(包括T-DNA和转座子)、化学诱变(如ethylmethane sulfonate,EMS)和高能射线方法构建的突变体库中假阳性突变体多,难以高效筛选到受精和早期胚胎发生相关基因的突变体。为解决这一难题,本研究建立了一种构建T-DNA插入突变体文库的新方法。即在载体p CAMBIA1302的T-DNA元件上增加花粉特异荧光标记基因(p LAT52∷EGFP),并遗传转化具有四分体花粉的Columbia野生型拟南芥突变体qrt1-2;对获得的突变体库可利用花粉荧光快速排除假阳性突变体,并采用反向PCR(inverse-PCR)扩增技术确定突变位点。此方法在筛选拟南芥受精和早期胚胎发生相关基因突变体上的成功应用表明,其是一种效率高、针对性强、操作相对快捷方便的拟南芥突变体筛选方法。     

提高水稻插入突变体库利用效率的尝试

T-DNA标签法是一种以农杆菌介导的遗传转化为基础来创造插入突变体库, 从而高通量地分离和克隆植物功能基因的方法。但由于种种原因, 水稻插入突变体库的利用效率较低。为了提高水稻插入突变体库的利用效率, 结合水稻一个双拷贝T-DNA插入突变体的发现和鉴定研究, 通过特异PCR检测、侧翼序列与目标性状的共分离分析, 在1个双插入位点均为杂合的植株的后代株系中分拆了2个插入事件, 分离出目标性状存在遗传分离且只带有1个插入事件的后代株系, 为后续的共分离检测和基因克隆研究打下了重要的基础。由此产生了对插入突变体库中的非串联多拷贝插入标签系进行研究的一些思路和方法, 提出来与同行商榷。     

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。     

快速徒手切片法观察谷子和水稻叶片显微结构

水稻是世界上一半以上人口的主食,且是禾本科植物分子生物学研究的模式植物。谷子是狗尾草属植物,是我国北方重要的杂粮,是典型的C4植物,谷子与水稻基因组共线性很高。对谷子和水稻两种作物的对比研究能够对研发C4水稻提供理论依据。突变体库是研究功能基因组学的重要材料,目前对突变体库的构建技术已经很成熟,但是对目的突变体,尤其是对解剖学方面的突变体材料筛选主要方法是进行石蜡切片,该过程费时费力。本研究旨在改进徒手切片方法对材料进行预筛选,能大大缩减工作量,提高工作效率,该方法得到结构完整、清晰的水稻和谷子叶片徒手切片。     

模式植物短柄草研究新进展

短柄草(Brachypodium distachyon)株型矮小,易于种植栽培,生长周期短,自花授粉,容易繁殖。另外,短柄草基因组比较小,易于转化,与小麦具有比较近的亲缘关系,是理想的草类特别是禾本科模式植物。近年来,短柄草的研究工作在细胞遗传学、基因组学、比较基因组学、植物-病原菌相互作用、功能基因组学等研究领域取得了许多进展,包括完成了Bd-21全基因组的测序工作、构建了T-DNA插入突变体库、用遗传学的方法首次研究短柄草基因的生物学功能等。本文综述了近年来特别是2009年以来短柄草的研究进展,并对未来的研究工作做了展望。     

插入突变在水稻功能基因组学中的研究进展

构建饱和的基因突变体库是最直接、有效的分析鉴定基因功能的方法.根据插入突变源不同可分为T-DNA插入突变、转座子插入突变等.主要介绍这两种方法的原理及其在水稻功能基因组学研究中的应用和进展,并分析和讨论了插入突变在水稻功能基因组学研究中存在的困难和发展趋势.     

RNAi for plant functional genomics

A major challenge in the post-genome era of plant biology is to determine the functions of all the genes in the plant genome. A straightforward approach to this problem is to reduce or knock out expression of a gene with the hope of seeing a phenotype that is suggestive of its function. Insertional mutagenesis is a useful tool for this type of study, but it is limited by gene redundancy, lethal knock-outs, nontagged mutants and the inability to target the inserted element to a specific gene. RNA interference (RNAi) of plant genes, using constructs encoding self-complementary 'hairpin' RNA, largely overcomes these problems. RNAi has been used very effectively in Caenorhabditis elegans functional genomics, and resources are currently being developed for the application of RNAi to high-throughput plant functional genomics.     

Genetic resources offer efficient tools for rice functional genomics research

Rice is an important crop and major model plant for monocot functional genomics studies. With the establishment of various genetic resources for rice genomics, the next challenge is to systematically assign functions to predicted genes in the rice genome. Compared with the robustness of genome sequencing and bioinformatics techniques, progress in understanding the function of rice genes has lagged, hampering the utilization of rice genes for cereal crop improvement. The use of transfer DNA (T‐DNA) insertional mutagenesis offers the advantage of uniform distribution throughout the rice genome, but preferentially in gene‐rich regions, resulting in direct gene knockout or activation of genes within 20–30 kb up‐ and downstream of the T‐DNA insertion site and high gene tagging efficiency. Here, we summarize the recent progress in functional genomics using the T‐DNA‐tagged rice mutant population. We also discuss important features of T‐DNA activation‐ and knockout‐tagging and promoter‐trapping of the rice genome in relation to mutant and candidate gene characterizations and how to more efficiently utilize rice mutant populations and datasets for high‐throughput functional genomics and phenomics studies by forward and reverse genetics approaches. These studies may facilitate the translation of rice functional genomics research to improvements of rice and other cereal crops.     

插入突变在功能基因组学研究中的应用

插入突变库的构建是功能基因组学研究的一个重要内容,可为确定基因的功能提供最直接的证据。构建插入突变库的方法有T-DNA插入突变、转座子插入突变和质粒介导的插入突变。本文分别介绍三种方法的原理及其在功能基因组学研究中的应用和研究进展。     

How Can We Use Genomics to Improve Cereals with Rice as a Reference Genome?

Rice serves as a model crop for cereal genomics. The availability of complete genome sequences, together with various genomic resources available for both rice and Arabidopsis, have revolutionized our understanding of the genetic make-up of crop plants. Both macrocolinearity revealed by comparative mapping and microcolinearity revealed by sequence comparisons among the grasses indicate that sequencing and functional analysis of the rice genome will have a significant impact on other cereals in terms of both genomic studies and crop improvement. The availability of mutants, introgression libraries, and advanced transformation techniques make functional genomics in rice and other cereals more manageable than ever before. A wide array of genetic markers, including anchor markers for comparative mapping, SSRs and SNPs are widely used in genetic mapping, germplasm evaluation and marker assisted selection. An integrated database that combines genome information for rice and other cereals is key to the effective utilization of all genomics resources for cereal improvement. To maximize the potential of genomics for plant breeding, experiments must be further miniaturized and costs must be reduced. Many techniques, including targeted gene disruption or allele substitution, insertional mutagenesis, RNA interference and homologous recombination, need to be refined before they can be widely used in functional genomic analysis and plant breeding.     

Insertional mutagenesis in mice: new perspectives and tools

Insertional mutagenesis has been at the core of functional genomics in many species. In the mouse, improved vectors and methodologies allow easier genome-wide and phenotype-driven insertional mutagenesis screens. The ability to generate homozygous diploid mutations in mouse embryonic stem cells allows prescreening for specific null phenotypes prior to in vivo analysis. In addition, the discovery of active transposable elements in vertebrates, and their development as genetic tools, has led to in vivo forward insertional mutagenesis screens in the mouse. These new technologies will greatly contribute to the speed and ease with which we achieve complete functional annotation of the mouse genome.     

Rice functional genomics research in China

Rice functional genomics is a scientific approach that seeks to identify and define the function of rice genes, and uncover when and how genes work together to produce phenotypic traits. Rapid progress in rice genome sequencing has facilitated research in rice functional genomics in China. The Ministry of Science and Technology of China has funded two major rice functional genomics research programmes for building up the infrastructures of the functional genomics study such as developing rice functional genomics tools and resources. The programmes were also aimed at cloning and functional analyses of a number of genes controlling important agronomic traits from rice. National and international collaborations on rice functional genomics study are accelerating rice gene discovery and application.