水稻MITEs类转座子mPing研究进展

Miniature Ping(mPing)是小型反向重复转座子(Miniature Inverted-Repeat Transposable Elements,MITEs)类转座子Tourist-like超家族重要成员,是水稻基因组内检测到的第一个活跃的MITEs,是MITEs大家族中少数低拷贝且可以在自然状态下维持转座活性的成员之一,因此,mPing是转座子相关领域研究的良好素材。该文综合阐述了近年来国内外有关mPing的结构、转座酶供体、激活特性以及对基因组的影响等方面的研究进展,为进一步深入探究MITEs的转座机制以及mPing转座子的开发利用提供资料。     

水稻转座子研究进展

转座子是植物基因组的重要组成部分,对于研究植物基因组进化等具有重要意义。随着水稻全基因组测序计划的开展和完成,水稻转座子研究取得了极大进展,目前已经在水稻基因组中发现了几乎所有类型的转座子,约占水稻基因组的35%。在正常情况下,大多数水稻转座子不具有转座活性,但是在特定的条件下（如组织培养或辐射等）,水稻基因组中沉默的转座子可以被激活,从而可能导致插入突变并影响基因的表达。在水稻中已鉴定出6个有活性的转座子,其中Tos17已被应用到水稻功能基因组研究中。转座子序列的新的分子标记转座子展示（transposon display,TD）现已被开发,并在水稻遗传作图和遗传分化研究中得到应用。     

水稻转座子研究进展

转座子是植物基因组的重要组成部分, 对于研究植物基因组进化等具有重要意义。随着水稻全基因组测序计划的开展和完成, 水稻转座子研究取得了极大进展, 目前已经在水稻基因组中发现了几乎所有类型的转座子, 约占水稻基因组的35%。在正常情况下, 大多数水稻转座子不具有转座活性, 但是在特定的条件下(如组织培养或辐射等), 水稻基因组中沉默的转座子可以被激活, 从而可能导致插入突变并影响基因的表达。在水稻中已鉴定出6个有活性的转座子, 其中Tos17已被应用到水稻功能基因组研究中。转座子序列的新的分子标记转座子展示(transposon display, TD)现已被开发, 并在水稻遗传作图和遗传分化研究中得到应用     

Ac/Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(AcTPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ac×Ds的杂交组合354个.检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性.结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4%.检测到T-DNA可插入到编码蛋白的基因中.在Ac×Ds的F2代中,Ds因子的转座频率为22.7%.对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制--转座和不完全切离等现象.获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录.探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略.     

二化螟piggyBac类转座子的克隆与分析

piggyBac转座子是DNA型转座子, 广泛分布于生物体内。基于piggyBac转座子超家族成员IFP2开发的转基因工具载体是目前转基因研究中使用最广泛的载体之一, 因此piggyBac转座子的研究受到广泛的关注和重视。本文是对二化螟Chilo suppressalis内源性piggyBac类转座子（piggyBac-like element, CsuPLE）的首次报道。克隆的CsuPLE（GenBank登录号： JX392388）全长2 537 bp, 包含一个长1 914 bp的完整开放阅读框（open reading frame, ORF）, 编码含637个氨基酸残基的转座酶, 转座酶中含有piggyBac家族保守的“DDD-domain”。CsuPLE全长序列具有完全对称的13 bp反向末端重复序列（inverted terminal repeats, ITRs）以及非完全对称的21 bp内部重复序列（internal repeats, IRs）, 在二化螟基因组上插入在特征性的“TTAA”靶位点重复（target site duplication, TSD）处。在我国地理跨度很大的不同二化螟种群中均存在结构完整的CsuPLE序列。本研究结果为深入研究piggyBac转座子的结构与功能的关系提供了新的素材, 也为评价利用转座子载体系统在二化螟体内进行转基因操作的可行性和安全性提供了重要的理论基础。     

利用Ac/Ds转座子系统在水稻中获得无选择标记转基因植株的方法

获得无选择标记转基因植株是进行重复转基因及消除转基因植株中标记基因潜在危害性的关键。实验采用了Ac／Ds转座子系统在水稻(Oryza sativa,L．)中进行无hpt选择标记的转基因。将含有目的基因bar的Ds元件和hpt标记基因置于同一个T-DNA中,通过农杆菌(Agrobacterium tumefaciens)EHAl05介导将Ac-T-DNA及Ds-T-DNA分别转入到不同的水稻植株,再将单拷贝的Ac-T-DNA植株与单拷贝的Ds-T-DNA植株杂交得到同时含有Ac和Ds元件的F1植株,Fl自交产生F2后代,F2植株中转座后的Ds元件与T-DNA独立分离,在总共100株F2水稻植株中筛选得到2株只含有Ds元件插入而无hpt标记基因的转基因水稻植株。结果表明,利用Ac／Ds转座子系统在水稻中获得无选择标记的转基因植株是可行的。     

转座子PiggyBac在哺乳动物中的应用

DNA转座子作为一种遗传工程工具已广泛应用于多物种的转基因及产生插入突变等研究。目前,在哺乳动物中有转座活性的转座子可分为三类：1）hAT样转座子;2）Tcl样转座子包括Sleeping Beauty和FrogPrince;3）PiggyBac转座子家族。其中甘蓝蠖度尺蛾（Cabbage looper moth Trichoplusia ni）来源的PiggyBac转座子是目前在哺乳动物中活性最高的转座子,并且可以携带十几kb的外源基因转座而不影响其效率,使其在哺乳动物的转基因、癌基因的发现、基因治疗研究方面具有巨大的应用潜力。此外,PB的无痕迹转座对于无转基因、无遗传物质改变的诱导多潜能干细胞（iPS）研究也具有非常重要的意义。本文主要对针对PB在哺乳动物中的应用现状及前景作一介绍。     

Ac／Ds转座系统在水稻转化群体中的转座活性及转座子插入位点旁侧序列的分析

利用本实验室构建的转Ac(Ac TPase)及Ds(Dissociation)的水稻(Oryza sativa L.)转化群体,配置了Ae×Ds的杂交组合354个。检测了转基因植株的T-DNA插入位点右侧旁邻序列,研究了Ac/Ds转座系统在水稻转化群体中的转座活性。结果表明,有些转化植株T-DNA插入位点相同或相距很近,插入位点互不相同的占65.4％。检测到T-DNA可插入到编码蛋白的基因中。在Ac×Ds的F2代中,Ds因子的转座频率为22.7％。对Ac×Ds杂交子代中Ds因子旁侧序列的分析,进一步表明了Ds因子在水稻基因组中的转座活性,除了从原插入位点解离并转座到新的位点之外,还有复制——转座和小完全切离等现象。获得的旁侧序列中,有些序列与GenBank中的数据没有同源性,目前有2个DNA片段在GenBank登录。探讨了构建转座子水稻突变体库进行水稻功能基因组学研究的策略。     

MITE转座子在不同甘蓝型油菜品种中的多态性研究

微型反向重复转座因子(MITE)在真核生物基因组中普遍存在,其活动能够在物种内形成丰富的多态性,在基因组进化和基因调控中均发挥重要作用。该研究利用286对不同MITE家族侧翼序列筛选的特异引物,对101个中国油菜、27个加拿大油菜和29个合成油菜品系(共157个品系)的基因组DNA进行多态性研究,以明确MITE家族的插入/缺失多态性以及在不同油菜品种之间插入的遗传多样性,进而探讨他们之间的亲缘关系。结果显示：(1)286对引物中筛选出60对条带清晰重复性高的多态性引物,多态性比例21.0%;其中Stowaway like家族和Tourist like家族特异引物的多态性比例分别为24.6%和20.0%。(2)PCR扩增结果显示,60对多态性引物对中国、加拿大、合成油菜品系的基因组DNA分别扩增出4 029、1 044、1 087条清晰可辨的条带。(3)UPGMA聚类显示,中国油菜品种间遗传相似系数在0.59~0.95,加拿大油菜品种间遗传相似系数在0.73~0.95;合成油菜品种间遗传相似系数值在0.64~0.93。研究表明,MITE在基因组中大量随机插入,形成种内丰富的多态性,利用MITE家族引物检测不同地区的油菜种质资源的多态性,可为油菜的育种找到许多尚未被开发的遗传变异。     

籼,粳稻及其杂种粗线期的核型分析

研究了典型籼,粳稻及其杂种粗线期的核型,结果表明：（１）籼稻品种南特号具有两对随体染色体,分别为第十和第十二染色体,粳稻品种巴利拉具１对随体染色体,为第十染色体,杂种南特号／巴利拉的随体染色体与南特号相同;（２）在粗线期,两品种均可见单核仁和双核仁的细胞存在,说明细胞贩核仁数目与随体数目并不对应;（３）两品种的对应染色体之间,在染色体相对长度,臂比及染色粒分布上均无明显差异。     

Mobilization of the active MITE transposons mPing and Pong in rice by introgression from wild rice (Zizania latifolia Griseb.)

Hybridization between different species plays an important role in plant genome evolution, as well as is a widely used approach for crop improvement. McClintock has predicted that plant wide hybridization constitutes a "genomic shock" whereby cryptic transposable elements may be activated. However, direct experimental evidence showing a causal relationship between plant wide hybridization and transposon mobilization has not yet been reported. The miniature-Ping (mPing) is a recently isolated active miniature inverted-repeat transposable element transposon from rice, which is mobilized by tissue culture and gamma-ray irradiation. We show herein that mPing, together with its putative transposase-encoding partner, Pong, is mobilized in three homologous recombinant inbred lines (RILs), derived from hybridization between rice (cultivar Matsumae) and wild rice (Zizania latifolia Griseb.), harboring introgressed genomic DNA from wild rice. In contrast, both elements remain immobile in two lines sharing the same parentage to the RILs but possessing no introgressed DNA. Thus, we have presented direct evidence that is consistent with McClintock's insight by demonstrating a causal link between wide hybridization and transposon mobilization in rice. In addition, we report an atypical behavior of mPing/Pong mobilization in these lines, i.e., the exclusive absence of footprints after excision.     

淹水条件下籼稻与粳稻苗期根际土壤硝化作用的时空变异

由于硝态氮（NO3^--N）对于水稻的生长起到非常重要的作用,所以发生在水稻根际的硝化作用越来越受到人们的重视.试验采用根盒（3室）--速冻切片技术研究了常规籼稻（扬稻6号）和常规粳稻（农垦57）苗期根际土壤矿质态氮、硝化作用和氨氧化细菌数量的时空变异.结果表明,在淹水条件下,土壤矿质态氮主要为铵态氮（NH4^＋-N）,NH4^＋含量随水稻生育期的推进变化不大,但随着距根区的距离增加其含量随之增加,两个水稻品种之间差异不显著;而NO3-的变化趋势与NH4^＋不一致,NO3^-含量随水稻生育期的延长而显著下降,在培养58d时其平均含量约为0.05 mg kg^-1,同时在整个土体内呈均匀分布,两个水稻品种之间差异显著.土壤的硝化强度随水稻的生长而增强,且两种水稻的硝化强度均为根际土壤最高,然后依次为土体土壤和根区土壤.扬稻6号和农垦57硝化强度最大值分别出现在距根6 mm和2 mm处,最大值分别为0.88 mg kg^-1h^-1和0.73 mg kg^-1h^-1.土壤氨氧化细菌（AOB）数量随水稻生长时间的增加而增加,且其水平变异趋势与土壤的硝化强度一致,根际土壤AOB数量最多,土体土壤次之,根区土壤最少.相关分析结果表明,硝化强度和AOB数量呈显著正相关关系（r=0.86,p＜0.01）.种植扬稻6号的土壤NO3^-浓度、硝化强度以及AOB数量总是高于农垦57.     

Integration of genomic tools to assist breeding in the <Emphasis Type="Italic">japonica</Emphasis> subspecies of rice

The cultivated rice (Oryza sativa L.) has two subspecies, indica and japonica. The japonica rice germplasm has a narrower genetic diversity compared to the indica subspecies. Rice breeders aim to develop new varieties with a higher yield potential, with enhanced resistances to biotic and abiotic stresses, and improved adaptation to environmental changes. In order to face some of these challenges, japonica rice germplasm will have to be diversified and new breeding strategies developed. Indica rice improvement could also profit from more “genepool mingling” for which japonica rice could play an important role. Interesting traits such as low-temperature tolerance, and wider climate adaptation could be introgressed into the indica subspecies. In the past decade, huge developments in rice genomics have expanded our available knowledge on this crop and it is now time to use these technologies for improving and accelerating rice breeding research. With the full sequence of the rice genome, breeders may take advantage of new genes. Also new genes may be discovered from the genepool of wild relatives, or landraces of the genus Oryza, and incorporated into elite japonica cultivars in a kind of “gene revolution” program. Expectedly, new technologies that are currently being optimized, aiming for novel gene discovery or for tracking the regions under selection, will be suggested as new breeding approaches. This paper revisits breeding strategies successfully employed in indica rice, and discusses their application in japonica rice improvement (e.g. ideotype breeding, wide hybridization and hybrid performance).     

Pyramiding transgenes for multiple resistance in rice against bacterial blight,yellow stem borer and sheath blight

The present investigation revealed that the alk and gel(t) genes, which cause the differences between a japonica rice variety Nipponbare and an indica rice variety Kasalath in terms of the disintegration of endosperm starch granules in alkali solution and their gelatinisation in a 4 M urea solution, respectively, cosegregated in backcross inbred lines derived from a cross between the two varieties. The segregation pattern of the profile for amylopectin chain-length, which was distinguished by enrichment in short chains of DP≦11 and depletion in intermediate-size chains of 12≦DP≦24 in japonica as compared with indica, was exactly the same as those of the above physico-chemical properties of starch granules, and the gene was designated as acl(t). Gene-mapping analysis showed that the starch synthase IIa (SSIIa) gene is located at the alk locus on chromosome 6 in the rice genome. These results lead us to the possibility that different alleles of the SSIIa gene are responsible for differences in amylopectin structure between the two varieties, in that SSIIa plays a distinct role in the elongation of short chains within clusters (A+B₁ chains) of amylopectin. It is proposed that the activity of SSIIa in japonica rice is reduced in amount or functional capacity relative to the activity of this enzyme in indica rice. This, in turn, would explain why starch from japonica rice has a lower gelatinisation temperature than starch from indica rice and is more susceptible to disintegration in alkali or urea. The evidence for this hypothesis is that the alk(t), gel(t), acl(t) and SSIIa genes all map to the same locus. Received: 29 January 2001 / Accepted: 12 April 2001     

不同栽培方式对籼、粳稻根表铁膜和根铁、镉含量的影响

在Cd污染土壤中研究淹水、覆膜、覆草和湿润栽培对籼、梗稻不同生育时期根表铁膜Fe、Cd含量和根Cd含量的影响．结果表明,在分蘖期,梗稻根表铁膜Fe含量和根Cd含量在4种栽培方式中平均为6．37和25．49mg．kg^-1。分别比籼稻的4．52和16．37mg·kg^-1增加1．85和9．12mg·kg^-1．在孕穗期,粳稻根表铁膜Fe、Cd含量和根Cd含量在4种栽培方式中平均为1．60、16．35和54．68mg·kg^-1,分别比籼稻的1．06、9．56kg和43．31mg·kg^-1增加0．54、6．79和11．37mg·kg^-1．在灌浆期,梗稻根表铁膜Fe含量覆草和湿润栽培为0．89和1．00mg·kg^-1,分别比籼稻的0．63和0．30mg·kg^-1增加0．26和0．70mg·kg^-1;梗稻根表铁膜Cd和根Cd含量在4种栽培方式中平均为15．23和73．68mg·kg^-1,分别比籼稻的3．46和52．38mg·kg^-1增加11．77和21．30mg·kg^-1收获时。根表铁膜Fe含量淹水栽培的籼稻为1．21mg·g^-1,比梗稻的0．65mg·g^-1增加0．56mg·g^-1,覆草栽培的梗稻为0．94mg·g^-1,比籼稻0．55mg·g^-1增加0．39mg·g^-1;根表铁膜Cd含量湿润栽培的梗稻为7．96mg·kg^-1,比籼稻的5．09mg·kg^-1增加2．87mg·kg^-1;根Cd含量梗稻在4种栽培方式中平均为54．53mg·kg^-1,比籼稻的35．91mg·kg^-1增加18．62mg·kg^-1。     

Comparison of promoters and selectable marker genes for use in Indica rice transformation

The effectiveness of different promoters for use in Indica rice transformation was compared. Plasmids encoding the Escherichia coli uidA (gus) gene under the control of CaMV 35S, Emu, Act1 or Ubi1 promoters were delivered into cell suspension cultures by particle bombardment. Transient gene expression, 48 h after delivery, was greatest from plasmids utilising the constitutive promoters, Act1 and Ubi1. Gene expression in stably transformed tissue was examined by bombarding embryogenic Indica rice calli with a pUbi1-gas plasmid and a plasmid containing either the selectable marker gene, hph, which confers hygromycin resistance, or bar, which confers resistance to the herbicide phosphinothricin (BASTA) each under the control of the CaMV 35S, Emu, Act1 or the Ubi1 promoters. The bombarded calli were placed on the appropriate selection media and stained for GUS activity at 1 day, 3 weeks and 5 weeks after shooting. Callus bombarded with the pUbi1-hph or the pEmu-hph constructs gave a dramatic increase in the size of the GUS staining areas with time. No such increase in the size of GUS staining areas was observed in calli co-bombarded with pUbi1-gus and any of the bar containing constructs.Co-bombardment of calli with either the pEmu-hph or pUbi1-hph construct and a virus minor coat protein (cp) gene construct resulted in many fertile transgenic Indica rice plants, containing one to eight copies of both the hph and cp genes. These genes were stably inherited by the T1 generation.     

中国栽培稻的起源与演化

中国栽培稻起源地主要有4种假说:华南起源说、云南起源说、长江中下游说和长江中游-淮河上游说。对其进行了简要概括,并进行评述:目前没有一种假说能完全符合水稻起源的4个条件,此外有些学者提出水稻的多起源中心;从分子生物学角度综述了栽培稻籼粳分化研究进展,主要有两个起源说:一源论和二次(或多次)起源论,大量研究支持籼稻和粳稻多起源论;从古DNA、水稻落粒性与驯化、植硅石等方面探讨栽培稻起源演化的研究方法。旨在能为水稻的起源演化研究提供有价值的参考。     

北方粳稻品种识别数据库系统的设计

为借助Internet技术进行数据的共享以及开发一套基于DNA指纹信息来识别粳稻品种的计算机功能平台,设计一套北方粳稻品种识别数据库系统。根据该数据库的设计所包含的信息含量,确定本数据库包含的四个表,并根据功能需求分析,设计了用户管理,北方粳稻DNA指纹数据管理,粳稻DNA指纹查询与粳稻品种识别分析四个功能模块,同时设计了该系统的界面设计图,为北方粳稻品种识别数据库系统的构建奠定基础。     

栽培稻及其近缘野生种间杂交揭示杂草稻的起源

通过水稻种间、亚种间和品种间的杂交,在套袋隔离和自然授粉两种条件下,对其杂交亲本主要农艺性状类型及杂交后代群体中杂草稻发生频率、类型及主要特征以及杂草稻的发生趋势进行调查分析,直接验证和重演杂草稻起源的主要路径。杂草稻的主要特征为:颖壳褐色或金色,种皮红色,散穗、易落粒,中长芒或无芒。结果表明,如果以近缘野生种为亲本,在其F2群体中比较容易发生类似杂草稻的单株;杂交组合中杂草稻出现频率的大小依次为:杂草稻或野生稻/粳稻(44.16%)>杂草稻或野生稻/籼稻(27.84%)>籼稻/粳稻(3.30%)>籼稻/籼稻(1.41%)>粳稻/粳稻(0)。这一结果显示杂交亲本间遗传差异越大,在后代中出现杂草稻类型植株的频率就越高。套袋条件下,后代中杂草稻出现的频率为10.70%,而不套袋情况下为4.90%。