植物茎分枝的分子调控

植物茎分枝结构决定了不同植物的不同形态结构.本文从腋生分生组织的发生、腋芽的生长两个方面综述了近年来植物分枝发生发育相关的分子机理研究及其进展.发现在不同植物中腋分生组织形成的基本机制是相似的,LS（lateral suppressor）及其同源基因在不同植物中都参与腋生分生组织的形成,而BL(blind)及其同源基因也参与调控腋生分生组织的形成.腋生分生组织的形成可能也是受激素调控的.目前,对腋芽生长的分子调控机制的认识主要集中于生长素通过二级信使的作用调控腋芽的生长.而生长素调控腋芽生长的机制已经较为清楚的有两条途径：一是生长素通过抑制细胞分裂素合成来调控腋芽的生长;另一途径是一种类胡萝卜素衍生的信号物质参与生长素的运输调控(MAX途径)来调控腋芽的生长.最新研究表明,TB1的拟南芥同源基因在MAX途径的下游负调控腋芽的生长.此外,增强表达OsNAC2也促进腋芽的生长.     

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。     

LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems

Aerial architecture in higher plants is dependent on the activity of the shoot apical meristem (SAM) and axillary meristems (AMs). The SAM produces a main shoot and leaf primordia, while AMs are generated at the axils of leaf primordia and give rise to branches and flowers. Therefore, the formation of AMs is a critical step in the construction of plant architecture. Here, we characterized the rice (Oryza sativa) lax panicle2 (lax2) mutant, which has altered AM formation. LAX2 regulates the branching of the aboveground parts of a rice plant throughout plant development, except for the primary branch in the panicle. The lax2 mutant is similar to lax panicle1 (lax1) in that it lacks an AM in most of the lateral branching of the panicle and has a reduced number of AMs at the vegetative stage. The lax1 lax2 double mutant synergistically enhances the reduced-branching phenotype, indicating the presence of multiple pathways for branching. LAX2 encodes a nuclear protein that contains a plant-specific conserved domain and physically interacts with LAX1. We propose that LAX2 is a novel factor that acts together with LAX1 in rice to regulate the process of AM formation.     

激素对水稻分蘖芽生长和分蘖相关基因表达的调控效应

以水稻品种‘南粳44＇为材料,研究了植物激素对水稻分蘖芽生长及OsTB1、OsD3、OsD10和OsD27等基因表达的调控。结果表明,水稻分蘖芽生长的调控至少存在两条途径,一条通过调控分蘖芽中CTK含量进而调控OsTB1等基因表达来实现,另一条通过调控OsD3、OsD10和OsD27等系列基因表达来实现。外源IAA和GA_3对水稻分蘖芽生长的调控是通过这两条途径共同实现的。     

The PIN1 family gene PvPIN1 is involved in auxin-dependent root emergence and tillering in switchgrass

Switchgrass (Panicum virgatum L.; family Poaceae) is a warm-season C4 perennial grass. Tillering plays an important role in determining the morphology of aboveground parts and the final biomass yield of switchgrass. Auxin distribution in plants can affect a variety of important growth and developmental processes, including the regulation of shoot and root branching, plant resistance and biological yield. Auxin transport and gradients in plants are mediated by influx and efflux carriers. PvPIN1, a switchgrass PIN1-like gene that is involved in regulating polar transport, is a putative auxin efflux carrier. Neighbor-joining analysis using sequences deposited in NCBI databases showed that the PvPIN1gene belongs to the PIN1 family and is evolutionarily closer to the Oryza sativa japonica group. Tiller emergence and development was significantly promoted in plants subjected toPvPIN1 RNA interference (RNAi), which yielded a phenotype similar to that of wild-type plants treated with the auxin transport inhibitor TIBA (2,3,5-triiodobenzoic acid). A transgenic approach that inducedPvPIN1 gene overexpression or suppression altered tiller number and the shoot/root ratio. These data suggest that PvPIN1plays an important role in auxin-dependent adventitious root emergence and tillering.     

Molecular analysis of the NAC gene family in rice

Genes that encode products containing a NAC domain, such as NO APICAL MERISTEM (NAM) in petunia, CUP-SHAPED COTYLEDON2 (CUC2) and NAP in Arabidopsis thaliana, have crucial functions in plant development. We describe here molecular aspects of the OsNAC genes that encode proteins with NAC domains in rice (Oryza sativa L.). Sequence analysis revealed that the NAC genes in plants can be divided into several subfamilies, such as the NAM, ATAF, and OsNAC3 subfamilies. In rice, OsNAC1 and OsNAC2 are classified in the NAM subfamily, which includes NAM and CUC2, while OsNAC5 and OsNAC6 fall into the ATAF subfamily. In addition to the members of these subfamilies, the rice genome contains the NAC genes OsNAC3, OsNAC4 (both in the OsNAC3 subfamily), OsNAC7, and OsNAC8. These results and Southern analysis indicate that the OsNAC genes constitute a large gene family in the rice genome. Each OsNAC gene is expressed in a specific pattern in different organs, suggesting that this family has diverse and important roles in rice development.     

LAZY1 controls rice shoot gravitropism through regulating polar auxin transport

Tiller angle of rice （Oryza sativa L.） is an important agronomic trait that contributes to grain production, and has long attracted attentions of breeders for achieving ideal plant architecture to improve grain yield. Although enormous efforts have been made over the past decades to study mutants with extremely spreading or compact tillers, the molecular mechanism underlying the control of tiller angle of cereal crops remains unknown. Here we report the cloning of the LAZY1 （LA1） gene that regulates shoot gravitropism by which the rice tiller angle is controlled. We show that LA1, a novel grass-specific gene, is temporally and spatially expressed, and plays a negative role in polar auxin transport （PAT）. Loss-of-function of LA1 enhances PAT greatly and thus alters the endogenous IAA distribution in shoots, leading to the reduced gravitropism, and therefore the tiller-spreading phenotype of rice plants.     

插入玉米Ds转座因子的水稻转化群体及其分子分析

转座子标签法是一种利用转座因子插入高等植物基因组中造成基因突变,然后通过分离转座因子插入的旁邻顺序,进而克隆出突变基因的策略。这种策略在高等植物功能基因组学的研究中是十分有用的。为此目的,将玉米的Ds因子及bar基因连接至载体pCAMBIA1300的T-DNA区域中,构建成重组Ti质粒pDsBar1300。pDsBar1300中T-DNA区域中的潮霉素抗性基因可在转化过程中用作水稻转化植株的选择标记。插入在Ds因子中的bar基因可追踪转化后代的Ds因子。pDsBar1300通过根瘤农杆茵介导引入水稻品种中花11号的幼胚组织。从各转化愈伤组织中获得了1400株独立的Ds水稻转化植株。通过PPT抗性检测和PCR分析证明了水稻转化植株中Ds因子的整合。Southernblot分析了转化植株基因组中Ds因子的插入拷贝数,其中单拷贝插入比率约占70％。这些插有Ds因子的水稻转化植株,当引入自主型的Ac因子反式活化Ds因子后,可使Ds因子跳跃到不同位点上,就可得到更多的突变植株。     

Large-scale systematic study on stability of the Ds element and timing of transposition in rice

Activator/Dissociation (Ac/Ds) transposon mutagenesis is a widely used tool for gene identification; however, several reports on silencing of the Ac/Ds element in starter lines and in stable transposants question the applicability of such an approach in later generations. We have performed a systematic analysis on various aspects of the silencing phenomenon in rice (Oryza sativa ssp. japonica cv. Nipponbare). High somatic and germinal transposition frequencies observed in earlier generations were maintained as late as T4 and T5 generations; thus the propagation of parental lines did not induce transposon silencing. Moreover, the stably transposed Ds element was active even at the F5 generation, since Ac could remobilize the Ds element as indicated by the footprint analysis of several revertants. Expression of the bar gene was monitored from F3 to F6 generations in >1,000 lines. Strikingly, substantial transgene silencing was not observed in any of the generations tested. We analyzed the timing of transposition during rice development and provide evidence that Ds is transposed late after tiller formation. The possibility, that the independent events could be the result of secondary transposition, was ruled out by analyzing potential footprints by reciprocal PCR. Our study validates the Ac/Ds system as a tool for large-scale mutagenesis in rice, since the Ds elements were active in the starter and insertion lines even in the later generations. We propose that harvesting rice seeds using their panicles is an alternative way to increase the number of independent transposants due to post-tillering transposition.     

Over-expression of OsPIN2 leads to increased tiller numbers, angle and shorter plant height through suppression of OsLAZY1

Crop architecture parameters such as tiller number, angle and plant height are important agronomic traits that have been considered for breeding programmes. Auxin distribution within the plant has long been recognized to alter architecture. The rice (Oryza sativa L.) genome contains 12 putative PIN genes encoding auxin efflux transporters, including four PIN1 and one PIN2 genes. Here, we report that over-expression of OsPIN2 through a transgenic approach in rice (Japonica cv. Nipponbare) led to a shorter plant height, more tillers and a larger tiller angle when compared with wild type (WT). The expression patterns of the auxin reporter DR5::GUS and quantification of auxin distribution showed that OsPIN2 over-expression increased auxin transport from the shoot to the root-shoot junction, resulting in a non-tissue-specific accumulation of more free auxin at the root-shoot junction relative to WT. Over-expression of OsPIN2 enhanced auxin transport from shoots to roots, but did not alter the polar auxin pattern in the roots. Transgenic plants were less sensitive to N-1-naphthylphthalamic acid, an auxin transport inhibitor, than WT in their root growth. OsPIN2-over-expressing plants had suppressed the expression of a gravitropism-related gene OsLazy1 in the shoots, but unaltered expression of OsPIN1b and OsTAC1, which were reported as tiller angle controllers in rice. The data suggest that OsPIN2 has a distinct auxin-dependent regulation pathway together with OsPIN1b and OsTAC1 controlling rice shoot architecture. Altering OsPIN2 expression by genetic transformation can be directly used for modifying rice architecture.     

用无启动子的GUS报告基因捕获水稻基因启动子

构建了嵌合质粒p13DGUTs,它是在Ds转座子中插入了无启动子的B．葡萄糖醛酸酶报告基因(GUS),用于分离水稻基因启动子。将p13DGUTs转化粳稻品种中花11的胚性愈伤组织,获得了496个转基因植株。抗性愈伤组织与转基因植株的GUS染色与PCR分析表明整合在水稻染色体上的Ds因子都发生了随机跳跃。转基因植株T0代与部分T1代的GUS染色结果表明,M92转基因植株中Ds转座子整合位置上游的水稻基因启动子指导GUS基因的表达及表达的特性是可遗传的。文章对此方法在分离水稻基因启动子与基因上的应用进行了讨论。     

Ac/Ds(GUS)结构介导的水稻启动子捕获系统的建立

构建了基于Activator/Dissociation(β-glucuronidase)[简称Ac/Ds(GUS)]结构的捕获质粒p13B,用于分离水稻基因启动子.以此质粒用衣杆菌介导的方法转化粳稻品种中花11的胚性愈伤组织,对获得的18个独立转化株的T2代植株进行了抗除草剂筛选,从141个抗除草剂转基因植株中用PCR方法检测到其中37株是Ds因子发生了转座的植株,而且这种转座到新位置上的Ds因子是遗传的.初步观察到其中5株的GUS染色呈阳性.     

New branching inhibitors and their potential as strigolactone mimics in rice

Strigolactones (SLs) are rhizosphere communication chemicals. Recent studies of highly branched mutants revealed that SL or its metabolites work as a phytohormone to inhibit shoot branching. When SLs are exogenously applied to the rice d10-1 mutant that has a highly branched phenotype caused by a defect in the SL biosynthesis gene (CCD8), they inhibit tiller bud outgrowth (branching in rice) of the mutant. We focused our attention on the SL function as a phytohormone and tried to find new chemicals mimicking the hormonal action of SL by screening chemicals that inhibit branching of rice d10-1 mutant. Fortunately, we found 5-(4-chlorophenoxy)-3-methylfuran-2(5H)-one (3a) as a new chemical possessing SL-like activity against the rice d10-1 mutant. Then, we prepared several derivatives of 3a (3b-3k) to examine their ability to inhibit shoot branching of rice d10-1. These derivatives were synthesized by a one-pot coupling reaction between phenols and halo butenolide to give 5-phenoxy 3-methylfuran-2(5H)-one (3) derivatives, which possess a common substructure with SLs. Some of the derivatives showed SL-like activity more potently than GR24, a typical SL derivative, in a rice assay. As SLs also show activity by inducing seed germination of root parasitic plants, the induction activity of these derivatives was also evaluated. Here we report the structure-activity relationships of these compounds.     

水稻短根毛突变体Ossrh2的表型分析与基因定位

在粳稻品种中花11为遗传背景的T-DNA突变体库中筛选获得一个遗传稳定的水稻（Oryzasativa）短根毛突变体Ossrh2（Oryza sativa short root hair2）。突变体在苗期表现为根毛数量减少,为野生型的61.4%,根毛长度明显变短,只有野生型的22.8%,同时根毛增粗,根毛形态也发生了变异,局部扭曲膨胀和分叉,除此之外突变体的地上部和根部生长情况与野生型相比没有显著差异。遗传分析表明,该突变性状受1对隐性单基因控制。通过对突变体T2和F2代的分子检测发现,该突变体表型非T-DNA插入引起。利用Ossrh2纯合体和籼稻品种Kasalath杂交构建的F2群体对OsSRH2进行基因定位,发现其与第10号染色体短臂上的SSR（simple sequence repeat）标记RM6370和RM474连锁,遗传距离分别为1.1cM和3.0cM。通过在两标记间发展3个新的STS（sequence-taggedsite）标记,将OsSRH2基因定位于标记S1227和S1531之间,物理距离约为304kb,为进一步克隆OsSRH2打下了基础。