Duplication and Divergence of Floral MADS-Box Genes in Grasses： Evidence for the Generation and Modification of Novel Regulators

The process of flowering is controlled by a hierarchy of floral genes that act as flowering time genes, inflorescence/floral meristem Identity genes, and/or floral organ-identity genes. The most important and well-characterized floral genes are those that belong to the MADS-box family of transcription factors. Compelling evidence suggests that floral MADS-box genes have experienced a few large-scale duplication events. In particular, the precore eudicot duplication events have been considered to correlate with the emergence and diversification of core eudicots. Duplication of floral MADS-box genes has also been documented in monocots, particularly In grasses, although a systematic study is lacking. In the present study, by conducting extensive phylogenetlc analyses, we identified pre-Poaceae gene duplication events in each of the AP1, P1, AG, AGL11, AGL2/3/4, and AGL9gene lineages. Comparative genomic studies further indicated that some of these duplications actually resulted from the genome doubling event that occurred 66-70 million years ago （MYA）. In addition, we found that after gene duplication, exonization （of intron sequences） and pseudoexonization （of exon sequences） have contributed to the divergence of duplicate genes in sequence structure and, possibly, gene function.

A MADS-box gene is involved in soybean resistance to multiple Soybean mosaic virus strains

Soybean mosaic virus(SMV)is a member of the genus Potyvirus that extensively impairs global soybean production.The full-length coding sequence of the MADS-box transcription factor Gm CAL was cloned from the SMV-resistant soybean cultivar Kefeng 1.SMV-induced expression analysis indicated that Gm CAL responded quickly to SMV-SC8 infection in Kefeng 1 but not in NN1138-2.Gm CAL was expressed at high levels in flowers and pods but at lower levels in leaves.The gene was localized to the nucleus by subcellular localization assay.Virus-induced gene silencing did not increase the accumulation of SMV in Gm CAL-silenced Kefeng 1 plants(with silencing efficiency~80%)after SC8 inoculation.Gm CAL-silencing plants still conferred resistance to SC8 that might be owing to incomplete silencing of genes with lower expression.SMV content decreased significantly in Gm CAL-overexpressing NN1138-2 plants after SMVSC3,SMV-SC7,and SMV-SC8 inoculation in comparison with a vector control,showing that overexpression of Gm CAL conferred broad-spectrum resistance to multiple SMV strains.These results confirm that Gm CAL,a key regulator but not a specific SC8 resistance gene(Rsc8),is a positive regulatory transcription factor involved in soybean resistance to SMV.

Construction of a Full-Length cDNA Library of Gossypium hirsutum L. and Identification of Two MADS-Box Genes

A full-length normalized cDNA library for the flower development stages of short-season cotton （Gossypium hirsutum L.） （CCRI36） was constructed. A total of 3 421 clones were randomly selected for sequencing, with a total of 3 175 effective sequences obtained after removal of empty-carriers and low-quality sequences. Clustering the 3 175 high-quality expressed sequence tags （ESTs） resulted in a set of 2 906 non-redundant sequences comprised of 233 contigs and 2 673 singletons. Comparative analyses indicated that 913 （43.6%） of the unigenes had homologues with function-known genes or functionassumed genes in the National Center for Biotechnology Information. In addition, 763 （36.4%） of the unigenes were functionally classified using Gene Ontology hierarchy. Through EST alignment and the screening method, the full-length cDNA of two MADS-box genes viz., GhMADSll and GhMADS12 were acquired. These genes may play a role in flower development. Phylogenetie analysis indicated that GhMADS11 and GhMADS12 had high homology and close evolutionary relationship with AGL2/SEP-type and PI-type genes, respectively. The expression of both GhMADSll and GhMADS12, genes was high in reproductive organs. In floral organs, GhMADSll expression was high in petals （whor12） and ovules, while GhMADS12 expression was high in petals （whor12） and stamens （whor13）. Results show that the EST strategy based on a normalized cDNA library is an effective method for gene identification. The study provides more insights for future molecular research on the regulation mechanism of cotton flower development.

Gene mapping and candidate gene analysis of aberrant-floral spikelet 1(afs1) in rice(Oryza sativa L.)

The spikelet is a unique inflorescence structure in grasses. However, the molecular mechanism that regulates its development remains unclear, and we therefore characterize a spikelet mutant of rice(Oryza sativa L.), aberrant-floral spikelet 1(afs1), which was derived from treatment of Xinong 1 B with ethyl methanesulfonate. In the afs1 mutant, the spikelet developed an additional lemma-like organ alongside the other normally developed floral organs, and the paleae were degenerated to differing degrees with or without normally developed inner floral organs. Genetic analysis revealed that the afs1 phenotype was controlled by a single recessive gene. The AFS1 gene was mapped between the insertion/deletion(In Del) marker Indel19 and the simple sequence repeat marker RM16893, with a physical distance of 128.5 kb on chromosome 4. Using sequence analysis, we identified the deletion of a 5-bp fragment and a transversion from G to A within LOC_Os04 g32510/LAX2, which caused early termination of translation in the afs1 mutant. These findings suggest that AFS1 may be a new allele of LAX2, and is involved in the development of floral organs by regulating the expression of genes related to their development. The above results provide a new view on the function of LAX2, which may also regulate the development of spikelets.

Characterization of the Promoter of a Homolog of Maize MADS-Box Gene m18

Maize （Zea mays L.） is one of the world’s major food crops, and often suffers from tremendous yield loss caused by abiotic stresses. The MADS-box genes are known to play versatile roles in plants, controlling plant responses to multiple abiotic stresses. However, understanding of regulation of their expressions by the conventional loss-of-function approach is very dififcult. So far, regulation of MADS-box gene expression is little known. The best approach to retrieve expression regulation of this category of genes is to characterize expression of their promoters. In this study, the promoter of a homolog （GenBank accession no. EC864166） of maize MADS-box gene m18 was cloned by way of genome-walking PCR, named Pro66. Predicative analysis indicated that Pro66 contains more than one TATA box and multiple cis-acting environmental conditions-responsive elements （ECREs）. Pro66 could drive expression of theβ-glucuronidase （GUS）-encoding gene in maize, and heterologous expression of GUS in red pepper stressed by water deifcit, salt, copper, iron deifciency, heat, cold, and grown under short and long photoperiods, echoing predicative ECREs. Conclusively, maize MADS-box gene m18 likely plays versatile functions in maize response to multiple abiotic stresses due to the promoter with multiple cis-acting elements. The complex arrangement of multiple cis-acting elements in the promoter features meticulously regulated expression of m18. The results give informative clues for heterologous utilisation of the promoters in monocot and dicot species. The copy of the ECREs and heterologous expression of the promoter in dicot species are also discussed.

The Influence of Photoperiod on the Regulation of Root and Callus Initiation of Perle Noir (<i>V. vinifera</i>L.): Expression of MADS-Box Gene

To study the influence of photoperiod on roots differentiation in the Tunisian grapevine (Vitis vinifera L.) cultivar Perle noir, roots and callus initiation were analyzed under three different conditions of day length: long day (LD), short day (SD) and darkness (D). The photoperiod influenced the number of callus and roots per cuttings;it has a significant effect on the roots and callus initiation. Expression profile analysis of six MADS-box genes (VTM8, VSEP2, VAG12, VAG17-1, VAG17-2 and VSOC1.3) during root and callus development is in agreement with the above-mentioned observation. The expression of the MADS-box genes during root and callus development fluctuated in a tissue-dependent manner. These data suggest that all genes are expressed in roots under three photoperiods. Total darkness gives the number of the most important root per cutting compared to the other two conditions. This photoperiodic condition gave the most important expression of the studied genes VAG12, VAG17-2, VAG17-1, VTM8 and VSEP2 transcripts were not found in callus grown in the dark or in LD conditions, respectively. VSOC1.3 transcripts were not found in callus grown in the dark or in SD conditions, respectively. Transcript abundance of VTM8 and VSOC1 was highest in LD.

MADS-box基因家族调控毛茛目植物花器官发育的研究进展

毛茛目植物花器官具有复杂多样的特点,是研究花器官形态与遗传进化相关问题的理想植物类群。通过综述毛茛目植物花器官形态的多样性,结合毛茛目基因组、转录组以及芸香唐松草(Thalictrum thalictroides)、蓝花耧斗菜(Aquilegia coulela)、黑种草(Nigella damascena)、花菱草(Eschscholzia californica)等几个代表物种的花发育研究现状,阐述了当前MADS-box基因家族A、B、C、D、E类基因的功能及其下游靶基因的研究成果,并对未来毛茛目植物花发育的研究切入点和可能面临的挑战进行展望,旨在丰富对基部真双子叶植物花器官发育分子调控途径的理解。

板栗MADS-box蛋白基因(CmMADS3)的克隆和表达分析

根据MADS-box基因保守区结构,设计简并引物,从板栗(Castaneamollissima)中分离出花特异表达基因的cDNA片段。并通过5’RACE方法获得了全长cDNA,命名为CmMADS3。该片段全长1016bp,包含一个729bp的开放阅读框,推导的氨基酸序列(243个氨基酸)与拟南芥的SEP1、SEP2和SEP33类MADS-box蛋白有很高的序列相似性。系统进化分析同样将CmMADS3基因归入MADS-box基因家族的AGL2组。RT-PCR分析显示,该基因在板栗的花和幼果中表达丰度高,在茎中有微弱的表达,在叶中不表达,研究结果表明CmMADS3基因是板栗花器官发育中具有E功能的功能基因。

洋葱花器官B类MADS-box基因AcPI的克隆及表达分析

【目的】克隆洋葱花器官B类PI/GLO家族MADS-box基因,分析其序列特征及时空表达模式,为探讨其在洋葱花发育过程中的分子遗传机制奠定基础。【方法】以洋葱花蕾总RNA为模板,根据同源克隆策略设计简并引物,利用RT-PCR结合RACE技术,获得AcPI的全长cDNA序列。用生物信息学方法对其基因序列特征进行分析;利用RT-PCR和Real-time PCR分析AcPI在花蕾整个生长过程中的时空表达模式。【结果】克隆获得洋葱AcPI基因(GenBank登录号:JX679083)的cDNA全长931 bp,包含615 bp的完整开放阅读框,编码205个氨基酸。蛋白分析表明,AcPI蛋白具有植物MADS-box蛋白典型的MADS和K结构域;与水仙的NTPI、风信子的HoMADS2有78%、75%的相似性,与金鱼草的GLO也有52%的相似性。系统进化树分析表明,AcPI属于B类MADS-box蛋白家族的PI亚家族。RT-PCR表达分析表明,AcPI只在生殖器官花蕾中表达,但主要在花的第一、二、三轮花器官中表达,而在营养组织根、茎和叶中不表达。Real-time PCR进一步分析表明,AcPI在花芽整个生长过程中,在心皮中微弱表达,但表达丰度呈递增趋势;而在外轮被片、内轮被片和雄蕊中强烈表达,其表达丰度除了在外轮被片中呈先增后减的趋势外,在内轮被片和雄蕊中都呈递增趋势。【结论】AcPI在洋葱第一轮花器官中的表达支持了van Tunen提出的修正的ABC模型;但AcPI在洋葱第四轮花器官中也有表达,这表明AcPI除了调控外轮被片、内轮被片和雄蕊发育外,还可能在心皮的形成发育过程中起着重要作用。

大花蕙兰MADS-box基因ChMADS1的克隆及表达分析

根据MADS-box基因保守区结构,设计简并性引物,从大花蕙兰(Cymbidium hybridium)子房中分离和克隆到一个MADS-box基因全序列cDNA(ChMADS1),序列分析表明该基因长871 bp,含一个编码228个氨基酸的完整开放读码框,具有典型的植物MADS-box蛋白结构,由MADS盒、I、K、C区四部分组成.该基因编码的蛋白质与另两种兰花的AP3类MADS盒基因蛋白质PeAP3(89%/94%)和DcOAP3A(89%/95%)同源性较高,属于B组AP3基因家族中的paleoAP3基因.RT-PCR和反Northern杂交分析进一步证实其在子房以及花的各个器官中表达,是B组AP3基因家族中具特殊表达功能的一个新成员.

植物MADS-box基因多样性及进化研究进展

MADS-box基因是一类序列特异的调节基因家族,在植物生长发育和信号传导过程中起着重要的作用。现综述了MADS-box基因特性、功能、进化等方面的研究进展,有助于深入了解基因重复及随后其功能分化的过程和机制,对于解释物种起源,遗传、演化乃至资源保护利用具有重要的理论指导意义,并探讨了MADS-box基因将来的发展趋势。

Floral-dip转化法将PEPC基因ihpRNA干扰表达载体导入甘蓝型油菜

根据磷酸烯醇丙酮酸羧化酶(PEPC)基因控制油菜籽中蛋白质与脂肪酸合成的原理,构建了PEPC基因片段ihpRNA干扰表达载体,该载体包含了一个PEPC基因片段正反向序列的回文结构.表达RNA干扰的载体结构(ihpRNA),在大肠杆菌体内进行克隆的过程中易被大肠杆菌自身携带的某种酶剪切,剪切位点不确定,可能是一种随机性的剪切.通过反复实验,最终获得了一个与我们设计一致的PEPC基因片段正反向序列结构,RNA干扰表达载体构建获得成功.利用花序浸渍法(floral-dip)转基因将构建的PEPC基因ihpRNA干扰表达载体转移到甘蓝型油菜中,并通过抗性筛选、PCR特异扩增、PCR-Southern杂交和克隆测序,对获得的转基因植株进行了鉴定,转化率达到了0.69%,说明floral-dip方法有效地实现了ihpRNA干扰表达载体的遗传转化.

兰花MADS-box基因研究进展

MADS-box基因是一类非常重要的转录调控因子,在植物发育和信号传导中起着关键性作用。包括花器官发育基因,花分生组织特异性基因,促进开花的基因。研究表明,A类基因可能与花器官发育和控制开花有关;B类基因可能与花被片形态和结构特异有关,并且有些基因在营养器官中也有表达。

小麦B类MADS-box基因WPI1和WPI2的克隆及表达分析

为探究小麦雌蕊化现象的分子遗传机制,本文从CSTP和HTS-1 2个小麦品系的幼穗中克隆得到了2个B类MADS-box基因WPI1和WPI2。WPI1基因c DNA序列长816 bp,ORF长627 bp,编码208个氨基酸残基;WPI2基因c DNA序列长983 bp,ORF长630 bp,编码209个氨基酸残基。WPI1和WPI2基因均含有典型的MADS-MEF2-like和K-box结构域,C端均具有PI结构基序。系统进化树分析表明WPI1和WPI2基因属于PI/GLO亚家族成员。Real-time PCR分析表明WPI1和WPI2基因在CSTP和HTS-1小穗中的表达模式明显不同。雌雄蕊原基形成期,WPI1和WPI2基因在HTS-1中的表达量较高,而在CSTP中表达量较低,接近为0;药隔期,WPI1和WPI2基因在HTS-1中的表达水平明显低于CSTP中的表达。WPI1和WPI2基因表达模式的改变与HTS-1雌蕊化表型有关。本研究结果为进一步探讨B类基因WPI1和WPI2在HTS-1雌蕊化中的作用奠定了基础。

MADS-box基因家族调控植物花器官发育研究进展

花器官的发生发育与形态构造是一个高度复杂的项目,涉及众多因素及其相互作用,MADS-box基因作为其中的关键转录因子可以改变整个发育过程,因而成为花器官研究最广泛的基因家族。通过梳理目前MADS-box基因对花器官发生、分化和形态建成方面的调控作用,为了解花发育程序和该基因家族的一般转录调控机制提供新见解,也为进一步深入挖掘该家族基因和完善花发育调控理论提供参考。

GmNMH7, a MADS-box transcription factor, inhibits root development and nodulation of soybean(Glycine max [L.] Merr.)

As an important food crop and oil crop, soybean(Glycine max [L.] Merr.) is capable of nitrogen-fixing by root nodule. Previous studies showed that GmNMH7, a transcription factor of MADS-box family, is associated with nodule development, but its specific function remained unknown. In this study, we found that GmN MH7 was specifically expressed in root and nodule and the expression pattern of GmNMH7 was similar to several genes involved in early development of nodule(GmENOD40-1, GmENOD40-2, GmNFR1 a, GmNFR5 a, and GmNIN) after rhizobia inoculation. The earlier expression peak of GmNMH7 compared to the other genes(GmENOD40-1, GmENOD40-2, GmNFR1 a, GmNFR5 a, and GmNIN) indicated that the gene is related to the nod factor(NF) signaling pathway and functions at the early development of nodule. Over-expression of GmNMH7 in hairy roots significantly reduced the nodule number and the root length. In the transgenic hairy roots, overexpression of GmN MH7 significantly down-regulated the expression levels of GmE NOD40-1, GmE NOD40-2, and GmN FR5α. Moreover, the expression of GmNMH7 could respond to abscisic acid(ABA) and gibberellin(GA_3) treatment in the root of Zigongdongdou seedlings. Over-expressing GmNMH7 gene reduced the content of ABA, and increased the content of GA_3 in the positive transgenic hairy roots. Therefore, we concluded that GmNMH7 might participate in the NF signaling pathway and negatively regulate nodulation probably through regulating the content of GA_3.

向日葵MADS-Box基因HAM23-like克隆和表达分析

为了解MADS-box基因在向日葵(Helianthus annuus)花发育过程中的作用,采用RT-PCR技术克隆了1个MADS-box基因新成员HAM23-like,开放阅读框为831bp,编码276个氨基酸,相对分子量为30.52k D,理论等电点为9.42。系统发育分析表明,HAM23-like与拟南芥的AGL18聚于同一分支,具有较近的亲缘关系。qRT-PCR分析表明,HAM23-like基因在花和成熟果实(籽粒饱满期)中的表达量较高;HAM23-like在开花当天的雄蕊中的表达量最高;随着花的发育,HAM 23-like表达量逐渐升高,在开花后5 d (果实形成早期)达到最高表达水平。因此,推断HAM23-like基因可能与向日葵花器官后期发育和瘦果早期发育相关。

Gene cloning and expression analysis of limonene synthase in Syringa oblata and S.oblata var.alba

Syringa species are important ornamentals with strong floral scent,of which monoterpenes are the main component.In this study,a new monoterpene synthase gene,named SoLIM,was collected from the flowers of Syringa oblata and S.oblata var.alba using a homologous cloning method.The full-length cDNA of SoLIM was1746 bp and encoded 581 amino acids.Sequence analysis showed that SoLIM contained the DDxxD and RRx8 W motifs,which are two typical conserved monoterpene synthase motifs,and was thus classified as belonging to the Tpsb subfamily.Using quantitative reverse-transcription PCR,SoLIM was significantly expressed in the petals and pistils of S.oblata and S.oblata var.alba,respectively.SoLIM expression peaked earlier than the D-limonene emissions in the diurnal experiments,but occurred later when D-limonene had peaked during the flowering phase,indicating that differences in SoLIM gene expression and D-limonene emissions existed.The synthesis of floral scent is thus associated with diverse regulatory mechanisms that require further investigation.

Reference Gene Selection for qRT-PCR Normalization in Iris germanica L.

Quantitative real-time PCR(qPCR)is an effective and widely used method to analyze expression patterns of target genes.Selection of stable reference genes is a prerequisite for accurate normalization of target gene expression by qRT-PCR.In Iris germanica L.,no studies have yet been published regarding the evaluation of potential reference genes.In this study,nine candidate reference genes were assessed at different flower developmental stages and in different tissues by four different algorithms(GeNorm,NormFinder,BestKeeper,and RefFinder).The results revealed that ACT11(Actin 11)and EF1α(Elongation factor 1 alpha)were the most stable reference genes in different tissues,whereas TUA(Tubulin alpha)and UBC9(Ubiquitin-protein ligase 9)were the most stable ones in different flower developmental stages.UBC9 and ACT11 were the most stable reference genes in all of the tested samples,while the SAMDC(S-Adenosylmethionine decarboxylase)showed the least stability.Finally,to validate the suitability of the selected reference genes,the relative expression level of IgTPS(beta-caryophyllene synthase)was assessed and highlighted the importance of suitable reference gene selection.This work constitutes the first systematic evaluation of potential reference genes in I.germanica and provides guidelines for future research on gene function and molecular mechanisms on I.germanica and related species.

Identification and validation of a major gene for kernel length at the P1 locus in Triticum polonicum

Kernel size, one of the traits that determine wheat yield, is controlled by multiple quantitative trait loci.Polish wheat(Triticum polonicum) has elongated and plump kernel and is a valuable material for breeding high-yielding wheat cultivars. However, genes or loci determining kernel length(KL) in Polish wheat are unknown. We identified and validated a major KL gene, KL-PW, at the P1 locus in Polish wheat. KL-PW is VRT-A2, which encodes a MIKC-type MADS-box protein(MADS55). An insertion/deletion mutation in intron 1 of VRT-A2;led to an alternatively spliced transcript, VRT-A2;. Quantitative PCR analysis showed that VRT-A2;was more highly expressed in developing seeds than was VRT-A2 Ailanmai.Brassinosteroid(BR) sensitivity experiment and the expression of BR-related genes indicated that VRTA2;functions as a positive regulator of BR responses. VRT-A2;significantly increased KL of wheat.These findings not only reveal the molecular basis of KL-PW in controlling KL, but also provide a valuable genetic resource for increasing kernel size in wheat.