棉花MADS框蛋白基因(GhMADS1)的克隆

作为转录因子,MADS框蛋白基因在植物花器官发育中有着重要的功能。为研究棉花花器官发育的分子机理,以棉花花器官突变体CHV1(cotton homeotic variant)和徐州142正常植株为材料,利用棉花EST数据库资料,通过EST序列整合,从陆地棉徐州142花蕾中克隆出一个MADS框蛋白的编码区段,GenBank登录号为AF538965。该片段(GhMADS1)长713bp,包含一个711bp的开放阅读框,推导的氨基酸序列(236个氨基酸)与葡萄、烟草、矮牵牛、拟南芥和金鱼草等的AGL2组MADS框蛋白有很高的序列相似性。系统进化分析同样将GhMADS1基因归人AGt2组MADS框蛋白。RT-PCR分析显示,该基因在陆地棉的花瓣、雄蕊、胚珠和纤维中表达,特别是在花瓣中表达量最高,而在根、茎、叶等营养器官和棉花同源异型突变体CHV1(所有花器官均变为苞叶状叶性器官)的变异花蕾中不表达。这些结果说明GhMADS1基因可能在棉花花器官发育中有着重要的功能。     

Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus.

In Antirrhinum, floral meristems are established by meristem identity genes. Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes. Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost. In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes.     

Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species

APETALA1 (AP1) and its homologue SQUAMOSA (SQUA) are key regulatory genes specifying floral meristem identity in the model plants Arabidopsis and Antirrhinum. Despite many similarities in their sequence, expression and functions, only AP1 appears to have the additional role of specifying sepal and petal identity. No true AP1/SQUA-functional homologues from any other plant species have been functionally studied in detail, therefore the question of how the different functions of AP1-like genes are conserved between species has not been addressed. We have isolated and characterized PEAM4, the AP1/SQUA-functional homologue from pea, a plant with a different floral morphology and inflorescence architecture to that of Arabidopsis or Antirrhinum. PEAM4 encodes for a polypeptide 76% identical to AP1, but lacks the C-terminal prenylation motif, common to AP1 and SQUA, that has been suggested to control the activity of AP1. Nevertheless, constitutive expression of PEAM4 caused early flowering in tobacco and Arabidopsis. In Arabidopsis, PEAM4 also caused inflorescence-to-flower transformations similar to constitutive AP1 expression, and was able to rescue the floral organ defects of the strong ap1-1 mutant. Our results suggest that the control of both floral meristem and floral organ identity by AP1 is not restricted to Arabidopsis, but is extended to species with diverse floral morphologies, such as pea.     

Conservation and divergence of APETALA1/FRUITFULL-like gene function in grasses: evidence from gene expression analyses

Duplicated APETALA1/FRUITFULL (AP1/FUL) genes show distinct but overlapping patterns of expression within rice (Oryza sativa) and within ryegrass (Lolium temulentum), suggesting discrete functional roles in the transition to flowering, specification of spikelet meristem identity, and specification of floral organ identity. In this study, we analyzed the expression of the AP1/FUL paralogues FUL1 and FUL2 across phylogenetically disparate grasses to test hypotheses of gene function. In combination with other studies, our data support similar roles for both genes in spikelet meristem identity, a general role for FUL1 in floral organ identity, and a more specific role for FUL2 in outer floral whorl identity. In contrast to Arabidopsis AP1/FUL genes, expression of FUL1 and FUL2 is consistent with an early role in the transition to flowering. In general, FUL1 has a wider expression pattern in all spikelet organs than FUL2, but both genes are expressed in all spikelet organs in some cereals. FUL1 and FUL2 appear to have multiple redundant functions in early inflorescence development. We hypothesize that sub-functionalization of FUL2 and interaction of FUL2 with LHS1 could specify lemma and palea identity in the grass floret.     

Characterization of SaMADS D from Sinapis alba suggests a dual function of the gene: in inflorescence development and floral organogenesis

SaMADS D gene of Sinapis alba was isolated by screening a cDNA library from young inflorescences with a mixture of MADS-box genes of Antirrhinum majus (DEF, GLO, SQUA) as probe. Amino acid sequence comparison showed a high degree of similarity between the SaMADS D and AGL9, DEFH200, TM5, FBP2 and DEFH 72 gene products. Analysis of the SaMADS D gene expression by in situ hybridization reveals a novel expression pattern for a MADS-box gene and suggests a dual function for this gene: first, as a determinant in inflorescence meristem identity since it starts to be expressed directly beneath the inflorescence meristem at the time of initiation of the first floral meristem, is no longer expressed in the inflorescence meristem forced to revert to production of leafy appendages, and is expressed again when the reverted meristem resumes floral meristem initiation, and, second, as an interactor with genes specifying floral organ identity since it is expressed in the floral meristem from the stage of sepal protrusion.     

棉花MADS框基因GhMADS3的克隆及其组成型表达对烟草花器官决定的影响

MADS框基因在植物花器官发育中发挥着关键性作用。为研究棉花花器官发育的机理,以徐州142花蕾为材料,利用EST数据库资料,通过EST序列整合,克隆出了一个MADS域蛋白的编码区段,GenBank登录号为AY083173。该片段(GhMADS3)包含一个732 bp的开放阅读框,推导的氨基酸序列(244氨基酸)与可可,黄瓜,烟草,矮牵牛,金鱼草等的AG亚家族基因的序列相似性高。进化树重建分析将GhMADS3基因归入MADS框基因AG亚家族C功能分支的euAG分支。RT-PCR分析显示,该基因在雄蕊和心皮中表达,在根、茎、叶等营养器官,萼片,花瓣,花器官变异体chv1(所有花器官均变为苞叶状器官)的花蕾中不表达。将GhMADS3与35S启动子融合构建成嵌合基因转化烟草,转基因烟草植株花朵出现萼片(轮1)向心皮,花瓣(轮2)向雄蕊的转变,花器官表现明显的白化倾向。同时,在轮1观察到丝状结构的出现,该结构在此前类似的研究中尚无报道。这些结果说明,实验中克隆了一个有生物学功能的棉花的AG亚家族MADS框基因,该基因可能在棉花花器官发育中有重要的功能。     

Regulation of Flowering Time by MicroRNAs

The shoot apical meristem (SAM) continuously produces lateral organs in plants.Based on the identity of the lateral organs,the life cycle of a plant can be divided into two phases:vegetative and reproductive.The SAM produces leaves during the vegetative phase,whereas it gives rise to flowers in the reproductive phase (reviewed in Poethig,2003).The floral transition,namely the switch from vegetative to reproductive growth,is controlled by diverse endogenous and exogenous cues such as age,hormones,photoperiod,and temperature (reviewed in B(a)urle and Dean,2006;Srikanth and Schmid,2011;Andres and Coupland,2012). The model annual Arabidopsis thaliana has been extensively used for the dissection of the molecular mechanism underlying the floral transition during the last two decades.The molecular and genetic analyses have revealed five flowering time pathways,including age,autonomous,gibberellins (GAs),photoperiod and vernalization (reviewed in Amasino and Michaels,2010).Growing lines of evidence indicate that there are extensive crosstalks,feedback or feed-forward loops between the components within these pathways,and that these multiple floral inductive cues are integrated into a set of floral promoting MADS-box genes including APETALA 1 (AP1),SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1),FRUITFULL (FUL) and LEAFY (LFY) (Amasino and Michaels,2010;Lee and Lee,2010;Srikanth and Schmid,2011).     

Patterns of molecular evolution among paralogous floral homeotic genes.

The plant MADS-box regulatory gene family includes several loci that control different aspects of inflorescence and floral development. Orthologs to the Arabidopsis thaliana MADS-box floral meristem genes APETALA1 and CAULIFLOWER and the floral organ identity genes APETALA3 and PISTILLATA were isolated from the congeneric species Arabidopsis lyrata. Analysis of these loci between these two Arabidopsis species, as well as three other more distantly related taxa, reveal contrasting dynamics of molecular evolution between these paralogous floral regulatory genes. Among the four loci, the CAL locus evolves at a significantly faster rate, which may be associated with the evolution of genetic redundancy between CAL and AP1. Moreover, there are significant differences in the distribution of replacement and synonymous substitutions between the functional gene domains of different floral homeotic loci. These results indicate that divergence in developmental function among paralogous members of regulatory gene families is accompanied by changes in rate and pattern of sequence evolution among loci.     

Patterns of gene duplication and functional diversification during the evolution of the AP1/SQUA subfamily of plant MADS-box genes

Members of the AP1/SQUA subfamily of plant MADS-box genes play broad roles in the regulation of reproductive meristems, the specification of sepal and petal identities, and the development of leaves and fruits. It has been shown that AP1/SQUA-like genes are angiosperm-specific, and have experienced several major duplication events. However, the evolutionary history of this subfamily is still uncertain. Here, we report the isolation of 14 new AP1/SQUA-like genes from seven early-diverging eudicots and the identification of 11 previously uncharacterized ESTs and genomic sequences from public databases. Sequence comparisons of these and other published sequences reveal a conserved C-terminal region, the FUL motif, in addition to the known euAP1/paleoAP1 motif, in AP1/SQUA-like proteins. Phylogenetic analyses further suggest that there are three major lineages (euAP1, euFUL, and AGL79) in core eudicots, likely resulting from two close duplication events that predated the divergence of core eudicots. Among the three lineages, euFUL is structurally very similar to FUL-like genes from early-diverging eudicots and basal angiosperms, whereas euAP1 might have originally been generated through a 1-bp deletion in the exon 8 of an ancestral euFUL- or FUL-like gene. Because euFUL- and FUL-like genes usually have broad expression patterns, we speculate that AP1/SQUA-like genes initially had broad functions. Based on these observations, the evolutionary fates of duplicate genes and the contributions of the frameshift mutation and alternative splicing to functional diversity are discussed.