Gene Cloning And Functional Analysis Of Med8 And Other Mediator Subunits In Tobacco And Rice

Mediator complex is an important component of general transcriptional machinery in eukaryotes. It provides an interface for activator and repressor proteins to transmit information from regulatory DNA elements to core promoters. The composition and functions of Mediator has been clearly studied in yeast (Saccharomyces cerevisiae) and relatively deeply researched in metazoan and human, but still remained elusive in plants until recently. Mediator complex is conserved in all eukaryotes, from yeast to humans, but huge differences still exist among different species in respect to its component, structure and function. These differences might have, to some extent, maintained the stability of heredity and the diversity of evolution among species.Studies have shown that the Mediator component Med8 plays important roles in both yeast and Arabidopsis. Loss-of-function mutation of Med8 gene causes temperature-sensitive lethality in yeast as well as a strong delay in flowering and increase of leaf number in Arabidopsis. The molecular mechanism of Med8 has been well understood in yeast and human, but still remains unclear in plants. So, it is necessary to study the roles of Med8 in plants, whill will be helpful to understand the regulation mechanism of Mediator in eukaryotes and offer theoretical foundation for the genetic improvment of crops.In this study, we cloned the homologous genes of Med8 in tobacco (Nicoliana tabacum; named as NtMed8) and rice (Oryza sativa:named as OsMed8). characterized their functions by RNAi and over-expression, investigated their temporal and spatial expression patterns by qRT-PCR or by Med8 promoter-driven GUS expression, detected their subcellular locations by transient transformation assays, and analysed their interactions with several other Mediator subunits by BiFC. The main results are as follows:1. Cloning and functional analysis of NtMed8 gene Several tobacco EST sequences highly homologous to the tomato LeMed8 gene were found by online BLAST in the NCBI website (http://blast.ncbi.nlm.nih.gov/Blast. cgi). These ESTs were assembled into a long cDNA sequence, which contains an open reading frame (ORF) of 1,593 bp in length, encoding a putative protein of 530 amino acids. The deduced amino acid sequence shares high similarities with Med8 homologs from eight other flowering plant species, containing two conserved motifs similar to yeast Med8C and Med8N, respectively. This result suggested that the ORF should be of NtMed8. This putative-NtMed8 ORF was then isolated and cloned. qRT-PCR analysis indicated that NtMed8 is expressed in both vegetative and floral tissues, especially in floral tissues as well as leaves, but very weak in senescing leaves. To investigate the function of NtMed8, we created transgenic tobacco plants with depressed-NtMed8 expression mediated by RNA interference (RNAi). Compared with the wild type, the NtMed8-RNAi plants exhibited more leaves with smaller but thicker blades; larger cells and vasculars with lower stomata density in leaves; swelled chloroplasts with thicker and lumen-enlarged thylakoids; weaker root system with fewer branch roots; larger flowers and floral organs; flowering earlier under long-day but later under short-day conditions; and male sterile with larger but less germinable pollens.2. Subcellular locations and expression profiles of several Mediator subunits of tobacco and riceWe cloned NtMed18,NtMed6 and NtMed21 by homology cloning method, constructed the expression vectors of these three genes as well as the previously cloned NtMed8 fused with enhanced green fluorescent protein (EGFP) and enhanced yellow fluorescent protein (EYFP) genes driven by 35S promoter, and then detected the subcellular locations of these fused genes by transient transformation assays performed with Agrobacterium-infiltrated tobacco leaves. The results showed that NtMed8. NtMed18,NtMed6 and NtMed21 were all located in both nucleus and cytoplasm,but mainly in nucleus. We also cloned OsMed6, OsMed8 and OsMed22 from rice by homology cloning and analyzed their subcellular locations by constructing their expression vectors fused with EYFP gene driven by maize ubiquitin promoter. The results showed that 1) OsMed6 was specifically located in nucleus; 2) OsMed8 was located in both nucleus and cytoplasm, but mainly in nucleus, similar to NtMed8 and other tobacco subunits; and 3) OsMed22 appeared to be located in cell wall as some discrete segments, and its subcellular location is still unclear. In addition, using rice microarray data downloaded from NCBI, we analyzed the expression profiles of 23 putative Mediator subunits of rice. The results indicated that the 23 subunits showed different expression patterns in 21 tissues or developmental stages.OsMed12 showed the highest specificity in tissue expression, the next is OsMedll. However, other 21 subunits showed irregular expression level in different tissues, were close to constitutive expressed genes. High expressions of OsMed15 were observed in almost all of 21 tissues, on the contrary, the low expressions OsMed10 were obersved in all tissues. OsMed12, OsMed11, OsMed28, OsMed31, OsMed17, OsMed18 and OsMed19b were genes which played crucial roles in clustering 21 tissues into 4 groups.3. Analysis of interactions between NtMed8 and other Mediator subunitsTo validate that NtMed8 is a tobacco Mediator subunit and to investigate the interactions between NtMed8 and other tobacco Mediator subunits, we constructed BiFC vectors containing coding sequence of EYFP N-terminus from amino acids 1-173 (pNYE and pNeYE) and coding sequence of YFP N-terminus from amino acids 151-238 (pCYE and pCeYE) driven by 35S promoter. Then, we subcloned NtMed8, NtMed6, NtMedl8 and-NtMed21 into the BiFC vectors respectively, and detected the subcellular locations with different combinations of the vectors by transient cotransformation assays performed with Agrobacterium-infiltrated tobacco leaves. In the coexpression experiments, two different bacterial cultures were 1:1 mixed prior to the leaf infiltration, with the inoculum of each construct adjusted to the required final OD600; the explant was incubated under normal growing conditions and analyzed 24 to 72 h after infiltration. The results showed that interactions exist between NtMed8 and NtMed18, and between NtMed8 and NtMed6, NtMed8 and NtMed21, and NtMed18 and NtMed6.4. Preliminary functional analysis of OsMed8To study the spatial and temporal expression pattern and function of OsMed8, we constructed a GUS expression vector driven by the promoter (an 1183-bp upstream sequence) of NtMed8, an OsMed8 over-expression vector and an OsMed8-RNAi vector, respectively, and transferred them into rice cultivar Zhonghua-15. The results showed that OsMed8 is expressed in all organs, but particularly strong in those tissues exhibiting vigorous growth in vegetative or reproductive development, including the lateral root primordium, ovary, the base of pistil, preliminary differentiational spikelet and young stem..OsMed8 over-expression caused early flowering, longer panicles and increased number of seeds per panicle. On the contrary,OsMed8-RNAi resulted in late flowering, reduced number of seeds per panicle and decreased male fertility.