A Full-Length Dof1 Transcription Factor of Finger Millet and its Response to a Circadian Cycle

DOF1 (DNA binding with one finger) plays an important role in regulating C/N metabolism in cereals. In order to validate its role in the regulation of nitrogen use efficiency (NUE) and photosynthetic efficiency in finger millet, 5′–3′ RACE PCR was performed to obtain and characterize full-length Dof1 genes of high and low grain protein finger millet genotypes. The full-length DOF1 ORFs were both 1,284 nt long and were 98.8 % similar over 427 amino acids containing the characteristic Dof domain. Comparison of both the EcDof1 protein sequences with the Dof1 of other cereals revealed high sequence similarity to the Dof1 of rice. Southern hybridization carried out using the probe developed from the region encoding the highly variable C-terminal region of EcDof1 showed the presence of four copies of the DOF1 gene in finger millet, which might explain the high NUE and photosynthetic performance of finger millet. Since the genes involved in C/N metabolism are regulated diurnally and play crucial roles in determining grain protein content during grain filling, the diurnal expression of EcDOF1 was assessed in two finger millet genotypes (GE 3885 and GE 1437) with differing grain protein content (13.8 % and 6.15 % respectively). It was found that EcDOF1 exhibited diurnal regulation and peak differential pattern expression with early phasing in GE3885 and late phasing in GE1437. Differential expression of DOF1 might alter the regulation of genes involved in C/N metabolism affecting grain protein composition of finger millet genotypes.     

Nitrate signals determine the sensing of nitrogen through differential expression of genes involved in nitrogen uptake and assimilation in finger millet

In order to understand the molecular basis of high nitrogen use efficiency of finger millet, five genes (EcHNRT2, EcLNRT1, EcNADH-NR, EcGS, and EcFd-GOGAT) involved in nitrate uptake and assimilation were isolated using conserved primer approaches. Expression profiles of these five genes along with the previously isolated EcDof1 was studied under increased KNO₃ concentrations (0.15 to 1,500 μM) for 2 h as well as at 1.5 μM for 24 h in the roots and shoots of 25 days old nitrogen deprived two contrasting finger millet genotypes (GE-3885 and GE-1437) differing in grain protein content (13.76 and 6.15 %, respectively). Time kinetics experiment revealed that, all the five genes except EcHNRT2 in the leaves of GE-3885 were induced within 30 min of nitrate exposure indicating that there might be a greater nitrogen deficit in leaves and therefore quick transportation of nitrate signals to the leaves. Exposing the plants to increasing nitrate concentrations for 2 h showed that in roots of GE-3885, NR was strongly induced while GS was repressed; however, the pattern was found to be reversed in leaves of GE-1437 indicating that in GE-3885, most of the nitrate might be reduced in the roots but assimilated in leaves and vice-versa. Furthermore, compared with the low-protein genotype, expression of HNRT2 was strongly induced in both roots and shoots of high-protein genotype at the least nitrate concentration supplied. This further indicates that GE-3885 is a quick sensor of nitrogen compared with the low-protein genotype. Furthermore, expression of EcDof1 was also found to overlap the expression of NR, GS, and GOGAT indicating that Dof1 probably regulates the expression of these genes under different conditions by sensing the nitrogen fluctuations around the root zone.     

Cloning, In Silico Characterization and Prediction of Three Dimensional Structure of SbDof1, SbDof19, SbDof23 and SbDof24 Proteins from Sorghum [Sorghum bicolor (L.) Moench]

In the present study, four full-length Dof (DNA-binding with one finger) genes from Sorghum bicolor namely SbDof1, SbDof19, SbDof23, and SbDof24 were PCR amplified, gel eluted, cloned, and sequenced (accession number HQ540084, HQ540085, HQ540086, and HQ540087, respectively). These sequences were further characterized in silico by subjecting them to homology search, multiple sequence alignment, phylogenetic tree construction, and protein functional analysis, revealing their identity to Dof like proteins. Phylogenetic analysis of cloned SbDof genes along with other reported Dof proteins revealed existence of two major groups A and B, while group A was further bifurcated into two sub-groups (viz., I and II). Motif scan analysis of SbDof proteins revealed the presence of glycine- and alanine-rich profiles in SbDof1, while proline-rich profile was observed in SbDof23. Asparagines, methionine, and serine-rich profiles were common in case of both SbDof19 and SbDof24 proteins. The three dimensional structures of SbDof proteins were predicted by I-TASSER server based on multiple threading method. The modeled structures were refined by energy minimization and their stereo chemical qualities were validated by PROCHECK and QMEAN server indicating the acceptability of the predicted models. The final models were submitted to PMDB database with assigned PMDB IDs, i.e., PM0077395, PM0077396, PM0077397, PM0077398, and PM0076448 for SbDof1, SbDof19, SbDof23, SbDof24, and Dof domain, respectively. Based on gene ontology (GO) terms in I-TASSER server putative functions of modeled SbDof proteins were also predicted.     

Agrobacterium-mediated transformation of finger millet (Eleusine coracana (L.) Gaertn.) using shoot apex explants

A new Agrobacterium-mediated transformation system was developed for finger millet using shoot apex explants. The Agrobacterium strain LBA4404 harboring binary vector pCAMBIA1301, which contained hygromycin phosphotransferase (hptII) as selectable marker gene and β-glucuronidase (GUS) as reporter gene, was used for optimization of transformation conditions. Two finger millet genotypes, GPU 45 and CO 14, were used in this study. The optimal conditions for the Agrobacterium-mediated transformation of finger millet were found to be the co-cultivation of explants obtained on the 16th day after callus induction (DACI), exposure of explants for 30 min to agrobacterial inoculum and 3 days of co-cultivation on filter paper placed on medium supplemented with 100 μM acetosyringone (AS). Addition of 100 μM l-cysteine in the selection medium enhanced the frequency of transformation and transgenic plant recovery. Both finger millet genotypes were transformed by Agrobacterium. A frequency of 19% transient expression with 3.8% stable transformation was achieved in genotype GPU 45 using optimal conditions. Five stably transformed plants were fully characterized by Southern blot analysis. A segregation analysis was also performed in four R₁ progenies, which showed normal Mendelian pattern of transgene segregation. The inheritance of transgenes in R₁ progenies was also confirmed by Southern blot analysis. This is the first report on Agrobacterium-mediated transformation of finger millet. This study underpins the introduction of numerous agronomically important genes into the genome of finger millet in the future.     

Dof基因家族调节植物生长发育功能的研究进展

Dof（DNA binding with one finger）蛋白是一类植物特异性转录因子,通常含有200～400个氨基酸和2个主要结构域。该家族成员的N 末端为高度保守的单锌指Dof结构域,具有与DNA和蛋白质相互作用的双重功能,其C末端的氨基酸序列则较为多变,是Dof蛋白重要的特异转录调控结构域。研究表明,Dof蛋白作为转录激活物或阻遏物参与了多方面的植物生长发育过程。随着基因组测序技术的发展,已有大量的Dof基因从植物基因组数据库中鉴定出来。该文对近年来国内外有关Dof基因家族的结构特点、全基因组鉴定、蛋白互作以及生物学功能等方面的研究进展进行综述,以期为Dof转录因子的深入研究提供参考。     

Comparative analyses reveal high levels of conserved colinearity between the finger millet and rice genomes

Finger millet is an allotetraploid (2n = 4x = 36) grass that belongs to the Chloridoideae subfamily. A comparative analysis has been carried out to determine the relationship of the finger millet genome with that of rice. Six of the nine finger millet homoeologous groups corresponded to a single rice chromosome each. Each of the remaining three finger millet groups were orthologous to two rice chromosomes, and in all the three cases one rice chromosome was inserted into the centromeric region of a second rice chromosome to give the finger millet chromosomal configuration. All observed rearrangements were, among the grasses, unique to finger millet and, possibly, the Chloridoideae subfamily. Gene orders between rice and finger millet were highly conserved, with rearrangements being limited largely to single marker transpositions and small putative inversions encompassing at most three markers. Only some 10% of markers mapped to non-syntenic positions in rice and finger millet and the majority of these were located in the distal 14% of chromosome arms, supporting a possible correlation between recombination and sequence evolution as has previously been observed in wheat. A comparison of the organization of finger millet, Panicoideae and Pooideae genomes relative to rice allowed us to infer putative ancestral chromosome configurations in the grasses. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

cDNA cloning and gene expression pattern following bacterial challenge of peroxinectin in Chinese shrimp <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis>

Peroxinectin, a cell-adhesive hemoperoxidase that binds superoxide dismutase and mediates blood cells adhesion and migration in invertebrate, is believed to play an important role in cellular immune reaction. In this study, we reported a new peroxinectin gene homologue from Chinese shrimp Fenneropenaeus chinensis. Based on expressed sequence tags (ESTs) of haemocyte cDNA library, we cloned a 2,611 bps full-length cDNA of peroxinectin gene homologue encoded 801 amino acids. Motif scanning of the predicted polypeptide revealed a peroxidase domain and an integrin binding motif (Lys-Gly-Asp, KGD). Peroxinectin gene expressed constitutively in haemocyte as determined by quantitative real-time RT-PCR, the expression level varied following bacterial challenge. These findings suggested that peroxinectin expression is susceptible to exterior stimulus and maintains at a high expression level during bacterial infection.     

Development of transgenic finger millet (<Emphasis Type="Italic">Eleusine coracana</Emphasis> (L.) Gaertn.) resistant to leaf blast disease

Finger millet plants conferring resistance to leaf blast disease have been developed by inserting a rice chitinase (chi11) gene through Agrobacterium-mediated transformation. Plasmid pHyg-Chi.11 harbouring the rice chitinase gene under the control of maize ubiquitin promoter was introduced into finger millet using Agrobacterium strain LBA4404 (pSB1). Transformed plants were selected and regenerated on hygromycin-supplemented medium. Transient expression of transgene was confirmed by GUS histochemical staining. The incorporation of rice chitinase gene in R₀ and R₁ progenies was confirmed by PCR and Southern blot analyses. Expression of chitinase gene in finger millet was confirmed by Western blot analysis with a barley chitinase antibody. A leaf blast assay was also performed by challenging the transgenic plants with spores of Pyricularia grisea. The frequency of transient expression was 16.3% to 19.3%. Stable frequency was 3.5% to 3.9%. Southern blot analysis confirmed the integration of 3.1 kb chitinase gene. Western blot analysis detected the presence of 35 kDa chitinase enzyme. Chitinase activity ranged from 19.4 to 24.8. In segregation analysis, the transgenic R₁ lines produced three resistant and one sensitive for hygromycin, confirming the normal Mendelian pattern of transgene segregation. Transgenic plants showed high level of resistance to leaf blast disease compared to control plants. This is the first study reporting the introduction of rice chitinase gene into finger millet for leaf blast resistance.