Expression pattern and activity of six glutelin gene promoters in transgenic rice

The shortage of strong endosperm-specific expression promoters for driving the expression of recombinant protein genes in cereal endosperm is a major limitation in obtaining the required level and pattern of expression. Six promoters of seed storage glutelin genes (GluA-1, GluA-2, GluA-3, GluB-3, GluB-5, and GluC) were isolated from rice (Oryza sativa L.) genomic DNA by PCR. Their spatial and temporal expression patterns and expression potential in stable transgenic rice plants were examined with beta-glucuronidase (GUS) used as a reporter gene. All the promoters showed the expected spatial expression within the endosperm. The GluA-1, GluA-2, and GluA-3 promoters directed GUS expression mainly in the outer portion (peripheral region) of the endosperm. The GluB-5 and GluC promoters directed GUS expression in the whole endosperm, with the latter expressed almost evenly throughout the whole endosperm, a feature different from that of other rice glutelin gene promoters. The GluB-3 promoter directed GUS expression solely in aleurone and subaleurone layers. Promoter activities examined during seed maturation showed that the GluC promoter had much higher activity than the other promoters. These promoters are ideal candidates for achieving gene expression for multiple purposes in monocot endosperm but avoid promoter homology-based gene silencing. The GluC promoter did not contain the endosperm specificity-determining motifs GCN4, AACA, and the prolamin-box, which suggests the existence of additional regulatory mechanism in determining endosperm specificity.     

Towards a better bowl of rice: assigning function to tens of thousands of rice genes

Rice, one of the most important food crops for humans, is the first crop plant to have its genome sequenced. Rice whole-genome microarrays, genome tiling arrays and genome-wide gene-indexed mutant collections have recently been generated. With the availability of these resources, discovering the function of the estimated 41,000 rice genes is now within reach. Such discoveries have broad practical implications for understanding the biological processes of rice and other economically important grasses such as cereals and bioenergy crops.     

Inflorescence meristem identity in rice is specified by overlapping functions of three AP1/FUL-like MADS box genes and PAP2, a SEPALLATA MADS box gene

In plants, the transition to reproductive growth is of particular importance for successful seed production. Transformation of the shoot apical meristem (SAM) to the inflorescence meristem (IM) is the crucial first step in this transition. Using laser microdissection and microarrays, we found that expression of PANICLE PHYTOMER2 (PAP2) and three APETALA1 (AP1)/FRUITFULL (FUL)-like genes (MADS14, MADS15, and MADS18) is induced in the SAM during meristem phase transition in rice (Oryza sativa). PAP2 is a MADS box gene belonging to a grass-specific subclade of the SEPALLATA subfamily. Suppression of these three AP1/FUL-like genes by RNA interference caused a slight delay in reproductive transition. Further depletion of PAP2 function from these triple knockdown plants inhibited the transition of the meristem to the IM. In the quadruple knockdown lines, the meristem continued to generate leaves, rather than becoming an IM. Consequently, multiple shoots were formed instead of an inflorescence. PAP2 physically interacts with MAD14 and MADS15 in vivo. Furthermore, the precocious flowering phenotype caused by the overexpression of Hd3a, a rice florigen gene, was weakened in pap2-1 mutants. Based on these results, we propose that PAP2 and the three AP1/FUL-like genes coordinately act in the meristem to specify the identity of the IM downstream of the florigen signal.     

Expression of iron-acquisition-related genes in iron-deficient rice is co-ordinately induced by partially conserved iron-deficiency-responsive elements

Rice plants (Oryza sativa L.) utilize the iron chelators known as mugineic acid family phytosiderophores (MAs) to acquire iron from the rhizosphere. Synthesis of MAs and uptake of MA-chelated iron are strongly induced under conditions of iron deficiency. Microarray analysis was used to characterize the expression profile of rice in response to iron deficiency at the genomic level. mRNA extracted from iron-deficient or iron-sufficient rice roots or leaves was hybridized to a rice array containing 8987 cDNA clones. An induction ratio of greater than 2.0 in roots was observed for 57 genes, many of which are involved in iron-uptake mechanisms, including every identified or predicted step in the methionine cycle and the biosynthesis of MAs from methionine. Northern analysis confirmed that the expression of genes encoding every step in the methionine cycle is thoroughly induced by iron deficiency in roots, and almost thoroughly induced in leaves. A promoter search revealed that the iron-deficiency-induced genes related to iron uptake possessed sequences homologous to the iron-deficiency-responsive cis-acting elements IDE1 and IDE2 in their promoter regions, at a higher rate than that showing no induction under Fe deficiency. These results suggest that rice genes involved in iron acquisition are co-ordinately regulated by conserved mechanisms in response to iron deficiency, in which IDE-mediated regulation plays a significant role.     

The sgp gene modulates stress responses of Streptococcus mutans: utilization of an antisense RNA strategy to investigate essential gene functions

The sgp gene from Streptococcus mutans has been previously isolated, characterized, and demonstrated to encode a G-protein. In order to investigate the function of this gene, a novel antisense RNA strategy was developed. Expression of sgp antisense RNA in Escherichia coli led to transient inhibition of growth. In addition, sgp antisense RNA expression in S. mutans resulted in decreased growth under environmental stress conditions (44°C, acidic pH, and high osmolarity). Therefore, these results suggest that the sgp gene plays a role in modulating the stress responses of S. mutans. This approach could be applicable for investigating the function of essential genes in other organisms for which mutants are not available.     

不同启动子调控外源基因在斑玉蕈中的表达分析

启动子是控制基因转录的重要顺式元件,也是遗传转化实验中驱动外源基因表达的重要工具.在同一真菌中,不同启动子驱动外源基因表达水平可能存在明显差异.因此,选择合适的启动子是提高外源基因表达水平的关键.本研究分别应用花椰菜病毒35S RNA (cauliflower mosaic virus 35S RNA,CaMV35S)...     

Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice

A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively. Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA(1). These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem.     

Topological characteristics of target genes regulated by abiotic-stress-responsible miRNAs in a rice interactome network

A great number of microRNAs (miRNAs) have been identified in responding and acting in gene regulatory networks associated with plant tolerance to abiotic stress conditions, such as drought, salinity, and high temperature. The topological exploration of target genes regulated by abiotic-stress-responsible miRNAs (ASRmiRs) in a network facilitates to discover the molecular basis of plant abiotic stress response. This study was based on the staple food rice (Oryza sativa) in which ASRmiRs were manually curated. After having compared the topological properties of target genes (stress-miR-targets) with those (non-stress-miR-targets) not regulated by ASRmiRs in a rice interactome network, we found that stress-miR-targets exhibited distinguishable topological properties. The interaction probability analysis and k-core decomposition showed that stress-miR-targets preferentially interacted with non-stress-miR-targets and located at the peripheral positions in the network. Our results indicated an obvious topological distinction between the two types of genes, reflecting the specific mechanisms of action of stress-miR-targets in rice abiotic stress response. Also, the results may provide valuable clues to elucidate molecular mechanisms of crop response to abiotic stress.     

Expression of expansin genes is correlated with growth in deepwater rice.

Expansins are a family of proteins that catalyze long-term extension of isolated cell walls. Previously, two expansin proteins have been isolated from internodes of deepwater rice, and three rice expansin genes, Os-EXP1, Os-EXP2, and Os-EXP3, have been identified. We report here on the identification of a fourth rice expansin gene, Os-EXP4, and on the expression pattern of the rice expansin gene family in deepwater rice. Rice expansin genes show organ-specific differential expression in the coleoptile, root, leaf, and internode. In these organs, there is increased expression of Os-EXP1, Os-EXP3, and Os-EXP4 in developmental regions where elongation occurs. This pattern of gene expression is also correlated with acid-induced in vitro cell wall extensibility. Submergence and treatment with gibberellin, both of which promote rapid internodal elongation, induced accumulation of Os-EXP4 mRNA before the rate of growth started to increase. Our results indicate that the expression of expansin genes in deepwater rice is differentially regulated by developmental, hormonal, and environmental signals and is correlated with cell elongation.     

Assessment of the utilization of the antisense RNA strategy to identify essential genes in heterologous bacteria

We employed an antisense RNA approach to identify essential genes common in both Gram-positive and Gram-negative bacteria by cloning a random library of Streptococcus mutans chromosomal DNA into an expression vector and transforming Escherichia coli. Twelve out of 27 E. coli transformants with growth defective phenotypes contained individual structural genes of S. mutans in the antisense orientation relative to the E. coli promoter. Thirty-three percent of these transformants (4/12) corresponded to the genes (gyrA, ileS, rplE and yihA orthologs) which are essential for bacterial viability.     

An effective strategy to establish a male sterility mutant mini-library by CRISPR/Cas9-mediated knockout of anther-specific genes in rice

Male sterility(MS),characterized by functional defects in male organs or gametes,is an important agronomic trait for hybrid seed production,especially for self-pollinated crops such as rice(Oryza sativa L)(Chen and Liu,2014).Spontaneous MS mutants are rare and difficult to maintain in nature,thus limiting basicresearch and breeding.Artificial mutants are typically generated by physical,chemical,or biological mutagenesis(Wei et al,2013).Recently developed genome editing systems such as CRISPR/Cas9 allow efficient and timesaving knockout of endogenous genes at specific sites(Smith et al,2000;Moscou and Bogdanove,2009;Gasiunas et al.,2012).