Zongxiu Sun

Phosphomannose-isomerase (pmi) gene as a selectable marker for rice transformation via Agrobacterium

A new selectable marker system was adapted for use in Agrobacterium-mediated transformation of rice (Oryza sativa L.). This selection system utilizes the pmi gene encoding for phosphomannose-isomerase that converts mannose-6-phosphate to fructose-6-phosphate. Only transformed cells were capable of utilizing mannose as a carbon source. Transgenic rice plants regenerated from selected transformed immature embryo-derived calli on media containing various concentrations of mannose. The highest transformation frequency of 6.0% was obtained when a combination of 25 g/l mannose and 5 g/l sucrose was used. Molecular and genetic analyses showed that the plants contained the pmi gene and the gene was transmitted to the progeny in Mendelian fashion. The results indicated that the mannose selection system, which was devoid of the disadvantages of antibiotic or herbicide selection, could be used for rice Agrobacterium-mediated immature embryo transformation.

Dlf1, a WRKY transcription factor, is involved in the control of flowering time and plant height in rice.

Flowering time and plant height are important agronomic traits for crop production. In this study, we characterized a semi-dwarf and late flowering (dlf1) mutation of rice that has pleiotropic effects on these traits. The dlf1 mutation was caused by a T-DNA insertion and the cloned Dlf1 gene was found to encode a WRKY transcription factor (OsWRKY11). The dlf1 mutant contains a T-DNA insertion at the promoter region, leading to enhanced accumulation of Dlf1 transcripts, resulting in a semidominant mutation. The dlf1 mutation suppressed the transcription of Ehd2/RID1/OsId1 and its downstream flowering-time genes including Hd1, Ehd1 and Hd3a under both long-day (LD) and short-day (SD) conditions. Knock-down of Dlf1 expression exhibited early flowering at LD condition related to the wild-type plants. Accumulation of Dlf1 mRNA was observed in most tissues, and two splicing forms of Dlf1 cDNAs were obtained (OsWRKY11.1 and OsWRKY11.2). These two proteins showed transactivation activity in yeast cells. Dlf1 protein was found to be localized in the nucleus. Enhanced expression of OsWRKY11.2 or its 5′ truncated gene showed similar phenotypes to the dlf1 mutant, suggesting that it might function as a negative regulator. We conclude that Dlf1 acts as a transactivator to downregulate Ehd2/RID1/OsId1 in the signal transduction pathway of flowering and plays an important role in the regulation of plant height in rice.

The Effect of the Crosstalk between Photoperiod and Temperature on the Heading-Date in Rice

Photoperiod and temperature are two important environmental factors that influence the heading-date of rice. Although the influence of the photoperiod on heading has been extensively reported in rice, the molecular mechanism for the temperature control of heading remains unknown. This study reports an early heading mutant derived from tissue culture lines of rice and investigates the heading-date of wild type and mutant in different photoperiod and temperature treatments. The linkage analysis showed that the mutant phenotype cosegregated with the Hd1 locus. Sequencing analysis found that the mutant contained two insertions and several single-base substitutions that caused a dramatic reduction in Hd1mRNA levels compared with wild type. The expression patterns of Hd1 and Hd3a were also analyzed in different photoperiod and temperature conditions, revealing that Hd1 mRNA levels displayed similar expression patterns for different photoperiod and temperature treatments, with high expression levels at night and reduced levels in the daytime. In addition, Hd1 displayed a slightly higher expression level under long-day and low temperature conditions. Hd3a mRNA was present at a very low level under low temperature conditions regardless of the day-length. This result suggests that suppression of Hd3a expression is a principle cause of late heading under low temperature and long-day conditions.

Characterization and fine mapping of a novel rice narrow leaf mutant nal9.

A narrow leaf mutant was isolated from transgenic rice (Oryza sativa L.) lines carrying a T-DNA insertion. The mutant is characterized by narrow leaves during its whole growth period, and was named nal9 (narrow leaf 9). The mutant also has other phenotypes, such as light green leaves at the seedling stage, reduced plant height, a small panicle and increased tillering. Genetic analysis revealed that the mutation is controlled by a single recessive gene. A hygromycin resistance assay showed that the mutation was not caused by T-DNA insertion, so a map-based cloning strategy was employed to isolate the nal9 gene. The mutant individuals from the F₂ generations of a cross between the nal9 mutant and Longtepu were used for mapping. With 24 F₂ mutants, the nal9 gene was preliminarily mapped near the marker RM156 on the chromosome 3. New INDEL markers were then designed based on the sequence differences between japonica and indica at the region near RM156. The nal9 gene was finally located in a 69.3 kb region between the markers V239B and V239G within BAC OJ1212_C05 by chromosome walking. Sequence and expression analysis showed that an ATP-dependent Clp protease proteolytic subunit gene (ClpP) was most likely to be the nal9 gene. Furthermore, the nal9 mutation was rescued by transformation of the ClpP cDNA driven by the 35S promoter. Accordingly, the ClpP gene was identified as the NAL9 gene. Our results provide a basis for functional studies of NAL9 in future work.

Rapid Generation of Selectable Marker-Free Transgenic Rice with Three Target Genes by Co-Transformation and Anther Culture

The ‘double T-DNA’ binary vector p13HSR which harbored two independent T-DNAs, containing hygromycin phosphotransferase gene (hpt) in one T-DNA region and three target genes (hLF, SB401, RZ10) in another T-DNA region, was used to generate selectable marker-free transgenic rice by Agrobacterium-mediated transformation. The regenerated plants with both the three target genes and the selectable marker gene hpt were selected for anther culture. RT-PCR analysis indicated that target genes were inserted in rice genomic DNA and successfully transcribed. It took only one year to obtain double haploid selectable marker-free transgenic plants containing the three target genes with co-transformation followed by anther culture technique, and the efficiency was 12.2%. It was also noted that one or two target genes derived from the binary vector were lost in some transgenic rice plants.

Resistance of Antimicrobial Peptide Gene Transgenic Rice to Bacterial Blight

Antimicrobial peptide is a polypeptide with antimicrobial activity. Antimicrobial peptide genes Np3 and Np5 from Chinese shrimp (Fenneropenaeus Chinensis) were integrated into Oryza sativa L. subsp. japonica cv. Aichi ashahi by Agrobacterium mediated transformation system. PCR analysis showed that the positive ratios of Np3 and Np5 were 36% and 45% in T0 generation, respectively. RT-PCR analysis showed that the antimicrobial peptide genes were expressed in T1 generation, and there was no obvious difference in agronomic traits between transgenic plants and non-transgenic plants. Four Np3 and Np5 transgenic lines in T1 generation were inoculated with Xanthomonas oryzae pv. oryzae strain CR4, and all the four transgenic lines had significantly enhanced resistance to bacterial blight caused by the strain CR4. The Np5 transgenic lines also showed higher resistance to bacterial blight caused by strains JS97-2, Zhe 173 and OS-225. It is suggested that transgenic lines with Np5 gene might possess broad spectrum resistance to rice bacterial blight.

Identification and Fine Mapping of a Gene Related to Pale Green Leaf Phenotype near the Centromere Region in Rice (Oryza sativa)

A thermo-insensitive pale green leaf mutant (pgl2) was isolated from T-DNA inserted transgenic lines of rice (Oryza sativa L. subsp. japonica cv. Nipponbare). Genetic analysis indicated that the phenotype was caused by a recessive mutation in a single nuclear-encoded gene. To map the PGL2 gene, an F2 population was constructed by crossing the mutant with Longtefu (Oryza sativa L. subsp. indica). The PGL2 locus was roughly linked to SSR marker RM331 on chromosome 8. To finely map the gene, 14 new InDel markers were developed around the marker, and PGL2 was further mapped to a 2.37 Mb centromeric region. Analysis on chlorophyll contents of leaves showed that there was no obvious difference between the mutant and the wild type in total chlorophyll (Chl) content, while the ratio of Chl a / Chl b in the mutant was only about 1, which was distinctly lower than that in the wild type, suggesting that the PGL2 gene was related to the conversion between Chl a and Chl b. Moreover, the method of primer design around the centromeric region was discussed, which would provide insight into fine mapping of the functional genes in plant centromeres.

Genetic analysis and identifi- cation of a large leaf angles (lla) mutant in rice

Mutants are essential genetic materials to elucidation of biological functions of genes involved. Characterization and isolation of genes in mutants is one of the research tasks in functional genomic era. T-DNA insertional mutagenesis has provided an efficient way to identify genes in plant species, in which the mutated genes could be rapidly isolated once the mutant was confirmed by T-DNA insertion. About 40% of 620 cloned Arabidopsis genes with mutant phenotypes were determined by T-DNA tagging. In recent years, a large number of T-DNA insertional rice mutant pools were established. Moreover, three genes were successfully isolated from rice. Leaf angles, the angles between main culms and leaves, are one of the important features of the plant types. Investigation on the mechanism of leaf angle formation will probably provide the basic knowledge of plant breeding on plant architectures. Several QTLs of leaf and flag leaf angles in rice were identified, which would provide the useful materials for manipulating these QTLs in a marker-assisted selection program. However, the major genes, which control leaf angles, have not been reported yet and very little is known about the mechanism of leaf angle formation. Based on T-DNA inserted rice (Oryza sativa L. subsp. Japonica cv. Zhonghua11) mutant pool, which contains about 10000 insertional mutant lines, a large leaf angles (lla) mutant T429 was found in T1 lines. At seeding stage, the leaf angles of lla mutant were larger than those of the wild type. In addition, the mutant seedlings showed semi-dwarf. At heading stage, the abnormal phenotypes of lla mutants were observed in plant height, leaf angel, leaf blade length and leaf blade width. The plant of lla was shorter than the wild type (Fig. 1). The length and width of the lla mutant leaves were shorter and wider than those of the wild type (unpublished data). Under natural long day conditions, the heading date of lla was about two weeks later than that of the wild type. Under winter natural short day conditions in greenhouse, the heading date of lla was about one week later than that of the wild type while the plant height and leaf angles remained significantly different from the control. This indicated that the mutant phenotype was slightly influenced by environmental conditions such as photoperiod and temperature. Different concentrations of GA3 were used to treat the plants at both seeding and heading stages; the results showed that the plant heights of wild type and the lla mutant were all sensitive to exogenous GA3 at different concentrations. But plant heights and leaf angles of GA3 treated mutant plants differed significantly from the corresponding wild types. At the same time, leaf angles of GA3 treated wild type and lla did not show any visible differences from the corresponding untreated plants.