Guangming Ao

Seed-specific expression of a lysine rich protein sb401 gene significantly increases both lysine and total protein content in maize seeds

The sb401 gene from potato (Solanum berthaultii) encoding a pollen-specific protein with high lysine content was successfully integrated into the genome of maize plants and its expression was correlated with increased levels of lysine and total protein content in maize seeds. A plasmid vector containing the sb401 gene under the control of a maize seed-specific expression storage protein promoter (P19z) was constructed and introduced into maize calli using microprojectile bombardment. The integration of the sb401 gene into the maize genome was confirmed by Southern blot analysis and its expression was confirmed by Western blot analysis. Quantification of lysine and protein content in R1 maize seeds showed that, compared to the non-transgenic maize control, the lysine content increased by 16.1% to 54.8%, and total protein content increased by 11.6% to 39.0%. There was no visible morphological change in vegetative parts and seeds of the transgenic maize plants. Lysine and protein analysis of the transgenic maize grains showed that the levels of lysine and total protein remained high for six continuous generations, indicating that the elevated lysine and total protein levels were heritable. These results indicate that the sb401 gene could be successfully employed in breeding programmes aimed at improving the nutritional value of maize.

Zm401, a short‐open reading‐frame mRNA or noncoding RNA, is essential for tapetum and microspore development and can regulate the floret formation in maize

In flowering plants, pollen formation depends on the differentiation and interaction of two cell types in the anther: the reproductive cells, called microsporocytes, and somatic cells that form the tapetum. Previously, we cloned a pollen specific gene, zm401, from a cDNA library generated from the mature pollen of Zea mays. Expression of partial cDNA of zm401 in maize and ectopic expression of zm401 in tobacco suggested it may play a role in anther development. Here we present the expression and functional characterization of this pollen specific gene in maize. Zm401 is expressed primarily in the anthers (tapetal cells as well as microspores) in a developmentally regulated manner. That is, it is expressed from floret forming stage, increasing in concentration up to mature pollen. Knockdown of zm401 significantly affected the expression of ZmMADS2, MZm3‐3, and ZmC5, critical genes for pollen development; led to aberrant development of the microspore and tapetum, and finally male‐sterility. Zm401 possesses highly conserved sequences and evolutionary conserved stable RNA secondary structure in monocotyledon. These data show that zm401 could be one of the key growth regulators in anther development, and functions as a short‐open reading‐frame mRNA (sORF mRNA) and/or noncoding RNA (ncRNA). J. Cell. Biochem. 105: 136–146, 2008.

Several cis-elements including a palindrome involved in pollen-specific activity of SBgLR promoter

SBgLR (Solanum tuberosum genomic lysine-rich) is a pollen-specific gene cloned from potato (Solanum tuberosum L.). The region from −269 to −9 (The A of translation start site “ATG” as +1) of the SBgLR promoter was identified as critical for gene specific expression in pollen grains. Sequence analysis indicates a palindromic sequence “TTTCTATTATAATAGAAA” in the −227 to −209 region, in which two pollen-specific motifs TTTCT and AGAAA surround a unique putative TATA box. Moreover, nine putative pollen-specific motifs are located in the region between the TATA box and ATG. We placed the −227 to −9 region (reserving the palindrome) and the −222 to −9 region (breaking the palindrome) downstream of the CaMV35S enhancer, respectively, to construct two fusion promoters. Histochemical assays in transgenic plants demonstrated that the region from −222 to −9 is necessary and sufficient for pollen-specific expression of the uidA gene. However, the region of −227 to −9 is incapable of driving GUS expression in pollen grains and parts of vegetative tissues. A series of 5′ deletions from −269 to −9 of SBgLR promoter were constructed. A transient expression assay indicated that the region from the −227 to −9 suppressed gfp gene expression in pollen, and a positive regulatory element was present in the region of −253 to −227. The function of the palindromic sequence as a repressor inhibiting gene expression in pollen was further confirmed by the mutated promoter, breaking the palindrome by substituting its 3′-flanking five base pairs, which resumes the reporter gene expression in mature pollen.

Particle-Bombardment-Mediated Co-Transformation of Maize with a Lysine Rich Protein Gene ( sb401 ) from potato

SummaryLittle work has been reported on genetic transformation with maize inbred lines, especially elite inbred lines used in breeding. In this work, 7 self-pollinated inbred lines and 4 hybrid lines have been screened. The results revealed that calli derived from immature embryos from two inbred lines X333 and X301 were compact, hyperhydric and unsuitable for transformation, but the calli induced from other inbred lines and all the hybrid lines were friable and yellow and could be used for genetic transformation. The sb401 gene isolated from potato (Solanum berthaultii) encodes a protein with a high lysine content. Maize calli from 5 self-pollinated inbred lines and 4 hybrid lines were transformed using particle-bombardment with different plasmids to simultaneously introduce the sb401 lysine rich gene and the selectable gene hpt respectively. Two hundred and sixty-eight regenerated plants were obtained from these genotypes. Co-insertion was confirmed in 29 regenerated plants by PCR and Southern blot analysis. Transgene segregation of the R1 plants was observed and one marker-free transgenic maize line was recovered. Analysis of the crude protein content in mature seeds of R1 transgenic plants also showed an increase from 36.8% to 48.2%. This study thus provides a workable system for generating transgenic maize free from selectable marker genes and generates valuable resources for obtaining marker free transgenic maize with a high-lysine protein content.

Ectopic expression of a maize pollen specific gene, zm401, results in aberrant anther development in tobacco

Our previous study shows that the maize zm401 cDNA is specifically expressed in maize pollens. This evidence suggests that zm401 likely functions in pollen growth and/or development. To confirm its possible involvement in pollen development, the full length cDNA of zm40l was ectopically expressed in tobacco plants under the control of a pollen specific promoter ZM13 (from maize). RT-PCR amplification demonstrated that the ZM13-driven zm401 gene was spatially expressed in tobacco pollens. It was found that all transgenic tobacco plants expressing zm401 showed various levels of sterility, ranging from abortive flower development to male sterility. Further analyses on anther development of transgenic plants indicated multiple abnormalities in the late stages of anther development. These abnormalities include lagged degradation of the tapetum and connective tissue, failed deposition of fibrous bands in endothecium cells, and aborted pollen grain development. These results strongly suggest that zm401 plays an essential role in anther development. However the exact functions of zm401 is still unclear, and further analysis of zm401 is required to determine the exact mechanism involved in anther development.

Cloning and characterisation of ZmZLP1, a gene encoding an endoplasmic reticulum-localised zinc transporter in Zea mays

The ZmZLP1 (ZmZIP-like protein) gene was isolated from a cDNA library of Zea mays L. (maize) pollen. Bioinformatics analysis indicated that ZmZLP1 shares many characteristics of the ZIP (ZRT/IRT-like protein) family of metal ion transporters. Under general nutrient conditions, the expression of ZmZLP1 was detected in both mature pollen and, less strongly, in male inflorescences, whereas an induction of the ZmZLP1 transcript was observed in roots after 12 h of zinc deprivation. The visualisation of GFP showed that ZmZLP1 was targeted to the endoplasmic reticulum (ER). To investigate the gene’s functions, we fused ZmZLP1 with the signal peptide of the plasma membrane-localised protein AtIRT1 and transformed this fusion protein into the zinc uptake-deficient yeast (Saccharomyces cerevisiae) strain ZHY3 and the wild-type strain DEY1457. The IRT1-ZmZLP1 transformants grew poorly on zinc-limited medium, and this growth defect was rescued by zinc supplementation, suggesting that ZmZLP1 is responsible for transporting zinc from the ER to the cytoplasm. Further research indicated that ZmZLP1 is involved in the unfolded protein response (UPR) pathway and enhances the heat resistance of yeast cells.

Ectopic expression of foxtail millet zip-like gene, SiPf40, in transgenic rice plants causes a pleiotropic phenotype affecting tillering, vascular distribution and root development

Plant architecture determines grain production in rice (Oryza sativa) and is affected by important agronomic traits such as tillering, plant height, and panicle morphology. Many key genes involved in controlling the initiation and outgrowth of axillary buds, the elongation of stems, and the architecture of inflorescences have been isolated and analyzed. Previous studies have shown that SiPf40, which was identified from a foxtail millet (Setaria italica) immature seed cDNA library, causes extra branches and tillers in SiPf40-transgenic tobacco and foxtail millet, respectively. To reconfirm its function, we generated transgenic rice plants overexpressing SiPf40 under the control of the ubiquitin promoter. SiPf40-overexpressing transgenic plants have a greater tillering number and a wider tiller angle than wild-type plants. Their root architecture is modified by the promotion of lateral root development, and the distribution of xylem and phloem in the vascular bundle is affected. Analysis of hormone levels showed that the ratios of indole-3-acetic acid/zeatin (IAA/ZR) and IAA/gibberellic acid (IAA/GA) decreased in SiPf40-transgenic plants compared with wild-type plants. These findings strongly suggest that SiPf40 plays an important role in plant architecture.