Evaluation of seed storage-protein gene 5' untranslated regions in enhancing gene expression in transgenic rice seed.

5' untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the ß-glucuronidase (GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5'-UTRs of six seed storage-protein genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5'-UTRs significantly enhanced the expression level of the GluC promoter without altering its expression pattern. The 5'-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5'-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore, the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing the translational efficiencies of the mRNA. The 5'-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds.

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.

[Advances in research on lignin biosynthesis and its genetic engineering].

Lignin, one of the main components in vascular plants, is important for the adaptation of terrestrial plants to environment during evolution. However, its presence in plants has negative effects on wood processing during pulping and stock breeding. Therefore much attention has been focused on the regulation of lignin biosynthesis. The pathways leading to the synthesis of lignin polymers have been studied for decades. Much understanding of lignin biosynthesis has been advanced. This paper reviewed the recent progress made in the various steps associated with monolignol biosynthesis. It includes the catalysis by three enzymes, i.e. p-coumarate-3-hydroxylase (C3H), ferulate-5-hydroxylase (F5H) and caffeic acid 3-O-methyltransferase (COMT); the multiform biosynthetic pathway of syringyl (S) lignin in angiosperms; the biosynthesis route of guaiacyl (G) and syringyl (S) lignin specifically regulated by cinnamyl alcohol dehydrogenase (CAD) and sinapyl alcohol dehydrogenase (SAD) and the formation of the lignin macromolecule. Based on the elucidation of lignin biosynthesis pathway, it has also been given the achievements in lignin gene engineering. Many studies were concentrated on the modification of lignin content and composition. In some cases, the potential value of transgenic plants with modified lignin beneficial for pulping has been demonstrated. To better understand the mechanism of lignin biosynthesis and improve the properties of plants, new biotechnological strategies can be developed, which include combinatorial modification of multiple lignin traits in plants through multigene cotransformation, transcriptional control of lignin biosynthesis and the application of RNA interference. The identification of novel genes by molecular and genetic approaches will be useful in opening up new avenues of lignin modification in the future.