Resistance function of rice lipid transfer protein LTP110.

Plant lipid transfer proteins (LTPs) are a class of proteins whose functions are still unknown. Some are proposed to have antimicrobial activities. To understand whether LTP110, a rice LTP that we previously identified from rice leaves, plays a role in the protection function against some serious rice pathogens, we investigated the antifungal and antibacterial properties of LTP110. A cDNA sequence, encoding the mature peptide of LTP110, was cloned into the Impact-CN prokaryotic expression system. The purified protein was used for an in vitro inhibition test against rice pathogens, Pyricularia oryzae and Xanthomonas oryzae. The results showed that LTP110 inhibited the germination of Pyricularia oryzae spores, and its inhibitory activity decreased in the presence of a divalent cation. This suggests that the antifungal activity is affected by ions in the media; LTP110 only slightly inhibited the growth of Xanthomonas oryzae. However, the addition of LTP110 to cultured Chinese hamster ovarian cells did not retard growth, suggesting that the toxicity of LTP110 is only restricted to some cell types. Its antimicrobial activity is potentially due to interactions between LTP and microbe-specific structures.

Cloning of an alkaline lipase gene from Penicillium cyclopium and its expression in Escherichia coli.

The gene encoding an alkaline lipase of Penicillium cyclopium PG37 was cloned with four steps of PCR amplification based on different principles. The cloned gene was 1,480 nucleotides in length, consisted of 94 bp of promoter region, and had 6 exons and 5 short introns ranging from 50 to 70 nucleotides. The open reading frame encoded a protein of 285 amino acid residues consisting of a 27-AA signal peptide and a 258-AA mature peptide, with a conserved motif of Gly-X-Ser-X-Gly shared by all types of alkaline lipases. However, this protein had a low homology with lipases of P. camembertii (22.9%), Humicola lanuginosa (25.6%), and Rhizomucor miehei (22.3%) at the amino acid level. The mature peptide-encoding cDNA was cloned and expressed in Escherichia coli on pET-30a for confirmation. A distinct band with a M.W. of 33 kDa was detected on SDS-PAGE. Results of a Western blot analysis and an enzyme activity assay verified the recombinant 33-kDa protein as an alkaline lipase. Its catalytic properties were not changed when compared with its natural counterpart.

Purification and Characterization of an Alkaline Lipase from Penicillium cyclopium PG37.

An extracellular, novel alkaline lipase produced by Penicillium cyclopium PG37 was purified by centrifugation, ammonium sulfate precipitation, and phenyl-Sepharose CL-4B, DEAE Sepharose fast flow and Sephadex G-75 column chromatographies. A 16.5-fold purification of the enzyme was achieved which had a specific activity of 5 200 u/mg protein, and the recovery of the activity was 33.2%. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and polyacrylamide gel electrophoresis (PAGE). The molecular weight of the native lipase was estimated to be about 29 kD by gel filtration using Sephadex G-150, and that of the denatured lipase was determined to be about 27.5 kD by its mobility on SDS-PAGE, indicating that the lipase was a monomer. The N-terminal amino acid sequence was determined with automatic protein sequencer to be ATADAAAFPD, which has no homology with other sequences of known lipases. The optimum temperature of the action of this enzyme was 25 degrees and the lipase was stable below 30 degrees, but only 30% of its activity remained after 20 min incubation at 40 degrees. The enzyme was stable at pH from 6.5 to 10.5, and its optimal pH for activity is 10.0. Low concentration of alkaline proteinase has little effect on the lipase PG37, therefore these two enzymes can be used as ingredients that are added to commercial detergents simultaneously.

[Expression, purification and function of rice nonspecific lipid transfer protein].

Plant nonspecific lipid transfer protein(nsLTP) is a class of protein which has in vitro lipid transferring activity between biomembranes. In order to study the antimicrobial function of rice nonspecific lipid transfer protein, a gene LTP110 encoding rice nsLTP was cloned into ThioFusion expression vector pET32a (+) and expressed in host strain Bl21(DE3)trxB-. After induction by IPTG at 30 degrees C for 5 h, the fusion protein thio-LTP110 was in large amount produced. The expressed protein was purified by Ni2(+)-chelating Sepharose fast flow column, then digested by enterokinase. By passing through nickel affinity column again, the cleavage product, LTP110, was obtained. CD spectrum scanning from 185 nm to 250 nm showed that the recombinant protein LTP110 had similar secondary structure with the nsLTP purified from rice etiolated seedlings. Activity determination by fluorescent lipid P-96 showed that it had lipid binding activity. Microbial inhibition test results revealed that LTP110 deterred germination of the spores of rice pathogen P. oryzae, showing it might be involved in plant microbial resistance function. Therefore, it has the potential to be used in plant transgene engineering to improve plant resistance.

Control of petal shape and floral zygomorphy in Lotus japonicus.

Zygomorphic flowers, with bilateral (dorsoventral) symmetry, are considered to have evolved several times independently in flowering plants. In Antirrhinum majus, floral dorsoventral symmetry depends on the activity of two TCP-box genes, CYCLOIDEA (CYC) and DICHOTOMA (DICH). To examine whether the same molecular mechanism of floral asymmetry operates in the distantly related Rosid clade of eudicots, in which asymmetric flowers are thought to have evolved independently, we investigated the function of a CYC homologue LjCYC2 in a papilionoid legume, Lotus japonicus. We showed a role for LjCYC2 in establishing dorsal identity by altering its expression in transgenic plants and analyzing its mutant allele squared standard 1 (squ1). Furthermore, we identified a lateralizing factor, Keeled wings in Lotus 1 (Kew1), which plays a key role in the control of lateral petal identity, and found LjCYC2 interacted with Kew1, resulting in a double mutant that bore all petals with ventralized identity to some extents. Thus, we demonstrate that CYC homologues have been independently recruited as determinants of petal identities along the dorsoventral axis in two distant lineages of flowering plants, suggesting a common molecular origin for the mechanisms controlling floral zygomorphy.

Regulation of biosynthesis and intracellular localization of rice and tobacco homologues of nucleosome assembly protein 1.

The nucleosome assembly protein 1 (NAP1) is considered to be a conserved histone chaperone, facilitating the assembly of nucleosomes in all eukaryotes. However, studies in yeast and animal cells also indicated that NAP1 proteins have diverse functions likely independent of nucleosome-assembly activity. Here, we describe the isolation and characterization of cDNAs encoding NAP1-like proteins from the monocotyledon rice ( Oryza sativa L.) and the dicotyledon tobacco ( Nicotiana tabacum L.). Northern-blot analysis demonstrated that the two rice NAP1-like genes are predominantly expressed in stem tissues such as root and shoot apical meristems as well as in young flowers. During the cell cycle, all four tobacco NAP1-like genes are highly expressed, with one of them showing a slightly increased expression at the G1/S transition. These results are consistent with a role for plant NAP1-like proteins in cell division. In vitro binding assays revealed that different NAP1-like proteins bind, with distinct relative binding strengths, to different classes of histone. Intracellular localization analyses showed that some NAP1-like proteins could be targeted into the nucleus whereas others are exclusively cytoplasm-localized. It is thus likely that different plant NAP1-like proteins have distinct functions in vivo. Plant NAP1-like proteins were observed to concentrate around the metaphase plate and in the phragmoplast, suggesting a role in mitotic events and cytokinesis.

The subcellular localization of an unusual rice calmodulin isoform, OsCaM61, depends on its prenylation status.

Calmodulin (CaM) is a small Ca2+-binding protein highly conserved in eukaryotes. We have reported previously a novel rice CaM-like protein (OsCaM61) which contains an N-terminal CaM domain and a C-terminal extension with a potential prenylation site. Here we report in vitro activity assays confirm OsCaM61 as a functional CaM. Using the green fluorescent protein (GFP) as a visual marker, we further studied the subcellular localization of OsCaM61 in stably transformed tobacco cells. The GFP-OsCaM61 fusion protein was membrane-associated whereas OsCaM61-GFP was mainly detected in the nucleoplasm. GFP-OsCaM61 was transported into the nucleoplasm upon a block in isoprenoid biosynthesis by mevinolin treatment of cells. These results indicate that the prenylated OsCaM61 molecules are mainly membrane-associated whereas its unprenylated counterparts are transported into the nucleoplasm. Thus, OsCaM61 may play functions in co-ordinating Ca2+ signaling with isoprenoid metabolism.

[Construction of the mutants of rice nonspecific lipid transfer protein and expression comparison in two kinds of thioredoxin fusion expression vectors].

Five structural important residues of rice nonspecific lipid transfer protein LTP110 were mutated by site-directed mutagenesis. Sequence results showed that they were all mutated successfully. After trying various E. coli expression systems, thioredoxin fusion expression system was found to be a proper system to express wild type and mutant LTP110. cDNA sequences encoding wild type LTP110 and the mutants Y17A, P72L, R46A, D43A, C50A were cloned into two kinds of thioredoxin fusion expression vectors. The expression results were compared. In pTrxFus/GI724 expression system, wild type LTP110 and the mutants Y17A, P72L, R46A could be expressed at low level while D43A and C50A could not be expressed normally; in pET32a(+)/BL21 (DE3) trxB- expression system, wild type LTP110 and all mutant proteins could be expressed very well and the levels were higher than that in pTrxFus/GI724 system. LTP110 fusion protein expressed in pET32a(+) vector was purified and its activity was checked by fluorescence labeled fatty acid. Results indicated that the recombinant LTP110 fusion protein has lipid binding activity. This work provides good basis for the further study.