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• Aqueous enzymatic extraction (AEE) is performed for oil extraction from Schizochytrium sp. • AEE process is optimized by response surface methodology. • Microalgal oil extracted by AEE has high contents of PUFA, tocopherols and phenolics. • AEEO exhibits considerable antioxidant activity as compared with SEO. Schizochytrium sp., a marine microalga, is a potential source of edible oil due to its short growth cycle and rapid lipid accumulation, especially of docosahexaenoic acid. An approach to isolate edible microalgal oil from Schizochytrium sp. using aqueous enzymatic extraction (AEE) was developed. Parameters were optimized by single-factor experiments followed by Box-Behnken design. Proteases were effective in extracting oil. The maximum free oil recovery (49.7%±0.58%) and total oil recovery (68.1%±0.94%) were obtained under optimum conditions of liquid-to-solid ratio of 4.8:1, a 2.5% enzyme concentration of papain and an extraction time of 2.2 h. There was a significant difference (P

Summary: We have previously reported that three distinct patterns of waxy (Wx) gene transcript accumulation were present in 31 rice cultivars. The cultivars with high amylose content (group I) contain a 2.3 kb mature Wx mRNA, cultivars with intermediate amylose content (group II) produce both a 3.3 kb Wx pre-mRNA, which contains intron 1, and the 2.3 kb Wx mature mRNA, and cultivars with no amylose (group III) accumulate only the 3.3 kb Wx pre-mRNA. Analyses of the cDNAs reveals that four splice donor sites and three splice acceptor sites in intron 1 give rise to six splicing patterns in 2.3 kb Wx mRNA of group II cultivars. In addition, aberrant intron 1 excision causes either deletion of 4 or 5 nucleotides, or addition of 7 and 13 nucleotides at the junction of exon 1 and exon 2 of the 2.3 kb mRNA. In contrast, only one normal splicing pattern (one splice donor site and one splice acceptor site) was found in the 2.3 kb mRNA of group I cultivars. Nucleotide sequences of the Wx intron 1 in group I and group II cultivars differ by 16 individual bases. We suggest that these deletions or additions contribute to inefficient splicing of intron 1 from the 3.3 kb Wx pre-mRNA, as well as an aberrant splicing of the Wx intron 1 to produce the 2.3 kb mRNA with a heterogeneous 5′ untranslated region (5′-UTR). As a consequence, the total amount of translatable Wx mRNA, and therefore the Wx protein and amylose content, are reduced in the group II cultivars compared with the group I cultivars. [ABSTRACT FROM AUTHOR]

Transgenic plants with Ds element distributed over different loci on chromosome 4 (Fig. 1) and the homozygous transformants with Ac transposase gene were established through Agrobacterium-mediated approach. In this study, the plants carrying Ds element from different loci were crossed with the plant carrying Ac transposase individually. The plants of F(1) generation carrying both Ds element and Ac transposase were used to produce the F(2) populations (Table 1). Analysis of the F(2) generation by the PCR method revealed that the excision frequencies of Ds element were higher in the telomeric region of chromosome 4 than in the centromeric region (Fig. 4). These results showed that the insertion site of Ds element has strong effect on its excision frequency. We suggest that the special construct of chromosome near the insertion site of Ds element is related to the excision frequency of the Ds element.

Gama-glutamylcysteine synthetase (GCS) is a rate-limiting enzyme in GSH biosynthesis. The GCS gene has been cloned in Arabidopsis thaliana and other plants, but has still not been reported in rice. From rice mutant population generated from T-DNA insertion, we cloned the rice GCS gene from mutant L395 by T-DNA tag cloning method, and named it OsGCS (Genbank accession No. AJ508915). Full length OsGCS cDNA clones were obtained from a rice cDNA library by the PCR method. A comparison of the genome and cDNA sequence (Genbank accession No. AJ508916) shows that OsGCS gene is composed of 15 exons and 14 introns and coding a 493-amino acid protein. The OsGCS gene is highly homologous with the AtGCS gene in the coding region but completely different in the promoter region. The putative transcription start site (TSS) confirmed by RT-PCR was located 211 bp upstream of the translation start codon "ATG". In mutant L395, a single T-DNA copy was integrated between the second intron and second exon of OsGCS gene, causing one nucleotide deletion in the second exon and two nucleotide deletions in the second intron. No significant differences were found in Cd(2+) stress tolerance, rice GCS gene expression level and GSH content between mutant L395 and Zhonghua 11. It is possible that another GCS gene on chromosome 7 might complement function of OsGCS gene on chromosome 5.

Transposon tagging was used to isolate genes in higher plant. In this study, a delayed heading mutant caused by T-DNA insertion in rice was identified. Genetic analysis of the mutant showed that the three types of phenotype, normal heading, delayed heading and overly delayed heading in the segregating populations derived from the T-DNA heterozygotes fit the ratio of 1:2:1. Test for Basta resistance showed the delayed heading plants were all resistant while the normal heading plants were susceptible, and the ratio of resistant and susceptible plants was 3:1, which indicated that the delayed heading mutant was co-segregated with Basta resistance. The delayed heading mutant caused by T-DNA insertion was confirmed by T-DNA detection using PCR method. This delayed heading mutant will be used for isolation of the tagged gene in rice.

A rolled-leaf mutant was obtained in a T-DNA(containing bar gene and Ds element) insertion population, which consist of transgenic japonica rice Zhonghua 11 mediated by Agrobacterium tumefaciens. Through self-hybridization of three generations, one of trait-purified mutants (R1-A2) was obtained and used as parent to cross with variety Zhonghua 11. The leaves of 36 F1 plants investigated were rolled and resistant to herbicide Basta. Among 852 F2 plants, the segregation ratio of rolled leaves to normal leaves(645:207) was consistent with 3:1. All rolled-leaf plants were resistant to herbicide Basta, and all normal leaf plants were sensitive to herbicide Basta. These results showed that the trait of rolled-leaf is co-segregated with Basta resistance. The total DNA of 45 rolled-leaf plants and 30 normal leaf plants in F2 population were amplified to test the presence of T-DNA by Ds primers. The results showed that the positive band were amplified in all rolled-leaf plants, but not in every normal leaf plant. In F1B1 progenies, all plants which derived from backcross parent R1-A2 were rolled leaves; while variety Zhonghua 11 was used as backcross parent, the segregation ratio of rolled-leaf to normal leaf was consistent with 1:1. Taking these data together, it indicated that the rolled-leaf mutant was co-segregation with T-DNA and controlled by single dominant gene.

A rice brittle culm mutant bcm581-1 which derived from the Ds transposone transformation population was found, but the mutant was identified that it was not to be induced by Ds transposone insertion through PCR. The examination of the vascular bundle and cortical fibre cells in culm under the light and electron microscope showed that, the number of cortical vascular bundle of mutant was much more, the hollow among the cortical vascular bundle was deeper, and the cell walls of cortical fibre cells were thinner than the normal. The test of culm mechanics intensity showed that the load, elongation, strain, and stress of bcm581-1 were 5-9 times lower than normal. The moisture content and the wide fibre content of culm were test, the former was 3.5% higher, but the latter was 8.12% lower than normal. The analysis of genetic segregation in F2 and F1B1 population indicated that the brittle culm mutant was controlled by one recessive gene.