Shao Ping Fu

The effects of calcium on the in vitro cassava storage root formation

Calcium can affect in vitro cassava storage roots formation and starch accumulation. Low concentration of calcium stimulates to induce in vitro cassava storage roots formation and the accumulation of starches. With the addition of calcium concentration, the diameter of the in vitro cassava storage roots was increased, but the induction rate and starch content was decreased. The scanning electron microscope observations SC124 in vitro cassava storage roots starch and field cultivation of cassava root starch, starch grains formed by these two different ways is very similar in size and shape. Our findings show that, apply tissue culture techniques to study the cassava starch synthesis mechanism is feasible.

Cloning and sequence analysis of two cDNA encoding invertase inhibitors from cassava (Manihot esculenta Crantz)

In this study, two encoding invertase inhibitors (INH) named as MeINH1 and MeINH2 were isolated from cassava (Manihot esculenta Crantz). TheMeINH1 contains a 583 bp fragment in length and codes a region of 172 amino acids; and the MeINH2 contains a 538 bp fragment in length and codes a region of 169 amino acids. The amino acid sequence analysis showed that both MeINH1 and MeINH2 have typical structure of invertase inhibitor proteins with a N-terminal signal peptide, four conserved cysteine residues and two disulfide bridges, respectively; whereas, they showed low homology to other organisms. This research provides a foundation for further study of the cassava INH.

Cloning and Analysis of Vacuolar Invertase Gene (MeVINV2) Promoter from Cassava (Manihot Esculenta Crantz)

Vacuolar invertases play a vital role in the progress of cassava tuber roots starch accumulation. In order to study the regulating mechanism of cassava vacuolar invertases, the promoter of cassava vacuolar invertase 2 (MeVINV2) was isolated using the PCR amplification approach, starting with a part of coding sequences. Sequencing result showed that 47 bp MeVINV2 gene CDS sequence and 1242 bp potential promoter sequence was obtained. PlantCARE analysis revealed that the MeVINV2 gene promoter contains typical eukaryotic elements CAAT box and TATA box, and also several light-responsive elements and stress-responsive elements. These cis-acting regulatory elements might be associated to the vacuolar invertase gene function of cassava starch accumulation and biological stress defense.

Cloning of MeGolS5 Promoter from Cassava (Manihot esculenta Crantz) and Expression Analysis in Abiotic Stress of MeGolS5

The MeGolS5 promoter fragment (1492 bp) was amplified from the genomic DNA of Manihot esculenta Crantz by inverse polymerase chain reaction. Promoter sequence analysis by PLACE and PlantCARE showed that the cloned fragment contained several putative cis-elements, such as abscisic acid response element (ABRE), heat shock elements (HSE), as well as TATA-Box and CAAT-Box. The expression prfile of MeGolS5 shows that the gene is induced by several abiotic stresses, such as salt, drought, H2O2 and ABA.

Cloning of MeCWINV3 Promoter from Cassava and Transient Expression Analysis in Tobacco

In order to study the inducement pattern and regulating mechanism of MeCWINV3 in Cassava. An 1160 bp promoter region upstream of the MeCWINV3 gene (GenBank Accession No. KC905170) was isolated from Cassava (Manihot esculenta) genomic DNA using PCR methods. Sequence analysis found that it contains typical TATA box and CAAT box, and several cis-acting elements that related plant stress responses, such as ABRE, ARFAT, GAREAT, MYB and MYC transcription factors. Furthermore, transient expression in transgenic tobacco was analyzed by inserting upstream of GUS gene in expressing vector. The results showed that GUS was mainly expressed in tobacco veins. This will be the basis for further investigating the function of the MeCWINV3 gene promoter.

Expression of CP:CBF3-35S:ICE1 enhances low temperature tolerance in transgenic Arabidopsis

We have constructed a vector pCAMBIA1300-CP:CBF3-35S:ICE1 and transformed into Arabidopsis. Results of PCR proved that the target genes had integrated into Arabidopsis genome. Transgenic Arabidopsis showed a bit slow growth, earlier flowering, but normal at other phenotype under 22°C with 8 h daily lights. In vitro low temperature stress tests showed that the transgenic lines were survival while the wild type was nearly dead. The transgenic plants also showed an increased proline content, SOD and POD activities under low temperature stress. The phenotype and physical evidence indicated that expression of CP:CBF3-35S:ICE1 under low temperature enhances the cold tolerance in transgenic plants.

Optimization of the Heterologous Expression of Sesuvium portulacastrum Betaine Aldehyde Dehydrogenase in Escherichia coli

A full-length sequence coding for a betaine aldehyde dehydrogenase gene from S. portulacastrum was cloned into expression vector pGEX-4T-1, and named pGEX-4T-SpBADH. The GST-SpBADH fusion protein was expressed and the expression conditions were optimized. Through the research on optimization of expression the concentration of IPTG, concentration of bacterium, induction time and temperature and so on, the results showed, the expression of GST-SpBADH increased accompany with the induction time. The expression level of GST-SpBADH fusion protein reached the highest for 5 h cultured and for OD600 is about 0.6 at 37°C, 0.2 mmol/L IPTG can effectively induce the expression of GST-SpBADH in Escherichia coli expression system.

Somatic Embryogenesis and Organogenesis of Biofuel Plant Cassava (Manihot esculenta Crantz) Chinese Cultivars

2, 4-D and picloram were compared for their ability to the induction of somatic embryogenesis in Chinese cassava (Manihot esculenta Crantz) cultivars SC5, SC6, SC7 and SC8. In all four cultivars tested, both 2, 4-D and picloram had the capacity to induce primary somatic embryos from axillary buds. And the two hormones were also suitable for subculture of somatic embryos of three cultivars SC5, SC6 and SC8. However both 2, 4-D and picloram can not keep the activity of somatic embryos of cultivar SC7. For organogenesis, cotyledon matured for 10~15 days were better than others.