[Transformation of Volvariella volvacea with a thermal hysteresis protein gene by particle bombardment].

A cDNA encoding a thermal hysteresis protein was isolated from the Swedish Arctic insect spruce budworm by RT-PCR amplification. Volvariella volvacea strain V34 was transformed with this cDNA through particle bombardment. PCR detection and Southern blotting analysis show that the thermal hysteresis protein gene is integrated into Volvariella volvacea genome. Cold stress assay reveals that transgenic Volvariella volvacea lines exhibit stronger cold tolerance than host strain. The morphological observation of transgenic Volvariella volvacea lines shows that growth rates of most Volvariella volvacea transformants are significantly slower than that of negative control strain. And hypha of most Volvariella volvacea tansformants is thinner than host strain's hypha. Transformant screening result indicates that three-round of selection procedure with first selection on PDSA solid selective medium followed by second and third selection in PDSB liquid selective medium is favorable to get genuine transformants and to eliminate false transformants. Cold tolerance assay of transgenic Volvariella volvacea F1 generation demonstrates that the progeny of transgenic Volvariella volvacea still possesses stronger cold tolerance than non-transformed host strain. This suggests that the cold tolerant characteristic of transgenic Volvariella volvacea is meiotically stable between generations.

Constitutive expression of human angiostatin in Pichia pastoris using the GAP promoter.

The GAP gene promoter was amplified from P. pastoris GS115 and used to replace the AOX1 promoter (P(AOX1)) on pPIC9K resulting in plasmid pGAP9K. The recombinant expression vector pGAP9K-AS was constructed by inserting the angiostatin gene(AS) into pGAP9K. pGAP9K-AS was then transformed into P. pastoris GS115. The multi-copy integration transformant P. pastoris GS115 (pGAP9K-AS) was used to investigate the constitutive expression of angiostatin in P. pastoris. The expression of angiostatin reached its peak after 4 d of culture in P. pastoris GS115 (pGAP9K-AS) while the angiostatin expressed in P. pastoris GS115 (pPIC9K-AS) after 4 d of induction or 5 d of culture is only 70% of that expressed by P. pastoris GS115 (pGAP9K-AS). The AS expression in inducible system reached the peak after 6 d of induction but the expressed AS was only 86% of that from constitutive system. The results of anti-angiogenic and antitumor activity assay showed that AS expressed from both constitutive and inducible system inhibited the CAM angiogenesis and suppressed B16 melanoma in C57BL/6J mouse and that the tumor inhibition rates reached 90.63% and 90.54%, respectively. The above data indicates that the constitutive promoter P(GAP) can served as an effective alternative to the inductive promoter P(AOX1) to express AS and other proteins in P. pastoris.

[Expression and cloning of cDNA encoding 43 kD rubber particle membrane protein of Hevea brasiliensis].

A rubber particle protein with apparent molecular mass of 43 kD as determined by SDS-PAGE was purified. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified protein was used to amplify a 1385 bp cDNA by 3' rapid amplification of cDNA ends (3'RACE). The cDNA contains five repeats in a head-to-tail arrangement without intervening sequences, each encoding a ubiquitin unit of 76 amino acids. The last ubiquitin unit is followed by an extraphenylanaline residue at the carboxyl-terminal end. The structure of the cDNA is consistent with the structure of other known polyubiquitin genes. Western blot demonstrated that 43 kD rubber particle protein might be a polyubiquitin. Southern blot analysis revealed that there were multiple copies of gene encoding 43 kD rubber particle protein in Hevea brasiliensis. The results of Northern blot analysis indicated that the gene was expressed in latex, young leaves and bark tissue.

HbMyb1, a Myb transcription factor from Hevea brasiliensis, suppresses stress induced cell death in transgenic tobacco.

Tapping panel dryness (TPD) is a complex physiological syndrome found widely in rubber tree (Hevea brasiliensis) plantations that causes severe yield loss in natural rubber-producing countries. In an earlier study, we confirmed that there is a negative correlation between HbMyb1 expression and TPD severity. To further investigate the function of HbMyb1 in TPD, HbMyb1 was over-expressed in tobacco controlled by a CaMV 35S promoter. In transgenic plants expressing HbMyb1, cell death induced by UV-B irradiation, paraquat and the hypersensitive reaction to necrotrophic fungal infection (Botrytis cinerea) was suppressed with a close correlation between HbMyb1 protein levels and the extent of suppression. In addition the nuclear condensation and degradation were observed in laticifer cells of TPD trees, while the nucleus of laticifer cells of healthy trees was morphologically normal. On the basis of the results described above, we propose that HbMyb1 maybe suppress stress induced cell death in rubber trees.