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.

Identification and characterization of genes associated with tapping panel dryness from Hevea brasiliensis latex using suppression subtractive hybridization.

BACKGROUND: Tapping panel dryness (TPD) is one of the most serious threats to natural rubber production. Although a great deal of effort has been made to study TPD in rubber tree, the molecular mechanisms underlying TPD remain poorly understood. Identification and systematical analyses of the genes associated with TPD are the prerequisites for elucidating the molecular mechanisms involved in TPD. The present study is undertaken to generate information about the genes related to TPD in rubber tree. RESULTS: To identify the genes related to TPD in rubber tree, forward and reverse cDNA libraries from the latex of healthy and TPD trees were constructed using suppression subtractive hybridization (SSH) method. Among the 1106 clones obtained from the two cDNA libraries, 822 clones showed differential expression in two libraries by reverse Northern blot analyses. Sequence analyses indicated that the 822 clones represented 237 unique genes; and most of them have not been reported to be associated with TPD in rubber tree. The expression patterns of 20 differentially expressed genes were further investigated to validate the SSH data by reverse transcription PCR (RT-PCR) and real-time PCR analysis. According to the Gene Ontology convention, 237 unique genes were classified into 10 functional groups, such as stress/defense response, protein metabolism, transcription and post-transcription, rubber biosynthesis, etc. Among the genes with known function, the genes preferentially expressed were associated with stress/defense response in the reverse library, whereas metabolism and energy in the forward one. CONCLUSIONS: The genes associated with TPD were identified by SSH method in this research. Systematic analyses of the genes related to TPD suggest that the production and scavenging of reactive oxygen species (ROS), ubiquitin proteasome pathway, programmed cell death and rubber biosynthesis might play important roles in TPD. Therefore, our results not only enrich information about the genes related to TPD, but also provide new insights into understanding the TPD process in rubber tree.

[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.

[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.

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.

Association of decreased expression of a Myb transcription factor with the TPD (tapping panel dryness) syndrome in Hevea brasiliensis.

TPD (tapping panel dryness) is a complex physiological syndrome widely found in rubber tree (Hevea brasiliensis) plantations, which causes severe yield and crop losses in natural rubber-producing countries. The molecular mechanism underlying TPD is not known and there is presently no effective prevention or treatment for this serious disease. To investigate the molecular mechanism of TPD, we isolated and characterized genes for which the change of expression is associated with TPD. We report here the identification and characterization of a Myb transcription factor HbMyb1. HbMyb1 is expressed in leaves, barks, and latex of rubber trees, but its expression is significantly decreased in barks of TPD trees. Our results suggest that the expression of HbMyb1 is likely associated with TPD and that the function of HbMyb1 is associated with the integrity of bark tissue of rubber trees.

[Re-cloning of THP gene and construction of high efficient expression yector of Volvariella volvacea].

PCR technique was used for amplifying THP gene in an unknown vector with primer AFP1 and AFP2. Then THP gene was ligated to pGEM T-Vector to be the plasmid pGTHP4. The plasmid pCAMBIA1301 was digested with restriction enzyme BstE II and Nco I, and digestion product was separated with 1% of agarose gel, then big fragment containing promoter was isolated and purified with the Agarose Gel DNA Extraction Kit. At the same way, the plasmid pGTHP4 was digested with restriction enzyme BstZ I and Nco I, and the small fragment containing THP gene was purified from 1% agarose gel with the Agarose Gel DNA Extraction Kit. The big fragment and the small fragment were ligated at Nco I digested cohesive-end. The ligation product was re-ligated to be cyclic plasmid by addition to a specific adapter, resulting in the pCTH823, a expression vectorof V. volvacea.