Study And Application On The Latex Cell-free Apoptosis System In Hevea Brasilinesis

Latex cell-free apoptosis system of Hevea brasiliensis was studied by using the latex and tobacco as research materials.HbMybl gene transformed tobacco was identified by morphological identification, PCR detection and Southern blot. It showed that the tobacco used in experiments was HbMybl gene transformed tobacco.The tobacco cell suspension culture system was established. Experimental results showed that:MS+2,4-D 0.5mg/L+KT 0.5mg/L+Agar 7.5g/L was optimal for tobacco callus induction. The optimal subculture medium was MS+2,4-D 0.3mg/L+KT 0.3mg/L +Agar 7.5g/L. Loose calli were obtained after four generations’subculture. Then the loose calli were cultured in the liquid medium, MS+2,4-D 0.2mg/L+KT 0.2 mg/L+0.1% hydrolyzed casein. So the yellow tobacco suspension system was obtained, which grew rapidly and had good dispersion and homogeneity. It was optimal to subculture the tobacco suspension cells 7d later.Different ways were used to collect latex and to extract latex DNA.The results showed that different collection methods had no significant effect on the integrity of latex DNA. The latex was divided into three main fractions by ultracentrifugation (Upper layer mainly contains of rubber particles. The middle layer includes soluble cytoplasmic components of the latex.The contents of lower layer mostly were lutoids).DNA was extracted from the three layers separately. It was found that almost all of the DNA was located in the lutoid layer. It indicated that the soluble cytoplasm of latex contained little DNA and wouldn’t interfere with subsequent experiments.Cytochrome c was added to a final concentration of 2.5μmol/L, to the latex cell-free system, to convert the cell-free system to apoptosis-inducing system. Tobacco cell nuclear apoptosis could be induced after adding the tobacco cell nuclear into the induction system when bathing at 23℃.TUNEL detection was positive. Agarose gel electrophoresis produced typical DNA Ladder. We found that the nuclear apoptosis time of HbMybl gene transformed tobacco was later than that of wild tobacco in the nuclear apoptosis inducing process.In-gel nuclease assay analysis results of latex cell-free system of healthy trees and tapping panel dryness trees indicated that the two nuclease band types were different. Tapping panel dryness tree latex nuclease had two specific bands between 33-48KD.