| Spinach (Spinacia oleracea L , 2n=2x=24 (XY)) is an annual (rarely biennial) flowering plant in the family of Amaranthaceae. It was cultivated widely as a common vegetable gardening. Spinach is a diploid dioecious plant, which possesses a pair of heteromorphic sex chromosome X and Y. Breeding of new variety, optimizing cultivation technology and the mechanism of sex determination were being studied in the past several decades. The research that focused on epigenetics characters especicially involved in epigenetics regulating mechanism of sexual development was rarely been reported. Plant DNA methylation was one of the first discovering genome epigenetic modification ways, and it was proved to succeed in regulating the plant gene expression. In order to explore the affection on DNA methylation in spinach growth and sex development stage, the following experiments were performed. The epigenetics phenotype and sex ratio were been investigated after treating use the methylation inhibitor 5-azaC. Meanwhile, the DNA methylation pattern and methylation level were detected by MS-ISSR,MS-RAPD and HPLC methods. The result showed as follow:1. Different 5-azaC concentration was used to treat the spinach feed and its affection on germination energy, germination percentage, Plant height, leaf length, root length, flowering time and sex ratio of spinach was explored. The result showed germination energy, germination percentage, root length, Plant height and leaf length are negative correlated with the 5-azaC concentration among 5-15μM compared with the control plants, and the flowering a little earlier than control plants. The spinach was not sensitive to 30μM 5-azaC and every growth indices was almost same as the control group. The spinach was treated by 5-azaC whose concentration was 50-1000μM and its growth indices decreased as the concentration increased. The high concentration inhibited obviously growth indices, for example the plant height was lower than the control group and the the flowering earlier more than one day. However it has little affection on spinach sex ratio and 1000- grain weight.2. The spinach was treated by 30,100,500μM methylation inhibitor, then its leaf and the normal one gene group were digested by methylation-sensitive restriction endonuclease. The change between them was investigated by MS-RAPD and MS-ISSR technology. The result showed that 20 polymorphism MS-RAPD primers were selected from 400 primers and 35 polymorphism MS-ISSR primers from 65 primers. 667distinct bands were obtained from 12 samples. The percentage of polymorphic loci were respectively 86.88% and 86.24%, respectively.Through the analysis on specific endonuelease sites, it found methylation and demethylation appeared during 30,100,500μM treat and demethylation play a important role.3. The extend of total DNA methylation of different spinach tissue were detected by HPLC method, the material was obtained from the treat plant by 30,100,500μM methylation inhibitor and the untreated control plants. The result showed that extend of gene methylation decreased obviously with the concentration increased in 30μM. However the changed was not obviously when the concentration exceed 500μM. Among the different tissue detection, the mature leaf had the highest DNA methylation extend, then spire, cotyledon and hypocotyl in sequence, and radicle has the lowest (the average value was 4.61%).The same trend that the gene methylation extend decreased as the concentration increased were observed. |
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