Stylosanthes Induced Resistance And The Expression Characteristics Of Disease Resistance Gene Analogues

Stylosanthes which is grown in tropical is a kind of legumes Pasture of high quality.Reyanâ…¡was collected and stored in the seed bank by agronomists of the International Center for Tropical Agriculture (CIAT), the number of which is CIAT184. Stylo anthracnose is a serious diseases hazard the production and marketing of Stylosanthes.In this investigation, diseased leaves and stems were collected from Nanning. Strains [Colletotrichum gloeosporioides(Penz.) Penz.and Sacc] named GX021001 were isolated from single spore and then were purified and cultivated in the laboratory. Three species of Stylosanthes tested showed symptoms coincide with the disease resistance when them was infected by the strains.In this research, the disease index of Stylosanthes resistant to the anthracnose was analysed by 0.0l mmol/L and 0.5 mmol/L concentrations of both salicylic acid.The enzyme activity of POD, CAT, PPO was determinated. The impact of salicylic acid on enzyme activity related of stylosanthes for anthracnose resistant was also analysed. The results showed that: The disease index of Stylosanthes after 0.0l mmol/L salicylic acid treatment was 42.0. The disease index of Stylosanthes after purified water treatment was 56.0. Experiments show that salicylic acid have the property of induced disease resistance, and it appear better effect of disease resistance from 0.0l mmol/L enriched of salicylic acid. After 72 h inoculation of anthrax, the ions and covalence of POD was significantly less than the standard sample after salicylic acid treatment, which prove that salicylic acid has inhibitory effect on POD. The CAT of 0.0l mmol/L and 0.5 mmol/L salicylic acid were 0.44 times and 0.82 times as standard sample, which prove that salicylic acid has inhibitory effect on catalase. The PPO of 0.5mmol/L and 0.01 mmol/L salicylic acid were 1.11 times and 2.56 times as standard sample, which prove that salicylic acid treatment could induce the production of key enzymes of PPO in defense reaction.The Modified SDS/ phenol extraction method , Modified CTAB method , common Trizol and Column Plant RNAout were employed to extract total RNA in leaf of Stylosanthes spp.The quality and quantity of total RNA were compared among five methods. The results indicated that the value of OD260nm/ OD230nm of RNA extracted with modified CTAB method and with Column Plant RNAout method were greater than 2.0 and the value of OD260nm/ OD230nm of RNA extracted with modified CTAB method and with Column Plant RNAout were 1.85 and 1.93, respectively. Which show high purity. Gelelectrophoresis showed that RNA extracted with modified CTAB method had clear bands of 28S rRNA and18S rRNA and was not degraded , RNA extracted with Column Plant RNAout had four clear bands of 28S rRNA and18S rRNA and was not degraded , RNA extracted with other two methods was degraded and dispersed in some degrees. RNA extracted with modified CTAB method and with Column Plant RNAout could be reversed to cDNA. The cDNA was amplify and one clear bands ofβ-actin gene fragment could be observed in agarose gel. These results further demonstrated that the quality and purity of the total RNA extracted with modified CTAB method and with Column Plant RNAout method can meet the demands of molecular biology experiment . and quality and purity of the total RNA with Column Plant RNAout method were better than with modified CTAB method.In this study,β-actin gene was used as housekeeping gene, designed primers according to the genome of NBS class of disease resistance gene analogues (RGA) sequenced in the laboratory,and then amplificated the target gene from stylosanthes plant cDNA by semi-quantitative RT-PCR technology. The optimal Mg²⁺ concentration of housekeeping gene and target gene, the amount of the best template and the number of platform cycles,the same tube amplification and different tube amplification were discussed,and it can be concluded that the optimal Mg²⁺ concentration of housekeeping gene and target genes was 3 mmol/L, the cycle number of housekeeping gene was 28, and the cycle number of target gene was 30. Different tube amplification were confirmed by layering experiment. Stylosanthes plants were sprayed with 1×106 spores/mL solution of anthrax and 0.0l mmol / L solution of salicylic acid to do semi-quantitative PCR. Taking spraying distilled water as a control group, sprayed RNA to the plants for 1 d, 2 d, 3 d ,4 d, 5 d, and amplified housekeeping gene and the target gene in accordance with the semi-quantitative RT-PCR, then compared the full band optical density (IOD).RNA were extracted from aseptic roots, flowers, cotyledons, leavesof stylosanthes, and the target gene were amplified. The result showed no gene expression dig up the roots and flowers, but the other organizations have the gene expression.