|
In recent years , the southwest karst region deteriorating ecological , soil erosion , the rocky desertification heavier , has seriously hampered the development of the local economy . The use of genetic engineering rocky desertification repair is an important means of karst vegetation recovery as typical vegetation of southwest China karst area , one of the South China honeysuckle study to adapt the karst environment , especially karst high calcium environment adaptation mechanisms , genetic engineering can repair rocky desertification provide important theoretical basis . This study to simulate the growth of the South China honeysuckle environment by GeneFishing technical analysis of genes differentially expressed under different concentrations of calcium salt processing , and its changes in expression level of analysis and validation . In addition, by means of the use of bioinformatics described the function of these genes . In this study, the following results were obtained : (1 ) the establishment of a technology system in southern honeysuckle GeneFishing . 2μg total RNA by reverse transcription primer dT- ACP , 42 ° C reaction 1 h, cDNA first strand synthesis , reverse transcription time can not be too short . cDNA first strand was diluted 5 - fold for GeneFishing PCR, PCR template 3μl, two-step PCR Forward temperatures were 50 ℃ and 65 ° C. The PCR product is required at least in a 2% agarose gel electrophoresis for 1 h , to get clearer Strip . (2 ) by GeneFishing technical analysis of different concentrations of calcium (0 and 125 mg / L ) treatment under the South China honeysuckle differences in gene expression , using a total of 20 pairs of primers , found 24 differentially expressed genes ( DEGs ) . By designing different primers in six different the concentration ( 0,25,50,75,100,125 mg / L ) calcium processed material , the validation of the semi-quantitative PCR , and observe the changes in the expression amount . (3) bioinformatic analysis DEGs adapt to the role in the process of high-calcium environment in southern honeysuckle . Blastn comparison found that 24 DEGs in a coded channel protein , three related to photosynthesis , 3 protein sorting and secretion , five plants oxidation -reduction reaction of two plant nutrition and substance relating to the synthesis , one associated with transcription factors , 8 no homology or unknown function , have reported that majority of genes are closely related and salt stress . Analysis of DEGs calcium target protein interactions , found that close contact the DEGs with calcium target proteins , and also calcium regulation .
|