Selection Of Appropriate Reference Genes For Expression Studies In Camellia Sinensis By Real-time Polymerase Chain Reaction

Tea plants (Camellia sinensis) is an important economic crop in china and originate in tropic and subtropical regions. The distribution of tea in China has a broad geographic range from18°to38°N,94°-122°E with complex climate and terrain. The quality of tea has been affected by.extreme climatic factors such as drought, low temperature, Mechanical damage and also heavy metal, which resulting in reduced production, decline in quality. Research on the resistance of tea is still at the begining, and most of the preliminary studies are focus on physiology and biochemistry. In this situation, plant molecular breeding is becoming an effective tool. With the separation of adversity genes and selection of molecular markers which linked to adversity traits, the early identification of adversity traits and analysis of hereditary stability could be carried on, which could shorten periods of breeding for stress tolerance in tea. Quantitative gene expression at the transcriptional level, such as real-time quantitative PCR, RNA blotting, RNase protection analysis, gene chips, etc., also quantitative methods at the protein level like Western blot both need the application of internal reference gene to normalize expression level of target gene, which lead to much more reliable results.Isolation of relative reference genes in tea using basic clone technology and real-time PCR assay for quantification of each reference gene respectively had been performed in this study, and then further research on the gene expression stability under different stress treatments of drought, aluminum, cold, salt and damage was carried on. The specific findings are as follows:(1) Using the technology of homologous clone, part of fragments of7reference genes(GAPDH、EF-1α、18S rRNA、TUAI、TUA2、ACT、UBI) in tea were selected. BLAST indicated that above genes showing highly homologous with similar genes in other species. All base homology were more than80%. On one hand explained the accuration of experimental results, on the other hand, it also proved the conservative characteristic of reference gene, and further verified the application universality in quantitative analysis.(2) Real-time PCR assays of each gene using QRT-PCR technology were established. Standard curves showed highly sensitivity and correlation while taking10-fold dilution of purified plasmid as templates, calculated the amount of gene in cDNA samples, and also provided more accurate data for subsequent experiment.(3) cDNA under different stress treatments as template was took to run the analysis of enpression stability of7reference genes uing QRT-PCR. Calculated by three biology software (geNorm, BestKeeper, NormFinder), the result showed that under aluminum stress,18S rRNA was the most stable reference; under cold stress, GAPDH and EF-1α can be used in related experiments; TUA1and GAPDH were the best choice in damage condition; GAPDH showed the high expression stability under drought treatment; besides, under NaCl treatment, Actin and GAPDH were the most stable reference.