Cloning And Screening Of Reference Genes Of Haloxylon Ammodendron

Haloxylon ammodendron is a windbreak and sand-fixation plant which grows in the desert, and it has very strong resistance. At present, the research of Haloxylon ammodendron has gradually shifted from the ecology and biology to the molecular level. However, the research of its molecular level is still relatively backward. If we want to study growing development and regulatory mechanism of moleculer of Haloxylon ammodendron, we must look for reference genes which can be steadly expressed as a contrast. According to different conditions to screen suitable reference genes, optimization of real-time quantitative expression system was gained. So the study of different reference genes has important theoretical and practical significance. According to partial sequences of reference genes which our lab has gained through transcriptome sequencing, we have designed primers of semi-quantitative and q RT-PCR. And the stability of expression of relevant reference genes were analyzed under different tissues and adversity stress. The followings are specific results of research.(1)According to Transcriptome Sequencing, we have chosen eight internal genes, and according to their nucleic acid sequences we have designed different primers to clone sequences of reference genes.Various reference genetic similarities of Haloxylon ammodendron were compared with corresponding reference genes of other plants. We found that various reference genetic similarities of Haloxylon ammodendron with the corresponding internal genetic similarities of other plants were over 80%. These results conform that the eight reference genes were highly conservative. On the basis of above-mention, we have gotten the full length of c DNA and genomic DNA sequences of Ha ACT1 and Ha ACT2. As a basic research of reference genes, the study provides more accurate data for subsequent experiment information,as well as gets laid the foundation to get the full length of c DNA and genomic DNA sequences of the eight reference genes.(2)q RT-PCR primers of eight reference genes of Haloxylon ammodendron were designed. With plasmid standard qualities of various reference genes as a template and according to the series of 10 times dilution is 8 concentration gradients. The technology of q RT-PCR was applied. Optimal reaction system of real-time fluorescent quantitative PCR of the reference genes was set up. The standard curves were drawed according to Ct value of various concentration of plasmid as the ordinate and with different concentration of plasmid copy number asvertical coordinates. The result showed that the standard curves were relatively ideal, Various resistance genes of Haloxylon ammodendron can be used for subsequent analysis of the experimental data. Eight internal genes selected in theirselves concentration had good linear relation, and the linear ranges were better wider.(3)Expression of reference genes of different tiusses was analysed using semi-quantitative PCR. The eight reference genes could be expressed steadily in assimilating branch and flowers and roots and seeds.Absolute fluorescence quantitative PCR of reference genes of various resistance was started under various abiotic stress. And the stability of expression of various resistance genes under different adversity situation was analyzed by the biostatistics software Best Keeper, ge Norm and Norm Finder. This result was that under drought and heat and cold and salt and damage, the best reference genes of Haloxylon ammodendron spectively were Ha18 Sr RNA and Ha TUA5 and Ha UBQ10; Ha18 Sr RNA and Ha GAPDH; Ha ACT7 and Ha UBQ10; Ha Ha ACT1 and Ha TUA5; Ha TUA5 and Ha GAPDH.