| Telomere is a kind of special structure of the chromosomal end like a “cap”, mainlyincluding DNA and the telomere associated proteins. It can protect the chromosomal end fromdegradation of the nuclear enzyme and have a stability effect. The length of the telomereinfluences the aging and apoptosis of the cells, but the length of the telomere is mainlydepending on the telomerase to maintain. Telomerase is a special kind of reverse transcriptase(RT), mainly formed by template RNA and telomerase binding proteins. It can take their ownRNA as templates, add synthetic telomere to repeat sequence to prolong the end of chromosomes.At present, from protozoa, yeast and various higher animals as well as the human cells, thetelomerase reverse transcriptase protein and their RNA template had been cloned, and foundnormal cells lack of telomerase activity, but the telomerase activity of the cancer cells wasefficiently expressed. These studies give people hope to find new ways to cure cancer. Comparedwith animals and human, the research of plant telomere and telomerase is still tiny, especially inthe cloning of plant telomerase RNA template subunit. The latest reports demonstrate that twodifferent RNA templates (TER1and TER2) are found in Arabidopsis, and the two componentsare in an active telomerase. Hence, it is important for further researches, among them, theisolation technics of the break through telomerase is key. This paper is intended to use broccolifor material, build a plant telomerase separation technology, and lay the foundation for highseparation plant telomerase.This paper was designed with polyethylene glycol (PEG) to separate and enrich telomerase.Polyethylene glycol (PEG) is a kind of amylose macromolecular polysaccharide with no chargeand can cause protein precipitation nonspecificly. Precipitation is reversible but the biologicalactivity of the precipitated protein isn’t affected. This paper first take the bud of broccoli asmaterials, adopt PEG of different molecular weight and different concentration to extracttelomerase respectively, detect the telomerase activity in the traditional TRAP method andcompare. Finding out the PEG of the best molecular weight and concentration aims to explorethe best method to extract efficient plant telomerase efficiently.The results of the study indicate that: through extracting broccoli telomerase by using sixdifferent molecular weight PEG (PEG1000, PEG2000, PEG4000, PEG6000, PEG8000, PEG10000), we found that the activity that is extracted by PEG6000is the highest. Therefore, we select the telomerase extracted by PEG6000, according to different enzyme content (200μg,160μg,130μg,100μg,80μg,60μg,40μg,20μg,160μg inactivated and the negative control)for enzymatic reaction and succeed in detecting when telomerase content is100μg, its activity isthe best. Based on the former experiment, go on the enzymatic reaction to the telomeraseextracted by seven concentration gradient PEG6000, set the normal enzymatic reaction, go onenzymatic reaction after the enzyme is inactivated and normal enzymatic reaction addingdigestion RNase A, and determine the telomerase activity. The experimental data show that whenthe eluent amount is700μL, telomerase activity is the highest. At the same time, we improve thedetection method of telomerase. Based on the detection meyhod, we have established a set ofmethods to apply plant telomerase separation. We execute the telomerase separation technologyfor further inspection and confirmation in the rice callus and young ears of rice. The results showthat the method of created separation is completely feasible, and applied for the national patentof invention (Patents have been accepted, Number of the patent:201110357993.4).Theseexperiments to make clear the important function of the telomere and telomerase in plant and thebasic research on in-depth plant telomerase has important significance in theory and practice. |
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