| Lotus root, a kind of perennial aquatic herb, belongs to the Nelumbonaceae family. It is a medicinal and edible plant. It has the pharmacological effects of lowering blood pressure, antioxidant, anti-aging, sedation, inhibition of fatty liver, antibacterial, etc.Although recently the demands for lotus roots are rising home and abroad, there are some browning problems during the processing and storage, which have caused the decline of their nutritional value and commodity value and have greatly influenced the foreign exchange earnings through export and medical effects. Studies have shown that, polyphenol oxidase(PPO) is not only one of the key enzymes that cause lotus root’s browning by participating in the formation of pigment, but also plays a vital role in the process of protecting lotus roots from plant diseases and pests. However, the current studies on PPO concentrate on aspects of pharmacology and cultivation techniques, yet studies on gene functions and mechanisms are rare.Therefore, based on former researches, further molecular studies on polyphenol oxidase are carried out by using lotus roots as materials. We have successfully constructed the bait plasmid and the cDNA library, all of which provide sound foundations for the primary study of the protein and cis acting elements in lotus root that interact with polyphenol oxidase by yeast two hybrid technique. It is hoped that the browning problems of lotus root can be totally solved and the consequent economic problems can be decreased. We also hope that the foundations for Plant resistance research can be laid.The results are as follows.1. This experiment adopts the CTAB-Li Cl method to extract RNA from lotus bud. And then by taking the cDNA synthesized by RT-PCR as a template, this thesis clones the open reading frame of polyphenol oxidase gene which include the restriction enzyme cutting site of EcoRI and NdeI by the methods of PCR, Gel Extraction, Sequencing, etc. Next, the gene is connected to the bait plasmid pGBKT’7 after it has been digested with NdeI and EcoRI. We have successfully obtained the bait plasmid pGBKT7-PPO. After confirmation by ways of sequencing and double enzyme digestion, pGBKT7-PPO and yeast two hybrid plasmid pGBKT7 are separately transformed into the yeast strain AH109 with AD. Self-activation and toxicity test of.pGBKT7-PPO have been processed in SD autotrophic medium. The results show that the expression product of Bait gene cannot activate the expression of reporter gene, and is not toxic to yeast strain as well. Thus we have obtained a usable bait vector.2. This experiment adopts the Smart method to construct a cDNA library. Firstly, we use the kit to extract the RNA from lotus bud, lotus stem and leaf, in order to obtain the high-quality RNA. Then we mix the three species of RNA into a Centrifuge tube. Next, we synthesize double stranded cDNA by using the method of RT-PCR and LD-PCR,and then purify it in order that the size of the fragment is within the scope of 500bp-3000 bp. Sequently,we connect the purified double stranded cDNA to the pGADT to construct the cDNA library. Lastly, we have made quality and pollution tests of the cDNA Library and have got the following results: the titer of initial library is more than 7.5×1010 cfu/ml; the transformation efficiency is 2.5×106 cfu/μg; and the storage capacity is 7.5×106 cfu. Meanwhile, the yeast suspension we have collected that contains lotus cDNA is not polluted. The results indicate that this library can not only be used to isolate and screen target proteins, but also guarantee the acquisition and preservation of the full length cDNA library. |
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