The Preliminary Study On Expanding Growth-related Genes From Sweet Potato

The impact of energy shortage has become an important issue of global economic development,China is an energy shortage and consumption power,reasonably making use of energy especially grain alcohol could be a good means of empoldering renewable energy.Sweet potato has a high biomass of production,which would be unique advantages in the production of fuel.Therefore,improving the output of starch and doing in-depth study of sweet potato becomes very important.In this paper,we made a preliminary study on expanding growth related genes from sweet potato and finally we obtained the the results as follows:1.An improved CTAB method was established to isolate total RNA of sweet potato.because sweet potato are rich in Polysaccharides and PolyPhenol compounds, the CTAB method was improved by adding polyvinylpyrrolidone(PVP) andβ-ME to remove PolyPhenol;adding KAc and ethanol to remove polysaeeharides;using Bentonite to absorb protein and inhibit the activity of RNase,furthermore adding LiCl to deposit RNA.The electrophoretic bands of the isolated RNA were regular and clear,so which is suit for the further experiment.2.An optimized cDNA-AFLP system being suitable for sweet potato have been established.Throgh groping and optimizing the reaction system of AFLP,we find that the method of digestion is 3h in water at 65℃after 3h at 37℃;the optimum system of pre-amplification is 30μl and selective is 20μl;the PCR program was that the first cycle performed as 2 min at 94℃to predenature the templet,then keep 94℃for 30s to denature,65℃for 30s to anneal(dropped 0.7℃per cycle),72℃for 1 min to extend,preceding process for 12 cycles;then 94℃for 30s to denature, 56℃for 30s to anneal,72℃for 1 min to extend for 23 cycle and a final extension at 72℃for 5min after the last cycle.Then preserve at 4℃.3.Four ways of silver staining was compared,we find that first way is better than others.So we chose it to optimize by changing the temperature,pH and reagent. Finally we established a better and fast Silver staining procedure and got 31 groups of different fragments.4.Designing the degenerate primer according to the expansin aminoacid consensus sequence registed in the gene bank,we've got two probably fragments of expansion gene by PCR,the results we got would be a foundation for the farther study.