Genetic Diversity Of Polyporus Umbellatus And Cloning Of Pyrophosphorylase

Polyporus umbellatus (Pers.) Fr., a famous traditional Chinese medicinal fungus thatbelongs to polyporaceae, Basidiomycetes, is widely distributed in Shaanxi, Yunnan, Sichuanand Hebei. The potent pharmacological properties of Polyporus umbellatus have attractedworldwide interest, whereas over-digging wild materials has made its resources drasticallydecrease in China which has been listed as endangered species in China Red Book (Li2008).The knowledge of the extent and distribution of genetic variation within a species isfundamental for evolutionary comprehension, conservation of genetic resources andsubsequent breeding purpose. In this study of Polyporus umbellatus resources, the geneticdiversity was investigated by SRAP, in order to make further exploitation on Polyporusumbellatus and provide some insights on conservation and breeding research for the species.The main results showed as follows:1The optimization method of DNA extraction and purification from Polyporus umbellatus forSRAPThe activated mycelium cultivated in PDA liquid medium was collected. DNA wasextracted by improved CTAB method.(1) Grifola umbellate grew in PDA liquid mediumwere harvested by filtration and ground to a powder using liquid nitrogen. Then1mLRNAiso-mate for Plant Tissue was quickly added and buried the powder completely. Thesamples were placed quietly until melt completely. Continue grinding till the lysate showstransparent shape. The homogenate was centrifuged at12000rpm for5min at4℃.(2) Afterthe Phenol-chloroform-isoamylalcohol (25:24:1, by vol) was added and centrifuged,supernatant was collected and2μl RNase was added, the mixture was incubated at37℃for1h.(3) After the protein and polysaccharide was eliminated, the supernatant was collected and2/3volume of isopropanol was added to precipitated DNA. The integrity and quality ofisolated DNA was evaluated. The results of UV-spectrophotometric method show thatA260/A280is1.915and the content of DNA is149.65ng/μL. The electrophoresis shows thatDNA belts are good integrity, no trailing and without any contamination. So the DNAextracted by improved CTAB is suitable for SRAP. 2An optimization system of SRAP was establishedTo establish the optimal SRAP reaction system, primer selection, template DNAconcentration, primer concentration and amplified conditions were tested by single factorexperiment. The suitable SRAP reaction of Grifola umbellate: DNA concentration0.8ng/μL,forward and reverse primer concentration0.4μM,1×Taq MasterMix, RNase-freeWater was added up to25μL. The optimized program was predenaturing at94℃for5min,then denaturing at94℃for1min,annealing at35℃for1min and extending at72℃for1minfor the first five cycles; followed by another35cycles with annealing temperature at50℃and finally terminated by extending at72℃for10min. The electrophoresis on agarose andpolyacrylamide gels shows that the SRAP bands are clear, stable and high genetic diversity.3SRAP analysisEight wild P. umbellatus strains collected from seven provinces of China were subjectedto sequence-related amplified polymorphism (SRAP) markers to estimate the level andpattern of genetic diversity. Forty-nine primer combinations generated a total of1219highlyreproducible and discernible loci, among which1023were polymorphic. Percentpolymorphism varied from35.71to96.30with an average of83.92. Genetic identidy amongall strains ranged from0.15to0.78with an average of0.46. The UPGMA dendrogramclustered eight strains in three clusters and the clustering pattern showed three groups.Principal Coordinate Analysis (PCA) further indicated that the genetic diversity of P.umbellatus strains was not evenly distributed, instead, was presented by a clustereddistribution pattern. Similar to the results revealed by the dendrogram, three groupsrepresenting two branches in one group, as well as two special strains in the other two groupswere obtained. Neither the UPGMA dendrogram nor the PCA analysis exhibited strictrelationship with geographic distribution and mycelial growth among the strains.4Comparing different RNA extraction methodCTAB-LiCl, Trizol, Trizol reagent, RNAiso Plus and improved RNAiso Plus were usedto extract total RNA. RNAiso Plus combined with RNAiso-mate for Plant Tissue is the bestisolation method, as the RNA bands extracted by this method were the clearest with highestpurity and no trailing strip. The A260/A280ratio of RNA isolated by this method is2.01and thecontent of RNA is1122ng/μL. This improved method is suitable for RNA extraction of P.umbellatus5The enzyme involved in the process of polysaccharide synthesis was clonedPyrophosphorylase plays an important role in the process of polysaccharide synthesis.According to the sequence of similar species, several primers were designed. In this study,RNA was extracted from P. umbellatus and reverse transcribed cDNA. Then this first strand cDNA is used as a template for PCR amplification and then sequenced. Sequence comparisondemonstrated that it had97%homology with Escherichia coli and96%homology withShigella sonnei.