Prabhjot Kaur Gill

DNA isolation from dry and fresh samples of polysaccharide-rich plants

DNA extraction is difficult in a variety of plants because of the presence of metabolites that interfere with DNA isolation procedures and downstream applications such as DNA restriction, amplification, and cloning. The chemotypic heterogeneity among species may not permit optimal DNA yield with a single protocol; thus, even closely related species may require different isolating protocols. Here we describe a modified procedure based on the hexadecyltrimethylammonium bromide (CTAB) method to isolate DNA from tissues containing high levels of polysaccharides. The procedure is applicable to both dry and fresh tissues and was tested on chickpea seeds, soybean, and wheat leaves. This method solved the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides. The isolated DNA proved amenable to PCR amplification and restriction digestion.

Changes in germination, growth and soluble sugar contents of Sorghum bicolor (L.) Moench seeds under various abiotic stresses

The effect of various abiotic stresses on germination rate, growth and soluble sugar content in Sorghum bicolor (L.) Moench cv. CSH 6 seed embryos and endosperm during early germination was investigated. Under stress conditions germination, water potential and tissue water content decreased markedly. Subsequently, this reduction resulted in marked decreases in fresh weight both in embryos and endosperm. Conversely, a substantial increase in dry weight was observed. Furthermore, a considerable increase in the sugar contents in both embryo and endosperm was detected. The fructose level was always higher than glucose and sucrose in response to various stresses. However, as compared to the control the level of glucose and sucrose was higher in embryos and endosperm after stress treatments. Based upon these results a possible physiological role of sugars in the germination of sorghum seeds is discussed.

Effect of media supplements and culture conditions on inulinase production by an actinomycete strain

Streptomyces sp. GNDU 1 produced high levels of extra-cellular inulinase (0.552 IU/ml) after 24 h at pH 7.5, temperature 46 degrees C in the presence of 1% inulin. The optimum temperature and pH for enzyme activity were 60 degrees C and 5.5 respectively. Yeast extract as a nitrogen source was found to be most suitable one for inulinase production whereas ammonium ion was inhibitory to the enzymatic production. All these conditions make Streptomyces sp. GNDU 1, a potential candidate for industrial enzymatic production of fructose from inulin.

RNA isolation from plant tissues rich in polysaccharides

Extraction of high-quality RNA is necessary formakingcDNAlibraries,isolatinggenesbyRT-PCR,orinvestigatinggeneexpressionprofiles.SeveralmethodsarecommonlyusedforisolationoftotalRNA[1–3]andare being developed because plants contain highamountsofmanydifferentsubstances;therefore,justonenucleicacidisolationmethodsuitableforallplantscanneverexist[4].Evenplantspeciesbelongingtothesamegenusorrelatedgeneracanexhibitenormousvariabilityinthecomplexityofpathwaysofdispensablefunctions.Thus,thebiochemicalcompositionsinplanttissuesofdifferentspeciesareexpectedtovaryconsid-erably.ThechemotypicheterogeneityamongspeciesmaynotallowoptimalRNAyieldfromoneisolationprotocoland,perhaps,evencloselyrelatedspeciesmayrequiredifferentisolationprotocols[5].Asouraimhasbeentostudydrought-induceddifferentialexpressionofgenesduringearlyandlateseeddevelopmentalstagesinsorghum,wemayneedaprotocolthatnotonlycangivethesamequalityandquantityofRNAateachstagebutalsoishighyieldinginordernottomisstherarelyex-pressedgenes.PreviousstandardplantRNAisolationproceduresfailedtoworkwhenappliedtotissuesrichinsecondaryproducts[3,6–8].Inthisreport,aproceduredevelopedbyLogemannetal.[3]wasmodifiedandappliedtosorghumseeds,whichareknowntocontainhighlevelsofpolysaccharides[9].Theextractionmethoddescribedinthisstudyissimple,notrequiringultra-centrifugationoradditionalprecipitationsteps,andal-lowsRNAextractionfromplantspeciesinwhichotherprocedureshavepreviouslybeenunsuccessful.Further,thequalityoftheisolatedRNAwasconsistentlyhighasindicatedbyspectrophotometricreadingsanditssepa-rationondenaturingagarosegels.TheyieldandqualityweresuitableforRT-PCRandNorthernblothybrid-ization.Seeds of sorghum (Sorghum bicolor cv. CSH-6)werepurchasedfromtheNationalSeedCorp.,(Pusa,NewDelhi,India)andseedsofchickpea(Cicerarieti-num),andsoybean(Glycinemax)werepurchasedfromPunjabAgriculturalUniversity(Ludhiana,India).Theseedsweresurfacesterilizedwith1%(w/v)mercuricchloride and 70% ethanol followed by rinsing withdeionizedwater.Seedswereimbibedfor6hindouble-distilledwaterat37 Candusedforfurtherstudies.Leavesofsoybeanandchickpeawerecollectedfrom6-day-oldgerminatedseedlingsgrowninaseedgermin-ater.Tissueswerestoredinliquidnitrogenforfurtheranalysis.Solutionsandreagentsusedwereasfollows:ho-mogenization solution (Solution I), NaCl (5M),Sarkosyl(2%);guanidinehydrochloridebuffer(pH7.0),8Mguanidinehydrochloride,20mMEDTA,20mMMes,

Purification and characterization of an exoinulinase from Aspergillus fumigatus.

An extracellular exoinulinase was purified from the crude extract of Aspergillus fumigatus by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-Sephacel, Sephacryl S-200, concanavalin A-linked amino-activated silica, and Sepharose 6B columns. The enzyme was purified 25-fold, and the specific activity of the purified enzyme was 171 IU/mg of protein. Gel filtration chromatography revealed a molecular weight of about 200 kDa, and native polyacrylamide gel electrophoresis (PAGE) showed an electrophoretic mobility corresponding to a molecular weight of about 176.5 kDa. Sodium dodecyl sulfate-PAGE analysis revealed three closely moving bands of about 66, 62.7, and 59.4 kDa, thus indicating the heterotrimeric nature of this enzyme. The purified enzyme appeared as a single band on isoelectric focusing, with a pI of about 8.8. The enzyme activity was maximum at pH 5.5 and was stable over a pH range of 4.0–9.5, and the optimum temperature for enzyme activity was 60°C. The purified enzyme retained 35.9 and 25.8% activities after 4 h at 50 and 55°C, respectively. The inulin hydrolysis activity was completely abolished with 1 mM Hg++, whereas EDTA inhibited about 63% activity. As compared to sucrose, stachyose, and raffinose, the purified enzyme had lower Km (0.25 mM) and higher Vmax (333.3 IU/mg) values for inulin.

Ethanol mediated enhancement in bacterial transformation

In molecular biology, transformation using E. coli as a host plays a key role in synthesizing gene libraries. The present study demonstrated a new ethanol-based method for transformation of plasmid DNA to E. coli . Ethanol at 10% concentration (v/v) showed best results. Further, as compared with traditional CaCl 2 method, the transformation rate, using protocol outlined in this study, was very high, suggesting amenable for further applications.