【作者】 申斯乐；

【导师】 邹广田；

【作者基本信息】 吉林大学 ， 凝聚态物理， 2006， 博士

【摘要】 利用高压手段对种子植物及真菌进行处理,考察高压对种子植物和真菌的生理生化影响及诱变作用。发现高压对小麦种子萌发及幼苗生长有一定的抑制作用,但这种影响并非随着压力升高而增加,而在某一条件下出现好转。对高压诱变的水稻两个稳定的变异品系进行DNA分子水平上的分析,结果显示原种及两个变异品系不仅存在着基因组结构的变异,而且发生了DNA甲基化模式的改变。首次证明高压可导致水稻多种转座元件的可能激活及DNA甲基化模式的改变。高压对米曲霉形态特征、生理生化性质产生影响,并诱变获得一株理想的变异菌株HP300a,主要酿造指标明显优于对照株。利用高压诱变普鲁兰菌,筛选获得了一株产生物絮凝剂活性明显提高的变异株PB3201,其絮凝活性比出发菌株提高了近12.4%。并且发生了DNA分子水平上的变异,本研究首次为利用高压诱变筛选工业菌种提供了理论与实践依据。继各种化学制剂、各种射线诱变等方法之后,高压不失为一种行之有效的新诱变方法。更多还原

【Abstract】 To study effect and induced-mutant of high pressure on plant and fungi, thedisquisition deals with plant and fungi by high hydrostatic pressure treatment, and analyzeschanges at the level of DNA molecule by technique of ISSR, RAPD and Southern blot.By using high-pressure treatment, germination of wheat seed and growth of wheatseedling were restrained partially, and the inhibition relates with weight and time ofpressure. Induced-inhibition by pressure does not increase continually with increasing ofpressure, and weakens at a certain pressure. Changes on activity of amylase are asphenomena on growth of wheat seedling;activity of peroxidase of wheat seedling wasanalyzed at 20 day growth, and results demonstrated that activity of peroxidase of all oftreatments is better than the control. By low temperature treatment on wheat seedling,proline content does not change obviously;to indicate cold-resistant capability does notchanges visibly by pressure treatment. To verify if activity of α-amylase of wheat seedlingchanges immediately by high pressure, pure α-amylase were treated by high pressure, andwere analyzed by activity of α-amylase and circular dichroism spectrum, demonstratedactivity of α-amylase of wheat seedling by high pressure changes indirectly.By using high-pressure treatment, two genetically stable rice lines were obtained fromthe japonica rice cultivar Bijing38. Mutant 1 is characterized by its lower segregationfrequency and 20 days less of maturation period than the original cultivar. Mutant 2 ischaracterized by its significantly increased height, increased tillering efficiency andelongated kernels which is similar to what has been seen for indica rice. ISSR and RAPDanalysis of these two mutants and the original cultivar revealed that there exists significantgenetic variation between the two mutants and the original cultivar, each with 28.57% and45.98% polymorphic bands respectively. The genetic diversity in Mutant 2 is significantlyhigher than in Mutant 1. ISSR analysis indicated that the mean absolute distance coefficientbetween the original cultivar and the Mutant 1 is 0.0303, while that between the originalcultivar and the Mutant 2 is 0.2613. RAPD analysis demonstrated that the mean absolutedistance coefficient between the original cultivar and the Mutant 1 is 0.0845, while thatbetween the original cultivar and the Mutant 2 is 0.4167. These studies have providedstrong evidence that high pressure could induce mutagenesis during seed germination andthe phenotypes and genetic traits can be stably passed on to the progeny. This is the firsttime we have shown at molecular level that treatment by high pressure can inducegenetically stable mutations in higher plants. In addition to a variety of chemical and x-raymutagenic methods, high pressure might as well be proven as another efficient method formutagenesis.By using high-pressure treatment, two genetically stable rice lines Mutant 1 andMutant 2 were obtained from the japonica rice cultivar Bijing38. The genomic DNA,digested by HpaⅡ/MspⅠ on the original cultivar and two Mutants, were analyzed by themethods of ISSR and RAPD. Meanwhile, the changes of the transposon mPing in rice werestudied through specific primer. A further research was processed on potential activeretrotransposons LTR(long terminal repeat)Osr7,Osr36,Tos19(Osr54)and transposonsMITE(miniature inverted-repeat transposable element)mPing and Pong by Southern blotanalysis with specific fragments as probes. The results show that there are variances in notonly the structure of genome but also the DNA methylation pattern between the originalcultivar and the two mutants. In this work, we demonstrate that genetically stable rice lineson morphological changes could be obtained from seeds of rice treated by high hydrostaticpressure, and morphological changes are based on extensive changes at the level of DNAmolecule. It is first proven that high hydrostatic pressure might induce the activation ofsome transposable elements in rice, and change the DNA methylated patterns. As aconclusion, high hydrostatic pressure can be one of the important factors to induce variationof plants, and it may become a useful mutagenic technique.To study effects of high pressure on livability, morphological changes andphysiochemical properties of fungi, Aspergillus Oryzae was treated by high pressure at0.1-400MPa, for 20 min.;And activity of protease and amylase of Aspergillus Oryzae weredetermined;At the seam time, an pressure-induced excellent strain was screened. The resultshowed that: high pressure obviously influence the livability and modality of AspergillusOryzae. The effects on activity of protease and amylase of Aspergillus Oryzae by highpressure are manifest especial disciplinarian, which is that activity of protease decrease asincreasing of pressure at the bound of 0.1-200MPa and increase as increasing of pressure atthe bound of 200-400MPa. The activity of protease is larger at 300MPa than original strainand maximal at 400MPa. Activity of amylase decrease at 100MPa, is the strongest at200MPa, while it decrease when pressure increase above 200MPa. In addition, the activityof amylase at 400MPa is same as original strain. A pressure-induced mutant strain HP300aat 300MPa was screened which has a series of eximious characteristic: quick growth, morespores, higher activity of protease, and is rarely polluted by other strains. Several mainindexes of soy sauce brewaged by HP300a are all better than control strain without pressuretreated. The study first offer theory basis for inducing and screening excellent strain ofAspergillus Oryzae and for improving quantities and quality of soy sauce product too, andfind especial disciplinarian of changes of protease activity and amylase activity ofAspergillus Oryzae treated by high pressure.To study effects of high pressure on Aureobosidium Pullulans, the strain was treated byhigh pressure at the bound of 80-320MPa, for 20 min. The result show that: high pressureobviously influence the livability and modality of Aureobosidium Pullulans. In addition, apressure-induced mutant strain PB3201 was obtained by screening over 5 generations,which produce flocculation reagent with great efficiency. PB3201 efficiency offlocculation reagent is about 97.5 % , higher above 12.4 % than original strain.Physiochemical properties and DNA of PB3201 were analyzed. The results show that thereare variances in not only the morphological and physiochemical properties but also thestructure of DNA between the original strain and the mutant PB3201.The study at first offer more perfectly morphological, physiochemical and DNAmolecular evidence for pressure-induced mutants of plant and fungi. The results show thathigh pressure is a new induced technique with great efficiency after chemical reagents andradials.更多还原