Abstract:To clone a rice yield QTL,a comparative analysis on the region of the physical maps derived from rice varieties Zhenshan 97 and Minghui 63,the two parents for mapping the QTL,was performed.Physical gaps in this region of the two physical maps were closed.Putative SNPs of this region between Zhenshan 97 BAC end sequences and Nipponbare reference sequence,and that between Minghui 63 BAC end sequences and Nipponbare reference sequence were validated.Results showed that most putative SNPs are correct but some are from errors of BAC end sequence.The confirmed SNPs will be used as important markers in the positional cloning of gene or QTL.Furthermore,representative putative Zhonghua 11 specific repetitive sequences found in our laboratory through Zhonghua 11 BES analysis were comparatively analyzed via Southern hybridization.The results will be helpful in cloning of the rice yield QTL and realizing the evolution of rice repetitive sequences among different rice varieties.

Abstract:To develop a method for the production of avian influenza proteins in wheat as an oral vaccine,a gene encoding hemagglutinin (HA) of avian influenza H5N1 was constructed in a wheat expression vector with promoter of cereal crops. The constructed vector DNA was bombarded into immature embryos of an elite wheat cultivar Zheng 9023. Calluses induced on media supplemented with 5 mg/L phosphinothricin (PPT) were screened and transferred to media containing 4 mg/L PPT for rooting and regeneration. The PPT-resistant green plantlets derived from the rooting media were identified by PCR,and the transgenic wheat plants carrying the HA gene were obtained. Southern blot analysis of T1 transgenic plants demonstrated that the HA gene was indeed integrated into the wheat genome with a single copy integration pattern. These results provide materials for further investigating expression of avian influenza HA gene and accumulation of its protein in wheat seeds.

Abstract:Nine strains of endophytic fungi were isolated from rapeseed and classified into 4 genera through morphological and molecular biological identification.Plate confronting test showed that the endophytic Chaetomium globosum had different inhibition on Sclerotinia sclerotiorum,Rhizoctonia solani,Botrytis cinerea and Fusarium graminearum,etc.The mechanism of antagonism C.globosum and R.solani with confrontation on slide found that C.globosum had apparent process of mycoparasitism during the confrontation with R.solani.

Abstract:The favorable condition for the growth and antagonistic production of a Paenibacillus kribbensis PS04, a biological control agent,by single factor experiments was presented in this study. The results showed that the best carbon source was sucrose,the best nitrogen was NaNO3. The best composition of the media contained: sucrose 3%, NaNO3 0.15% and K2HPO4 0.1% by the orthogonal experiment. Subsequently the culture conditions were optimized and the results showed the medium initial pH was 7-8, the culture temperature was 30 ℃, the quantity of medium was 100 mL in a 1 000 mL flask, the culture cycle was 84 h, and the rotation speed was 180 r/min respectively.

Abstract:In present study,porcine ovaries were obtained and aspirated.Cumulus-oocyte complexes were followed by in vitro culture until the presence of the first polar body(pbⅠ).We investigated the effects of basic culture medium,hormone,follicle diameter,and oocytes wrapped cumulus cells on in vitro maturation of pig oocytes for establishing good in vitro culture system.The results showed that the mature rate of oocytes with mTCM199 was significantly higher than NCSU-23(P＜0.05).Based on the results of experiment 1,the maturation rate of cumulus-oocyte complexes in maturation media with PMSG,hCG and pFF were 55.90%,58.16%,and 51.50%,respectively,showing that the combined treatment group (80.63%) was significantly higher than those of single treatment groups.There were no significant differences in the maturation rates of the ovary follicles larger than 6 mm (76.43%) and 3-6 mm(83.33%) in diameter,but both were significantly higher than the ovary follicles less than 3 mm in diameter(30.94%,P＜0.01).The cumulus-oocyte complexes were divided into level A,B and C according to layer of cumulus cells,which were cultured in vitro matured for 44 h,the maturation rates of level A and B were not significant (P＞0.05),but the both were significantly higher than oocytes of level C (P＜0.01).

Abstract:The PCR-SSCP polymorphism of myostation (MSTN) gene sequence of intron 2 and its correlation with growth traits were investigated in 277 individuals from one cultivated yak breeds (Datong yak) and two native breeds（Gannan and Tianzhu White yak).The results showed that there were two alleles and three genotypes in the locus of MSTN gene intron 2.They were at Hardy-Weinberg equilibrium state in this locus while Datong yak obviously was unequilibrium.The least square analysis showed that the different genotype of MSTN intron 2 had a significant effect on partial traits of growth including heart girth,body weight,index of chest circumference,index of body slanting length,beef performance index(P＜0.05).However the genotype did not affect body height,cannon circumference,body slanting length,index of cannon circumference significantly(P＞0.05).In conclusion,the mutation of MSTN intron 2 could be used as the molecular genetic marker for yak breeding.

Abstract:Hybrids from Brassica napus×Brassica oleracea were obtained by embryo rescue techniques,which is an effective approach for increasing the resources of male sterility of Brassica oleracea.Both the DNA content and chromosome number of hybrids were the mid-parent values.Characteristics of the hybrid were investigated.The results showed that hybrids were morphologically similar to parents during the growing periods,while inclined to the female parent during flowering.But hybrids had strong heterosis of growth.The leaf structure,chloroplast number in stomata guard cell,the floral organ,and the early flowering periods of hybrids were mid-parent,except the mutated leaf structure of mosaic hybrid.Hybrids had their own unique peroxidase isozymes bands.Test of the pollen viability showed that hybrids bore only non-viability pollens with 100% sterility.Backcross generation could be obtained only through embryo rescue techniques to overcome barriers of hybridization.

Abstract:Paraffin sectioning and microscopic technique are used to reveal the mega-/micro-sporogenesis and female-/male-gametogenesis of Hosta plantagine Aschers.The relationship between the developmental stage of pistil and stamen and the size of bud were analysed to further study the molecular mechanisms of the double flower.The results showed that the form of host megaspore development was fritillaria typed embryonic.The anthers of the fertile line had four microsporangiates and the tapetum was belonged to the glandular type.Consecutive cytokinesis in microspore mother cell accompanies meiosis to produce tetrahedral tetrad.The mature pollen is 3-cellular type with two small sperms and one big vegetative cell.

Abstract:The genetic diversity of eight natural populations of Rosa odorata Sweet var.gigantea Rehd.et Wils.in Yunnan Province was analyzed with SSR markers.The result showed that 72 bands were amplified with twelve polymorphic primers.Polymorphic loci were 70 and percentage of polymorphism was 97.22%.At the level of species,the Nei’s genetic diversity was 0.301 2 and the Shannon information index was 0.460 0,showing that the genetic diversity of the populations of Rosa odorata Sweet var. gigantea in Yunnan Province was abundant.It was 69.49% of genetic variation within populations,and 30.51% of genetic variation among populations.So the genetic variation within populations was the main reason of the genetic variation.The Nei’s pairwise genetic identity and genetic distance were 0.761 5-0.934 8 and 0.067 4-0.272 4.The cluster analysis based on genetic distance showed that the genetic distance was not significantly related with the geographic location.

Abstract:Nematodes of the genus Bursaphelenchus collected from Guangdong Province were identified by using morphology, rDNA-ITS-RFLP and rDNA-ITS-PCR-sequencing. B.fungivorus and B.doui were isolated from a growing substrate for Ficus microcarpa in Panyu and wood packaging material in Dongguan respectively. B.fungivorus was a new geographic record species in China, and B.doui was a new geographic record species in Chinese Mainland.

Abstract:In this paper, a full cDNA encoding LAC-1 from Kaburagia rhusicola Takagi was obtain by RT-PCR and RACE techniques. The complete cDNA（2 327 bp）contains a 1 773 bp open reading fram encoding 597 amino acid residues and flanked with a 449 bp 5′UTR and a 105 bp 3′ UTR. The predicted molecular weight of this complete amino acid is about 67 ku with an isoelectric point of 6.31. Compared with other insect laccase, the cloned gene shares four conserved anino acid sequences motife HWHGHH, THFWHSH, HPFHLHGH and WLFHCHIEFH. The analysis of phylogenetic relationship showed that the clonde gene has high amino acid sequences homology with other insect laccase. 

Abstract:The natural dynamics of the population density and mound density of imported fire ants Solenopsis invicta Buren has been studied from April 2009 to March 2010 in Shenzhen City. The results were as follows: the population density of workers appeared in curve with multiple peaks, the primary peak during late April to early July, the second peak during early September to late October, and the rest during mid February to late March in order. The mound density began to increase in late February, with the peak during early May to late August, and it started to decrease in early September until the bottom in mid February of the next year; afterwards, it began to rise in late February. New mounds appeared respectively during April to July, in November and in February and March of the following year, with the highest density in mid April. The disappearance of the mounds occurred throughout the year, except in May, June, August and next February, March, and the highest number of mound disappearance occurred in early September. The population density and surface temperature, active mound density and ten day average temperature there have a positive correlation.

Abstract:On the basis of the data of the outbreak years of Nilaparvata lugens (Stl) in eastern Asia during 1912-2006, the outbreak years of N.lugens in southeastern Asia during 1959-2010, and the relative number of sunspots from 1880 to 2009, by adopting the methods of correlation analysis, phase analysis, key time analysis and cycle analysis, this paper studied the relationship between the sunspot cycle and the outbreak years of N.lugens in Asia. The results showed that there was a very significant positive correlation between the outbreak year (grade) of N.lugens in both eastern Asia and southeastern Asia and the relative number of sunspots in the 17th year before that (P<0.01). The outbreak years of N.lugens in both eastern Asia and southeastern Asia appeared mostly at the declining stage of sunspot activity cycle. Sunspot activity cycle had no significant influence on the outbreak year of N.lugens in both eastern Asia area and southeastern Asia area (P>0.10). The periodicity of the outbreak of N.lugens is 8-9 years in some parts of eastern Asia, while it is 1-2 years in parts of southeastern Asia.

Abstract:Fifty-one different plant samples from Shiwandashan Mountain were involved in screening new compounds against mites through methods such as the immersion of leaf disks, the glass piece method and so on. In this process, the toxic activities of the 51 samples′ methanol extracts on female adult mite of and Panonychus citri Me Gregor were determined. The research results are listed as follows: 11 kinds of plants showed good toxic activities on Tetranychina hart（Ewing）female adults. They are Rhamnella crenata,Psychotria ruba,Mallotus barbatus,Derris glauca,Rhus silvestris,Sophora flavescens,Alangium kurzii,Gnetum panvifolium,Trachelospermum jasminoides,Clemalis crassifolia and Breynia fruticosa. Meanwhile,Trachelospermum jasminoides,Rhus silvestris and Cassytha filiformis L. exhibited good toxic activity on Panonychus citri Me Gregor female adults.

Abstract:Erosion restoration measures such as,polyacrylamide (PAM),straw mulching,zonal forage (paspalum notatum),and the combination of them were used in the eroded plots of red soil rotated by peanut and wheat in southern area of Hubei Province.The water stable aggregates(more than 4.00,2.00-4.00,1.00-2.00,0.50-1.00,0.25-0.50 mm) and the content of available N,P and K in the eroded red soil was detected to reveal the effects of erosion restoration measures on aggregate stability and available nutrient.The results showed that straw mulching could significantly increase the contents of 1.00-2.00 and 2.00-4.00 mm water stable aggregate (WSA),and the increase rate was 40% for the contents of more than 0.25 mm WSA.In the straw mulching treatment,the contents of organic matter and available K separately increased 20% and 150% after planting wheat.The measures of PAM and the combination of PAM and zonal forage could increase the content of 0.50-1.00 mm WSA,and rates were 25% higher than that of the control,but they were not significant for the increase of the soil organic matter and available nutrients.The combination of zonal forage and straw mulching could significantly increase the content of more than 4.00 mm WSA by 500% compared to the control,but no obvious influences to the soil organic matter and available nutrients.The measure of straw mulching was better than the combination of straw mulching and zonal forage for increasing the content of 0.25-4.00 mm WSA.In improving the content of more than 0.25 mm WSA,the effect of the composition of PAM and zonal forage was better than the measure of zonal forage,but less than the measure of PAM for increasing the content of 0.25-1.00 mm WSA.

Abstract:A field experiment was carried out to study the effects of combining application of green manure and chemical fertilizer on the growth and development,yield and quality of flue-cured tobacco in dry year.The results showed that the growth of flue-cured tobacco was inhibited in the dry year,especially under no fertilizer applying.Meanwhile the disease of flue-cured tobacco severely occurred,the yield and output value were significantly decreased.In comparison with single chemical fertilizer,combination of green manure and chemical fertilizer significantly increased the plant height,the length and width of the max leaf and stem circumference of flue-cured tobacco,decreased the incidence of granville wilt and tobacco mosaic virus,enhanced the dry weight of root,stem and leaves of flue-cured tobacco.The higher the percentage of green manure accounted for the total rate of fertilizer application,the higher was the yield and output value of the flue-cured tobacco.To compare with single chemical fertilizer application,the yield and output value of flue-cured tobacco that the green manure were applied were 1.16-1.71 times and 1.09-2.16 times,respectively.Combining application of green manure and chemical fertilizer increased the content of nicotine and potassium in all leaves,decreased the ratio of reducing sugar to nicotine in all leaves and the ratio of total nitrogen to nicotine in the middle and lower leaves,and improved the ratio of total nitrogen to nicotine in upper leaves of the flue-cured tobacco.These suggested that the harmony of the chemical component of flue-cured tobacco would be raised when green manure was ploughed in.

Abstract:Three wild polygonaceae plants,Polygonum lapathifolium,Polygonum perfoliatum and Polygonum hydropiper,were used to study the physiological responses under five aluminum (AlCl3) concentrations (0,25,50,100,400 μmol/L) with solution culture method.It was found that relative root elongation was decreased with the increase of AlCl3 concentrations in polygonaceae plants,but the magnitude of decrease was higher in Polygonum perfoliatum than that in Polygonum lapathifolium and Polygonum hydropiper,suggesting that Polygonum lapathifolium and Polygonum hydropiper were much more tolerant to Al than Polygonum perfoliatum was.Al contents and oxalate secretion in roots of three polygonaceae plants were increased with increase of AlCl3 concentraions and oxalate secretion showed that a saturating phenomenon at approximately 50 μmol/L AlCl3 in Polygonum hydropiper and Polygonum perfoliatum,while Al contents were significantly lower and the amount of oxalate secretion significantly higher in Polygonum lapathifolium and Polygonum hydropiper than that in Polygonum perfoliatum.When compared with the control,high AlCl3 treatment (400 μmol/L) significantly decreased root activity,leaf activities of peroxides (POD) and catalase (CAT),increased malondialdehyde (MDA) content of three polygonaceae plants,especially in Polygonum perfoliatum.In the low concentration of AlCl3 treatments (25-50 μmol/L),root activity in Polygonum lapathifolium and Polygonum hydropiper showed an increasing trend,and leaf proline content,activities of POD and CAT significantly increased,MDA content significantly decreased compared with the control.These findings suggested that Polygonum lapathifolium and Polygonum hydropiper could enhance the tolerance to Al stress by increasing Al-induced oxalate secretion and maintaining higher proline content,higher activities of POD and CAT,lower MDA content under the low AlCl3 concentration.

Abstract:An 1HNMR method for quantitative detecting 2,4-dichlorophenoxyacetic acid in soil with 1,3,5-benzenetricarboxylic acid（BTC） as internal standard was developed.Proton nuclear magnetic resonance spectroscopy (1HNMR) spectra were obtained in methanol and acetone-d6 with a Bruker AV 600 MHz spectrometer at 25 ℃.For each sample，128 scans were recorded with the following parameters:observing frequency (600.13 MHz)，spectra width (9 001.95 Hz)，90° pulses width (12.10 μs) ，acquisition time (3 s) and delay time (3 s).The linearity range is 30.00-200.00 mg/L,the minimum detection of 2,4-D was 25.0 mg/kg.The average recoveries of 2,4-D in paddy soil and garden soil were 83.54%-90.26% and 85.26%-92.65% with CVs of 3.82%-5.15% and 2.32%-5.89%,respectively.

Abstract:Full length cDNA of silver carp transferrin,Hypophthalmichthys molitrix,was first cloned using reverse transcription polymerase chain reaction (RT-PCR) and SMART RACE methods.The entire transferrin cDNA sequence is 2 365 bp long and the open reading frame is 2 025 bp and encodes a protein with 674 amino acids.Molecular characteristics of the gene were forecasted by online molecular softwares.The results showed that it is composed of two domains,with a signal peptide of 15 amino acids which located at the 21st amino acid of N-terminal.Silver carp transferrin gene has high homology with the other species,sharing the highest identity of 74% with grass carp Ctenopharyngodon idella. Phylogenetic tree analysis revealed that transferrin of five Cyprinid fish,Danio rerio,Carassius auratus,Cyprinus carpio,Ctenopharyngodon idella and Hypophthalmichthys molitrix were classified together.Siler carp transferrin mRNA only expressed in liver and spleen (a higher expression level in liver).During embryogenesis,the expression of transferring mRNAs was first detected at the gastrula stage and the expression level increased steadily until the tail bud embryo.Transferrin mRNA showed marked reduction after the stage of tail bud embryo until larvae.Results from the present study indicated that transferrin played an important role in organogenesis during embryogenesis.

Abstract:Healthy red swamp crayfish,Procambarus clarkii were artificially infected by injected with Aeromonas hydrophila at the concentration of 1.5×104（GroupⅠ） and 1.5×106 cfu/mL（GroupⅡ）.Immunity factors including total haemocyte counts (THCs),phenoloxidase (PO) activity,superoxide dismutase (SOD),lysozyme activity and antibacterial activity in the haemolymph were examined at 1-96 h post infection at regular time.The results showed significant differences were observed in immune indicators of two infected groups during 8-48 h post infection.THCs and SOD activities of two treatment groups significantly decreased after 8-48 h compared to control,and THCs,SOD activities of groupⅠ decreased to the lowest level after 12，24 h,and returned to original level after 48，96 h respectively.However,THCs,SOD activities of groupⅡ decreased to the lowest level after 48 h,and recovered gradually after 96 h.Lysozyme activity，antibacterial activity and PO activity of groupⅠwere significantly higher than those of the control group after 12-48 h,and reached to the top level after 24，24 and 48 h,respectively,and finally returned to original level after 96 h.Significant decrease was observed in lysozyme activity，antibacterial activity and PO activity of groupⅡ after 12-48 h,and the lowest level was observed after 48 h,then gradually recovered after 96 h.

Abstract:The investigation of phytoplankton community characterization including species composition,population size,dominant species and saprobic indicators was conducted in order to evaluate the water quality of Majia and Tuhai River in Haihe River basin.The results revealed that 126 species of phytoplankton belonging to 7 phyla and 67 genera existed in Majia River,while 114 species of phytoplankton belonging to 7 phyla and 63 genera were found in Tuhai River.Chlorophyta was the richest algae in both rivers,followed by Bacillariophyta and Cyanophyta.Cyanophyta,Chlorophyta and Bacillariophyta were found in all the sampling sites (the frequency was 100%).Cyanophyta was absolute the majority in Majia River in population,which accounted for 53.59%,while the Chlorophyta was predominant in Tuhai River,which accounted for 35.73%.The average density of phytoplankton in Majia and Tuhai River were 22.424×106 ind./L and 13.950×106 ind./L,respectively.The Shannon-Weaver index,Margalef index and Pielou index varied among 2.48-4.64,3.62-9.91 and 0.47-0.84 in Majia River,while 3.04-4.57,2.94-10.15 and 0.63-0.82 in Tuhai River,respectively.The water quality of both rivers was preliminary evaluated based on above results.In conclusion water quality was good,can be labeled as mesotrophic,but had the potential to turn to eutrophication.So we should pay attention to protect the water quality and enhance the management of the both rivers.

Abstract:A green manure embedding machine was developed according to the operational principle of rotary spade. The main structure and working process of the machine were determined. The lengthwise edge of the blades can effectively prevent the blades from winding the grasses by using the sine index curve. A top plate was equipped to assist the furrow slice to turn over. A soil scraping device was equipped to scrape the soil adhering to the blade. The results of field tests showed as follows: the green manure coverage rate was more than 85%，the maximum tillage depth was 200 mm. The power consumption of the machine was lower. This machine meets the design requirements and returns green manure，rice straws and wheat straws to the field through mechanization operation. It would be important, practical and valuable to improve the utilization ratio of the existing straws.

Abstract:The roles of nitric oxide (NO),a significant gas signaling molecule,in regulating plant physiology progress and modulating signal transduction is being a novel hotspot of researches.In recent decades,a great deal of important progress have been achieved on the roles of NO in the generation and development of plant root,especially on the generation of endogenous NO from plant roots,the details of NO-participated progress of root generation and development,and the related signaling pathways.In order to get further understanding of the roles of NO in plants,the update advances in studying the generation of endogenous NO in plant root and the mechanisms and signaling pathways of NO in promoting generation and development of plant root are reviewed.

Abstract:Typical zonal soil such as Brown soil was sampled from Tianwai village,Taishan,Shandong Province in China.Two soil colloidal components i.e,fine clay (＜0.2 m) and coarse clay (0.2-2.0 m) were separated by centrifugation.Two treatments applied to fine and coarse clays were organic matter left on the samples (organic clays) and organic matter removed from the samples by H2O2 (inorganic clays).Brown soil was divided into four types of clays:coarse organic clay (0.2-2.0 μm,organo-mineral complexes),coarse inorganic clay (0.2-2.0 μm,H2O2-treated clay),fine organic clay (＜0.2 μm,organo-mineral complexes) and fine inorganic clay (＜0.2 μm,H2O2-treated clay).The adsorption,desorption and binding mechanism of DNA on Brown soil colloid or mineral such as montmorillonite,hydroxyaluminum-montmorillonite,kaolinite and goethite,the ability of transforming competent cells of bound DNA and the resistance to DNaseⅠ degradation,the PCR amplification of bound plasmid DNA and the effects of soil active particles on microbial metabolic activities were investigated.The main results were as following:1.Adsorption-desorptionof DNA,ATR/FTIR,circular dichroism (CD),fluorescence spectroscopy,microcalorimetry were used to clarify the adsorption mechanism of DNA on permanent-charge soil active particles.The maximum amount of DNA adsorbed followed the order:montmorillonitefine inorganic clay>fine organic clay>kaolinite>coarse inorganic clay>coarse organic clay.A marked decrease in the adsorption of DNA on organic clays and montmorillonite was observed with the increase of pH from 2.0 to 5.0.Little DNA was adsorbed by organic clays above pH 5.0.As for inorganic clays and kaolinite,a slow decrease in DNA adsorption was found with increasing pH from 2.0 to 9.0.Magnesium ion was more efficient than sodium ion in promoting DNA adsorption on soil colloids and minerals.DNA molecules adsorbed on soil colloids and minerals were desorbed by sequential washing with 10 mmol/L Tris-HCl,100 mmol/L NaCl and 100 mmol/L sodium phosphate buffer at pH 7.0.A percentage of 53.7%~64.4% of DNA adsorbed on organic clays and montmorillonite was released,while only 10.7%~15.2% of DNA on inorganic clays and kaolinite was desorbed by Tris-HCl and NaCl.The percent desorption of DNA from inorganic clays,organic clays,montmorillonite and kaolinite by sodium phosphate buffer was 39.7%~42.2%,23.6%~28.8%,29.7% and 11.4%~29.7%,respectively.DNA adsorption on organic clays was endothermic (1.1 ＜△Hads＜3.5 kJ/g),whereas that on inorganic clays was exothermic (-0.3kJ/g＜△Hads＜-0.1 kJ/g).Dehydration effects and electrostatic interactions dominated DNA adsorption on organic clays and montmorillonite,and DNA was adsorbed predominantly by ligand exchange and possibly hydrogen bonding on inorganic clays and kaolinite.ATR/FTIR spectra showed that the binding of DNA on kaolinite and inorganic clays changed its conformation from the B-form to the Z-form,whereas montmorillonite and organic clays retained the original B-form of DNA.A structural change from B- to C-form in DNA molecules desorbed from kaolinite was observed by CD spectroscopy and confirmed by fluorescence spectroscopy and DNA molecules desorbed from soil colloid or montmorillonite were still B-form.2.The effects of various organic and inorganic ligands on DNA adsorption on active soil particles were studied.An obvious decrease in DNA adsorption was observed on montmorillonite and kaolinite with increasing anion concentrations from 0 to 5 mmol/L.However,the amount of DNA adsorbed by montmorillonite and kaolinite was enhanced when ligand concentration was higher than 5 mmol/L.In the system of soil colloids,with the increase of anion concentrations,a steady decrease was found and the ability of ligands to depress DNA adsorption followed the order of phosphate>citrate>tartrate.Compared to inorganic clays,a sharp decrease in DNA adsorption was observed on organic clays with the increase of ligand concentration.The results indicated that the influence of anions on DNA adsorption varies with the type and concentration of anion as well as the surface properties of soil components.Introducing DNA into the system before the addition of ligands had the maximum amount of DNA adsorption on soil colloids.Organic and inorganic ligands promoted DNA adsorption on montmorillonite and kaolinite when ligands were introduced into the system before the addition of DNA.3.Adsorption and desorption of DNA on Bacillus thuringiensis and Pseudomonas putida and their composites with soil colloids or minerals were investigated.B.thuringiensis and P.putida did not show significant difference in the amount of DNA adsorption although the two bacterial cells had different surface properties.DNA was adsorbed on bacteria mainly through van der Waals force and electrostatic force.Compared with B.thuringiensis,DNA adsorbed by P.putida was desorbed more easily.There was no significant difference in the amount of DNA adsorption on kaolinite between the absence and the presence of P.putida.Except for the effect of P.putida on DNA adsorption on kaolinite,the presence of B.thuringiensis and P.putida significantly promoted DNA adsorption on soil colloids and minerals,and the promotion of B.thuringiensis was stronger than that of P.putida in the system of soil colloids. 4.Adsorption of DNA on different hydroxyaluminum-montmorillonite complexes (Al(OH)x-M) containing 2.5,10.0 and 20.0 mmol coated Al/g clay (AM2.5,AM10 and AM20) was studied in Tris-HCl and sodium phosphate buffers at pH 7.0.The coatings of montmorillonite by hydroxyaluminum species decreased the amount of DNA adsorption,but increased the affinity of DNA adsorption.At the same pH,the amount of DNA adsorption on montmorillonite or Al(OH)x-M complexes in sodium phosphate was greater than that in Tris-HCl,suggesting that the nature of a buffer solution strongly affected DNA adsorption on clays.As for Al(OH)x-M complexes,the higher the level of Al(OH)x coatings,the lesser the amount of DNA was adsorbed in sodium phosphate buffer.The reduction of DNA adsorption in sodium phosphate buffer with the increase of the level of Al(OH)x coatings may be ascribed to the strong competition of phosphate anions with DNA molecules on surface sites of Al(OH)x-M complexes.An increase of the concentration of Ca2+ and/or a decrease of the values of pH helped DNA adsorption on montmorillonite and Al(OH)x-M complexes.The desorption percent of DNA from montmorillonite,AM2.5,AM10 and AM20 was 65.01%,30.00%,8.04% and 5.18% by Tris-HCl buffer.It suggests that the larger the OH-Al loading on M surface,the greater the binding energy of DNA.DNA adsorption on montmorillonite was endothermic (△Hads=1.15 J/g),whereas that on Al(OH)x-M complexes was exothermic (-9.50 J/g＜△Hads＜-6.64 J/g).The bases and phosphate groups of DNA are involved in DNA adsorption on clays and DNA changes its conformation from the B-form to the Z-form as the result of its binding on AM10 and AM20.Electrostatic forces,hydrogen bonding and ligand exchange dominated DNA adsorption on Al(OH)x-M complexes.SEM showed that a thin layer was formed on the surface of AM10 after the binding of DNA,while that was not observed on montmorillonite surface.5.Electrophoresis and thermometric TAM III were used to investigate the degradation of chromosomal DNA in the system of active soil particles or bound on soil colloid or mineral by DNaseⅠ.When nuclease concentration was 2.0μg/mL,DNA was completely degraded.In systems of kaolinite and coarse and fine inorganic clays,DNA was degraded to 2-4 kb segments at 20 μg/mL of nuclease.For DNA in systems of montmorillonite and coarse and fine organic clays,no evident change was observed in the patterns with 40 μg/mL nuclease in comparison with no nuclease.The heat released from the hydrolysis of DNA,free or bound on coarse inorganic clay,coarse organic clay,kaolinite and montmorillonite by the nuclease was -4.76,-4.06,-2.38,-2.36,-0.22 mJ,respectively.These results indicated that soil colloids and minerals could exert an effective protection for DNA to resist degradation by the nuclease.Among the soil colloids and minerals studied,montmorillonite and organic clays provide more protection for DNA against degradation by DNaseⅠ than kaolinite and inorganic clays.The protection of DNA was not a result of the adsorption affinity of DNA for soil colloid or mineral and the changes in DNA structure.The presence of organic matter and an efficient adsorption of nucleases on soil colloids and minerals appeared to be responsible for the lower degradation of DNA in soil ecosystems.6.The ability of bound plasmid p34S DNA on soil colloids and minerals to transform competent cells of CaCl2-treated Escherichia coli,and the resistance of bound plasmid DNA to degradation by DNaseⅠ were investigated.The transformation efficiency of bound plasmid DNA increased with increasing concentrations of Ca2+ at which soil colloid or clay mineral-plasmid DNA complexes were formed.Plasmid DNA bound by kaolinite showed the lowest transformation efficiency,and especially no transformants were observed with kaolinite-plasmid DNA complex prepared at 5-100 mmol/L Ca2+.Compared with organic clays and fine clays,plasmid DNA bound on inorganic clays and coarse clays showed a lower capacity to transform E.coli at different Ca2+ concentrations.Transformation by 10 μg of free plasmid DNA was inhibited 99.8% by 10 ng of DNaseⅠ.As for the same amount of plasmid DNA bound by soil colloids and montmorillonite,100 ng of DNaseⅠ resulted in 92.3%-93.8% inhibition of transformation by plasmid DNA bound on inorganic clays,whereas 2000 ng of DNaseⅠ caused only 64.0%-98.0% inhibition of transformation by that on organic clays and montmorillonite.The percentage of reduction of transformants by plasmid DNA bound on coarse clays was higher than that on fine clays.Montmorillonite,organic clays and fine clays showed stronger protective effects for plasmid DNA than that of inorganic clays and coarse clays.The adsorption affinity of plasmid DNA for soil colloid or mineral and a conformational change in the plasmid DNA molecule bound on clays may determine the efficiencies of transformation.7.The polymerase chain reaction (PCR) was used to amplify a 600-base pair (bp) sequence of plasmid pGEX-2T DNA bound on soil colloidal particles and three different minerals (goethite,kaolinite,montmorillonite).DNA bound on soil colloids,kaolinite,and montmorillonite was not amplified when the complexes were used directly but amplification occurred when the soil colloid or kaolinite-DNA complex was diluted 10- and 20-fold.The montmorillonite-DNA complex required at least 100-fold dilution before amplification was detectable.DNA bound on goethite was amplified whether the complex was used directly,or diluted 10- and 20-fold.The PCR amplification of mineral-bound plasmid DNA was markedly influenced by the types and concentrations of minerals used.8.The thermodynamic data of the metabolic activity of E.coli as influenced by soil colloids and minerals were analyzed.The growth rate constant (k) of E.coli in LB was 0.074/min,and the k values of E.coli in the system of coarse inorganic clay,kaolinite,coarse organic clay,montmorillonite and goethite were 0.073,0.058,0.054,0.045 and 0.020/min,respectively.It suggested that the selected soil colloids and minerals inhibited significantly the exponential growth of E.coli.The inhibitory ability of the three minerals on metabolic activity of E.coli followed the sequence of goethite>montmorillonite>kaolinite.Compared with inorganic clay,organic clay showed a higher inhibitory effect on metabolic activity of E.coli.

Abstract:Studies on plant resistant genes are one of the hotspots in the field of plant molecular biology.Until now,among the over 50 isolated resistance genes,only 6 are recessive genes.The mechanisms of recessive genes can not be explained by those of dominant resistant genes,which have been universally accepted.Bacterial blight,caused byXanthomonas oryzae pv.oryzae (Xoo),is one of the most serious diseases of rice worldwide.Thirty-two bacterial blight resistance genes (23 dominant genes and 9 recessive genes) in rice have been identified.However the xa13,located on the distal end of the long arm of rice chromosome 8,was a typical recessive R gene,which is fully recessive and specifically confers resistance to the Philippine Xoo race 6 (PXO99).The xa13 gene can interact with other dominant or recessive R genes,indicated that the disease resistant mechanism of xa13 was special.To clone xa13 gene,a normalized whole-life-cycle cDNA library was firstly constructed with different tissues from 9 developmental stages using rice cultivar Minghui 63,based on the strategy of saturation hybridization with genomic DNA.Then a physical map covering the region flanked by markers E6a and S14003 was constructed by chromosome walking using Minghui 63 BAC clones.And finally xa13 was fine-mapped to a DNA fragment of 9.2 kb using a series of sequence-based molecular markers.Sequence analysis indicated that this region contain only one candidate gene,which was annotated by full length EST sequence that isolated from the constructed cDNA library.Transforming IRBB13 with the DNA fragment encompassing the Xa13 candidate that based on the optimized tissue cultural system,including the promoter region isolated from IR64,produced 45 independent transformants.Thirty of the 45 T0 transgenic plants were susceptible upon infection by PXO99.PCR analysis demonstrated that all of the susceptible transgenic plants had the band derived from the Xa13-candidate fragment,while none of the resistant plants amplified this band.T1 families derived from four of the single-copy T0 transgenic plants were further investigated.And the susceptibility cosegregated perfectly with the transgene in all four families,indicated that the clone of xa13 gene through map based cloning strategy has succeed,and the candidate gene in this fragment was indeed Xa13,and the allelic gene in recessive parent was xa13.At the same time,the expression of Xa13 or xa13 was suppressed in different rice lines using the RNA interference (RNAi) strategy.It was found that suppression of Xa13 expression in rice line Zhonghua 11 and MH63 significantly increased the resistance to PXO99,and the level of increased resistance was associated with the reduced accumulation of Xa13 transcripts.Suppressing the expression of xa13 in rice line IRBB13 further enhanced xa13-mediated resistance.Interestingly,the expression of Xa13 or xa13 was very low in leaves but quite high in panicles and anther.And it was found that some RNAi transgenic plants with significantly reduced expression of xa13 or Xa13 had reduced spikelet fertility.The reduced fertility could be ascribed to male sterility as the xa13- and Xa13-suppressed plants had smaller anthers than its wild types and produced mostly abortive pollen.Histological analysis showed that the development of the most of microspores stopped at the unicellular pollen grain stage or bicellular pollen grain stage and gradually degenerated afterwards in those transgenic plants.Coincided with the development stage of abnormal pollen appearance,the expression of Xa13 in anther was identified by in situ hybridization.Results showed that no transcripts were detectable at early stage and Xa13transcripts accumulated to high level at unicellular pollen grain stage.The coincidence in time between the accumulation of Xa13-transcripts in wild type plants and the appearance of pollen abortion in Xa13-suppressed plants during pollen development suggests an indispensable role of Xa13 in pollen development.Further researches showed that the expression of Xa13 in leaves was modulated by pathogen inoculation.The core mutation resulted in loss-of those functions in xa13 genes may occur at a 18 bp region in the promoter region,rather than on gene coding region.And this mutation wouldn’t influence the expression of xa13 in anther.Xa13 is a unknown function plasma membrane protein,belonging to the MtN3 gene family.It acts as important functions both during in the process of PXO99 aggression and anther development.Therefore it would mediate a new pathway of resistance independent from those by activated expression of pathogenesis-related genes or by increased thicken of secondary cell-wall in vascular bundle element of leaves.Above all,the cloned xa13 and its dominant allele Xa13 gene in this study,would not only display a new resistance mechanism:suppressing the expression level of resistance gene is the foundation of plant resistant to pathogen with high efficiency,but aslo provide a new jumping-off point for the study on the relationship between resistance to disease and anther development in plant.