Abstract:Plant height,heading date and number of spikelets per panicle are important agronomic traits of rice.Using BC3F2 population derived from the cross Zhenshan97/ Pokkali,a main effect QTL for plant height designated Qph1,significantly influenced the number of spikelets per panicle as well,was located on chromosome 1 near sd-1.Near isogenic line population,in which Ghd7 and Qph1 were simultaneously segregated,was constructed to analyze their interaction.The results showed Pokkali carried positive alleles at both Ghd7 and Qph1 loci,epistatic interaction between Ghd7 and Qph1 affected plant height.Further analysis found that additive×dominance and additive×additive effect affected plant height.Comparing the phenotypes of nine genotypes in BC3F3 population,the genotype of Zhenshan 97 homozyogous at Qph1 but Pokkli homozyogous at Ghd7 was found to well coordinate the relationship among plant height,heading date and spikelets per panicle,and the yield could reach to a reasonable level.

Abstract:Simple and useful evaluation indexes for the high temperature stability of photosynthesis (HTSP) in different rice varieties was studied for screening and identifying germplasm resources and new variety breeding.The diurnal changes of photosynthesis were tested under high temperature both in natural and illuminating incubator conditions using CIRAS-I portable photosynthesis system.Meanwhile,the genetic characters of HTSP were analyzed using F2 population derived from Texianzhan,a worse HTSP variety,and Shuhui 527,a better HTSP variety,respectively.The results showed that the difference value (D-value) of net photosynthetic rate (Pn) at 09:00 a.m and 13:00 p.m,as well as stomatal conductance (Sc),in flag leaf could be regarded as the evaluation indexes for high temperature stability of photosynthesis (HTSP) in the natural condition.The D-value of Pn of individual plant from F2 population of Shuhui 527/Texianzhan showed a continuous distribution at 09:00 a.m and 13:00 p.m under high temperature.Based on the proportion of D-value of individual plant to population,10%～30% accounted for the maximum part.In addition,the aptness test of D-value distribution curve and theoretic normal distribution of Pn in F2 generation indicated that,the trait of midday depression in photosynthesis was inherited as quantitative character controlled by polygene,other than a simple qualitative character.It was suggested that the identification of and screening HTSP should be carried out in advanced generation populations.

Abstract:An E.coli-expression mutant library containing 1 000 cry mutant genes was constructed using DNA family shuffling method with five initial cry genes cry1Ac,cry1Ab,cry1C*,cry2A* and cry9C*.The 1 000 clones were expressed in E.coli and their toxicity was tested upon Helicoverpa armigera (cotton bollworm) larvae.The expression level of mutation clones were quantified by ELISA.Compared with the original Cry1Ac,there were 122 clones with significantly enhanced toxicity,38 clones with similar toxicity; 232 clones with decreased toxicity; and 608 clones with lost toxicity.All 1 000 cry mutant genes in the library were sequenced and amino acid sequence homology of all cry mutant genes was analyzed by multiple comparisons.Gene sources of 945 clones were found and divided into 5 classes based on homology,55 clones were unrecognized.According to toxicity level,the 1 000 clones were further divided into 3 classes:increased toxicity,similar toxicity and decreased toxicity.Analyzing the amino acid sequences of the 1 000 clones,the homology in 5 decreased toxicity classes was lower than increased toxicity classes and similar toxicity classes.

Abstract:AsE246 and AsIB259 were two root nodule-specific expressed nodulin genes encoding nsLTP1 (non-specific lipid transfer protein 1).The temporal-spatial expression characteristics of two target genes during root nodule development and nitrogen fixation process were investigated with real-time quantitative PCR.Meanwhile,the dynamic changes of expression level of target genes under heavy metal cadmium stress condition were also examined.The results showed that two target genes were strictly expressed in symbiotic organ-root nodules,and reached the peak level at about 22 days after inoculation of Mesorhizobium huakuii 7653R.It was also found that the expression levels of AsE246 and AsIB259 were significantly increased during the early stage as a response to low-level-cadmium stress.However,either at low- or high-level-cadmium stress conditions,their expression were significantly suppressed during the interim stage of Cd2+ stress treatment comparied with those in normal nodules,and continued to maintain a relative lower level until the final stage.These indicated the two nsLTP1 genes could play a fundamental role in the response mechanism of A.sinicus to cadmium stress.In this work,some convincing data were obtained to confirm that AsE246 and AsIB259 were specific expressed nsLTP1 genes in A.sinicus root nodules. 

Abstract:Using reverse transcriptase PCR method and a bacterial fosmid library screening,an arsenite oxidase gene cluster were isolated from an arsenite-oxidizing bacterium Acidovorax sp.GW2.There are seven genes including aoxRSXABCD putatively encoding the transcriptional regulator AoxR of a two-component signal transduction system (68% identity),a periplasmic sensor histidine kinase AoxS (55% identity),a periplasmic binding protein AoxX (55% identity),arsenite oxidase AoxAB(74% and 71% identity,respectively),nitroreductase AoxC (46% identity) and cytochrome c AoxD (63% identity) respectively.According to the reverse transcriptase PCR experiments,aoxR and aoxS encoding for a two-component system proteins are co-transcribed and located in opposite to structural genes aoxABCD.aoxX and aoxRS are not in the same operon.Functional analyses through gene knock-out of aoxS,aoxX and aoxD showed that aoxS and aoxX are the essential genes in arsenite oxidation of GW2,and the loss of aoxD did not show significant effects on arsenite oxidation.

Abstract:Thirty bacterial strains were isolated from soil using nicotine as the only carbon source and a high nicotine degradation bacterium strain,named as DBA5,was screened out from them.Based on standard morphologica1,physiological properties and 16S rDNA analysis,DBA5 was identified as Arthrobacter nicotianae.When the medium with 4.0 g/L nicotine,DBA5 could grow well and degrade 93.32% of nicotine in 48 h.When the medium with 5.0 g/L nicotine,it degrade 65.10% of nicotine in 48 h.The growth and nicotine degradation of DBA5 was declined when nicotine concentration was more than 6.0 g/L,and it degraded less than 7.56% of nicotine.DBA5 showed high ability to degrade nicotine.

Abstract:Three hundred and twenty one-day-old healthy Arbor Acres(AA) broilers were randomly divided into two groups,i.e.,experimental group and control group,and later bred in the chicken houses of different sanitary environmental management conditions.To better understand the impacts of different raising environments on broilers’ immune function,the concentrations of aerobic bacteria,fungi,and endotoxins in the air of the chickens’ houses were determined periodically.Meanwhile,newcastle disease antibody(ND-Ab) levels and immune organ indexes of broilers were detected.The results showed that the microbial concentrations of the experimental group were significantly higher than those of the control group (P＜0.05).Newcastle antibody levels of the control group were higher than those of the experimental group significantly (P＜0.05) at 35,42 and 49 days.The spleen index was influenced significantly (P＜0.05) compared with the control one and very significant impact on the thymus (P＜0.01) was identified,while little effect on the bursa (P＞0.05) was observed.Therefore,the poor sanitary condition and high concentration of microbial aerosols may have a bad effect on the immune functions of broilers,from which it could be concluded that keeping good sanitary condition of raising environments is one of the important measures to keep animals healthy.

Abstract:The distribution of GHS-R1a in hypothalamus-pituitary-ovarian axis were detected by PV-9000 two-step immunohistochemical.Real-time quantitative PCR was used to detect expression of GHS-R1a mRNA during estrous cycle.The results showed that GHS-R1a were mainly distributed in hypothalamus of the arcuate nucleus,ventromedial nucleus,median eminence,the chromophil cells of anterior pituitary,luteal cells,hilus interstitial cells,and oocyte.GHS-R1a mRNA were expressed in hypothalamus and pituitary of rats throughout the estrous cycle and regulated by estrous cycle.GHS-R1a mRNA was not observed in ovary of rats.

Abstract:The effects of 4 color shading-nets (red:shading rate 45.0%,blue:shading rate 51.1%,grey:shading rate 47.2%,black:shading rate 47.3%) on plant growth and photosynthetic characteristics of flowering Chinese cabbage (Brassica campestris L.ssp.chinensis (L.) var.utilis Tsen et Lee) were studied.The results showed that red-net covering increased plant height，stem diameter,total plant fresh mass and leaf area of flowering Chinese cabbage significantly,while blue,grey and black net covering decreased plant growth characteristics comparing with the control (no shading).The 4 shading nets covering significantly reduced plant specific leaf mass,while the contents of chlorophyll a,b,total chlorophyll and carotene were increased with the tendency black net>red net>grey net>blue net>control.The ratio of chlorophyll a/b was decreased by shading treatments compared with the control.In the light density range of 0-2 000 μmol/(m2·s),net photosynthetic rate (Pn) of flowering Chinese cabbage leaf increased steadily with the increase of light density,and the Pn value among treatments showed control≈red net>blue net>grey net>black net.Apparent quantum yield (AQY) of flowering Chinese cabbage in red-net shading treatment increased by 1.1%,compared with the control,but decreased by 30.3%,44.0%and 54.2% in blue,grey and black net shading treatments,respectively.Red-net shading could promote plant growth of flowering Chinese cabbage significantly by improving its photosynthetic performance,indicating that it was suitable for popularization and application.

Abstract:To obtain the suitable culture system of bulblets growth and enlargement,the effects of different culture systems with appending sucrose,AC,Me-JA to the MS medium on the growth and enlargement of Lilium lancifolium bulblets in vitro were studied.The results showed that bulblets are significantly heavy in the liquid culture system compared with solid and solid-liquid.In the liquid culture system,L.lancifolium can culture bulblets with fresh mass of 249.84 mg and percentages of bulblets (diameter＞8 mm) of 54.05% on the average.The concentration of 90.0 g/L sucrose and 3.0 g/L AC was optimal to enlarge bulblets in vitro with the average fresh mass of 479.17 mg and the biggest of 870 mg.The concentration of 10-7 mol/LMe-JA are most favorable for enlargement of bulblets with the average fresh weight of 180 mg.The comparison trials of planting using bulblets of different diameters indicated that the survive of big bulblet were easier than that of small bulblet.

Abstract:Ubiquitin is a small protein that widely exists in all eukaryote whose basic function is through the ubiquitin-proteasome pathway (UPP）to let the targeting protein degradation effective and highly selectivity. A cDNA fragment encoding ubiquitin-52aa extension protein from the third instar larvae of Plutella xylostella was amplified through RT-PCR and RACE method. The ubiquitin-52aa extension gene in P.xylostella, named Px-ubi, was 537 bp in total length and 387 bp in ORF. It encoded a peptide of 129 amino acid residues (GeneBank No.FJ527489), in which the relative quality of molecules is 16.7 ku and the isoelectric point is 9.1.Multiple sequence alignment indicated that Px-ubi was similar to the homologous proteins of other eukaryotic species and it shared 92% to 98% amino acid sequence identity with other species. The theoretical three dimensional structure of Px-ubi was displayed by homology modeling. Through the RT-PCR, it was found that Px-ubi was expressed at different times in the P.xylostella, and its content was higher in the egg stage, third instar larvae and pre-pupal stage than in the adult stage. The Px-ubi was then inserted into expression pET-32a (+) and transformed into E.coli DE3. Western blotting indicated that the Px-ubi was expressed successfully in the BL21 strain of Escherichia coli induced with IPTG. The results provide some helpful information for the further study of the function of ubiquitin gene in insect.

Abstract:A taro strain (compatible or pathogenic), a pomegranate（Punica granatum) strain (compatible or pathogenic) and a sweet potato strain (incompatible or non-pathogenic) of Ceratocystis fimbriata Ellis et Halsted, were inoculated into taro (Colocasia esculenta Schott) tubers. Subsequently, the changes of the activities of peroxidase (POD), catalase (CAT), superoxide dismutase (SOD) and the content of malondialdehyde (MDA) in inoculated taro disks were observed. The results showed that the surfaces of the taro tuber inoculated by all the strains turned reddish under the same conditions after 10 h of inoculation. The taro strain and the pomegranate strain were pathogenic to and compatible with taro, while the sweet potato strain was non-pathogenic to and incompatible with taro. POD, SOD, CAT increased to a higher level in incompatible (sweet potato) strain than those in compatible (pomegranate and taro) strains after 25-30 h inoculation with those pathogens respectively. The surfaces of the taro tuber inoculated by all the strains turned mahogany after 10 h inoculation, whose MDA increased up to a peak at 20 h. 

Abstract:In this research,shipborne samples were randomly selected from imported Canadian rapeseeds to investigate the occurrence of phoma stem canker(Leptosphaeria maculans) by PCR amplification,pathogen isolation and pathogenicity test. The results showed that all fifteen rapeseed samples from three ships were positive in PCR detection of Leptosphaeria maculans.The results from pathogen isolation, identification and pathogenicity testing of three randomly selected samples confirmed the PCR detection.

Abstract:Two species of genus Merlinius Siddiqi,1970: Merlinius montanus Maqbool & Shahina,1987 and Merlinius brevidens (Allen, 1955) Siddiqi,1970 were described in this paper. They were all extracted from rhizosphere soil of potatos in China. M.montanus is a new record species in China and M.brevidens a new record species in Shaanxi Province,China.

Abstract:The occurrence and distribution of this nematode was investigated by random sampling in 16 cities of five regions of Ningxia Hui Autonomous Region. The pathogen was identified with the morphological and metrological examination.The results showed that the cereal cyst nematode distributed in most areas of five the regions of Ningxia Hui Autonomous Region and the detection ratio of CCN was 59.8% in all samples. The highest cyst number existed in Yinchuan City and the lowest one existed in Shizuishan City. The number of eggs per cyst in Guyuan City,Zhongwei City,Wuzhong City and Qingtongxia City were significantly higher than that of Shizuishan City. The pathogen of wheat was identified as H.avenae by morphological examination.

Abstract:Analysis on the quantity,time and spatial patterns of threet pests of Lepidoptera and their natural enemies were conducted with grey system analysis,ecological niche analysis and collective intention analysis of spatial framework in order to protect and use the natural enemies of spiders rationally. The three pests of Lepidoptera were Ectropis obliqua,Eterusia aedea and Homona coffearia. The synthetic ranking results indicated that the dominant natural enemies of Ectropis obliqua were in order of Theridion octomaculatum,Clubiona reichini,Erigonidium graminicolum,Xysticus ephippiatus and Neoscona theisi in order; the dominant natural enemies of Eterusia aedea were in order of Clubiona reichini,Xysticus ephippiatus,Theridion octomaculatum,Neoscona theisi and Misumenops tricuspidatus in order；the dominant natural enemies of Homona coffearia were in order of Misumenops tricuspidatus,Clubiona reichini,Xysticus ephippiatus,Neoscona theisi and Theridion octomaculatum in order.Gathering averages of the three pests and their natural enemies were less than two due to the environmental factors.

Abstract:The leaf and root tip tissues of Alternanthera philoxeroides were treated with vulculic acid of different concentrations to study the effects of vulculic acid on the ultrastructure.The results showed that the damages appeared as the disruption of plasma membrane,digestion of the membrane of chloroplast and mitochondria,disorder of chloroplast lamellae,digestion of mitochondria ridge in leaf tissue,disruption of plasma membrane,digestion of mitochondria membrane and ridge,vacuolation and shrinking of meristematic and epidermal cells in root tip tissue.The severity of damages was increased with the increasing concentration of toxin and time of treatment.

Abstract:The aims of this study were to investigate the development,stability,cross-resistance and preservation of Aeromonas hydrophila resistance to tetracyclines and fluoroquinolones. A.hydrophila was grown at 28 ℃ for 72 h and used it to test for the development of resistance after 9 sequential subcultures in sub-inhibitory concentrations of two tetracyclines(doxycycline and tetracycline) and two fluoroquinolones (norfloxacin and levofloxacin).After 9 subcultures the minimal inhibitory concentrations (MIC) to tetracyclines were 8-32 times greater than the initial values，MIC values to fluoroquinolones were 125-7 997 times greater than the initial values,and drug-resistance were stable.The doxycycline-resistant isolate was resistant to tetracycline and fluoroquinolones,and the levofloxacin-resistant isolate was resistant to tetracyclines and norfloxacin.The drug-resistance stability test indicated that long-term storage of the bacteria at 4℃ could reduce their resistance to antimicrobials.

Abstract:Genetic diversity of a wild population and a cultured population from Anabarilius grahami was measured based on cytochrome b gene of the mitochondrial.Fourteen and fifteen unrelated individuals were chosen in wild and cultured populations,respectively.Twenty-one haplotypes were identified.The cultured population exhibited higher average nucleotide diversity (π) and haplotype diversity (Hd) than the wild population.The genetic distances of the wild and the cultured populations were 0.003 39 and 0.003 43,respectively.The distance of inter-population (0.003 64) was larger than that of intra-population.AMOVA analysis demonstrated that Fst was 0.064 1(P<0.01).Small variance occurred between the two populations (6.41%) and large part of the variance occurred within populations (93.59%).It implied that genetic variance mainly existed among intra-populations.No significant genetic differentiation was observed between the wild and cultured populations.The values from Tajima’s D,Fu and Li’s D and F of neutrality tests were all negative.It suggested that the test results of two A.grahami populations departured from neutrality model,which indicated that populations of A.grahami were possibly experienced population expansion and natural selection.

Abstract:To understand anti-infectious response to bacteria in the Asian yellow pond turtle (Mauremys mutica),a full length cDNA library was constructed for it by SMART technique experimentally infected with Serratia marcescens.Firstly,the double-strand cDNA was synthesized using SMART TM PCR cDNA systhesis kit.Second,the ds cDNA was separated into two parts based on the size distribution of amplified ds cDNA by agarose gel size fractionation.The part shorter than 500 bp was discarded and the other one longer than 500 bp was ligated to the pGEM-T vector.The ligation mixture was transformed into E.coli JM109 by electroporation.The cDNA library contained 1.8×105 independent clones with DNA inserts of 0.5-3.0 kb.The recombination rate was 90.3%.We sequenced 80 cDNA clones about 1 kb and most of the genes were found the first time in reptiles.We classified these clones in functions with 9 in immunity,6 in cell signaling,8 in catalytic activity,2 in sugar/glycolysis metabolism,1in transport metabolism,and 2 in cell structure.The successfully constructed cDNA library will be essential for rapid isolation of differentially expressed genes related to Serratia marcescens infection and useful for understanding the anti-infectious molecular mechanism in the Asian yellow pond turtle.

Abstract:Five shRNAs targeting different sites in white spot syndrome virus (WSSV) genome,respectively designated as RR9,RR1,DP1,DP2 and PK1,were designed and constructed.Followed by WSSV infection,each of which was introduced into Litopenaeus vannamei shrimp by injection.The result demonstrated that both the proliferation of WSSV in vivo and the shrimp mortality were decreased in different degrees in all of the shRNAs-treated groups.Based on the analysis,the RR9 targeting WSSV ribonucleotide reductase gene had the best protective effect against WSSV,which would be used for a further research on anti-WSSV by RNAi.

Abstract:The effects of fermentation temperature,the initial pH of fermentation broth,shaking speed and feeding time on the cell dry weight and yield of S-adenosyl-L-methionine (SAM) produced by Saccharomyces cerevisiae (A12A,A18A) selected from 45 yeast strains were studied,and the fermentation conditions was optimized.The results showed that cell dry weight and SAM production were significantly affected (P<0.05) by fermentation temperature,the initial pH,rotation speed and the feeding time.With the orthogonal optimization,the most suitable conditions for SAM accumulation are temperature 25 ℃,initial pH 6.0,shaking speed 225 r/min,and feeding time 108 h.Under fermentation conditions mentioned above,the SAM production of Saccharomyces cerevisiae A18A was up to 2.54 g/L broth,1.17-fold higher than before optimization.

Abstract:To alleviate the toxic effects of heavy metals on Pleurotus ostreatus,detoxification of rice bran and Swida wilsoniana oilcake to heavy metals was studied through a series of plate cultivation and sawdust cultivation.The results showed that the mycelium growth of Pleurotus ostreatus were all significantly inhibited when the basic cultivating medium was contaminated by 50 mg/L Pb2+ or 15 mg/L Hg2+,respectively.Both of rice bran and Swida wilsoniana oilcake can promote the mycelium growth of Pleurotus ostreatus remarkably when they was added to the basic medium,and the best concentration was 20 g/L.Meanwhile,rice bran and Swida wilsoniana oilcake can take chelation on Pb2+ or Hg2+.The chelation capacity of rice bran on Hg2+ is one order of magnitude stronger than that of Swida wilsoniana oilcake.When rice bran and Swida wilsoniana oilcake are added in the medium contaminated by Pb2+ or Hg2+ respectively,the toxicity of Pb2+,Hg2+ to mycelium growth was decreased and the growth ofPleurotus ostreatus was recovered.In cultivation experiments,rice bran and Swida wilsoniana oilcake showed a very strong effects on chelate detoxification and increased yield as they were added in cultivation materials.Analyzing concentration of heavy metals in fruiting bodies of Pleurotus ostreatus by different cultivation methods,it was found that the concentration of Pb2+or Hg2+ were decreased when rice bran and Swida wilsoniana oilcake were added in corresponding cultivation materials,especially the detecting results of tests about lead conformed to the hygienic standard for edible fungi in China.

Abstract:Rice bacterial blight disease caused by Xanthomonas oryza pv.oryza and fungal blast disease caused by Magnaporthe grisea,are two of the most devastating diseases of rice worldwide.These two diseases can lead to tremendous yield loss every year.Efficient control of disease through improving rice defense system is economic and environment friendly.Characterizing rice disease resistance related genes and elucidating the mechanism of rice disease resistance are important both in scientific theory and in rice improvement.The signaling induced by the plant growth hormoneauxin is generally recognized to regulate plant growth and development.Here we report rice GH3-8,an auxin-responsive gene functioned in auxin-dependent development,activates disease resistance in a salicylic acid–and jasmonic acid–signaling-independent pathway.Bacteria induce accumulation of indole-3-acetic acid (IAA),the major type of auxin in rice.IAA induces the expression of α- and β-expansins,the proteins that are known to loose cell wall,the native barrier of biotic intruder,to facilitate the growth of cells.In rice resistance variety carrying Xa21 or Xa26,the infection of bacteria induce rice to synthesize GH3-8 in infection site of rice. GH3-8 encodes an IAA-amino synthetase that prevents free IAA accumulation and looseness of cell wall.Over-expression of GH3-8 enhanced disease resistance and delayed growth and development,which is partly due to inhibiting the expression of α- and β-expansins via suppressing auxin signaling.Here we show the mechanism of bacteria hijacks auxin as virulence factor to infect rice,and the regulating pathway of rice to the virulence factor; in addition to,explain the cause that plants growth was restrained in disease resistance. Overexpression of GH3-8 results in sterility of plants.Analysis of forward and reverse cross showed that GH3-8-overexpressing plants were male sterility and female sterility.We found that the stigmas of GH3-8-overexpressing plants are abnormal by morphological observation.We observed the mature embryo sac of GH3-8-overexpressingplants using laser scanning confocal microscopy.The result showed that the mature embryo sacs of GH3-8-overexpressing plants were abnormal.This may be the reason of female sterility.No obvious difference was observed in stamen between GH3-8-overexpressing plants and wide-type plant,but the most of pollen of GH3-8-overexpressing plants were sterile.This may be the reason of male sterility.GH3-8 had high expression level in stamen,and the expression of GH3-8 changes as the development of flower.Tissue and time differential expression confirmed the role of GH3-8in regulating flower development.We identified several auxin responsive factors (ARFs) that interacted with the promoter of GH3-8 by analysis of yeast one hybrid.Overexpression of OsARF8 in Mudanjiang 8 activated the expression of GH3-8.This result suggested that OsARF8 is the transcription factor in regulating the expression of GH3-8.OsARF8 expressed highly in pistil,but lowly in stamen.Fertility of GH3-8-overexpressingplants was lower than that of wide type.The most of pollen of OsARF8-overexpressing plants were sterile.The overexpression of auxin signaling genes (OsARF8 and GH3-8) resulted in decrease of rice fertility.This result suggested that auxin plays a critical role in regulating flower development.The detection of the auxin distribution in panicle development showed that auxin is affinitively related with panicle development.The transgenic plants with repressed expression of OsDR8 showed reduced resistance or susceptibility to Xanthomonas oryzae pv.oryzae and Magnaporthe grisea causing bacterial blight and blast,respectively.The putative product of OsDR8 was highly homologous to an enzyme involved in the biosynthesis of the thiazole precursor of thiamine.Exogenous application of thiamine could complement the compromised defense of the OsDR8-silenced plants.The expression level of several defense-responsive genes including the earlier function genes of defense transduction pathway,OsPOX and OsPAL,and the downstream genes of the pathway,OsPR1a,OsPR1b,OsPR4,OsPR5 and OsPR10,was also decreased in the OsDR8-silenced plants.These results suggest that the influence of OsDR8 on disease resistance in rice may be through the regulation of expression of other defense-responsive genes and the site of OsDR8 function is on the upstream of the signal transduction pathway.In addition,the accumulation of thiamine may be essential for bacterial blight resistance and blast resistance.A mutant with lesion mimics on the leaves was found through screening a rice T-DNA inserted pool.The T-DNA was inserted into the open reading frame (ORF) of a gene named OsDR9.The predicted encoding product of OsDR9 consists of 180 amino acids with unknown function.OsDR9 had very low expression level in stem and young panicle but higher level in seedling,flag leaf,sheath and callus; no OsDR9 expression was detected in the root.In addition,OsDR9 had higher expression level in old leaf than young leaf.The mutant was highly resistant to Magnaporthe grisea causing fungal blast disease and Bipolaris oryzae causing Cochliobolus miyabeanus disease in field.Histochemical detection and DNA fragmentation of the leaves developed lesion mimics showed that the cell death had the same features of apoptosis.In addition,the expression of pathogenesis related (PR) proteins genes PR4 and PR8 as well as a blast resistance related gene AOS2 was upregulated in the mutant.The mutant also accumulated autofluorescent materials,salicylic acid and phytoalexins (both momilactone A and sakuranetin).The mutant contained elevated levels of superoxide and H2O2.A 10.5-kb fragment harboring the OsDR9 gene from rice variety Nipponbare was transferred into the mutant.Lesion mimic phenotype was disappeared in the transgenic plants,indicating that knockout of OsDR9 by T-DNA insertion caused the lesion mimic mutant phenotype.These results suggest that OsDR9 is a negative regulator in rice disease resistance and apoptosis.

Abstract:Chestnut rose(Rosa roxburghii Tratt),belonging to Rosa genus of Rosaceae family,is a new promising fruit crop in China due to its fruits having high content of vitamin C and displaying high levels of superoxide disomutase (SOD) activity,which can delay senescence and prevent cancer.However,powdery mildew disease is common in the production area,especially when large areas of chestnut rose are cultivated.Damage caused by powdery mildews can be stunting and distort leaves,buds,growing tips,and fruit,the symptoms become more serious in areas with high humidity and hot weather.Powdery mildew resistance breeding becomes one of the most important goals for various economical crops within Rosaceae family; understanding the molecular mechanism underlying powdery mildew resistance in a relative wild crop would be beneficial to the molecular breeding of disease resistant cultivars of fruit and ornamental crops in Rosaceae family.Using the powdery mildew resistant and susceptible genotypes as materials,phytopathological,cytological,genetics,and genomics researches were carried out to understand the mechanism involved in powdery mildew resistance in chestnut rose.The main results are as following:1.The annual life-cycle of powdery mildew fungi and the shapes of fungi in different stages,the host-microbe interaction,and the expression of defense-related enzymes upon powdery mildew infection were studied.The experiments included the observation or determination of conidiophores,conidia,ascospore,hyphae and ascocarps.Moreover,H2O2 were observed accumulating near around the hyphae attacking sites,and the callose was detected by fluorescence analysis.Chitinase and glucanase were significantly responded to the innoculation of powdery mildew.The biological habits and morphological characterization of powdery mildew fungi in chestnut rose studied herein provided evidence for the correct classification of this fungus.2.One hundred and twenty six resistance gene analogs (RGAs) were cloned from chestnut rose genome.The RGA genes,clustered in the genome,are rapidly evolved,meiotically instable,and evolutionarily complex.The reasons for these characteristics and the generation of new resistance specificity could be the positive selection,balancing selection,recombination,point mutation,and even transposable elements.From chestnut rose,96 resistance gene aanalogues (RGAs) were cloned and characterized,of which 34 were derived from resistant parent,30 from susceptible parent,32 from F1 progeny.Comparison revealed that the nucleotide similarity between the resistant and susceptible parent is averaged at 54%,higher than that within resistant parent.Phylogenetic analysis divided these 96 genes into two groups; one group showed high homology with non-TIR resistance genes,the other is highly homologous to TIR resistance gene.Genetic mapping divided these 96 genes into 3 linkage groups:the biggest group contained 23 genes,designated as CR1; the second one contained 12 genes,all were from susceptible parent; the last group CR3 contained 6 genes.Comparing the positions of genetic map with phylogenetic tree,the RGA gene from a phylogenetic clade tended to cluster in genetic map,which was caused by tandem duplication and diversification.The other case is RGA genes from different clades clustered together in the genetic map and formed heterogeneous cluster,this might caused by ecotopic recombination.From the parents to F1 progeny,deletion of RGA gene’s fragment was observed,which may related with meiotic instability.Using TAIL-PCR strategy,a retrotransposon-like gene flanking RGA gene was isolated,Southern analysis revealed that the copy number of retrotransposon-like gene is relative large with great difference among different materials.Based on the published RosaceaeRGA genes from chestnut rose,apple,peach,pear,strawberry,apricot and plum,were comparatively analyzed on genus and species level.The synteny of RGA gene between different genus were discussed,and the 125 H site was identified to be under positive selection by evolutionary prediction analysis.This is the first report of using overlap extension strategy for target isolation of RGA genes in plants; this method can overcome the problem of bias amplification of degenerate PCR.Also this is the first report of meiotic instability for RGA gene.3.The downstream components of plant immunity system in chestnut rose,including the PTO-like protein kinase and defense-related genes,were investigated.The immunity related genes mostly exist in the genome as gene family.Among the members,single nucleotide polymorphisms (SNPs) are more prevalent than other sequence polymorphisms.Several members are obviously responded to the powdery mildew attack.From the resistant cultivar,30 defense-related genes (DR genes) were cloned,including 9 PTO-like kinase genes,21 pathogenesis-related genes (12 PR2 genes and 9 PR5 genes).The polymorphism of gene family was mainly composed of single nucleotide polymorphisms (SNPs).The average frequency for PR5 gene was one SNP per 59 bp,64 bp for PR2 genes.Based on the SNPs,SNAP markers were developed，23 primer pairs were designed and 17 markers were finally mapped in F1 population.Reverse Northern revealed that all PR2 genes were not induced significantly after innoculation,while one PR5 gene’s expression was significantly enhanced.The immunity related genes in chestnut rose genome from upstream to downstream,i.e.R gene→STK gene→PR2 gene,were predicted to be involved in a co-evolution system.4.Highly expressed genes activated by powdery mildew pathogen attack were cloned and characterized.Suppression subtraction hybridization (SSH) library which enriched powdery mildew responded genes was constructed,and reverse Northern technology was used to screen the clones from the library.Sequencing the differentially expressed clones revealed many genes highly homologous to resistance-related genes reported previously,such as PR10、P450、STK-like kinase gene,Cf-9-like,LRR receptor-like genes,transcription factor NAC gene,and even the transposon elements.The most noticeable genes appeared in the library is photorespiratory-related genes,such as ribulose bisphosphate carboxylase (Rubisco),Rubisco activase,glyoxylate aminotransferase,glutamine synthetase,glyceraldehyde dehydrogenase,ferredoxin,transport protein,etc.Real-Time PCR were used to verify the three most important photorespiratory genes,which revealed that expression levels of Rubisco activase,glyoxylate aminotransferase were significantly enhanced after innoculation.The enzymatic activities of these three photorespiratory genes were also verified to be enhanced significantly after innoculation by spectrophotometric analysis.For example,the transcription level of aminotransferase gene in powdery mildew infected material was as high as 40 folds of that in control material,and the enzymatic activity of aminotransferase in treated material was 25 folds higher than that in control material.Genetic mapping showed that the three photorespiratory genes exist in the chesnut rose genome as gene cluster.The full length cDNA of these three genes were further obtained by RACE strategy.Moreover,the DNA regions of these genes among the genomes of chestnut rose with different genotypes and rosaceae fruit crops were compared.Interestingly,the sequence of ChrRBCs in Guinong No.6,which is highly resistant to powdery mildew,is identical with that in Wuzi Cili,a genotype immune to powdery mildew.5.A new mechanism for powdery mildew disease resistance in plants,i.e.photorespiration,was proposed.Photorespiratory genes are not only highly responded to powdery mildew pathogen attack with the transcription and the enzyme activites highly induced; but also,we found that the transcriptions of photorespiratory genes are significantly induced by resistance signal salycilic acid,and the salycilic acid with another signal peroxide was accumulated significantly in the powdery mildew infected samples.Altogether,photorespiratory gene may be a kind of new resistance gene; therefore photorespiration maybe a new mechanism for plant to defense against pathogen attack were proposed. We are now carring out a further research on Arabidopsis to verify the new function of photorespiratory genes from chestnut rose,and to investigate the molecular mechanism of photorespiratory gene functioned in powdery mildew resistance.