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Genetic and Proteomic Analyses of a Xanthomonas campestris *** purC Mutant Deficient in Purine Biosynthesis and Virulence

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Journal of Genetics and Genomics 2013年第9期40卷 473-487页

作者： Zhihui Yuan Li Wang Shutao Sun Yao Wu Wei Qian State Key Laboratory of plant Genomics Institute of MicrobiologyChinese Academy of Sciences University of Chinese Academy of sciences Center of Core Facility Institute of MicrobiologyChinese Academy of Sciences

Bacterial proliferation in hosts requires activation of a number of housekeeping pathways, including purine de novo biosynthesis. Although inactivation of purine biosynthesis genes can attenuate virulence, it is unclear which biochemical or virulence factors are associated with the purine biosynthesis pathway in vivo. We report that inactivation of purC, a gene encoding phosphoribosylaminoimidazole-succinocarboxamide synthase, caused complete loss of virulence in Xanthomonas campestris pv. cam- pestris, the causal agent of black rot disease of cruciferous plants. The purC mutant was a purine auxotroph; it could not grow on minimal medium, whereas addition of purine derivatives, such as hypoxanthine or adenine plus guanine, restored growth of the mutant. The purC mutation also significantly enhanced the production of an unknown purine synthesis associated pigment and extracellular polysaccharides by the bacterium. In addition, comparative proteomic analyses of bacteria grown on rich and minimal media revealed that the purC mutation affected the expression levels of diverse proteins involved in purine and pyrimidine synthesis, carbon and energy metabolisms, iron uptake, proteolysis, protein secretion, and signal transduction. These results provided clues to understanding the contributions of purine synthesis to bacterial virulence and interactions with host immune systems.

关键词： Purine biosynthesis Virulence Xanthomonas campestris pv. campestris Proteomics

Polycalin is involved in the action mechanism of Cry2Aa toxin in Helicoverpa armigera(Hübner)

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Journal of Integrative Agriculture 2019年第3期18卷 627-635页

作者： WANG Bing-jie WANG Ya-nan WEI Ji-zhen LIU Chen CHEN Lin Myint Myint Khaing LIANG Ge-mei State Key Laboratory for Biology of plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Environment and plant Protection institute Chinese Academy of Tropical Agricultural Sciences Key Laboratory of Integrated Pest Management of tropical Crops Ministry of Agriculture

Receptor proteins on the brush border membrane of the insect midgut epithelium are involved in the mode of action of insecticidal Cry proteins from Bacillus thuringiensis(Bt). Polycalin has been identified as a binding protein of the Bt Cry1 Ac toxin in several Lepidoptera including Helicoverpa armigera, but its role in the action mechanism of Cry2 Aa is still unclear. In this study, we investigated the binding characteristics of polycalin from the midgut of H. armigera with Cry2 Aa and its role in the toxicity of Cry2 Aa. The results demonstrated that heterologously expressed H. armigera polycalin peptide could bind with Cry2 Aa with high affinity(K_d=32 nmol L^(–1)). The toxicity of Cry2 Aa decreased by 27% after H. armigera larvae ingested polycalin antisera. These results suggested that polycalin could be a potential functional receptor for Cry2 Aa, and it plays an important role in the susceptibility of H. armigera to Cry2 Aa.

关键词： Helicoverpa armigera polycalin insecticidal toxicity antisera binding affinity

Improvement of xylanase production by Aspergillus niger XY-1 using response surface methodology for optimizing the medium composition

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2008年第7期9卷 558-566页

作者： Yao-xing XU Yan-li LI Shao-chun XU Yong LIU Xin WANG Jiang-wu TANG institute of plant Protection and microbiology Zhejiang Academy of Agricultural Sciences Hangzhou 310021 China

Objective: To study the optimal medium composition for xylanase production by Aspergillus niger XY-1 in solid-state fermentation (SSF). Methods: Statistical methodology including the Plackett-Burman design (PBD) and the central composite design (CCD) was employed to investigate the individual crucial component of the medium that significantly affected the enzyme yield. Results: Firstly, NaNO3, yeast extract, urea, Na2CO3, MgSO4, peptone and (NH4)2SO4 were screened as the significant factors positively affecting the xylanase production by PBD. Secondly, by valuating the nitrogen sources effect, urea was proved to be the most effective and economic nitrogen source for xylanase production and used for further optimization. Finally, the CCD and response surface methodology (RSM) were applied to determine the optimal concentration of each sig-nificant variable, which included urea, Na2CO3 and MgSO4. Subsequently a second-order polynomial was determined by multiple regression analysis. The optimum values of the critical components for maximum xylanase production were obtained as follows: x1 (urea)=0.163 (41.63 g/L), x2 (Na2CO3)=?1.68 (2.64 g/L), x3 (MgSO4)=1.338 (10.68 g/L) and the predicted xylanase value was 14374.6 U/g dry substrate. Using the optimized condition, xylanase production by Aspergillus niger XY-1 after 48 h fermentation reached 14637 U/g dry substrate with wheat bran in the shake flask. Conclusion: By using PBD and CCD, we obtained the optimal composition for xylanase production by Aspergillus niger XY-1 in SSF, and the results of no additional expensive medium and shortened fermentation time for higher xylanase production show the potential for industrial utilization.

关键词： Xylanase Optimization of medium composition Plackett-Burman design (PBD) Central composite design (CCD) Response surface methodology (RSM). Aspergillus niger XY- 1

Effects of Powdery Mildew on 1 000-Kernel Weight, Crude Protein Content and Yield of Winter Wheat in Three Consecutive Growing Seasons

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Journal of Integrative Agriculture 2014年第7期13卷 1530-1537页

作者： CAO Xue-ren YAO Dong-ming DUAN Xia-yu LIU Wei FAN Jie-ru DING Ke-jian ZHOU Yi-lin State Key Laboratory for Biology of plant Diseases and Insect Pests Institute of Plant Protection Chinese Academy of Agricultural Sciences Key Laboratory of Integrated Pest Management on tropical Crops Ministry of Agriculture/Environment and Plant Protection InstituteChinese Academy of Tropical Agricultural Sciences College of plant Protection Anhui Agricultural University

In order to clarify the impact posed by wheat powdery mildew （Blumeria graminis f. sp. tritici） on the yield and yield components in different epidemic seasons, field trials were conducted in three growing seasons, 2009-2010, 2010-2011 and 2011-2012, in Langfang City, Hebei Province, China. The relationships between 1000-kernel weight, crude protein content of grain and yield and disease index （DI）, as well as area under disease progress curve （AUDPC） were studied. The models of the percentage of loss of 1000-kernel weight, crude protein content and yield were constructed using DI at critical point （CP） of growth stages （GS） and AUDPC in the three growing seasons, respectively. The CPs for estimating 1 000-kernel weight, crude protein content of grain and yield of wheat caused by powdery mildew were GS 11.1, GS 10.5.3 and GS l 0.5.3, respectively. Models based on DI at CP to estimate the percentage of loss of 1000-kernel weight, crude protein content of grain and yield were better than models based on AUDPC. And models of the percentage of loss of 1000-kernel weight, crude protein content and yield for 2011-2012 season were significant different from these for 2009-2010 and 2010-2011 seasons. These results indicated that besides powdery mildew, weather conditions also had influence on 1 000-kernel weight, crude protein content of grain and yield loss of wheat when powdery mildew occurred.

关键词： wheat powdery mildew yield 1000-kemel weight crude protein content loss

A novel chorismate mutase effector secreted from root-knot nematode Meloidogyne enterolobii manipulates plant immunity to promote parasitism

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Journal of Integrative Agriculture 2024年第12期23卷 4107-4119页

作者： Tuizi Feng Yuan Chen Zhourong Li Ji Pei Deliang Peng Huan Peng Haibo Long Key Laboratory of Integrated Pest Management on tropical Crops Ministry of Agriculture and Rural AffairsEnvironment and Plant Protection InstituteChinese Academy of Tropical Agricultural SciencesHaikou 571101China Hainan Key Laboratory for Monitoring and Control of tropical Agricultural Pests Environment and Plant Protection InstituteChinese Academy of Tropical Agricultural SciencesHaikou 571101China State Key Laboratory for Biology of plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China

Meloidogyne *** an economically important plant-parasitic nematode distributed *** fight with host immune system for successful parasitism,plant parasitic nematodes secrete effectors to promote *** this study,we identified one chorismate mutase(CM)effector from ***,named *** and temporal expression assays exhibited Me-cm is expressed in esophageal glands and up-regulated at parasitic-stage ***-CM affects the pathogenicity of *** based on the reduced infection rate,number of galls,egg masses,eggs per mass and multiplication rate collected from RNA silencing *** showed that Me-CM localized in the cytoplasm and nucleus of plant cells and decreased the expression level of the marker gene PR1 of salicylic acid(SA)***,constitutive expression of Me-cm in Arabidopsis thaliana significantly reduced salicylic acid *** results suggested that *** may secrete effector Me-CM to fight with plantimmunesystemsvia regulating SA signaling pathway when interacting with host plants,ultimately facilitating parasitism.

关键词： Meloidogyne enterolobii effector chorismate mutase salicylic acid plant immunity

Identification and Characterization of ABA-Responsive MicroRNAs in Rice

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Journal of Genetics and Genomics 2015年第7期42卷 393-402页

作者： Caijuan Tian Zhangli Zuo Jin-Long Qiu State Key Laboratory of plant Genomics Institute of MicrobiologyChinese Academy of Sciences University of Chinese Academy of sciences

MicroRNAs （miRNAs） are endogenous non-coding small RNAs that silence genes through mRNA degradation or translational inhibition. The phytohormone abscisic acid （ABA） is essential for plant development and adaptation to abiotic and biotic stresses. In Arabidopsis, miRNAs are implicated in ABA functions. However, ABA-responsive miRNAs have not been systematically studied in rice. Here high throughput sequencing of small RNAs revealed that 107 miRNAs were differentially expressed in the rice ABA deficient mutant, Osabal. Of these, 13 were confirmed by stem-loop RT-PCR. Among them, miR1425-5P, miR169a, miR169n, miR390-5P, miR397a and miR397b were up-regulated, but miR162b reduced in expression in Osabal. The targets of these 13 miRNAs were predicted and validated by gene expression profiling. Interestingly, the expression levels of these miRNAs and their targets were regulated by ABA. Cleavage sites were detected on 7 of the miRNA targets by 5＇-Rapid Amplification of cDNA Ends （5＇-RACE）. Finally, miR162b and its target OsTRE1 were shown to affect rice resistance to drought stress, suggesting that miR162b increases resistance to drought by targeting OsTRE1. Our work provides important information for further characterization and functional analysis of ABA-responsive miRNAs in rice.

关键词： Rice Abscisic acid High throughput sequencing MicroRNA Target gene

Selection of reference genes for RT-qPCR analysis of Phenacoccus solenopsis(Hemiptera: Pseudococcidae) sex-dimorphic development

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Journal of Integrative Agriculture 2019年第4期18卷 854-864页

作者： ZHENG Lu-ying ZHANG Zhi-jun ZHANG Jin-ming LI Xiao-wei HUANG Jun LIN Wen-cai LI Wei-di LI Chuan-ren LU Yao-bin College of Agriculture Yangtze University institute of plant Protection and microbiology Zhejiang Academy of Agricultural Sciences

Mealybugs, such as Phenacoccus solenopsis, are highly sexually dimorphic. Winged adult males present such remarkable morphological differences from females that, to the untrained eye, conspecific adults of both sexes of P. solenopsis may be considered as two different insect species. A method to investigate sex-dimorphic mechanisms is by evaluating gene expression using RT-qPCR. However, the accuracy and consistency of this technique depend on the reference gene(s) selected. In this study, we analyzed the expression of 10 candidate reference genes in male and female P. solenopsis at different development stages, using common algorithms including the ?Ct method, NormFinder, geNorm, BestKeeper, and a web-based analysis tool, RefFinder. The results showed that EF1-β, RP-L32 and RP-18 S were selected as the most stable genes by both the ?Ct method and NormFinder; TUB-α was the most stable gene identified by BestKeeper; and RP-L40 and RP-L32 were the most stable genes ranked by geNorm. RefFinder, a comprehensive analysis software, ranked the ten genes and determined EF1-β and RP-L32 as the most suitable reference genes for the various developmental stages in male and female P. solenopsis. Furthermore, the two most suitable reference genes were validated by examining expression of the juvenile hormone acid O-methytransferase(JHAMT) gene. Results of the validation portion of the study showed that JHAMT expression was sex-biased towards males and exhibited a dynamic and classic expression pattern among the P. solenopsis developmental stages. The results can help further our knowledge on the molecular mechanisms underlying sexual dimorphic development in P. solenopsis.

关键词： Phenacoccus solenopsis RT-qPCR sexual dimorphic development reference gene gene stability

Expressing activity of promoter elements of large intergenic region from cotton leaf curl virus in host plant

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Science China(Life sciences) 2001年第1期44卷 8-17页

作者：谢迎秋刘玉乐朱祯 Laboratory of plant Genetic Manipulation Institute of Genetics Chinese Academy of Sciences Beijing China institute of microbiology Chinese Academy of Sciences Beijing China

Cotton leaf curl virus (CLCuV) is a type of single-stranded DMA virus, belonging to geminivirus of subgroup Ⅲ. In order to determine the function of CLCuV large intergenic region (LIR), total DNA of CLCuV-infected cotton leaves was used as template, and fragment of LIR was obtained by PCR and inserted into clone vector. The fragment of LIR was fused with gus reporter gene and nos terminator in the orientation of transcription of virion sense and complementary sense respectively, and the plant expression vectors were constructed. GUS activity of Agrobacte-rium-mediated transgenic tobacco was measured. The result indicated that LIR showed strong promoter activity in complementary sense gene orientation. Average GUS activity of the complementary sense promoter was 5-6 times that of CaMV 35S promoter, and the highest GUS activity of individual plant was ten times of that of CaMV 35S promoter. Histochemical localization confirmed its activity in both mesophyll and vascular tissues. Activity of virion sense of LIR was rather low. Thus LIR isolated from CLCuV could be used as a novel strong promoter in plant genetic manipulation.

关键词： geminivirus large intergenic region promoter tobacco.

The two domains of cotton WLIM1a protein are functionally divergent

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Science China(Life sciences) 2016年第2期59卷 206-212页

作者： Libo Han Yuanbao Li Yongduo Sun Haiyun Wang Zhaosheng Kong Guixian Xia State Key Laboratory of plant Genomics Institute of Microbiology Chinese Academy of Sciences College of Life sciences University of Chinese Academy of Sciences

Our previous study demonstrated that WLIMla has dual roles in fiber elongation and secondary cell wall synthesis in upland cotton, and the protein acts either as an actin-binding protein or as a transcription factor. Because WLIMla consists of two different LIM domains, it is possible that these elements contribute differentially to the dual functions of the protein. In this study, we dissected the two LIM domains and characterized their biochemical functions. By using red fluorescent protein （RFP） fusion, co-sedimentation, and DNA binding methods, we found that the two domains of WLIM 1 a, domain 1 （D 1） and domain2 （D2）, possessed different biochemical properties. While D1 contributed primarily to the actin filament-bundling activity of WLIMla, D2 contributed to the DNA-binding activity of the protein; both D1 and D2 relied on a linker sequence for their ac- tivities. In addition, we found that WLIMla and its two LIM domains form dimers in vitro. These results may lead to a better understanding of the molecular mechanisms of dual functions of WLIMla during cotton fiber development.

关键词： WLIMla actin bundle transcription factor dual function