Identification of potential internal control genes for real-time PCR analysis during stress response in Pyropia haitanensis

Pyropia haitanensis has prominent stress-resistance characteristics and is endemic to China. Studies into the stress responses in these algae could provide valuable information on the stress-response mechanisms in the intertidal Rhodophyta. Here, the effects of salinity and light intensity on the quantum yield of photosystem II in Py. haitanensis were investigated using pulse-amplitude-modulation fluorometry. Total RNA and genomic DNA of the samples under different stress conditions were isolated. By normalizing to the genomic DNA quantity, the RNA content in each sample was evaluated. The cDNA was synthesized and the expression levels of seven potential internal control genes were evaluated using qRT-PCR method. Then, we used geNorm, a common statistical algorithm, to analyze the qRT-PCR data of seven reference genes. Potential genes that may constantly be expressed under different conditions were selected, and these genes showed stable expression levels in samples under a salinity treatment, while tubulin, glyceraldehyde- 3-phosphate dehydrogenase and actin showed stability in samples stressed by strong light. Based on the results of the pulse amplitude-modulation fluorometry, an absolute quantification was performed to obtain gene copy numbers in certain stress-treated samples. The stably expressed genes as determined by the absolute quantification in certain samples conformed to the results of the geNorm screening. Based on the results of the software analysis and absolute quantification, we proposed that elongation factor 3 and 18S ribosomal RNA could be used as internal control genes when the Py. haitanensis blades were subjected to salinity stress, and that a-tubulin and 18S ribosomal RNA could be used as the internal control genes when the stress was from strong light. In general, our findings provide a convenient reference for the selection of internal control genes when designing experiments related to stress responses in Py. haitanensis.

Identification of stable internal control genes for accurate normalization of real-time quantitative PCR data in testicular tissue from two breeds of cattle

Objective:To assess the stability of 10 candidate internal control genes(ICGs),namely GAPDH,ACTB,RPL23,RPS15A,ATPSF1,GLUT5,HMBS,ATP2B4,PPIA,and BRP to normalize the transcriptional data from testes samples of Zebu and crossbred bulls.Methods:Total RNA was isolated from testicular tissue of Zebu and crossbred bulls(n=6 each)between 2-8 years of age.cDNA was synthesized,and the quantitative real-time polymerase chain reaction(PCR)was performed.The cycle threshold values were used for the analysis of the stability of ICGs.Four different statistical algorithms:geNorm,Normfinder,BestKeeper,and RefFinder,were used to assess the stability of these genes.Results:ATPSF1,HMBS,PPIA,and RPS15A were the most reliable and stable ICGs for Zebu testes,and ATPSF1,RPL23,and PPIA for crossbred testes.Conclusions:A panel of stable ICGs(ATPSF1,HMBS,PPIA,RPS15A for Zebu and ATPSF1,RPL23,and PPIA for crossbred)for normalization of gene expression data in testes samples can be helpful for researchers to conduct functional genomics studies at the testicular level in cattle bulls.

Validation of housekeeping genes as internal controls for studying the gene expression in Pyropia haitanensis(Bangiales, Rhodophyta) by quantitative real-time PCR

Pyropia haitanensis is an economically important mariculture crop in China and has a high research value for several life phenomena, for example environmental tolerance. To explore the mechanisms underlying these characteristics, gene expression has been investigated at the whole transcriptome level. Gene expression studies using quantitative real-time PCR should start by selecting an appropriate internal control gene; therefore, the absolute expression abundance of six housekeeping genes （18S rRNA （18S）, ubiquitin-conju-ating enzyme （UBC）, actin （ACT）, β-tubulin （TUB）, elongation factors 2 （EF2）, and glyceraldehyde-3-phos- phate dehydrogenase （GAPDH） examined by the quantitative real-time PCR in samples corresponding to different strains, life-cycle stages and abiotic stress treatments. Their expression stabilities were assessed by the comparative cycle threshold （Ct） method and by two different software packages： geNorm and NormFinder. The most stable housekeeping gene is UBC and the least stable housekeeping is GADPH. Thus, it is proposed that the most appropriate internal control gene for expression analyses in P. haitanensis is UBC. The results pave the way for further gene expression analyses of different aspects of P. haitanensis biology including different strains, life-history stages and abiotic stress responses.

Selection of suitable internal controls for gene expression normalization in rats with spinal cord injury

There is a lack of systematic research on the expression of internal control genes used for gene expression normalization in real-time reverse transcription polymerase chain reaction in spinal cord injury research.In this study,we used rat models of spinal cord hemisection to analyze the expression stability of 13 commonly applied reference genes:Actb,Ankrd27,CypA,Gapdh,Hprt1,Mrpl10,Pgk1,Rictor,Rn18s,Tbp,Ubc,Ubxn11,and Ywhaz.Our results show that the expression of Ankrd27,Ubc,and Tbp were stable after spinal cord injury,while Actb was the most unstable internal control gene.Ankrd27,Ubc,Tbp,and Actb were consequently used to investigate the effects of internal control genes with differing stabilities on the normalization of target gene expression.Target gene expression levels and changes over time were similar when Ankrd27,Ubc,and Tbp were used as internal controls but different when Actb was used as an internal control.We recommend that Ankrd27,Ubc,and Tbp are used as internal control genes for real-time reverse transcription polymerase chain reaction in spinal cord injury research.This study was approved by the Administration Committee of Experimental Animals,Jiangsu Province,China(approval No.20180304-008)on March 4,2018.

Identification of a suitable endogenous control gene in porcine blastocysts for use in quantitative PCR analysis of microRNAs

To obtain reliable results in quantitative PCR （qPCR） reactions, an endogenous control （EC） gene is needed to correct for systematic variations. In this study, a TaqMan low density array was used to quantify the expression levels of microRNA （rniRNA） genes in in vivo fertilized, in vitro fertilized, parthenogenetic and somatic cell nuclear transfer blastocysts. The aim was to identify suitable EC genes for the qPCR analysis of rniRNAs in porcine blastocysts. The results showed that thirty-six miRNAs were commonly expressed in the four kinds of embryos and the expression levels of eleven miRNAs were similar in the different embryo types （P-value〉0.05）. These 11 miRNAs were selected as candidate EC genes for further analysis and, of these, miR-16 was identified as the most stable EC gene by the GeNorm （a tool based on a pair-wise comparison model that calculates the internal control genes stability measure and determines the most reliable pair of EC genes） and NorrnFinder （an excel plug-in that uses an ANOVA-based model to estimate intra- and inter- group variation to indicate the single most stable EC gene） programs. In addition, a cell number normalization method validated miR-16 as a suitable EC gene for use in future qPCR analysis of miRNAs in porcine blastocysts.

Myeloidcell-lineage and premylocytic-stage-specific-expression of the mouse myeloperoxidase gene is controlled at initiation as well as elongation levels of transcription

The myeloperoxidase (MPO) is an important microbicidal protein present at high concentration in the primary granule of mature granulocyte and its expression is regulated in both myeloidcell-lineage and premyelocytic-stagespecific manners. A better understanding of the underlying control mechanisms should provide insights into the temporal and co-ordinate regulation of the gene expression during granulopoiesis. We have identified its promoter by mapping the start(s) of transcription using various molecular approaches together with demonstrating the promoter function of the relevant DNA segment in a transient transfection reporter assay. Besides the major start of transcription mapped at G residue, 11 nucleotide upstream of the 3’ end of exon 0, the usage of that is specific to the MPO expressing cell lines, we have shown that irrespective of the MPO-expression status of the hematopoietic cells, transcription occurs broadly within a two kb region upstream of the 5’ proximity of the gene, and is largely terminated in nitron 2. These data support a model of the pre myelocytic-stage-specific MPO expression, the control of which is operated at initiation as well as elongation levels of transcription.

Hypoxic response elements and Tet-On advanced double-controlled systems regulate hVEGF_(165) and angiopoietin-1 gene expression in vitro

Angiogenesis in ischemic tissue is a complex and multi-gene event. In the study, we constructed hypoxic re-sponse elements （HRE） and the Tet-On advanced double-controlled systems and investigated their effects on the expression of hVEGF165 and angiopoietin-1 （Ang-1） genes in rat cardiomyocytes exposed to hypoxia and pharma-cologic induction. We infected neonatal rat cardiomyocytes with recombinant rAAV-rtTA-Rs-M2/rAAV-TRE-Tight-Ang-1 and rAAV-9HRE- hVEGF165. Our results indicated that the viral titer was 1×1012 vg /mL and the viral purity exceeded 98%. hVEGF165 expression was induced by hypoxia, but not by normoxia （P 0.001）. Ang-1 expression was evident under doxycycline induction, but undetectable without doxycycline induction （P 0.001）. Immunofluorescence staining showed that positively stained hVEGF165 and Ang-1 protein appeared only under both hypoxia and doxycycline induction. We demonstrate here that HRE and the recombinant Tet-On advanced double gene-controlled systems sensitively regulate the expression of hVEGF165 and Ang-1 genes in an altered oxygen environment and under pharmacological induction in vitro.

Suitable internal control genes for qRT-PCR normalization in cotton fiber development and somatic embryogenesis

The mechanisms of cotton fiber development and somatic embryogenesis have been explored sys-tematically with microarray and suppression subtractive hybridization. Real-time RT-PCR provides the simultaneous measurement of gene expression in many different samples,with which the data from microarray or others can be confirmed in detail. To achieve accurate and reliable gene expression re-sults,normalization of real-time PCR data against one or several internal control genes is required,which should not fluctuate in different tissues during various stages of development. We assessed the gene expression of 7 frequently used housekeeping genes,including 18S rRNA,Histone3,UBQ7,Actin,Cyclophilin,Gbpolyubiquitin-1 and Gbpolyubiquitin-2,in a diverse set of 21 cotton samples. For fiber developmental series the expression of all housekeeping genes had the same down tendency after 17 DPA. But the expression of the AGP gene(arabinogalactan protein) that has high expression level at the later fiber development stage was up-regulated from 15 to 27 DPA. So the relative absolute quanti-fication should be an efficient and convenient method for the fiber developmental series. The expres-sion of nonfiber tissues series varied not so much against the fiber developmental series. And three best control genes Histone3,UBQ7 and Gbpolyubiquitin-1 have to be used in a combinated way to get better normalization.

The Genetic Structure and Diversity of Repomucenus curvicornis Inhabiting Liaoning Coast Based on Mitochondrial COⅠ Gene and Control Region

[Object] This study was conducted to explore the genetic diversity and structure of the wild Repomucenus curvicornis inhabiting Liaoning Coast, China. [Method] The mitochondrial COⅠ gene and control region（CR） were PCR amplified from the wild R. curvicornis populations from the Liaodong Bay（n=22） and the northern Yellow Sea（n=18）, sequenced and analyzed for genetic diversity. [Result] The contents of A, T, C and G of 624 bp COⅠ gene were 24.09%, 31.04%, 25.28%, and 19.59%, and those of 460 bp CR fragment were 32.96%, 32.80%, 14.86% and 19.38%, respectively. The total number of variable sites, average number of nucleotide differences（ k）, haplotype diversity（H） and nucleotide diversity（π） based on COⅠ gene were 38, 4.67,（0.96±0.02） and（0.007 5±0.004 2）, and those based on CR fragment were 26, 3.35,（0.97 ±0.02） and（0.007 3±0.004 3）, respectively. Based on mitochondrial COⅠ gene and CR, the genetic diversity of Liaodong Bay population was lower than that of the northern Yellow Sea population. The AMOVA analysis based on CR fragments revealed almost significant genetic divergence between the Liaodong Bay and the northern Yellow Sea populations, while there was no significant genetic divergence based on COⅠ gene. The results showed that CR and COⅠ gene are effective molecular markers for detecting the genetic diversity of R. curvicornis population, while CR is more reliable than COⅠ gene in detecting the genetic structure. [Conclusion] CR is an appropriate marker for genetic analysis of marine fish population.

Controlling N-methyl-D-aspartate receptor subunit 1 with calcitonin gene related peptide after cerebral ischemic injury

BACKGROUND: Activation of N-methyl-D-aspartate receptor (NMDAR) is a key link of exitotoxicity at the phase of cerebral ischemic injury. Because NMDAR is a main way to mediate internal flow of Ca2+ among glutamic acid receptors, over-excitation can cause neuronal apoptosis. Calcitonin gene related peptide has a strongly biological activity. On one hand, it can protect ischemic neurons through inhibiting the expression of NMDAR1 mRNA; on the other hand, it can play the protective effect through down-regulating the expression of NMDAR1 mRNA by exogenous calcitonin gene related peptide. OBJECTIVE: To observe the expression of NMDAR1 and the regulatory effect of calcitonin gene related peptide on the expression of NMDAR1 mRNA and protein in the cerebral cortex of rats with focal cerebral ischemia/reperfusion (I/R). DESIGN: Randomized controlled animal study. SETTING: China Medical University. MATERIALS: A total of 216 healthy male Wistar rats, general grade, weighing 250-280 g, were selected in this study. Twelve rats were randomly selected to regard as control group; meanwhile, other 204 rats were used to establish middle cerebral artery occlusion/reperfusion (MACO) models. The main reagents were detailed as follows: calcitonin gene related peptide (Sigma Company); calcitonin gene related peptide kit (Boster Company); antibody Ⅰ, Ⅱ and antibody β-actin Ⅰ, Ⅱ of NMDAR1 mRNA and chemiluminescence reagent (Santa Cruz Company, USA). METHODS: The experiment was carried out in the Laboratory of Neurobiology of China Medical University from August 2005 to June 2006. ① Right MCAO models of rats were established to cause focal ischemia and scored based on Zea Longa five-grade scale. If the scores were 1, 2 and 3 after wakefulness, the MACO models were established successfully and involved in the experiment. A total of 120 rats with successful modeling were randomly divided into I/R group and administration group with 60 in each group. All rats in the both groups were observed at five time points, including 6, 12, 24, 48 and 72 hours after reperfusion and after 2-hour ischemia, with 12 experimental animals at each time point. Six rats were prepared for detection of hybridization in situ, and the other 6 were used for Western blotting histochemical detection. Rats in the control group were opened their skin to separate common carotid artery and not treated with line and drugs. In addition, rats in the I/R group were treated with 1 mL saline at 2 hours after focal cerebral ischemia, and then, rats in the administration group were treated with 1 mL (1 g/L) calcitonin gene related peptide at 2 hours after focal cerebral ischemia. ② The expression of NMDAR1 mRNA was detected with hybridization in situ at various time points; moreover, the expression of NMDAR1 protein was measured with Western blotting method at various time points. The results were analyzed with Metamoph imaging analytical system. MAIN OUTCOME MEASURES: The expression of NMDAR1 mRNA and its protein in cortical neurons of rats at various time points. RESULTS: A total of 84 rats were excluded because of non-symptoms, exanimation or death; and then, 132 rats were involved in the final analysis. The expression of NMDAR1 mRNA and its protein in cortical neurons of rats in the control group was 0.205±0.001 and 0.184±0.001, respectively; after I/R, expression of NMDAR1 mRNA and its protein was up-regulated, especially, expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.245±0.003, 0.287±0.004, 0.354±0.008, 0.284±0.002 and 0.217±0.006, respectively; moreover, expression of protein at 6, 12, 24, 48 and 72 hours was 0.222±0.003, 0.261±0.028, 0.311±0.004, 0.259±0.013 and 0.210±0.008, respectively. There was significant difference between the two groups (0.205±0.001, P < 0.01). The expression was up-related in the former 24 hours, reached peak at 24 hours, down-regulated, and decreased to the level of control group at 72 hours. Except 72 hours, the expression of NMDAR1 mRNA and its protein was lower in administration group than that in I/R group at other four time points. In addition, the expression of mRNA at 6, 12, 24, 48 and 72 hours was 0.223±0.005, 0.243±0.001, 0.292±0.002, 0.250±0.003 and 0.213±0.003, respectively; moreover, the expression of protein at 6, 12, 24, 48 and 72 hours was 0.216±0.006, 0.245±0.025, 0.276±0.003, 0.241±0.045 and 0.202±0.013, respectively. There was significant difference at various time points (P < 0.05). CONCLUSION: The expressions of NMDAR1 mRNA and its protein of peripheral cortical neurons are up-related in ischemic area after focal cerebral I/R. Meanwhile, exogenous calcitonin gene related peptide can protect cortical neurons through inhibiting expression of NMDAR1 mRNA and its protein after focal cerebral I/R.

柴胡皂苷A调节cAMP/PKA/CREB信号通路对失眠大鼠的改善作用及机制研究

目的基于cAMP/PKA/CREB通路探讨柴胡皂苷A对失眠大鼠的改善作用及机制。方法将75只SD大鼠随机分为空白组、模型组、柴胡皂苷A低剂量组(0.625 mg·kg^(-1))、柴胡皂苷A高剂量组(2.500 mg·kg^(-1))、艾司唑仑组(0.1 mg·kg^(-1)),每组15只。采用腹腔注射苯丙氨酸(PCPA,0.1 mg·kg^(-1))复制失眠大鼠模型。观察大鼠一般情况及昼夜节律;采用戊巴比妥钠翻正实验测定大鼠睡眠潜伏期及睡眠持续时间;观测大鼠睡眠时相,记录慢波睡眠第1期(SWS1)、慢波睡眠第2期(SWS2)、快速眼球运动睡眠期(REMS)时长以及总睡眠时长(TST);qRT-PCR法测定下丘脑节律基因Clock、Bmal1 mRNA及钟控基因Rev-erbα、RorαmRNA的表达水平;免疫荧光法测定海马组织NeuN表达水平;ELISA法测定脑组织中的cAMP水平;Western Blot法测定脑组织中Clock、Bmal1、Rev-erbα、Rorα及cAMP/PKA/CREB通路相关蛋白表达水平。结果与空白组比较,模型组大鼠昼伏夜出的节律紊乱,极度兴奋,易激惹,睡眠减少;睡眠潜伏期明显延长(P<0.05),睡眠持续时间及SWS1、SWS2、REMS、TST均明显缩短(P<0.05);神经元排列紊乱,NeuN阳性神经元IOD值明显降低(P<0.05);脑组织Clock、Bmal1、Rev-erbα、RorαmRNA及蛋白表达水平明显降低(P<0.05);脑组织cAMP、p-PKA/PKA、p-CREB/CREB蛋白表达水平明显降低(P<0.05)。与模型组比较,给药组大鼠的攻击性明显减弱,昼伏夜出有节律性,活动减少,睡眠增多;睡眠潜伏期明显缩短(P<0.05),睡眠持续时间及SWS1、SWS2、REMS、TST均明显延长(P<0.05);神经元排列紊乱情况有所恢复,NeuN阳性神经元IOD值明显升高(P<0.05);脑组织Clock、Bmal1、Rev-erbα、RorαmRNA及蛋白表达水平明显升高(P<0.05);脑组织cAMP、p-PKA/PKA、p-CREB/CREB蛋白表达水平明显升高(P<0.05)。结论柴胡皂苷A可能通过激活cAMP/PKA/CREB通路改善失眠大鼠的昼夜节律。

DNA条形码技术在桃蚜寄生蜂鉴定上的应用

桃蚜(Myzus persicae)是桃树上最重要的害虫之一,为明确其寄生性天敌的物种多样性,该研究对江苏沿海地区桃园内桃蚜僵蚜进行采集,待僵蚜中寄生蜂成虫羽化后采用形态学和DNA条形码的方法相结合进行鉴定。目前调查共发现该地区桃蚜寄生蜂有2科5种,分别为蚜小蜂科(Aphelinidae)的短矩蚜小蜂(Aphelinus abdominalis)和褐带蚜小蜂(Aphelinus maculatus);茧蜂科(Braconidae)的细长径蚜茧蜂(Lipolexis gracilis)、棉蚜茧蜂(Binodoxys communis)和烟蚜茧蜂(Aphidius gifuensis)。该研究提取了5种寄生蜂的COI基因,通过构建进化树的方法,发现分子鉴定与形态学鉴定结果一致,证明了DNA条形码技术在鉴定寄生蜂这类小型昆虫中的准确性和广阔的应用前景。

Construction and Control of Genetic Regulatory Networks:A Multivariate Markov Chain Approach

In the post-genomic era, the construction and control of genetic regulatory networks using gene expression data is a hot research topic. Boolean networks (BNs) and its extension Probabilistic Boolean Networks (PBNs) have been served as an effective tool for this purpose. However, PBNs are difficult to be used in practice when the number of genes is large because of the huge computational cost. In this paper, we propose a simplified multivariate Markov model for approximating a PBN The new model can preserve the strength of PBNs, the ability to capture the inter-dependence of the genes in the network, qnd at the same time reduce the complexity of the network and therefore the computational cost. We then present an optimal control model with hard constraints for the purpose of control/intervention of a genetic regulatory network. Numerical experimental examples based on the yeast data are given to demonstrate the effectiveness of our proposed model and control policy.

食源性金黄色葡萄球菌生物膜形成调控与生物防控策略研究进展

生物膜是很多食源性致病菌应对各种极端环境、杀菌理化因子以及维持菌体内环境稳定的重要基质屏障。数据显示,有超过80%的细菌感染由生物膜引起,尤以金黄色葡萄球菌感染多见。食源性金黄色葡萄球菌是引发细菌性食物中毒和食品安全事故的重要风险源,特别是多重耐药菌株。金黄色葡萄球菌的耐药性、致病性和免疫逃逸与生物膜复杂的三维结构有重大关系。由于生物膜结构基因和调控因子结构相对保守,现已成为金黄色葡萄球菌生物防控新的重要的效应靶点。本文从多糖细胞间黏附素、胞外DNA和生物膜形成相关蛋白等角度阐明了生物膜形成机制,从群体感应系统(如Agr系统和LuxS/AI-2群体感应系统)、全局性调控因子(如附属调节因子Sar和转录因子SigB)及双组分信号转导系统(如SrrAB系统、SaeRS系统、ArlRS系统、LytRS系统和WalKR系统)等角度系统阐述了生物膜形成调控机制。最后,从抗菌肽(蛋白)、植物源化合物、生物酶、抗菌药物等角度提出新的防控策略,以期为食源性金黄色葡萄球菌的生物防控提供指导。

福建省猪源葡萄球菌多重耐药基因的分子流行病学调查

【目的】明确多重耐药基因cfr、optrA和poxtA在福建省猪源葡萄球菌中的分子流行特征,为耐药葡萄球菌的控制提供科学依据。【方法】通过PCR扩增16S rRNA对2020-2022年分离自福建省福州市、厦门市、漳州市、三明市、龙岩市、南平市、宁德市不同猪场的107株菌(样品为鼻拭子、唾液、粪便和肺脏)进行鉴定。对分离菌株进行cfr、optrA和poxtA多重耐药基因检测;采用MEGA软件对cfr和optrA阳性菌株进行系统发育分析;采用琼脂稀释法或微量肉汤稀释法对cfr和optrA基因阳性菌株进行3种抗菌药物(酰胺醇类抗菌药物氟苯尼考和氯霉素,恶唑烷酮类抗菌药物利奈唑胺)最小抑菌浓度(minimum inhibitory concentration,MIC)测定。【结果】经鉴定107株菌均为葡萄球菌,分属18个种,其中以科氏葡萄球菌占比最高。流行病学调查结果显示,107株葡萄球菌主要来自猪鼻拭子样品(占67.3%),并且在福州市猪场的样品中分离率最高(36.4%)。PCR筛查结果显示,在107株葡萄球菌中未检出poxtA基因阳性菌株;检测出14株cfr阳性菌和6株optrA阳性菌,检出率分别为13.1%和5.6%,其中葡萄球菌FJNP2209-1和FJCT2212-1同时携带cfr及optrA基因。系统发育分析结果显示,14株cfr阳性葡萄球菌处于同一个分支内,其中菌株FJFQ2211与所研究的其他菌株亲缘关系稍远;6株optrA基因阳性葡萄球菌属于2个分支。药敏试验结果表明,携带多重耐药基因cfr或optrA的18株葡萄球菌对氯霉素和氟苯尼考100%耐药,对利奈唑胺的耐药率为83.33%。【结论】福建省不同猪场来源的葡萄球菌中多重耐药基因cfr和optrA阳性率较高,但poxtA基因未检出。福建省猪源葡萄球菌对氟苯尼考、氯霉素、利奈唑胺均表现出较高的耐药率,应加强抗菌药物在养猪场中的规范使用。

Characterization of reference genes for qPCR analysis in various tissues of the Fujian oyster Crassostrea angulata

Accurate quantification of transcripts using quantitative real-time polymerase chain reaction （qPCR） depends on the identification of reliable reference genes for normalization. This study aimed to identify and validate seven reference genes, including actin-2 （ACT-2）, elongation factor 1 alpha （EF-1α）, elongation factor 1 beta （EF-1β）, glyceraldehyde-3-phosphate dehydrogenase （GAPDH）, ubiquitin （UBQ）, β-tubulin （β-TUB）, and 18 S ribosomal RNA, from Crassostrea angulata, a valuable marine bivalve cultured worldwide. Transcript levels of the candidate reference genes were examined using qPCR analysis and showed differential expression patterns in the mantle, gill, adductor muscle, labial palp, visceral mass, hemolymph and gonad tissues. Quantitative data were analyzed using the geNorm software to assess the expression stability of the candidate reference genes, revealing that β-TUB and UBQ were the most stable genes. The commonly used GAPDH and 18S rRNA showed low stability, making them unsuitable candidates in this system. The expression pattern of the G protein β-subunit gene （Gβ） across tissue types was also examined and normalized to the expression of each or both of UBQ andβ-TUB as internal controls. This revealed consistent trends with all three normalization approaches, thus validating the reliability of UBQ and β-TUB as optimal internal controls. The study provides the first validated reference genes for accurate data normalization in transcript profiling in Crassostrea angulata, which will be indispensable for further fimetional genomics studies in this economically valuable marine bivalve.

砖红镰刀菌菌株Pa2对草莓灰霉病的抑制作用

由灰葡萄孢菌Botrytis cinerea引起的草莓灰霉病是草莓栽培中最具破坏性的病害之一,严重降低食用以及经济价值。本研究以“红颜”草莓为试验材料,测定砖红镰刀菌Fusarium lateritium Pa2对草莓灰霉病菌的抑菌活性、防治效果以及草莓病程相关基因表达水平的影响。结果表明,菌株Pa2对草莓灰霉病菌具有显著的抑菌活性,其上清液可以破坏草莓灰霉病菌菌丝细胞膜,导致细胞内容物泄露。离体接种试验结果表明菌株Pa2对草莓叶片灰霉病、草莓果实灰霉病防治效果分别为79.13%、54.29%。经菌株Pa2上清液处理24h后草莓果实病程相关基因FaPR1、FaPR4、FaGR分别上升19.05、24.64、3.69倍。综上所述,砖红镰刀菌菌株Pa2是一种防治草莓灰霉病的新型生防菌资源。

Advances in Research and Application of gE Gene/Protein in Prevention and Control of Swine Pseudora-bies

Research and application progresses of gE gene and its encoding gE protein in PR vaccines, diagnostic technique and epidemiological investigation are summarized, which have certain reference value for comprehensive prevention and control of PR and gradual purification of PR in different regions.

芽胞杆菌JZB30-1的鉴定及其对番茄灰霉病的生防作用

为获得对番茄灰霉病具有良好防效的生防芽胞杆菌,采用对峙培养、双层平板以及生防相关特性检测等方法筛选高活性拮抗菌株,根据形态学、生理生化特性及多基因测序进行菌株种类鉴定,通过离体果实和盆栽试验明确供试菌株的防病促生效果。结果表明,芽胞杆菌JZB30-1对番茄灰霉病菌等10余种植物病原真菌和细菌具有广谱抑菌活性;该菌株具有合成蛋白酶、纤维素酶、脂肽类物质,分泌铁载体、生长激素及溶磷等特性,经鉴定其为解淀粉芽胞杆菌Bacillus amyloliquefaciens;菌株JZB30-1无菌发酵滤液50倍液喷施处理,对番茄果实灰霉病防效为95.0%,发酵液10倍稀释液灌根,番茄株高、鲜重分别较对照增加25.7%和28.4%。相关结果为利用解淀粉芽胞杆菌JZB30-1进行番茄灰霉病生物防治提供理论依据。

抗香蕉枯萎病菌菌株F2-14的鉴定及其抑菌机制初探

香蕉枯萎病已严重威胁全球香蕉产业发展,生物防治是防控香蕉枯萎病的重要途径之一。本研究筛选香蕉土壤中的拮抗香蕉枯萎病菌(Fusarium oxysporum f.sp.cubense tropical race 4,Foc TR4)活性细菌,得到1株具有高拮抗活性的菌株F2-14。采用生理生化特性、形态特征和基因组ANI值分析鉴定该菌株,并进行其生物信息学分析,利用antiSMASH预测其次级代谢产物合成基因簇,与CAZy等数据库进行比对分析;并进行其抑菌物质粗提及其抑菌机制研究。结果表明,菌株F2-14鉴定为贝莱斯芽孢杆菌Bacillus velezensis。其基因组中具有产生几丁质酶、葡聚糖酶、蛋白酶等的基因,还含有编码杆菌溶素、儿茶酚型嗜铁素、丰原素、表面活性肽等7种抗菌活性的基因簇。菌株F2-14具有广谱抗真菌活性,能够平板抑制辣椒炭疽病菌Colletotrichum acutatum等15种病原真菌菌丝生长,且能通过产生次级代谢物致使Foc TR4病菌的细胞壁破裂和细胞膜受损,从而抑制Foc TR4病菌生长。说明贝莱斯芽孢杆菌菌株F2-14在植物病害防控方面有着较好的应用潜力。