小麦Glu-3位点基因拷贝数的变异分析

【目的】基因拷贝数变异是一种常见又重要的基因结构变异,往往影响个体表型。低分子量麦谷蛋白（low-molecular-weight glutenin subunit,LMW-GS）是小麦贮藏蛋白的主要组成部分,位于Glu-3位点。小麦作为异源六倍体,其庞大且复杂的基因组结构导致难以利用传统方法检测目的基因的拷贝数,针对小麦基因组,筛选可靠稳定的内参基因和体系,探索适合复杂基因组的拷贝数变异测定技术,测定Glu-3位点LWM-GS基因拷贝数。【方法】以Acc1为内参基因,根据基因序列设计内参引物和探针,通过定性和定量PCR测定内参基因在12个普通小麦品种中的拷贝数,分析该基因拷贝数在不同品种间的稳定性;又以小麦品种篙优2018的5个稀释浓度的基因组DNA为模板,利用qRT-PCR验证Acc1内参系统的重复性和准确性;根据Glu-A3位点LMW-GS基因序列设计特异性引物及探针,利用qRT-PCR和ddPCR 2种方法检测8个小麦品种Glu-A3位点基因拷贝数,比较后选择更优的高通量基因拷贝数检测方法;再根据Glu-B3和Glu-D3位点LMW-GS基因序列设计相应的特异性引物及探针,并利用ddPCR技术检测和分析了231份小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝数。【结果】Acc1在12个普通小麦品种间、同一品种5个DNA稀释浓度间的拷贝数测定结果一致,技术重复间的变异系数仅为0.07%—0.77%,所构建的Acc1内参系统稳定;比较qRT-PCR和ddPCR 2种拷贝数检测方法,8个品种所测的Glu-A3位点拷贝数结果一致,分别为3、5、3、4、3、3、3和3;且ddPCR检测重复间的变异系数为0.30%—1.67%,远低于qRT-PCR的3.14%—12.72%,更加可靠;利用ddPCR对231份普通小麦品种的Glu-A3、Glu-B3和Glu-D3位点上LMW-GS基因拷贝检测后分析发现,大多数小麦品种在3个位点上的拷贝数为4,所占频率分别为51.95%、32.03%和28.57%,Glu-3位点总拷贝数变异范围为10—21,变异系数为16.12%。【结论】Acc1内参系统具有良好的稳定性和重复性,可以用作小麦Glu-3位点和其他目的基因拷贝数检测的内参;qRT-PCR和ddPCR均可用于小麦基因拷贝数的检测,但后者更稳定、可靠,且操作简单、检测通量高。     

Effect of miRNA-33 on ABCA1/ABCG1 expression in RAW264.7 macrophage-derived foam cells after Hcy treatment

AIM:To study whether homocysteine (Hcy) inhibits the expression of ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) by microRNA-33 (miRNA-33) signaling, and reduces the efficiency of reverse cholesterol transport (RCT).METHODS:RAW264.7 macrophages were induced by oxidized low-density lipoprotein (ox-LDL) to establish foam cell model. Oil red O staining was used to determine whether the model was established successfully. miRNA-33 mimics and miRNA-33 inhibitor were transfected into the cells by Lipofectamine 2000, and the cells were exposed to Hcy at concentration of 5 mmol/L for 24 h. The intracellular lipid droplets were observed by Oil red O staining. The expression of ABCA1 and ABCG1 at mRNA and protein levels was determined by real-time PCR and Western blot. The cellular cholesterol content was analyzed by HPLC, and effluent rate of cholesterol was detected by the method of liquid scintillation counting.RESULTS:Compared with blank control group, the lipid content in miRNA-33 mimics group was increased, and the expression of ABCA1 and ABCG1 at mRNA and protein levels was decreased (P<0.05). The intracellular cholesterol content was increased gradually (P<0.05), and the cellular cholesterol efflux rate was gradually decreased (P<0.05) in miRNA-33 mimics group. Compared with blank control group, the testing results in miRNA-33 inhibitor group were the opposition of those in miRNA-33 mimics group (P<0.05). No diffe-rence of the above indexes among blank control group, miRNA-33 mimics-NC group and miRNA-33 inhibitor-NC group was observed.CONCLUSION:Hcy inhibits the mRNA and protein expression of ABCA1 and ABCG1 through miRNA-33 signaling, and reduces the efficiency of RCT in RAW264.7 macrophage-derived foam cells.     

SCFAs对奶牛瘤胃上皮细胞Ca2+信号通路相关基因表达的影响

为探究短链脂肪酸(SCFAs)对奶牛瘤胃上皮细胞(BRECs)Ca~(2+)信号通路相关基因表达的影响,试验分为3个处理组,分别为野生型BRECs组,含20mmol/L SCFAs BRECs组,含20mmol/L SCFAs且通过CRISPR/Cas 9系统敲除GPR41基因的BRECs组,每个处理组3个重复,每组细胞均培养24h后,收集细胞提取总RNA,通过qRT-PCR对Ca~(2+)信号通路相关基因的mRNA表达量和细胞内Ca~(2+)浓度进行测定。结果表明,与野生型BRECs相比,添加20mmol/L SCFAs可极显著增加PLCB2的mRNA表达量(P0.01),显著增加IP3R1的mRNA表达量(P0.05),对PLCE1、PLCL1、PKCB和PKCG的mRNA表达量无显著差异(P0.05),可增加细胞内Ca~(2+)浓度但无显著差异(P0.05);敲除SCFAs的受体GPR41后,添加20 mmol/L SCFAs可显著降低IP3R1的mRNA表达量(P0.05),极显著上调PLCE1和PLCB2的mRNA表达量(P0.01),但对PLCL1、PKCB和PKCG的mRNA表达量无显著影响(P0.05),细胞内Ca~(2+)浓度有降低趋势但无显著差异(P0.05)。综上,SCFAs可以通过激活其受体GPR41来调控BRECs内Ca~(2+)信号通路中相关基因的表达和细胞内Ca~(2+)的释放。     

加州鲈弹状病毒三水株全基因组序列测定及分析

【目的】对加州鲈弹状病毒弱毒三水株(MSRV-SS-7)及其母源株(MSRV-SS)进行全基因组克隆和测序分析。【方法】采用分段扩增方法对MSRV-SS-7及MSRV-SS基因组核心序列进行PCR扩增,第1轮根据鳜鱼弹状病毒(SCRV)基因序列(NC_008514.1)设计10对引物进行扩增并测序;在第1轮扩增产物测序、拼接基础上,设计8对引物进行第2轮扩增,拼接后获得基因组核心序列。同时用cDNA末端快速扩增法(RACE)获得3′和5′末端序列。采用Vector NTI 8.0对基因组核心序列和末端序列进行拼接,获得基因组全序列,使用Clone Manager 8.0对MSRV-SS-7和MSRV-SS进行序列比对分析;同时使用DNAMAN将基因组G基因序列转换为氨基酸序列,并通过MEGA 5.0与其他鱼类弹状病毒的G蛋白氨基酸序列进行进化树分析。【结果】MSRV-SS-7及其母源株MSRV-SS基因组全长均为11 548 bp,包含N、P、M、G、L等5个结构基因,基因组结构为3′Leader-N-P-M-G-L-Trailer 5′;MSRV-SS-7与MSRV-SS全基因组中共有10处核苷酸不同。G蛋白进化分析表明,MSRV-SS-7及母源株MSRV-SS与SCRV亲缘关系最近,且与鲈鱼弹状病毒属(Perhabdovirus)聚为一支。【结论】克隆分析了MSRV-SS-7的全基因组序列,可以用于后续活疫苗的开发。     

中国与“77国集团和中国”罗马分部在联合国粮农机构的合作:现状与未来

本研究旨在提高国内对"77国集团和中国"罗马分部重要性的认识,以期通过中国项目和经费支持加强"77国集团和中国"罗马分部的能力建设,进而推动提升发展中国家在联合国粮农机构的话语权和影响力。研究通过查阅有关"77国集团和中国"罗马分部会议纪要及相关文献,走访咨询"77国集团和中国"罗马分部主席、秘书处和有关人员,参加"77国集团和中国"全体会议发现,"77国集团和中国"罗马分部的重要性没有得到充分认识,该机制和平台在运营和管理中心面临较多的困难和挑战,需要并期待中国智慧和中国支持。建议应积极加强中国与"77国集团和中国"罗马分部在联合国粮农三机构框架下的合作。     

生防菌+恶霉灵最优组合的筛选及对苦瓜枯萎病的防治效果研究

苦瓜枯萎病是由尖孢镰孢菌苦瓜专化型（Fusarium oxysporum f. sp. momordicae）侵染引起的一种土传病害，目前市场上缺乏防治效果较好的农药或生防菌剂。为了更好地防治苦瓜枯萎病，通过盆栽试验筛选出绿针假单胞菌（Pseudomonas chlororaphis）G5 与恶霉灵复配施用的最优组合，并在苦瓜枯萎病发病样地对盆栽试验筛选出的最优组合进行防效验证。试验结果表明：菌药组合B+0.75Hym（1×10⁹ cfu · mL^-1 G5 菌悬液+187.5 mg · L-¹ 恶霉灵）处理对盆栽苦瓜枯萎病的防病效果为81.60%，明显高于其他处理；同时该组合对苦瓜幼苗的促生作用最为明显。接种苦瓜枯萎病菌后，菌药组合B+0.75Hym的苦瓜叶片中PAL、PPO、POD 防御酶活性较高。田间防效验证试验中苦瓜苗移栽30、60 d 后菌药组合B+0.75Hym 对苦瓜枯萎病的防治效果较高，分别达到了52.59%、32.13%，且苦瓜产量也高于其他处理。表明盆栽试验筛选出的菌药最优组合B+0.75Hym 不仅能够降低农药的使用量，而且能够有效防治苦瓜枯萎病，提高苦瓜产量，有一定的生产实用价值。     

Identification of sorghum genotypes suitable for specific end uses: Semolina recovery and popping

There has always been an interest in devising breeding programs for designer foods that would benefit both the producer and consumer. The challenge today is transformation of agriculture from “subsistence farming” to “market and income generation oriented” production system for which sorghum with its diverse end uses can assume significant role. Breeding for end-use identity-specific genotypes is needed for increased profitability to the farmers. In the present study, 60 sorghum genotypes were evaluated over two years to identify genotypes suitable for semolina recovery and popping properties, i.e. popping efficiency and pop volume expansion. Semolina recovery ranged from 20.7% to 48.3%, while popping efficiency ranged from 0 to 77.5%. Semolina recovery had positive and significant association with endosperm texture (r = 0.62), grain density (r = 0.49) and grain hardness (r = 0.55) indicating that genotypes with corneous endosperm yield high semolina. Also, semolina recovery had significant positive correlation with popping efficiency (r = 0.49) indicating that genotypes suitable for semolina can also be used for popping. Genetic divergence studies indicated that out of three clusters formed, cluster II having guinea race germplasm lines are suitable for semolina and popping. The information generated and the genotypes identified will help in enhancing the demand for sorghum as an industrial crop.     

Identification of resistance to Aspergillus flavus infection in cotton germplasm

Natural resistance in cottonseed to Aspergillus flavus infection has not been explored to date. A green fluorescent protein (GFP) expressing A. flavus strain was used to assess the resistance of seed from 35 cotton varieties from Gossypium arboreum, G. barbadense, and G. hirsutum. Mature cotyledons devoid of seed coat were wounded, inoculated, and assessed for innate resistance to A. flavus infection. Of the initial 35 varieties tested, we observed a range of resistance to infection in representatives of all three species. A subset of 15 representatives was further analyzed. Within this group, G. arboreum cultivar A2 186 and G. hirsutum cultivar SA 1582 were most resistant to fungal infection. The most susceptible cultivar in this group was G. hirsutum SA 1595. The remaining 12 representatives tested in the secondary screen (3 G. arboreum, 3 G. barbadense, and 6 G. hirsutum lines) exhibited intermediate resistance. We did not observe any relationship between species and resistance. Within each species, there was a range of responses to fungal infection. Future studies using this methodology to screen additional diploid and tetraploid cotton lines may enable us to identify naturally resistant germplasm that can be used to develop cotton with enhanced resistance to fungal infection.