Molecular identification of blast resistance genes in rice landraces from northeastern India

Rice blast disease caused by the fungus Magnaporthe oryzae is one of the most devastating diseases causing huge losses worldwide. In the present study, major blast resistance genes were investigated in landraces originating from northeastern India. Based on phenotypic evaluation, 288 landraces were classified into three distinct groups: resistant (75), moderately resistant (127) and susceptible (86). The genetic frequencies of the 18 major blast resistance genes were between 6.2% and 27.4%, with only two genotypes possessing a maximum of nine blast resistance genes. The cluster and population structure analysis grouped the landraces into two groups. Through principal coordinate analysis, the scatter plots partitioned the resistant and moderately resistant landraces into different groups. Analysis of molecular variance showed maximum (96%) diversity within populations and least (4%) diversity between populations. Association analysis identified six markers, CRG4_2, RM72, tk59-2, pi21_79-3, RM1233 and RM6648, that are significantly associated with blast disease and explained a phenotypic variance of 1.1–6.5%. The associated genes could be used in marker-assisted rice breeding programmes for gene pyramiding to develop rice varietal resistance against blast disease. The present study represents a valuable blast resistance genetic resource that could be used for identification of new R genes, donors for blast resistance, and genomic studies.     

Crude garlic extract significantly inhibits replication of grapevine viruses

Efforts to control viral diseases of grapevine include the production of certified material and development of virus-resistant transgenic grapevines. However, effective antiviral agents, once the viruses have infected the plants, are still lacking. This study shows that a crude garlic extract has significant antiviral activity against grapevine viruses. Replication of grapevine leafroll-associated virus 2 (GLRaV-2) was obviously inhibited in grapevine cv. Cabernet Sauvignon calli treated with diluted (1:100) garlic extract. The relative RNA levels of GLRaV-2 and grapevine fleck virus (GFkV) in cv. Summer Black grapevine in in vitro-grown plantlets 10 days after treatment with diluted (1:100) garlic extract were about 22% and 20%, respectively, of that in controls. The viral RNA accumulation of GLRaV-2, GFkV, grapevine virus A (GVA), grapevine fanleaf virus (GFLV) and grapevine rupestris stem pitting-associated virus (GRSPaV) in field-grown grapevine cv. Centennial Seedless plants sprayed with diluted (1:100) garlic extract were about 31–40%, 26–38%, 18–31%, 17–42% and 15–18%, respectively, of that in controls. Moreover, the garlic extract treatment led to a significant decrease in viral RNA accumulation of GLRaV-3, GLRaV-2, GVA, GFkV, GFLV, GRSPaV and grapevine Pinot Gris virus in pot-grown grapevine cv. Shine Muscat plants, and viral disease symptoms in these plants were obviously attenuated. In addition, this extract significantly induced expression of pathogenesis-related protein genes and stimulated activity of antioxidant enzymes in grapevines. Taken together, these results indicate that the crude garlic extract acts as a significant inhibitor against a broad range of grapevine viruses.     

人溶菌酶密码子优化及其在牛乳腺细胞中高效表达

【目的】分析牛乳腺中7种主要乳蛋白基因密码子使用偏好性,筛选高频密码子和低频密码子,依据其对人溶菌酶基因进行密码子局部优化和全局优化,通过牛乳腺上皮细胞等多种细胞对优化效果进行分析评价,为提高重组人溶菌酶表达量,开发新型、高效、安全的重组人溶菌酶提供理论依据。【方法】利用CodonW和EMBOSS等软件对7种主要牛乳蛋白和人溶菌酶基因密码子偏好进行生物信息学分析,筛选牛乳蛋白基因高频、低频密码子并根据牛乳蛋白基因密码子使用偏好对人溶菌酶基因翻译起始区前22位密码子（LYZop22）和全局密码子(LYZop)分别进行优化。构建人溶菌酶-荧光素酶融合表达载体（pGL3-LYZcw/op22/op）和人溶菌酶过表达载体（pcDNA-LYZcw/op22/op）,将上述载体分别转染牛乳腺上皮细胞(BMEC)、牛成纤维细胞（BFFC）和C127小鼠乳腺上皮细胞等3种细胞,通过荧光素酶、实时荧光定量PCR及Western-blot等方法检测密码子优化对溶菌酶表达的影响。【结果】牛乳蛋白基因密码子偏好以GC结尾,GC3s平均含量为0.537±0.062,而人溶菌酶GC3s含量为0.407,偏好以AT结尾;聚类结果表明,牛乳中酪蛋白与乳清蛋白类基因在密码子使用偏好性上也存在一定差异。依据筛选获得的牛主要乳蛋白基因5个高频密码子（RSCU>1.5）和7个低频密码子（RSCU<0.5）对人溶菌酶基因密码子进行优化并转染多种细胞进行表达效果分析。荧光素酶分析发现,相比野生型溶菌酶密码子（LYZcw）,翻译起始区密码子优化类型LYZop22分别在BMEC、BFFC细胞中提高1.48倍（P<0.01）和1.30倍（P>0.05）;而全局密码子优化类型LYZop分别在BMEC、BFFC中提高2.2倍（P<0.01）和2.44倍（P<0.01）,说明密码子优化能明显提高人溶菌酶在多种细胞中表达量。实时荧光定量PCR结果表明,LYZop22相比LYZcw在BMEC和BFFC细胞中分别提高了2.08倍（P<0.05）和1.5倍（P>0.05）,而LYZop则分别提高了22倍（P<0.01）和17.8倍（P<0.01）,mRNA表达水平与密码子优化后的mRNA二级结构稳定性呈正相关。Western-blot结果也进一步表明,密码子优化后的LYZop22和LYZop能明显提高重组人溶菌酶在牛乳腺上皮细胞中的表达量。上述结果表明,根据牛乳腺中主要乳蛋白基因密码子使用偏好进行密码子优化,能显著提高人溶菌酶在牛乳腺上皮细胞及成纤维细胞中的表达量,且溶菌酶基因全局密码子优化效果优于翻译起始区密码子优化效果。【结论】通过生物信息学分析获得了牛乳蛋白基因使密码子使用偏好及高低频密码子;依据牛乳蛋白密码子使用偏好性对人溶菌酶密码子优化能显著提高重组人溶菌酶mRNA水平和蛋白水平的表达量,为今后利用生物反应器高效生产重组人溶菌酶奠定基础。     

生物炭连续施用对农田土壤氮转化微生物及N2O排放的影响

【目的】研究连续添加生物炭6年后对农田土壤氮转化相关微生物功能基因的影响,揭示生物炭影响作物产量和N₂O排放的微生物学机制,并为生物炭的推广使用提供理论依据。【方法】通过在潮土农田设置0（BC0,对照)、2.25（BCL,低量）、6.75（BCM,中量）和11.25 t·hm^-2（BCH,高量）4个秸秆生物炭量处理的田间定位试验,采用田间观测、化学分析、荧光定量PCR（qPCR）技术,系统研究施用生物炭对氧化亚氮（N₂O）排放、氨单加氧酶（amoA）、亚硝酸还原酶（nirK、nirS）、氧化亚氮还原酶（nosZ）基因丰度及夏玉米产量的影响。【结果】与对照BC0处理相比,施用生物炭可显著提高夏玉米籽粒产量,且BCM处理籽粒产量达到最大值10 811 kg·hm^-2,显著降低夏玉米生育期N₂O累积排放量,并以BCM处理减少N₂O排放效果最优。研究还发现,在夏玉米多个生育时期,与对照比较,生物炭施用可以显著提高耕层土壤无机氮储量和土壤含水量。此外,随着生物炭施用量增加,土壤氨氧化古菌（AOA）基因拷贝数在夏玉米大喇叭口期和成熟期均表现为先上升后下降趋势,且两个时期均以BCM处理最高,而氨氧化细菌（AOB）基因拷贝数在夏玉米大喇叭口期和成熟期分别为BCH处理和BCM处理最高。与对照相比,中、高量生物炭施用（BCM、BCH处理）可显著提高夏玉米大喇叭口期和成熟期土壤反硝化作用功能相关基因（nirK、nirS、nosZ）拷贝数。相关性分析表明,夏玉米成熟期土壤N₂O排放通量与土壤硝态氮、土壤含水量、AOA、AOB、nirK、nirS、nosZ呈显著负相关关系。【结论】施用生物炭通过增加土壤微生物氮转化功能基因丰度进而降低土壤N₂O排放,通过增加土壤耕层无机氮储量和土壤水分含量进而提高作物产量,并以中等用量（6.75 t·hm^-2）施用效果最优。     

牦牛瘤胃微生物抗生素抗性基因对3种外源性刺激因子的响应

通过给牦牛投喂硫酸头孢喹肟(CEF)、盐酸二氟沙星(DIF)和黄曲霉毒素B1(AFB1),并进行瘤胃微生物宏基因组测序,旨在揭示这3种外源性刺激因子对抗生素抗性基因(ARGs)种类、抗性类型、抗性机制等的影响,对于深入研究微生物抗性组特征和抗性机制具有重要价值。选取15头牦牛,随机分5组。Cef组和Dif组分别根据说明书推荐剂量按体重计算、灌服CEF 1 mg·kg^-1和DIF 1 mL·kg^-1;E1组和E2组分别按采食量投喂AFB120、60μg·kg^-1;C组为对照组。处理7 d后,采集瘤胃液,提取DNA,Illumina HiSeq测序,对reads counts进行标准化得到TPM值,并进行方差分析。结果显示,对照组共获得132种ARGs,分属30种抗性类型,其中,四环素类tetQ和tetW基因丰度较高;Cef组tetW基因丰度增加(P<0.05),Dif组tetQ丰度增加(P<0.05);Cef组四环素类和头孢菌素类抗性基因丰度增加(P<0.05),Dif组四环素类和氨基香豆素类抗性基因丰度增加(P<0.05),E1组氨基香豆素和青霉烯类抗性基因丰度增加(P<0.05),E2组青霉烯类、头孢菌素类等9类抗性基因丰度均增多(P<0.05);Dif组Erm基因23S核糖体RNA甲基转移酶丰度增加(P<0.05),E2组中ATP结合盒超家族等3种抗性机制相关基因的丰度增加(P<0.05);3种因子均显著增加四环素类ARGs宿主的种类。结论:瘤胃是蕴含丰富ARGs的储藏库,其中,四环素类抗生素抗性基因tetQ和tetW是主要的ARGs。不仅CEF和DIF使部分ARGs的种类、抗性类型以及耐药机制相关酶等的丰度升高,增加瘤胃微生物的耐药性,而且AFB1也具有类似作用,且高剂量AFB1对抗性类型的影响范围较抗生素大。这3种因子还导致携带四环素类ARGs宿主微生物的种类数量增加,从而强化横向转移机制,加快ARGs传播,增强微生物对四环素类的耐药性。     

70份国外小麦品种(系)的苗期和成株期抗叶锈病鉴定

小麦叶锈病是小麦生产中的重要病害之一,培育持久抗病品种是最经济、有效和环保的方法。本研究用19个不同毒力的叶锈菌小种苗期接种70份国外引进小麦品种(系)及36个已知抗叶锈病基因的载体品种进行抗性鉴定,同时在2016—2017年度分别于河北保定和河南周口对70份国外引进品种进行田间抗叶锈性鉴定。为进一步检测材料中所携带的苗期和成株抗叶锈病基因,利用12个与已知基因紧密连锁的分子标记进行检测,综合基因推导、系谱分析和分子标记检测的结果,在33份材料中鉴定出15个抗叶锈病基因,包括Lr1、Lr2a、Lr26、Lr3ka、Lr11、Lr17、Lr30、Lr10、Lr14a、Lr2b、Lr13、Lr15、Lr21、Lr44和Lr45,田间鉴定筛选出39份品种表现慢锈性。苗期和田间表现表明,国外品种中含有丰富的对我国叶锈菌小种有效的苗期和成株期抗叶锈病基因,可作为小麦抗叶锈病抗源在抗病育种中加以利用。     

Effects of Estradiol and Testosterone on the Expression of Growth‐related Genes in Female and Male Nile Tilapia,Oreochromis niloticus

Nile tilapia exhibits strong sexual growth dimorphism. The potential role of sex steroid hormones in sexual growth dimorphism is not fully understood. We investigated the effects of estradiol (E₂) and testosterone (T) on growth rate, plasma sex hormones, and expression of growth hormone (GH)‐insulin‐like growth factor (IGF) axis genes and muscle regulatory factor (MRF) genes in female and male Nile tilapia. The results revealed that serum concentrations of E₂ and T were significantly higher after correlative injection (P < 0.05). Compared to male fish, female fish had lower growth rates. E₂ increased growth performance in females with no significant effects on males, whereas T significantly increased growth performance in males, with no significant effects on females. In females, E₂ significantly increased expression of ghr1, ghr2, igf1, and igf2, while T decreased igf2 and increased ghr1 and ghr2 expression. In males, T increased expression of igf1, igf2, ghr1, and ghr2, and E₂ decreased expression of igf1, ghr1, and ghr2. Additionally, E₂ and T enhanced the expression of MRF genes (myod1, myod2, myog, and myf5) in female and male fish, respectively. The results suggest that sex steroid hormones play a role in sexual growth dimorphism by regulating the expression of GH‐IGF axis and MRF genes.     

Cloning and expression analysis of innate immune genes from red sea bream to assess different susceptibility to megalocytivirus infection

As suggested by the Office International des Epizooties (OIE), fishes belonging to the genus Oplegnathus are more sensitive to megalocytivirus infection than other fish species including red sea bream (Pagrus major). To assess the roles of the innate immune response to these different susceptibilities, we cloned the genes encoding inflammatory factors including IL‐8 and COX‐2, and the antiviral factor like Mx from red sea bream for the first time and performed phylogenetic and structural analysis. Analysed expression levels of IL‐1β, IL‐8 and COX‐2 and the antiviral factor like Mx genes performed with in vivo challenge experiment showed no difference in inflammatory gene expression or respiratory burst activity between red sea bream and rock bream (Oplegnathus fasciatus). However, the Mx gene expression levels in red sea bream were markedly higher than those in rock bream, suggesting the importance of type I interferon (IFN)‐induced proteins, particularly Mx, during megalocytivirus infection, rather than inflammation‐related genes. The in vitro challenge experiments using embryonic primary cultures derived from both fish species showed no difference in cytopathic effects (CPE), viral replication profiles, and inflammatory and Mx gene expression pattern between the two fish species.     

Infection routes of Aeromonas salmonicida in rainbow trout monitored in vivo by real‐time bioluminescence imaging

Recent development of imaging tools has facilitated studies of pathogen infections in vivo in real time. This trend can be exemplified by advances in bioluminescence imaging (BLI), an approach that helps to visualize dissemination of pathogens within the same animal over several time points. Here, we employ bacterial BLI for examining routes of entry and spread of Aeromonas salmonicida susbp. salmonicida in rainbow trout. A virulent Danish A. salmonicida strain was tagged with pAKgfplux1, a dual‐labelled plasmid vector containing the mutated gfpmut3a gene from Aequorea victoria and the luxCDABE genes from the bacterium Photorhabdus luminescens. The resulting A. salmonicida transformant exhibited growth properties and virulence identical to the wild‐type A. salmonicida, which made it suitable for an experimental infection, mimicking natural conditions. Fish were infected with pAKgfplux1 tagged A. salmonicida via immersion bath. Colonization and subsequent tissue dissemination was followed over a 24‐h period using the IVIS spectrum imaging workstation. Results suggest the pathogen's colonization sites are the dorsal and pectoral fin and the gills, followed by a progression through the internal organs and an ensuing exit via the anal opening. This study provides a tool for visualizing colonization of A. salmonicida and other bacterial pathogens in fish.