色氨酰-tRNA合成酶基因WRS1调控水稻种子发育

【目的】氨酰-tRNA合成酶（aminoacyl-tRNA synthetases, aaRSs）与遗传信息传递密切相关,已发现植物中aaRSs家族蛋白在维持翻译功能之余,还参与配子发生与胚发育、质体的早期发育以及免疫信号的感知与病害防御等生物学过程。本研究利用水稻胚乳发育缺陷突变体,分析水稻色氨酰-tRNA合成酶（WRS1）在胚乳发育中的作用,证明WRS1基因编码一个影响水稻胚乳发育的关键因子。【方法】本研究通过甲烷磺酸乙酯（ethyl methane sulfonate, EMS）诱变籼稻（Oryza sativa subsp. indica）品种N22,筛选到一个稳定遗传的水稻粉质胚乳突变体（wrs1）,图位克隆获得目标基因。对wrs1成熟种子进行形态学观察以及淀粉相关理化性质测定,利用细胞学切片分析wrs1发育中胚乳的结构,利用实时荧光定量PCR（quantitative real-time PCR, qRT-PCR）和GUS活性染色分析基因表达模式,通过qRT-PCR比较野生型与突变体花后12 d胚乳中淀粉合成相关基因表达情况,免疫印迹检测野生型与突变体成熟种子中淀粉合成酶蛋白积累情况,使用全自动氨基酸分析仪测定游离氨基酸含量。【结果】 wrs1突变体幼苗表现出明显的发育滞后且逐渐蔫萎死亡,从杂合突变体（WRS1wrs1）中分离到的粉质籽粒呈现明显的腹部皱缩,粒厚、千粒重下降,同时总淀粉含量下降,糊化淀粉的峰值黏度和崩解值均低于野生型。wrs1突变体发育胚乳中复合淀粉颗粒变小,排列疏松。WRS1定位于第12染色体长臂约183 kb的区间内,测序发现编码色氨酰-tRNA合成酶（tryptophanyl-tRNA synthetase, WRS）基因的第6外显子上发生单碱基替换,导致一个保守位置上的甲硫氨酸被替换。wrs1突变体中大部分淀粉合成相关基因表达量下调,且野生型与突变体间基因表达的变化与相应蛋白积累的差异存在不一致的趋势。wrs1突变体籽粒中蛋白质积累降低,而游离氨基酸含量显著升高。【结论】 WRS1编码色氨酰-tRNA合成酶,该基因突变后通过影响氨基酸稳态和蛋白质合成,造成淀粉合成相关基因异常表达从而影响淀粉的合成与积累,导致种子发育缺陷。     

Responses of glutamine synthetase activity and gene expression to nitrogen levels in winter wheat cultivars with different grain protein content

Glutamine synthetase (GS) plays a central role in plant nitrogen (N) metabolism, which improves crops grain protein content. A pot experiment in field condition was carried out to evaluate GS expression and activity, and grain protein content in high (Wanmai16) and low grain protein (Loumai24) wheat cultivars under two N levels (0.05 and 0.15 g N kg⁻¹ soil). High nitrogen (HN) resulted in significant increases in GS1 and GS2 expression at 10 days after anthesis (DAA), and higher GS activity during the entire grain filling stage. HN also significantly increased yield, grain protein content and protein fraction (except for glutenin of Luomai24) in two wheat cultivars, which indicated that it increased grain yield and protein content by improving nitrogen metabolism. Wanmai16 showed higher grain protein content, gliadin and glutenin content, and had higher expression level of GS2 both in flag leaves and grains at early grain filling stage. However, Luomai24 had greater yield and higher expression level of GS1. The difference expression of GS2 and GS1 genes indicates they had various contributions to the accumulation of protein and starch in wheat grains, respectively. The results suggest that GS2 would be serving as a potential breeding target for improving wheat quality.     

两个褐飞虱海藻糖转运蛋白基因的结构及调控海藻糖代谢功能

【目的】海藻糖是昆虫中的主要血糖物质,在昆虫的发育及生理活动中发挥重要功能。其中,海藻糖转运蛋白（Tret）在将海藻糖从其生成组织（例如脂肪体）运输到其消耗组织的过程中起着重要作用。本研究通过分析褐飞虱（Nilaparvata lugens）两条Tret1的序列结构,进一步抑制NlTret1的表达,探讨这两个NlTret1在褐飞虱体内的生物学功能。【方法】以褐飞虱两条Tret1序列为研究对象,利用生物信息学技术分析其蛋白结构以及与其他昆虫之间的同源性。采用RNAi（RNA interference）技术将合成的外源dsRNA（double-stranded RNA）注射到实验室饲养褐飞虱种群体内,抑制其体内NlTret1的表达,分别在注射48 h后取材,抽取总RNA并用反转录试剂盒合成第一链cDNA,采用qRT-PCR（quantitative real-time PCR）技术检测dsNlTret1的干扰效果以及RNAi后褐飞虱体内海藻糖代谢通路中相关基因的表达,最后测定葡萄糖、海藻糖和糖原含量以及海藻糖酶活性。【结果】生物信息学分析表明,NlTret1-like X1和NlTret1-2 X1的开放阅读框长度分别为1 920和1 578 bp,分别编码639和525个氨基酸,预测蛋白分子量分别为69.29和58.71 kD,等电点分别为8.32和8.36;NlTret1-like X1和NlTret1-2 X1的二级结构主要包含螺旋和卷曲;保守结构域分析显示它们均属于MFS家族。进化分析结果显示,不同昆虫的Tret1蛋白具有较高的同源性,且褐飞虱与其他半翅目昆虫具有较近的亲缘关系;与注射dsGFP组相比,注射靶标基因的dsRNA后均能够显著沉默本基因的表达;褐飞虱体内糖原、葡萄糖在注射dsNlTret1-like X1和dsNlTret1-2 X1后,其含量均无显著变化,然而不同于注射dsNlTret1-2 X1组,在注射dsNlTret1-like X1后褐飞虱体内海藻糖含量极显著升高;干扰NlTret1-like X1 48 h后褐飞虱体内TPS1、TPS2、TRE1-1、TRE1-2和TRE2的表达均极显著下调;而干扰NlTret1-2 X1 48 h后褐飞虱体内TPS1、TPS2和TRE1-1的表达虽也极显著下降,但TRE1-2和TRE2极显著上调;在注射dsNlTret1-like X1后,试虫可溶性海藻糖酶和膜结合型海藻糖酶的活性均极显著降低,而在注射dsNlTret1-2 X1后无显著变化。【结论】褐飞虱的两个Tret1在不同组织间发挥着不同的功能,其中NlTret1-like X1在特异性转运海藻糖参与能量供应中起到更为显著的作用。研究结果有助于探索Tret1在昆虫或无脊椎动物中调控海藻糖代谢平衡的调节机制,可为将来通过调控血糖平衡来控制褐飞虱等害虫提供理论依据。     

Effect of trehalose supplementation in milk replacer on the incidence of diarrhea and fecal microbiota in preweaned calves

Trehalose, a nonreducing disaccharide consisting of d-glucose with α,α-1,1 linkage, was evaluated as a functional material to improve the gut environment in preweaned calves. In experiment 1, 173 calves were divided into two groups; the trehalose group was fed trehalose at 30 g/animal/d with milk replacer during the suckling period, and the control group was fed nonsupplemented milk replacer. Medication frequency was lower in the trehalose group (P < 0.05). In experiment 2, calves (n = 20) were divided into two groups (control group [n = 10] and trehalose group [n = 10]) based on their body weight and reared under the same feeding regimens as in experiment 1. Fresh feces were collected from individual animals at the beginning of the trial (average age 11 d), 3 wk after trehalose feeding (experimental day 22), and 1 d before weaning, and the fecal score was recorded daily. Fecal samples were analyzed for fermentation parameters and microbiota. The fecal score was significantly lower in the trehalose group than in the control group in the early stage (at an age of 14 to 18 d; P < 0.05) of the suckling period. Calves fed trehalose tended to have a higher proportion of fecal butyrate on day 22 than calves in the control group (P = 0.08). Population sizes of Clostridium spp. were significantly lower (P = 0.036), whereas those of Dialister spp. and Eubacterium spp. tended to be higher in the feces of calves in the trehalose group on day 22 (P = 0.060 and P = 0.083). These observations indicate that trehalose feeding modulated the gut environment and partially contributed to the reduction in medication frequency observed in experiment 1.     

Induction of Drought Tolerance in Maize (Zea mays L.) due to Exogenous Application of Trehalose: Growth,Photosynthesis, Water Relations and Oxidative Defence Mechanism

The present investigation was conducted to assess the ameliorative effects of foliar‐applied trehalose on growth, photosynthetic attributes, water relation parameters and oxidative defence mechanism in two maize cultivars under field water deficit conditions. Various components of the experiment comprised two maize cultivars (EV‐1098 and Agaiti‐2002), two water‐stress levels (irrigation after 2 weeks and irrigation after 3 weeks during the entire period of growth), and two levels of trehalose (0 and 30 mm ) and four replicates of each treatment. Water stress significantly reduced the plant biomass production, photosynthetic attributes and water relation parameters in both maize cultivars. In contrast, water stress considerably increased the leaf malondialdehyde (MDA) contents, the activities of antioxidant enzymes such as peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT), and the levels of non‐enzymatic compounds such as ascorbic acid and tocopherols. In contrast, water stress caused a marked reduction in leaf phenolic contents. Foliar‐applied trehalose significantly increased plant biomass production, and improved some key photosynthetic attributes and plant–water relation parameters. The ameliorative effect of exogenously applied trehalose was also observed on the activities of some key antioxidant enzymes (POD and CAT) and non‐enzymatic compounds (tocopherols and phenolics). Overall, exogenously applied trehalose considerably improved drought tolerance of maize plants by up‐regulating photosynthetic and water relation attributes as well as antioxidant defence mechanism.     

耕作与秸秆还田方式对稻田N_2O排放、水稻氮吸收及产量的影响

保护性耕作是改善农田土壤肥力的重要举措,然而其对作物氮吸收与产量的作用尚不明确。为此,本试验于2016—2017年稻季在湖北省武穴市花桥镇,设置常规翻耕与免耕两种耕作方式以及前茬作物秸秆全量还田与不还田两种秸秆还田方法,研究耕作与秸秆还田方式对稻田土壤N_2O排放、根系酶活性、水稻氮吸收与产量的影响。结果表明,耕作方式显著影响土壤N_2O排放,但不影响根系硝酸还原酶与谷氨酰胺合成酶活性、水稻氮吸收与产量。与翻耕处理相比,免耕处理2016年和2017年土壤N_2O排放量分别显著提高了12.5%～18.2%和21.1%～38.6%。秸秆还田显著影响土壤N_2O排放量、根系酶活性、水稻氮吸收与产量。相对于秸秆不还田处理,秸秆还田处理2016年和2017年土壤N_2O排放量分别显著提高了38.5%～45.5%和13.1%～29.5%。秸秆还田处理相对于不还田处理根系硝酸还原酶与谷氨酰胺合成酶活性分别显著增加了6.7%～45.9%和9.0%～46.7%,水稻氮吸收量提高了12.5%～26.0%,产量增加了9.4%～12.6%。本文认为,虽然秸秆还田提高了水稻氮吸收与产量,但也促进了土壤N_2O的排放,因此在评估保护性耕作稻田温室效应时应加强对温室气体(CH4和N_2O)排放和土壤碳固定影响的长期监测,以期为发展低碳稻作提供理论依据和技术支撑。     

Functional analysis of the venom allergen-like protein gene from pine wood nematode Bursaphelenchus xylophilus using a baculovirus expression system

The pine wood nematode, Bursaphelenchus xylophilus, infects pine trees, leading to fatal pine wilt disease. Here, recombinant venom allergen-like protein (VAP) was obtained by expressing Bx-vap-1 in insect cells. Three-year-old Pinus massoniana were inoculated with recombinant VAP, simulating B. xylophilus esophageal gland secretions. Recombinant VAP up-regulated α-pinene synthase gene expression, the trees showed disease symptoms 15 d after inoculation and the xylem pith revealed brown tissue discoloration, indicating that recombinant VAP could damage P. massoniana cells. Recombinant VAP did not, however, lead to cavitation, indicating that the VAP secreted from B. xylophilus acts as a defense response elicitor.     

SO2对家蚕血淋巴中谷胱甘肽含量及相关酶活性的影响

通过分析在SO2胁迫下家蚕5龄幼虫血淋巴中谷胱甘肽(GSH)含量及相关酶活性的变化,探讨SO2对家蚕的毒理作用机制。当对家蚕5龄幼虫每天以40 mg/m3SO2熏气刺激6 h,蚕体血淋巴中的GSH含量显著降低,雄蚕GSH的平均含量为49.8μmol/L,雌蚕为41.0μmol/L,分别为对照的73.80%和55.76%,而谷胱甘肽硫-转移酶(GST)活性显著升高,雄蚕GST的平均活性为31.86 U/L,雌蚕为31.80 U/L,分别高于对照46.15%和27.30%。与此同时,γ-谷氨酰转肽酶(γ-GT)活性在蚕体中也有不同程度的升高,其中在雄蚕体内的变化极为显著;谷胱甘肽过氧化酶(GSH-Px)和谷胱甘肽合成酶系的活性在蚕体内均无显著变化。以上结果表明:SO2刺激可对家蚕血淋巴中的GSH含量及其相关酶的活性产生影响,且雌、雄蚕对SO2的敏感性不同;GSH含量的减少及其相关酶活性的上升,说明SO2对蚕体的毒害作用与其降低蚕体抗氧化物质水平、削弱抗氧化防御系统有关。     

玉米蛋白多肽对鹅不同组织脂肪酸合成酶定位表达的影响

选用免疫组化技术,探讨玉米蛋白多肽对鹅不同组织脂肪酸合成酶（fatty acid synthase,FAS）定位表达的影响。选取1日龄昌图豁鹅240只,公母各半,随机分为4组,每组设6个重复,每个重复10只。对照组饲喂基础日粮低、中、高剂量组分别于基础日粮中添加玉米蛋白多肽100、300、500 mg/kg。于30和60日龄分别采集动物肝脏和皮下脂肪组织,运用免疫组化法分析两种组织中FAS的表达量及分布。结果表明,鹅两种组织中FAS的表达主要分布在肝脏内皮细胞胞质和胞核及脂肪细胞膜上,且随着鹅日龄增加,FAS含量均增加;玉米蛋白多肽的添加可促进鹅肝脏中FAS的表达,抑制脂肪组织中FAS的表达;高、中、低3个剂量组肝脏中FAS含量,鹅60日龄时分别比30日龄升高47.75%、36.21%、158.74%,增幅均高于对照组（11.00%）;而60日龄时,3个剂量组脂肪组织中FAS含量分别比30日龄降低22.34%、46.43%、49.17%。结果提示,玉米蛋白多肽呈现双重作用,在鹅日粮中较长时间低剂量水平（100 mg/kg日粮）添加,可有效促进肝脏中FAS表达,降低脂肪组织中FAS含量,从而有利于调控机体脂类代谢,提高动物胴体品质。     

Prd29A启动子在小麦中的诱导表达活性及大肠杆菌海藻糖合成酶基因的转化

以gus基因为报告基因，通过瞬时表达测定了Prd29A诱导型启动子在小麦愈伤组织中的诱导表达活性。结果表明，Prd29A-gus基因的表达水平可在不同浓度NaCl盐的胁迫条件下得到显著提高。由此证实，Prd29A启动子在小麦中具有较强的诱导表达活性。在此基础上，将Prd29A诱导型启动子驱动下的大肠杆菌海藻糖合成酶基因（otsA）采用花粉管途径导入目的小麦品系，经PCR筛选、Southern鉴定及转化后代海藻糖含量的测定，获得了一批otsA转基因植株及株系，为进一步选育耐盐抗旱的转基因小麦新品系奠定了基础．