In vivo growth and genomic characterization of rickettsia‐like organisms isolated from farmed Chinook salmon (Oncorhynchus tshawytscha) in New Zealand

A rickettsia‐like organism, designated NZ‐RLO2, was isolated from Chinook salmon (Oncorhynchus tshawytscha) farmed in the South Island, New Zealand. In vivo growth showed NZ‐RLO2 was able to grow in CHSE‐214, EPC, BHK‐21, C6/36 and Sf21 cell lines, while Piscirickettsia salmonis LF‐89^T grew in all but BHK‐21 and Sf21. NZ‐RLO2 grew optimally in EPC at 15°C, CHSE‐214 and EPC at 18°C. The growth of LF‐89 ^T was optimal at 15°C, 18°C and 22°C in CHSE‐24, but appeared less efficient in EPC cells at all temperatures. Pan‐genome comparison of predicted proteomes shows that available Chilean strains of P. salmonis grouped into two clusters (p‐value = 94%). NZ‐RLO2 was genetically different from previously described NZ‐RLO1, and both strains grouped separately from the Chilean strains in one of the two clusters (p‐value = 88%), but were closely related to each other. TaqMan and Sybr Green real‐time PCR targeting RNA polymerase (rpoB) and DNA primase (dnaG), respectively, were developed to detect NZ‐RLO2. This study indicates that the New Zealand strains showed a closer genetic relationship to one of the Chilean P. salmonis clusters; however, more Piscirickettsia genomes from wider geographical regions and diverse hosts are needed to better understand the classification within this genus.     

Replacement of de‐oiled rice bran by soaked and fermented sweet potato leaf meal: Effect on growth performance,body composition and expression of insulin‐like growth factor 1 in Labeo rohita (Hamilton), fingerlings

A 60‐day feeding trial was conducted to determine the growth performance and expression of insulin‐like growth factor 1 gene (IGF‐I gene) in Labeo rohita fingerlings fed with either raw, soaked or fermented sweet potato leaf meal (SPLM) by completely replacing de‐oiled rice bran (DORB), following a completely randomized design. Seven isonitrogenous (30%) and isocaloric (1.8 MJ/100 g) diets were prepared by replacing DORB with 50% and 100% raw, soaked and fermented sweet potato leaf meal, maintaining DORB‐containing diets as a control. Weight gain %, SGR (specific growth rate) and PER (protein efficiency ratio) were significantly (p < 0.05) higher when 100% DORB was replaced by fermented SPLM in comparison to other treatment groups. The fermented and soaked SPLM‐fed groups had registered with lower FCR value. The expression of growth regulating gene IGF‐I mRNA and RNA/DNA ratio was found to be significantly higher (p < 0.05) in soaked and fermented SPLM‐fed groups. In this study, the body protein and lipid composition did not vary significantly (p > 0.05). Hence, the study concludes that the fermented sweet potato leaf meal using Chaetomium globosum can replace 100% DORB in the diet of Labeo rohita without any detrimental effect on growth performance.     

基于单片机的仿人多指番茄采摘机械手设计

为了降低番茄采摘过程的破碎率,提高采摘效率,实现自动化采摘过程,利用单片机和PID控制技术,设计了一种新型的仿人多指采摘机械手,并采用反馈调节控制方案设计了仿人多指机械手的控制系统。基于AT8 9 S5 2单片机构建了单手指控制器,来对机械手手指夹紧力信号进行采集和处理,并发出夹紧指令。为了测试机械手的有效性和可靠性,首先对番茄的质量尺寸相关性和电压产生的夹紧力进行了测试,通过参数调整后得到了番茄夹紧力随时间变化曲线和破碎率随时间变化曲线,最后对果实采摘的采摘时间和漏采率进行了8次试验。由测试结果可以看出:对于单颗番茄的采摘最高用时仅为2.32s,最高漏采率仅为0.42%,采摘作业效率和采摘精度均较高。     

Competitive ability of ALS‐inhibitor herbicide‐resistant Fimbristylis miliacea

Fimbristylis miliacea, a weed in rice, has evolved resistance to acetolactate synthase (ALS) inhibitors. This study aimed to investigate the competitive abilities of ALS‐resistant (R) and ALS‐susceptible (S) F. miliacea with rice. A replacement series experiment was conducted in the glasshouse at the Federal University of Pelotas, Brazil. The proportions of rice to F. miliacea were 100:0, 75:25, 50:50, 25:75 and 0:100, with 1060 plants m^?2. The experimental units were arranged in a completely randomised design with four replications. A follow‐up study was conducted at the University of Arkansas, Fayetteville, USA, in a split‐plot design with four replications. The main plot was species mixture (rice × R, rice × S, R × S). The subplot was competition partitioning (below‐ and above‐ground, below‐ground only, above‐ground only and no interspecific competition). Leaf area, plant height and shoot dry mass were recorded. Rice was more competitive than the R or S F. miliacea. In equal proportions of rice and F. miliacea, regardless of ecotype, the relative leaf area, height and dry mass of rice were greater than that of F. miliacea. The ALS‐resistant ecotype was less competitive with rice than the S ecotype. Intraspecific competition among rice plants was stronger than rice competition with F. miliacea. Competition for below‐ground resources was the most critical aspect of interference among rice and F. miliacea. In production fields, high infestation levels of F. miliacea results in significant yield losses; thus, resistance to ALS inhibitors needs to be curtailed.     

Serendipitous identification of a new Iflavirus‐like virus infecting tomato and its subsequent characterization

The genomic sequence of a previously undescribed virus was identified from symptomless tomato plants (Solanum lycopersicum). The viral genome is a positive‐sense ssRNA molecule of 8506 nucleotides. It is predicted to encode a single polyprotein of 314·5 kDa that is subsequently processed into three coat protein components of 13·7, 17·9 and 13·5 kDa, and a viral replicase of approximately 207 kDa with conserved motifs for a helicase, a protease and RNA‐dependent RNA polymerase (RdRp). Pairwise analysis of the deduced amino acid sequence of the RdRp revealed that it shares closest identity with members of the family Iflaviridae, genus Iflavirus (19–47% identity). Evidence of replication in plants was detected by RT‐PCR of the viral replicative strand, and short interfering RNAs (siRNAs) matching the virus. The name Tomato matilda virus (TMaV) is proposed, and furthermore, that the genus Tomavirus (Tomato matilda virus) be created within the family Iflaviridae. This is the first report of a plant‐infecting virus resembling members of the Iflaviridae.     

紫花苜蓿盐诱导类受体蛋白激酶基因MsSIK1的克隆及功能分析

【目的】对紫花苜蓿（Medicago sativa L. cv. Zhongmu-1）stress-induced protein kinase gene 1（MsSIK1）进行克隆与表达研究,了解该基因的分子机制及其应用。【方法】以紫花苜蓿叶片总RNA为模板,根据同源克隆设计简并引物,利用RT-PCR结合RACE技术,获得MsSIK1的编码序列。利用同源性比对进行序列分析。通过SMART网站（http://smart.embl-heidelberg.de/）模拟该基因的蛋白结构。构建MsSIK1的亚细胞定位瞬时表达载体,使用基因枪转化法将MsSIK1与GFP在洋葱表皮细胞中融合瞬时表达并观察其亚细胞定位荧光信号。通过Real time-PCR分析MsSIK1在NaCl、ABA和干旱处理条件下的表达特征。利用农杆菌侵染方法获得转基因拟南芥植株,通过RT-PCR对转基因植株进行表达鉴定,获得转基因植株后,利用转基因株系进行盐处理进而对成苗期转基因拟南芥性状鉴定。在盐胁迫处理下,测定野生型与转基因株系的叶绿素含量、MDA含量进而验证该基因的抗盐功能。【结果】获得MsSIK1编码序列2 478 bp,编码825个氨基酸。该蛋白C端与多种植物激酶具有相当高的同源性,模拟蛋白结构发现该基因具有类受体蛋白激酶高度保守的丝氨酸/苏氨酸结构域、跨膜结构域和富含亮氨酸重复序列的膜外结构域。Real time-PCR分析表明该基因在NaCl、ABA和干旱处理条件下上调表达,其中在盐处理条件下,MsSIK1表达先升高后降低,在处理4 h时达到最大值（约为对照值的7倍）。在干旱胁迫处理时,MsSIK1受诱导表达增强明显,当处理2 h时表达量达到最大值（约为对照值的6倍）;ABA处理时,MsSIK1被诱导表达明显,当处理3 h时表达量达到最大值,约为对照值的6.8倍。MsSIK1与GFP融合瞬时表达的洋葱表皮细胞中的荧光信号主要集中于质膜附近,转化空载体的洋葱表皮细胞中的荧光信号分布于细胞各个部位。转基因植株的RT-PCR鉴定表明,T₁代6个株系中所得到的MsSIK1条带明显、亮度高,且T₁-10中表达量最高;但在野生型中检测不到该条带,说明外源基因已经整合到拟南芥染色体中并能遗传到子代。成苗期转基因拟南芥盐处理后发现T₃-2、T₃-6、T₃-10转基因株系较野生型植株长势好,说明MsSIK1的转入提高了拟南芥的抗盐性。与对照相比,转MsSIK1拟南芥在NaCl处理下,叶绿素含量下降较少,其中,野生型叶绿素含量降低了77%,T₃-3降低了53%,T₃-6降低了44%,T₃-10降低了35%;同样盐胁迫下,3个转基因株系的MDA含量积累较少,其中,野生型MDA的含量是T₃-10株系的1.3倍。【结论】MsSIK1作为一个类受体蛋白激酶受多种逆境胁迫诱导,该基因的过量表达提高了拟南芥的抗盐性。     

类蚕丝丝素结晶区蛋白表达载体的构建与表达

为探讨蚕丝结构与性能的关系,设计了几种类似蚕丝丝素结晶区高度重复序列结构(GAGAGX)n的基因序列,并构建了大肠杆菌表达载体,在BL-21菌株中成功诱导表达了4种约由110个氨基酸组成的小分子蛋白。对表达产物进行W estern印迹分析和ELISA检测的结果显示,(GAGAGA)n、(GAGAGY)n、(GAGAGV)n3种蛋白都有小于14.4 kD的大小相同的条带,而(GAGAGS)n有一条稍大于20.1 kD的条带。以超声波破菌的缓冲液作空白对照,4种蛋白的ELISA的结果均比对照的BL-21(无质粒)高,显示为阳性。     

金雀异黄素对雌性大鼠体内促性腺激素及胰岛素样生长因子表达的影响

本试验旨在研究金雀异黄素(genistein,GEN)对雌性大鼠体内促性腺激素及胰岛素样生长因子表达的影响。选取40只SD雌性大鼠[体重(200±20)g],随机分为5组,分别为阴性对照(NC)组、GEN低(L)、中(M)、高剂量(H)组及阳性对照(PC)组,每组8只,NC组灌胃花生油(其他组灌胃试剂以此为溶剂);L、M、H组分别灌胃15、30、60 mg/(kg BW·d)GEN,PC组灌胃己烯雌酚0.5 mg/(kg BW·d)。试验期30 d。采用酶联免疫吸附试验(ELISA)法检测血清中卵泡刺激素(FSH)、黄体生成素(LH)、胰岛素样生长因子-1(IGF-1)、胰岛素样生长因子结合蛋白-1(IGFBP-1)含量;实时定量PCR法检测卵巢IGF-1、IGFBP-1 mRNA表达水平。结果表明:与NC组比较,试验组血清中FSH、LH含量有升高趋势,但差异不显著(P0.05),作用效果与PC组一致;试验组血清IGF-1含量略有降低,但差异不显著(P0.05),PC组显著降低(P0.05);试验组血清IGFBP-1含量显著或极显著升高(P0.05或P0.01),PC组显著升高(P0.05);试验组卵巢组织中IGF-1、IGFBP-1 mRNA表达水平均升高,其中M、H组显著升高(P0.05),与PC组变化一致。由此可见,GEN能够提高雌性大鼠血清FSH、LH含量、降低血清IGF-1含量、提高血清IGFBP-1含量,同时提高卵巢中IG FBP-1、IG F-1 mRNA的表达水平,这些指标协同作用于卵巢,能够促进卵泡的成熟,调节卵巢功能。     

Conversion of Goat Fibroblasts into Lineage‐Specific Cells Using a Direct Reprogramming Strategy

Direct reprogramming is an efficient strategy to convert one cell type to another. In this study, due to the failure of maintaining the undifferentiated state of goat embryotic stem‐ and induced pluripotent stem‐like cells in vitro, we explored an alternative way to directly convert goat fibroblasts to lineage‐specific cells. The ‘Yamanaka factors’ was ectopically expressed in fibroblasts for a short term to situate cells in a metastable state. By culturing with lineage‐specific media for 1–2 weeks, the cardiomyocyte‐like cells and neurocyte‐like cells were generated and confirmed by the quantitative RT‐PCR and immunocytochemical staining. The metastable‐state cells could also be converted into oocyte‐like cells (OLCs) after culturing in media with retinoic acid (RA) and bovine follicular fluid (bFF) for 2–3 weeks. The generated OLCs were surrounded by cumulus granulosa cell‐like cells and formed a structure resembling goat cumulus‐oocyte complex from ovaries. This primary follicular structure could be developed further in oocyte mature medium and expressed germ cell‐specific markers. In addition, we found that the induction efficiency was higher and OLC cell size was bigger in bFF than in RA treatment. Altogether, the direct reprogramming of goat fibroblasts into lineage‐specific cells can facilitate stem cell research in domestic animals.