Sensitive detection of the c‐KIT c.1430G>T mutation by mutant‐specific polymerase chain reaction in feline mast cell tumours

Here, we describe the establishment of mutant‐specific polymerase chain reaction (PCR) for detection of a c‐KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c‐KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c‐KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant‐specific PCR but in only 7.1% (5 of 70) by PCR–restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin‐fixed paraffin‐embedded sections or cells collected by fine needle aspiration. Mutant‐specific PCR showed remarkably higher detection rate than did PCR–RFLP. DNA sequence analysis did not always yield identical results to those of mutant‐specific PCR, suggesting heterogeneity of tumour cells. Mutant‐specific PCR is a valid and efficient screening tool for detection of the c‐KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR–RFLP and sequencing analysis.     

Factors (type,colour, density,and shape) determining the removal of marine plastic debris by seabirds from the South Pacific Ocean: Is there a pattern?

1. While floating near the sea surface plastic debris interacts with a number of external factors, including many different organisms. Seabirds have the most extensive documented history of interactions with plastics, through ingestion, entanglement, and nest construction.
2. In the present study, eight seabird species from the South Pacific Ocean were used as a proxy to determine potential patterns of removal of marine plastic debris, and three hypotheses were tested in relation to their feeding habits and nesting areas.
3. Plastics from abiotic compartments (Chilean continental coast, South Pacific Gyre, and Rapa Nui beaches) and biotic compartments (surface-feeding seabirds, diving seabirds, and nesting areas) were compared, according to their type, colour, shape, and density.
4. Continental beaches had a relatively wide range of colours and shapes, with many non-buoyant plastics. Samples from the South Pacific Gyre (SPG) and Rapa Nui (Easter Island) beaches comprised mainly hard, rounded, buoyant, and white/grey plastics.
5. These results indicate that the composition of floating plastics from terrestrial sources changes during transport with oceanic currents, reducing the proportion of prey-like plastics present in the subtropical gyres.
6. The stomach contents of surface-feeding and diving seabirds were dominated by hard, white/grey, and round plastic items, similar to plastics from the SPG, suggesting non-selective (accidental or secondary) ingestion.
7. Nesting areas had a more variable composition of brightly coloured plastics, suggesting a pattern of selective removal of plastics by seabirds, probably from oceanic sources.
8. The present study reveals extensive interactions of seabirds with plastics on a broader scale, which is highly relevant given that the impacts of plastics on seabirds are increasing worldwide, compromising their efficient conservation.

     

Breeding and larval rearing of threatened, endemic fish stonesucker, Garra ceylonensis (Bleeker)

This study was aimed to develop captive breeding and nurture the young of stonesucker (Garra ceylonensis, Bleeker), a small threatened freshwater fish species endemic to Sri Lanka. Breeding of stonesucker was possible under indoor conditions without hormonal stimulation. They were single spawners having a fecundity of 562±176 eggs per fish (total length of 7.9±0.6 cm) with an egg size of 770–1877 μm. Duration of development stages were, egg to hatchling 36–48 h, hatchling to postlarva stage 3–4 days, postlarva to juvenile stage 5–64 days and adult stage in 300–365 days at 26–28°C. Juveniles exhibited bottom dwelling habit in 70–75 days. Postlarva fed with formulated feed exhibited the highest growth (P<0.05) compared with live diets, Artemia and microworm (Anguillula sp.). Although the juveniles revealed a significantly high growth performance (P<0.05) with swine liver diet up to day 45 compared with formulated feed and combination of detritus and plankton, formulated feed exhibited an increase in growth from day 50. Therefore, formulated feed could be used for rearing of postlarva and juvenile stages without any live feeds.     

生长素内流载体基因CkLAX3参与柠条锦鸡儿干旱胁迫响应的分析

植物干旱胁迫进程往往伴随着生长抑制类激素如脱落酸、乙烯的参与,而近年研究表明,生长素也可以响应干旱胁迫。本研究通过染色体步移、生信分析和亚细胞定位等方法,对CkLAX3基因在干旱响应中的作用进行了分析鉴定。研究经PCR扩增获得一个全长为1 398 bp的柠条生长素内流载体基因CkLAX3。生信分析发现,该序列编码465个氨基酸,分子量为52.47 kDa,其编码的蛋白为稳定的疏水性蛋白,二级结构主要由α螺旋组成,具有10重跨膜结构。亚细胞定位结果表明CkLAX3定位于细胞质膜。进化树分析结果表明,CkLAX3与红三叶、鹰嘴豆等植物LAX3基因亲缘关系较近。联合染色体步移和高效热不对称交错PCR (HiTail-PCR)方法克隆得到CkLAX3基因的未知启动子区序列总计1 352 bp。对启动子序列上的顺式作用元件进行分析发现,CkLAX3基因启动子区存在大量干旱响应元件、光响应元件、激素响应元件等。进一步对干旱处理的柠条锦鸡儿进行定量分析发现,CkLAX3的表达量受干旱胁迫诱导,推测该基因在干旱胁迫下发挥重要作用。本研究为进一步探索生长素在调节干旱胁迫应答过程中的作用机制提供了基础。     

番茄 SlMAPK7基因的亚细胞定位与组织表达特性

分离番茄 SlMA PK7基因,利用实时荧光定量反转录聚合酶链反应技术分析在番茄不同组织和花发育不同时期 SlMA PK7的表达特性,发现该基因在雄蕊的表达量明显高于其他组织,在长度4.6～6.5 mm 的花蕾中表达量最高;通过构建黄色荧光蛋白融合表达载体,利用基因枪介导洋葱内表皮细胞的瞬时表达,发现SlMA PK7基因表达蛋白定位在细胞核和细胞膜中;为进一步研究 SlMA PK7的表达特性,分离克隆了SlMA PK7基因的启动子序列,利用 PLACE 和 PlantCARE 软件预测出其含有多种典型的 SlMA PK 启动子顺式作用元件;通过瞬时表达分析该启动子活性,经农杆菌介导转化拟南芥,GUS(β‐glucuronidase ,β‐葡萄糖苷酶)组织化学染色发现,在幼苗期 SlMA PK7主要集中在顶端分生组织和根尖分生组织中表达,在成年植株中则集中在花器官中表达.以上结果表明,SlMA PK7可能在细胞核和细胞膜中参与番茄多种信号的传导,并可能在花器官的发育过程中行使功能.     

Molecular Characterization and Expression Profiles of Myrosinase Gene（RsMyr2） in Radish（Raphanus sativus L.）

Myrosinase is a defense-related enzyme and is capable of hydrolyzing glucosinolates into a variety of compounds, some of which are toxic to pathogens and herbivores. Many studies revealed that a number of important vegetables or oil crops contain the myrosinase-glucosinolate system. However, the related promoter and genomic DNA sequences as well as expression profiles of myrosinase gene remain largely unexplored in radish（Raphanus sativus）. In this study, the 2 798 bp genomic DNA sequence, designated as RsMyr2, was isolated and analyzed in radish. The RsMyr2 consisting of 12 exons and 11 introns reflected the common gene structure of myrosinases. Using the genomic DNA walking approach, the 5′-flanking region upstream of RsMyr2 with length of 1 711 bp was successfully isolated. PLACE and PlantCARE analyses revealed that this upstream region could be the promoter of RsMyr2, which contained several basic cis-regulatory elements including TATA-box, CAAT-box and regulatory motifs responsive to defense and stresses. Furthermore, recombinant pET-RsMyr2 protein separated by SDS-PAGE was identified as myrosinase with mass spectrometry. Real-time PCR analysis showed differential expression profiles of RsMyr2 in leaf, stem and root at different developmental stages（e.g., higher expression in leaf at cotyledon stage and lower in flesh root at mature stage）. Additionally, the RsMyr2 gene exhibited up-regulated expression when treated with abscisic acid（ABA）, methyl jasmonate（MeJA） and hydrogen peroxide（H2O2）, whereas it was down-regulated by wounding（WO） treatment. The findings indicated that the expression of RsMyr2 gene was differentially regulated by these stress treatments. These results could provide new insight into elucidating the molecular characterization and biological function of myrosinase in radish.     

福建主要茶区茶小绿叶蝉种名的存疑与鉴别

于2012和2013年在福建省闽北、闽南和闽中等茶区采集茶小绿叶蝉标本,同时结合2006-2008年在武夷山茶区采集的标本,进行物种形态鉴定.先后共鉴定514头雄性茶小绿叶蝉成虫,结果均为小贯小绿叶蝉,所有鉴定标本中未发现假眼小绿叶蝉.因此,笔者对福建省乃至全国各茶区的茶小绿叶蝉种名的归属问题提出质疑,并同意秦道正等(2014)提出的我国茶树小绿叶蝉种类仍需深入探究的观点,建议在全国各茶区展开大范围的茶小绿叶蝉种类调查.     

不同启动子在水稻悬浮细胞中诱导外源基因的表达

为进一步提高外源基因在水稻悬浮细胞中的表达水平,以花椰菜花叶病毒(Ca MV)35S启动子为对照,克隆了玉米泛素蛋白启动子(Ubi)、水稻肌动蛋白启动子(Actin)以及水稻Oscc1启动子,以GFP为报告基因,通过启动子串联的方式,构建了10个植物表达载体:p CAMBIA 1304 35S-GFP、Ubi-GFP、Actin-GFP、Oscc1-GFP、35S/Ubi-GFP、35S/Actin-GFP、35S/Oscc1-GFP、Ubi/Actin-GFP、Ubi/Oscc1-GFP、Actin/Oscc1-GFP,采用根瘤农杆菌介导的方法,将表达载体导入日本晴胚性愈伤,经悬浮培养分别获得转基因的悬浮细胞系,利用荧光显微镜、酶标仪检测GFP荧光强度,用Western blot检测GFP蛋白质的表达水平。结果发现串联启动子明显强于单个启动子驱动的GFP蛋白质表达水平。在10个表达载体中,35S/Ubi、Ubi/Actin和Ubi/Oscc1是3种优化的启动子组合,其中Ubi/Oscc1串联启动子驱动GFP的表达水平最高,约为对照35S启动子的3倍。     

棉花种子特异性启动子的克隆及序列分析

启动子是基因表达的重要顺式调控元件,在基因工程中,种子特异性启动子可以调控外源基因在种子中特异表达,提高表达效率,增强转基因的效果。本试验根据棉花LEA蛋白D34基因序列设计引物,以海岛棉基因组DNA为模板,通过touch down PCR技术,获得D34基因的种子特异性启动子片段。测序结果表明该片段长1 384 bp,与已发表的D34基因序列相似度为94.87%。该片段除了含有启动子的基本元件TATA框、CAAT框外,还含有种子特异性启动子元件E-box、G-box、B-box、AACA基序等。此外,对其顺式元件做了生物学功能分析。