H1N1猪流感广东株血凝素基因的克隆与序列分析

用RT-PCR方法扩增H1N1亚型猪流感病毒广东分离株A／Swine／GuangDong／711／2001HA基因，对其进行了克隆和测序。结果显示，HA基因全长1771bp．共编码579个氨基酸。A／Swine／Guang Dong／711／2001HA基因编码的氨基酸序列中有8个糖基化位点，5个位于HAl基因的第27、28、40、104和287位点，3个位于HA2基因的21、153和213位点。与H1N1亚型猪流感经典毒株比较后发现，血凝素糖基化位点并不是高度保守的。将A／Swine／Guang Dong／711／2001与自Gen Bank读取的1918年人流感毒株A／South Carolina／1／18、1991年人流感毒株A／MD／12／91和1997年猪流感毒株A／Swine／Wisconsin／238／97等进行核苷酸同源性比较分析，A／Swine／Guang Dong／711／2001与A／SouthCarolina／1／18、A／MD／12／91和A／Swine／Wisconsin／238／97等毒株的核苷酸同源性分别为93．9％、94．7％和93．9％。从系统发生树来看，A／Swine／Guang Dong／711／2001与A／South Carolina／1／18和A／Swine／Wisconsin／238／97的亲缘关系相近。     

UHPLC-Q/TOF MS结合HPLC-DAD定性和定量分析硫酸黏菌素可溶性粉中的未知添加物

定性和定量分析一批兽药硫酸黏菌素可溶性粉中的未知添加物。照《中国兽药典》2010年版一部对该批检品用微生物检定法进行含量测定时,发现该样品的抑菌圈为虚圈,用薄层色谱鉴别该样品,未显示与标准品溶液一致的主斑点,怀疑该样品中有处方外非法添加物。采用超高效液相色谱-四级杆-飞行时间质谱(UHPLC-Q/TOF MS)对该样品进行筛查,发现疑似添加物,并使用液相色谱-二极管阵列检测(HPLC-DAD)法进行了双重确证和含量测定。该样品中非法添加物确证为磺胺氯达嗪和甲氧苄啶,添加量分别为58.5 mg/g和13.4 mg/g。本研究通过建立筛查方法为监管部门提供技术支撑,通过分析非法添加物的可能原因为打击兽药处方外非法添加提供了思路。     

Effects of five cryoprotectants on proliferation and differentiation‐related gene expression of frozen‐thawed bovine calf testicular tissue

The cryopreservation of testicular tissue is a potential method for preserving male fertility. However, the effect of cryopreservation on bovine calf testicular tissue is scarce. This study investigated the effect of different cryoprotectants on bovine calf testicular tissue at the molecular level. Testicular tissue from ten immature bovine calves (6 months) was collected after slaughter and cryopreserved in an extender containing different concentrations of the following five cryopreservation solutions (CP): bovine serum albumin (BSA) with 5% dimethyl sulfoxide (DMSO), trehalose with 5% DMSO, DMSO and glycerol and ethylene glycol (EG). After 7‐day cryopreservation, the expression levels of three spermatogonial stem cell (SSC)‐related genes, octamer‐4 (OCT4), KIT ligand (MGF/SCF) and kit oncogene (C‐KIT), were investigated by quantitative PCR (qPCR). The cell viability was highest for the tissues preserved with 30 mg/ml BSA (77.82% ± 1.22) and 40 mg/ml trehalose (74.23% ± 1.16) compared with other groups (p < 0.05), and the level of expression of the three genes was highest with 30 mg/ml BSA (p < 0.05). Compared with other CPs, the 30 mg/ml BSA and 40 mg/ml trehalose have the better cryopreserve protection. The 30 mg/ml BSA is the most viable media for the cryopreservation of testicular tissue from cattle.     

发病鲆鲽类分离菌株的16S rRNA基因测序分析

细菌性疾病是中国海水养殖鲆鲽类的主要病害,为全面了解病原菌种类,本研究对1999~2012年从山东、江苏、河北、天津等沿海地区养殖场发病鲆鲽鱼类中分离得到的124株优势菌株进行了16S rRNA基因测序和系统发育学分析。将基因序列与GenBank核酸序列数据库进行相似度比对分析,结果显示,有83株与弧菌属(Vibriosp.)细菌相似度最高,11株与气单胞菌属(Aeromonas sp.)细菌相似度最高,4株与爱德华氏菌属(Edwardsiella sp.)细菌相似度最高,26株为其他15种属的细菌。根据系统发育学分析结果,进一步将66株菌鉴定为16个种,优势种为溶藻弧菌(V.alginolyticus)、哈氏弧菌(V.harveyi)、鳗弧菌(V.anguillarum)、杀鲑气单胞菌(A.salmonicida)和迟缓爱德华氏菌(E.tarda)。选择其中的9株鳗弧菌和4株迟缓爱德华氏菌进行人工感染实验,结果显示,其中7株鳗弧菌和3株迟缓爱德华氏菌对大菱鲆(Scophthalmus maximus)有较强的致病性。研究结果可为阐明中国海水养殖鲆鲽类的流行病发生历史、病原种类、病原监测及疾病控制提供重要参考。     

Genome Mining as an Alternative Way for Screening the Marine Organisms for Their Potential to Produce UV-Absorbing Mycosporine-like Amino Acid

Mycosporine-like amino acids (MAAs) are small molecules with robust ultraviolet (UV)-absorbing capacities and a huge potential to be used as an environmentally friendly natural sunscreen. MAAs, temperature, and light-stable compounds demonstrate powerful photoprotective capacities and the ability to capture light in the UV-A and UV-B ranges without the production of damaging free radicals. The biotechnological uses of these secondary metabolites have been often limited by the small quantities restored from natural resources, variation in MAA expression profiles, and limited success in heterologous expression systems. Overcoming these obstacles requires a better understanding of MAA biosynthesis and its regulatory processes. MAAs are produced to a certain extent via a four-enzyme pathway, including genes encoding enzymes dehydroquinate synthase, enzyme O-methyltransferase, adenosine triphosphate grasp, and a nonribosomal peptide synthetase. However, there are substantial genetic discrepancies in the MAA genetic pathway in different species, suggesting further complexity of this pathway that is yet to be fully explored. In recent years, the application of genome-mining approaches allowed the identification of biosynthetic gene clusters (BGCs) that resulted in the discovery of many new compounds from unconventional sources. This review explores the use of novel genomics tools for linking BGCs and secondary metabolites based on the available omics data, including MAAs, and evaluates the potential of using novel genome-mining tools to reveal a cryptic potential for new bioproduct screening approaches and unrevealing new MAA producers.     

新麦草YUCCA基因克隆及其表达特性分析

【目的】克隆新麦草分蘖关键基因YUCCA并明确其表达特性,为该基因功能验证和育种利用奠定基础。【方法】以不同分蘖类型的新麦草分蘖节为材料,采用PCR法克隆新麦草YUCCA基因并进行同源序列比对,构建1300-cYFP-YUCCA过表达载体,利用烟草瞬时转染及蛋白质印迹法分析新麦草YUCCA蛋白的表达位置及表达情况,通过RNA-Seq数据及qRT-PCR分析验证YUCCA基因的相对表达量。【结果】成功克隆了新麦草YUCCA基因全长,大小为1 336 bp,最大阅读框为1 236 bp,并成功构建了1300-cYFP-YUCCA过表达载体。多序列比对结果显示,新麦草YUCCA主要与麦类作物的亲缘关系较近。表达特性分析发现,YUCCA基因在多分蘖型新麦草中的相对表达量远低于少分蘖型新麦草;与叶、根等组织相比,YUCCA基因在分蘖节中的相对表达量最高。烟草瞬时转化和蛋白质印迹法分析均显示,YUCCA蛋白可以正常表达,其亚细胞定位于细胞质中。【结论】YUCCA基因在调控新麦草分蘖中起下调作用,可用于后续YUCCA基因功能的研究。     

基于基因编辑技术创制高亚油酸水稻材料

【目的】亚油酸是一种人体必需的脂肪酸,能加快体内脂肪代谢与分解、减少胆固醇在血管壁上形成积存、有效预防动脉硬化的发生、提高人体免疫力、促进骨骼发育、提高记忆力、预防脑部功能退化等功能。利用基因编辑技术对控制脂肪酸合成途径的关键酶ω-3脂肪酸去饱和酶基因进行精准编辑,关闭下游产物的合成途径,以便提高水稻上游亚油酸的含量,为创制富集亚油酸水稻材料提供依据。【方法】水稻脂肪酸合成途径中的关键酶ω-3脂肪酸去饱和酶基因(OsFAD3)存在2个拷贝,分别分布在第11和第12染色体,且两者cDNA同源性达97.32%。根据基因编辑原理(编辑的特异性取决于引导RNA(gRNA)的特性),在2个同源基因序列高度一致的外显子区域,分别设计合成2个gRNA,并分别构建植物基因编辑载体,利用农杆菌介导法转化本地受体材料(富源4号)。通过对基因编辑植株进行靶位点测序鉴定,分析2个位点的编辑效率和基因型。同时,对OsFAD3双突变体进行粒宽、粒长等主要籽粒农艺性状分析。采用气质联用检测法测量OsFAD3双突变体籽粒的37种脂肪酸含量。【结果】获得2个位点的纯合编辑材料,同时也获得了其他不同基因型组合的编辑材料;...     

籼稻9311HR-T显性高秆基因的初步定位

从转入bar基因的籼稻半矮秆材料9311HR的BC3F2中发现了1株高秆突变体9311HR-T,其株高和秆长分别比野生型9311HR增加60.8%和71.5%,其高秆性状受1对显性基因控制.以9311HR-T与02428杂交产生的F2群体为材料,利用微卫星标记将9311HR-T高秆基因定位在水稻第1染色体长臂上的SSR标记RM472和RM1387之间,距RM472和 RM1387的遗传距离分别为8.6 cM和12.3 cM,该基因暂命名为DT1.