利用酵母双杂交法检测甘蓝SCR与SRK之间的相互作用

为深入研究甘蓝S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白(SCR)间相互识别的分子机理,以具有典型自交不亲和性的甘蓝D3为材料,利用巢式PCR分别扩增SRK胞外域(eSRK)和SCR,通过酵母双杂交检测二者之间的相互作用。并利用同源重组技术将eSRK与GAL4报告基因DNA活化域融合(pGADT7eSRK)以及SCR与GAL4报告基因DNA结合域融合(pGBKT7SCR)。两种重组质粒分别转化酵母Y187和Y2HGold后未出现自激活现象。融合的二倍体酵母(pGADT7eSRK× pGBKT7SCR)能在选择性固体培养基(SD/–Ade/–His/–Leu/–Trp/X-a-Gal/AbA)上生长,并且菌落呈蓝色。结果表明, eSRK与SCR蛋白能够相互结合, 为深入研究二者作用位点成功建立了一个技术平台。     

Yeast extract, brewer’s yeast and spirulina in diets for Labeo rohita fingerlings affect haemato-immunological responses and survival following Aeromonas hydrophila challenge

A feeding trial was conducted for 60 days to study the immunomodulatory role of three different immunostimulants yeast extract (YE), brewer’s yeast (BY) and spirulina (SP) in Labeo rohita fingerlings. Four hundred and fifty fingerlings (avg. wt 3.35 ± 0.15 g) were randomly distributed in ten treatments and fed with either of ten iso-nitrogenous and iso-caloric semi-purified diets, prepared with three incremental levels (1%, 2% and 4%) of different immunostimulants except the control. Growth parameters did not vary significantly (p > 0.05) among the experimental groups. Haematology and serum parameters was performed before Aeromonas hydrophila challenge whereas respiratory burst activity was analysed following challenge. The respiratory burst activity, total leucocyte count, serum total protein and globulin was significantly higher (p < 0.05) in YE 1% supplemented group. The survival (%) after challenging with A. hydrophila was also highest in the YE fed groups. The results indicate that among the different sources and levels of immunostimulants, YE at lower inclusion level is more effective in promoting the immune status of L. rohita fingerlings.     

拟南芥抗灰霉病基因T1N6_22互作蛋白的筛选与分析

前期研究中从拟南芥突变体库中筛选获得一株对灰霉病菌侵染表现为感病的突变体。利用TAIL-PCR等技术克隆获得拟南芥抗灰霉病基因T1N6_22。为了进一步明确T1N6_22在拟南芥抗灰霉病过程中的调控机制,利用酵母双杂交技术(Y2H),以构建成功的p AS1/T1N6_22蛋白为诱饵,筛选拟南芥的cDNA文库。通过酵母双杂交筛选,共获得28个酵母克隆。利用T1N6_2基因特异性引物鉴定酵母阳性克隆,并进行测序。共获得了4个可能与T1N6_22互作的蛋白AT2G19480、AT1G06050、AT5G34780和AT1G21400。Blast分析发现,AT2G19480、AT1G06050、AT5G34780和AT1G21400分别为核小体装配蛋白、功能未知蛋白、假定的泛酸还原酶和硫胺二磷酸盐结合折叠超家族蛋白,分别参与到核糖体装配、泛酸盐合成、植物代谢等过程中。研究结果为确定T1N6_22蛋白的互作蛋白,阐明其调控拟南芥抗灰霉病的分子机制奠定了基础。     

利用酵母单杂交筛选拟南芥AtSTK基因表达调控蛋白的研究

拟南芥丝氨酸/苏氨酸蛋白激酶基因AtSTK的过量表达可以明显提高拟南芥的耐盐性。为进一步研究AtSTK基因表达的调控机制,以拟南芥基因组DNA为模板设计引物,扩增获得AtSTK基因的启动子序列,并构建到报告载体pAbAi,将重组报告载体质粒pAbAi-HYT利用BstbⅠ进行单酶切线性化,转化酵母菌株Y1H Gold,线性化的pAbAi-HYT整合到基因组中。然后,将纯化的拟南芥双链cDNA、pGADT7-Rec载体共转化含有报告载体pAbAi-HYT的酵母菌,将菌液涂布到含有100 ng/m L Ab A的SD/-Leu培养基上进行阳性克隆的酵母单杂交筛选。通过回复鉴定、序列测定,最终筛选获得了可能参与AtSTK基因表达调控的4个拟南芥基因。测序结果表明,酵母单杂交筛选获得的拟南芥基因AT3G32090含有WRKY转录因子保守结构域,为WRKY类转录因子的一员。因此,AtSTK基因的表达很可能接受MAPK信号传导途径中AT3G32090基因编码的WRKY类转录因子的调控,从而影响拟南芥植株的耐盐性。     

BrVIN3.1 互作蛋白BrZAT12 在大白菜春化途径中行使功能初探

BrVIN3.1 在春化过程中通过调控BrFLC1 的表观修饰状态,进而调控其表达控制大白菜花期。本试验以BrVIN3.1 （Bra020445）为诱饵,进行酵母cDNA 文库筛选,从中筛选到它的互作蛋白BrZAT12（Bra006691）;进一步进行酵母双 杂一对一验证,证明BrZAT12 能够与BrVIN3.1 互作。采用低温处理不同花期的大白菜材料,进行表达模式分析,发现 BrZAT12 的表达模式在抽薹性状不同的大白菜材料中存在差异,在易抽薹材料中的表达量明显高于耐抽薹材料,且在耐抽薹 材料中变化幅度较小,同时BrZAT12 的表达模式与BrVIN3.1 相关。推测BrZAT12 可能在大白菜春化调控的开花途径中行使 功能。     

酵母蛋白促杂交瘤细胞和骨髓瘤细胞克隆化和诱生腹水的作用

从隐球酵母科酵母菌提取的酵母蛋白对杂交瘤细胞和骨髓瘤细胞具有促生长、促克隆率和诱生腹水的作用.在单个细胞克隆化不加饲养细胞的情况下,克隆率在50％以上.酵母蛋白对抗李氏杆菌杂交瘤细胞促生长作用为对照组的9.8倍,应用ELISA法测定培养上清抗体反应结果,吸光度值平均增加0.20.在不预先注射液体石蜡的情况下,将杂交瘤细胞或骨髓癌细胞悬于含酵母蛋白的培养液中,直接注入BALB/c小鼠腹腔,无论腹水出现时间还是产量收获均与预先注射液体石蜡常规对照组相似(P>0.05),且腹水效价实验组较常规液体石蜡对照组增加近1倍.     

酵母双杂交表达载体pGBKT7_NPR1与pGADT7_NPR1的构建及酵母菌的转化

NPR1是水杨酸介导的系统获得性抗性（SAR）途径中不可或缺的一个转录辅助因子。在SA诱导下,NPR1被转运至细胞核内并与一些转录因子结合,以启动下游基因的表达。本研究通过PCR扩增获得拟南芥中NPR1基因CDS序列,经过限制性内切酶酶切后在T4连接酶作用下分别克隆至pGBKT7与pGADT7酵母表达载体上,并成功将其转化至Gold酵母菌株中。为利用酵母双杂交筛选NPR1参与形成的转录复合体中其它可能存在的蛋白质并阐明其在植物防御信号传导途径中的互作模式奠定了基础。     

酵母β-葡聚糖对早期断奶羔羊瘤胃氮代谢的影响

选用甘肃肉用绵羊新品种选育群初生公羔20只,随机分为5组,观测了饲粮中添加不同剂量（0、37．50、75．00、112．50、150．00mg／kg）酵母β-葡聚糖对早期断奶羔羊瘤胃氮代谢参数的影响,结果表明,添加酵母β-葡聚糖对日龄82d的羔羊瘤胃液中总氮、氨氮和蛋白氮含量均没有显著影响（P〉0.05）,75．00mg／kg的添加剂量组尿素氮含量有高于对照组的趋势（P=0．091）,食前150．00mg／kg添加剂量组血清尿素氮显著低于对照组（P〈．05）。     

Effect of concentrate level and live yeast (Saccharomyces cerevisiae) supplementation on Texel lamb performance and carcass characteristics

An alternative additive that improves the nutrient use by ruminants and reduces the use of antibiotics is desirable in order to promote a sustainable and ecological animal production. The objective of the study was to evaluate the supplementation of live yeast (Saccharomyces cerevisiae) in diets of finishing lambs fed with different concentrate levels on their performance, carcass traits, metabolic profile and eating pattern. The trial was carried out at Institute of Animal Science and Pastures, in Nova Odessa city, Brazil, with 24 Texel lambs, 18.0 Kg±1.14 Kg of initial live weight, and averaging 70 days-old, kept in individual pens. The animals were fed with diets comprising hay and concentrate rations in two different proportions (80 or 60%, on DM basis), supplemented or not with live yeast. A completely randomized blocks design, on a 2×2 factorial arrangement (2 concentrate levels with presence or absence of live yeast), was used to compare the means. Supplementation with live yeast did not affect (P>0.05) DM intake, feed conversion, daily live weight gain, total live weight gain and final live weight, although it increased cold carcass weight (P=0.0135) and external carcass length (P=0.0104) regardless of the concentrate feed proportion in the diet. On carcass traits, supplementation with live yeasts did not modify (P>0.05) cold carcass dressing percentage, compactness index, leg length, external chest depth, buttock circumference, carcass fat cover score, and conformation score. Lambs fed diets with greater concentrate level (80%) significantly increased (P<0.05) DM intake, feed conversion, daily live weight gain, total and final live weight gains, cold carcass weight, compactness index, external carcass length, external chest depth, and buttocks circumference and did not modify (P>0.05) the cold dressing percentage, degree of carcass fat cover score and conformation. The live yeast decreased plasmatic lactate (P<0.05), did not affect (P>0.05) plasmatic glucose, and reduced (P<0.05) serum urea, only on the higher concentrate diets. The proportion of concentrate did not affect (P>0.05) the plasmatic glucose and lactate. Supplementation of live yeasts did not improve (P>0.05) the rib eye dimensions and the carcass subcutaneous fat thickness measured by ultrasound. Lambs on diet with greater concentrate level had smaller (P<0.05) ingestion and rumination time. The supplementation with live yeast increased the rumination time and did not modify the ingestion time. It can be concluded that live yeast supplementation increases the weight and the carcass length, regardless of the amount of concentrate added to the diet.     

海藻中产油脂酵母菌的分离与鉴定

[目的]从湛江海藻中分离筛选产油脂酵母菌。[方法]通过稀释凃布法及苏丹黑染色法,从海藻中分离产油脂酵母菌,对目标菌进行菌落及显微形态观察,同时做碳源同化试验、氮源同化试验、产类淀粉化合物试验、产酯试验、产酸试验、脲酶试验等生理生化试验进行进一步分析,再通过26S rDNA D1/D2区域的系统发育树分析进行分子生物学水平鉴定。[结果]共筛选到2株油脂产生菌,经多相分类鉴定,菌株HY12属于Rhodotorula mucilaginosa;菌株HY16属于Pseudozyma antarctica。[结论]该研究可为开发海藻中产油脂酵母菌提供理论依据。