结球甘蓝雌蕊调控因子SPT与HEC1的克隆及相互作用分析

为研究SPT和HECs及其相互作用对甘蓝雌蕊发育的影响, 以结球甘蓝自交不亲和系E1为材料, 提取雌蕊总RNA, 根据拟南芥中SPT和HEC1基因设计引物, 采用同源克隆的方法克隆SPT基因, 其序列1085 bp, 开放阅读框(ORF) 1062 bp; HEC1基因ORF 696 bp。通过cDNA推导得到的氨基酸序列表明, SPT编码353个氨基酸残基, 预测分子量为37.67 kD, pI为6.83; HEC1编码231个氨基酸残基, 预测分子量为25.26 kD, pI为10.2。分析表明, 两基因在各器官中均表达, 但SPT在果实和雌蕊中表达量最高, 而HEC1在根和花蕾中的表达量最高。为检测两者的相互作用, 构建原核表达质粒pCold I-SPT和pGEX-HEC1, Pull-down试验表明两蛋白能够在体外相互作用。同时构建pGBKT7-SPT、pGADT7-HEC1及互换载体pGBKT7-HEC1和pGADT7-SPT酵母表达载体, 分别转化酵母Y2HGold和Y187感受态细胞后均未出现自激活和毒性现象, 融合后的二倍体酵母均能在SD/–Trp–Leu–Ade–His/X-α-gal/AbA板上长出蓝色菌斑, 表明SPT和HEC1能够相互作用激活下游的HIS3、AUR1-C、MEL1和ADE2报告基因, 酵母双杂交试验结果与Pull-down检测一致, 说明SPT和HEC1能够相互作用以调控雌蕊的发育。     

Effect of replacing soybean meal with yeast‐fermented canola meal on growth and nutrient retention of Nile tilapia,Oreochromis niloticus (Linnaeus 1758)

Yeast fermentation integrated with water soaking was applied as a method to reduce the levels of some anti‐nutritional factors in canola meal. The procedure completely eliminated glucosinolates and it reduced phytic acid content by 18%. It also led to increase of 9% of crude protein and 8–32% of some minerals in the meal. A 60‐day feeding trial was conducted to evaluate replacement of dietary soybean meal protein with yeast‐fermented canola meal for Nile tilapia at ratios of 0, 25, 50, 75 and 100% respectively. Three groups of fish with an initial weight of 10 g were fed with each diet twice a day to an apparent satiation. The results showed that there were non‐significant differences (P > 0.05) in growth, survival rate, feed intake and feed conversion ratio for all fish fed with the test diets. However, replacement with 75 and 100% levels significantly reduced (P < 0.05) protein efficiency ratio and nutrient digestibility of protein, lipid, ash, Ca, Mg and P of fish compared with the lower levels. The low retention of protein, lipid, ash, Ca, Mg and P were also observed. These effects seemed to be related to an increased dietary phytic acid.     

Immunostimulatory effect of mangrove‐derived marine yeasts in Penaeus monodon

This study attempted to assess the efficacy of mangrove‐derived marine yeasts as a source of immuno‐stimulant in the tiger shrimp, Penaeus monodon. The shrimp was fed on the diets in supplementation with yeast biomass at 10% for a period of 15 days and then challenged with the pathogenic Vibrio cholerae. The animals were assessed for immune responses in terms of total haemocyte counts, phenol oxidase and endobiotics. Among the yeasts tested, Rhodotorula minuta was found to have high immunostimulatory effect in the shrimps especially after challenge with the pathogenic vibrio.     

从小鼠17.5d胚胎cDNA文库中筛选Bax相互作用蛋白

为了研究Bax可能形成的在细胞凋亡中起作用的蛋白复合体,选择Bcl-2家族中的前凋亡因子Bax作为诱饵蛋白,对17.5 d小鼠cDNA文库进行酵母双杂交筛选和BLAST,找到1个RACK1蛋白。为进一步验证上述酵母中Bax与RACK1的结合,用Bax与RACK1的真核表达载体,以免疫共沉淀和免疫荧光染色验证其相互作用。利用293T细胞过量表达Bax和RACK1蛋白,收获裂解液后进行免疫共沉淀试验,结果表明,Bax和RACK1在体内仍可形成复合体;在293T细胞中共转Bax和RACK1质粒,用各自荧光抗体标记,然后用激光共聚焦显微镜观察,发现Bax和RACK1可以共定位。     

酵母双杂交技术及其在植物与病原物互作中的应用

介绍了酵母双杂交系统的基本原理及其优缺点,以及十几年来此项技术在植物与病原物互作中的应用情况和此项技术的发展前景。     

酵母培养物对肉仔鸡屠宰性能及胴体品质的影响

主要研究在玉米豆粕基础日粮中添加酵母培养物(YC)对肉仔鸡屠宰性能及胴体品质的影响。将960只1日龄肉仔鸡随机等分为4组,其中试验3组,对照1组,试验组分别添加2.5 g/kg,5 g/kg,7.5 g/kg的YC,试验期为1~42 d。结果表明:①试验各组肉仔鸡的胸肌率、腹脂率、半净膛率和全净膛率与对照组相比差异不显著(P>0.05),但添加2.5 g/kg、7.5 g/kg YC的试验组可显著提高肉仔鸡的腿肌率(P<0.05);②在肉鸡日粮中添加YC显著降低了胸肌、腿肌的剪切力(P<0.05);滴水率有下降趋势(P>0.05);pH值(45 min)亦有降低。研究证明:在日粮中添加一定量的YC能降低肉仔鸡胴体肌肉的剪切力及滴水损失,提高肉的嫩度及持水能力,达到改善肉鸡的屠宰性能和肌肉品质的目的。     

大豆(Glycine max) GmDREB5互作蛋白GmGβ1的筛选及鉴定

从大豆(Glycine max)中克隆了一个与抗逆相关的DREB (Dehydration Responsive Element Binding Protein) 基因GmDREB5。功能分析证明, GmDREB5基因能够显著提高烟草的抗旱性和耐盐性。为了筛选GmDREB5的互作蛋白, 采用酵母双杂交系统以GmDREB5蛋白73~226位氨基酸区段为诱饵筛选干旱处理的大豆cDNA文库, 发现一个互作蛋白含有保守的WD40结构域, 与棉花(Gossypium hirsutum)和水稻(Oryza sativa)的G蛋白β亚基分别具有61%和52%的同源性, 说明蛋白可能是一类新的大豆G蛋白β亚基, 将其定名为GmGβ1。将GmDREB5与GmGβ1共转化酵母菌株AH109, 转化的酵母能够在四营养缺陷型培养基上(SD/ trp- leu- his- ade-)正常生长, 而对照不能生长; 同时, 共转化的酵母能够激活LacZ报告基因的表达, 证明GmGβ1与GmDREB5之间存在相互作用。表达特性分析表明, GmGβ1基因受干旱、低温、高盐等胁迫和激素ABA处理的诱导而表达, 证明GmGβ1不仅参与植物对非生物胁迫的响应, 同时参与对GmDREB5蛋白水平的调控。     

Rheofermentometer parameters and bread specific volume of frozen sweet dough influenced by ingredients and dough mixing temperature

Response surface methodology described the effects of salt, lactic acid, shortening, and exogenous trehalose and dough mixing temperature (DMT) and their interactions on the three rheological and fermentation parameters. These included maximum dough height (Hm), maximum height of gas release (Hm′) and CO₂ production, measured by the Rheofermentometer F3, and bread specific volume (Sp. Vol.) of frozen sweet dough. The models could estimate the four parameters with R² values of 0.76, 0.69, 0.93, and 0.59, respectively. Salt significantly influenced all four parameters in a negative way. DMT affected positively the Hm and Sp. Vol. of bread. Lactic acid affected Hm only, but its interactions with other variables influenced all four parameters. Shortening level affected Hm′ and CO₂ production positively and Sp. Vol. negatively. The added exogenous trehalose improved Hm, Hm′, and CO₂ production significantly, but not the Sp. Vol. of bread. Among the three Rheofermentometer parameters, Hm showed the highest correlation with Sp. Vol. (R² = 0.75). DMT for the maximum Hm and Sp. Vol. varied with the level of other ingredients. Trehalose alone could not overcome the challenges in a sweet frozen dough system to improve the Sp. Vol., and its combined effects with other ingredients will need to be evaluated to restore the impaired gas retention of the frozen sweet dough.     

Puroindoline-a and puroindoline-b interact with the Saccharomyces cerevisiae plasma membrane through different amino acids present in their tryptophan-rich domain

Puroindolines are two small, basic cysteine-rich proteins isolated from Triticum aestivum seeds and characterized by a tryptophan-rich domain. They form the molecular basis of wheat grain hardness and display antimicrobial activity that may contribute to plant defence. Their antimicrobial activity is presumed to be due to their hydrophobic tryptophan-rich domain. However, little is known about their mode of action and there is no in vivo evidence that the binding of puroindolines to membranes is mediated by their tryptophan-rich domain. In this study, using a yeast complementation assay, we showed that puroindolines interact with the Saccharomyces cerevisiae plasma membrane. By site-directed mutagenesis of their tryptophan-rich domain, we determined that two tryptophan residues (W41 and W44) are mandatory for interaction of puroindoline-a with the yeast membrane whereas interaction of puroindoline-b depends on lysine residues. These results highlight that other residues than tryptophan play a critical role in the interaction of puroindolines with membranes, and probably their affinity for lipids and antimicrobial activities.     

利用大豆浓缩蛋白乳清制备水苏糖的发酵条件

利用酵母对大豆浓缩蛋白乳清进行发酵处理制备水苏糖.在确定最佳起始发酵液的糖度为31.8Brix后,对温度、pH、接种量、装液量等工艺条件进行了单因素试验及正交试验,结果表明最佳发酵条件为:温度32°C,pH5.5,接种量8%,在此发酵条件下利用酵母对大豆浓缩蛋白乳清进行发酵处理48 h,水苏糖的纯度达到了90%,保留率为68%.