RNAi沉默淀粉分支酶sbe3基因对水稻直链淀粉的影响

本研究利用RNAi技术,通过RT-PCR方法克隆出水稻淀粉分支酶sbe3基因片断,经两步组装法将sbe3基因片断分别正向、反向组装入pRNAi-ubi,成功构建sbe3基因RNAi转化载体pRNAi-ubi/sbe3。在农杆菌介导下,对粳稻中花11未成熟胚诱导的愈伤组织进行转化,通过PCR、Southern杂交鉴定获得一批转基因植株。半定量RT-PCR鉴定出转化苗T1代种子中sbe3基因表达被明显抑制,但只引起转基因水稻胚乳中直链淀粉含量少量的提高,说明采用RNAi仅沉默sbe3基因对水稻胚乳直链淀粉含量没有显著影响。     

大米抗性淀粉双酶制备及其结构表征

以大米淀粉为原料,α-淀粉酶与普鲁兰酶为酶解剂,利用单因素试验研究大米抗性淀粉的制备工艺条件;通过抗酶解试验研究了其抗酶解性;采用扫描电子显微镜(SEM)、差示扫描量热仪(DSC)和X-射线衍射(X-RD)表征了大米抗性淀粉的结构。确定最佳工艺条件为:pH 5.5、温度80℃、反应时间40 min、酶α-淀粉用量4 u/g,抗性淀粉得率为45.2%。利用抗酶解试验,通过与单酶法和湿热法制备得到的抗性淀粉相比较,发现双酶法制备的抗性淀粉具有较强的抗酶解性能,24 h时的酶解率为8.02%。DSC、SEM和X-RD分析表明:双酶解法制备所得抗性淀粉具有糊化热性能稳定、空间结构紧密以及结晶度高等特点。表明双酶法制备的大米抗性淀粉抗消化能力强。     

重组大肠杆菌产淀粉分支酶的发酵条件探索

淀粉分支酶可增加淀粉的α-1,6-糖苷键,从而改善淀粉的理化特性和生理功能。本文以可胞内表达Geobacillus thermoglucosidans淀粉分支酶的重组大肠杆菌BL21(DE3)(pET-20b(+)/be)为研究对象,首先研究了超声波提取胞内淀粉分支酶的条件,结果显示,当超声波输出功率为325W,总工作时间为15min,菌液OD600为1.4时,超声波破壁效果最佳。在此基础上,对重组大肠杆菌产淀粉分支酶的摇瓶发酵条件进行了探索,结果表明,最适发酵培养基为TB,初始pH为7.0,在25℃、IPTG浓度为0.01mmol/L下诱导培养12h,发酵所得菌体经超声后产生的淀粉分支酶总酶活最高为222.4U/mL。     

Thermomonospora curvata淀粉分支酶的过量表达及其催化反应机理研究

本文以质粒pET-22b(+)为载体,Thermomonospora curvata淀粉分支酶基因(Tc SBE)为客体,采用构建重组大肠杆菌BL21(pET-22b(+)-TcSBE)的方法,实现TcSBE过量表达。目标分支酶经分离纯化,酶活达90.28 U/mg,并分别以长直链淀粉和短直链糊精为底物,研究了TcSBE作用淀粉机理。结果表明:TcSBE以链间反应模式催化长直链淀粉生成大分子的支链淀粉和小分子的低聚糖;以短直链糊精为底物时,TcSBE通过水解作用和转糖基作用同时作用于底物,反应初期,水解作用较强,将底物随机水解成聚合度不等的小分子直链糊精,所需供体链的最低聚合度(DP)为12,随着反应的进行,水解作用不断减弱,转糖基作用增强,将DP 3~8的短链糊精通过α-1,6糖苷键与产物相连接,使产物中分支侧链含量增加,反应12 h后,TcSBE作用产物的α-1,6糖苷键含量达到0.9 m M。     

整粒大米蛋白质分离方法的研究

本文对整粒大米蛋白质的分离方法．在不破坏大米形态的前提下，将米粒于酸性溶液中浸泡，使米中蛋白质溶出或部分水解后溶出。酸浸温度50℃，酸浸时间24～30小时，酸浓度1．5mol／L时效果较好。     

不同灭酶处理对青稞的营养价值和蛋白质功能性质的影响

采用蒸制、炒制和微波烘烤三种灭酶方式处理青稞,以不灭酶的青稞为对照,研究灭酶方式对青稞营养价值和蛋白质功能性质的影响。结果表明,蒸制后青稞蛋白质和淀粉分别降低29%和23%,炒制后蛋白质降低26%,而微波烘烤对青稞基本组分影响较小。蒸制和炒制后青稞蛋白质的溶解度、起泡性及泡沫稳定性、乳化性和乳化稳定性降低,蒸制后蛋白质持水力降低,而炒制后增加,微波烘烤后青稞蛋白质的溶解度、持水力、起泡性和泡沫稳定性保持相对稳定,乳化性和乳化稳定性增加,灭酶后青稞蛋白质凝胶硬度增加,微波烘烤后最大,高于对照组18%。这表明,微波烘烤能保持青稞营养价值和提高青稞蛋白质功能性质,是青稞灭酶的最佳方式,适合青稞的制粉工艺和食品加工。      

利用酶制剂降解烤烟烟叶中淀粉和蛋白质的研究

为探索外加酶制剂施于调制后原烟发酵过程的效果,设计了不同加酶量、作用时间、相对湿度和温度处理的方法来降解烤烟烟叶中的淀粉和蛋白质的试验。结果表明,单一施用中性蛋白酶对烤烟蛋白质的降解率不理想,综合使用α-淀粉酶、葡萄糖淀粉酶针对不同的部位均有不同的相对最佳处理组合。在各等级相对最佳条件下施加α-淀粉酶、葡萄糖淀粉酶后再喷施中性蛋白酶发酵烟叶后针对3个等级烟叶的淀粉和蛋白质有所降解,氨基酸含量均有显著提高,但还原糖含量、总氮和烟碱含量变化不明显。     

高温高湿贮藏对玉米淀粉合成关键酶的影响

选用2013年收获的"农大709"玉米籽粒,根据吉林省夏季平均气温与相对湿度设定玉米的贮藏条件,将玉米籽粒分别贮藏于室温和恒温恒湿培养箱(35℃,75%)中,并测定分析了不同贮藏时间玉米籽粒可溶性淀粉合成酶(SSS)、束缚性淀粉合成酶(GBSS)、淀粉分支酶(SBE)以及淀粉脱支酶(DBE)活性的变化,进而分析玉米在不同贮藏条件下淀粉品质的变化。试验结果表明:常温条件下,只有SBE在贮藏第30天时有显著的下降(P0.05),其余3种淀粉关键酶(SSS、GBSS、DBE)活性均没有发生显著变化(P0.05)。高温高湿贮藏条件下,SSS、SBE、DBE酶活性均呈极显著下降(P0.01),且分别下降了42.1%、39.5%和33.7%。而GBSS酶活性则呈先上升后下降的趋势(P0.01),较常温贮藏,GBSS酶活性总体呈上升趋势。     

碳源诱导嗜热脂肪芽孢杆菌生产淀粉分支酶的研究

利用嗜热脂肪芽孢杆菌（Bacillus stearothermophilus）生产淀粉分支酶,研究不同碳源种类及浓度对嗜热脂肪芽孢杆菌产酶性能的影响。结果表明,以0.20%麦芽糊精为碳源时,淀粉分支酶的酶活最高,达20.02 U/mL,是其他碳源的2～10倍。采用最优培养基在小试试验的基础上进行了扩大培养中试试验,淀粉分支酶酶活稳定,为19.87 U/mL,是小试试验的1.1倍。     

两步酶解法制备香蕉抗性淀粉的工艺研究

本文主要研究果胶酶、纤维素酶和中温a-淀粉酶制备香蕉抗性淀粉的酶解工艺。研究发现：第一步酶解中果胶酶与纤维素酶配比1:2,酶添加量为0.22%,温度45 ℃,pH 5.0,时间35 min,第二步酶解添加中温a-淀粉酶0.35%、温度52 ℃、pH 6.3、时间3.5 h为酶解最适条件,此时的香蕉抗性淀粉含量达到81.24%。     

Evaluation of compositional and nutritional equivalence of genetically modified rice to conventional rice using in situ and in vitro techniques

BACKGROUND: Concerns that genetically modified (GM) rice may pose nutritional risks have led to the need for studies comparing its nutritional composition with that of its isogenic counterpart. The present study explored the compositional and nutritional equivalence of rice grains and straw derived from a glufosinate herbicide‐tolerant GM rice (Bar68), its non‐transgenic conventional counterpart (D68) and a transgenic hybrid generation (X125S/Bar68) of Bar68 with conventional rice X125S. RESULTS: The chemical and nutritional composition, in vitro fermentation and in situ nylon bag degradation parameters were employed. Statistical comparisons to test the equivalence between D68, Bar68 and X125S/Bar68 were made with a criterion of maximum differences (scaled by D68) not exceeding 20%. The chemical and amino acid components of Bar68 and X125S/Bar68 were equivalent to those of D68, with the exception of Ca, P, K, Zn, cysteine and phenylalanine contents of grains and P and Mn contents of straw. Bar68 and X125S/Bar68 were equivalent to D68 in terms of in vitro fermentation and in situ degradation parameters of grains and straw, with the exception of the rapidly degradable component of neutral detergent fibre and the potentially degradable component of acid detergent fibre of straw. The maximum differences in some chemical components and nutritional indices were noted between D68 and X125S/Bar68. CONCLUSION: The results of this study indicated that grains and straw of the glufosinate herbicide‐tolerant GM rice Bar68 and its transgenic hybrid generation X125S/Bar68 were essentially equivalent in chemical composition and nutritive value to those of its non‐transgenic counterpart D68. Copyright © 2009 Society of Chemical Industry     

转基因大米育种及其食用安全性研究进展

伴随着环境的恶化及资源的短缺,世界范围内正面临着一场粮食危机,转基因育种技术也因此得以迅速发展与应用。主要介绍了转基因大米的育种技术,同时对转基因大米的食用安全性进行了论述。     

转基因大米及其安全性评价研究进展

近年来,转基因稻米的迅速发展引起了广泛关注,尤其是与食品密切相关的营养与安全问题。分析转基因大米的基因来源及对大米品质的影响,总结转基因大米可能存在的危害及其安全性评价的方法,可使人们科学的对待转基因大米,并为研究与开发新型转基因大米提供参考。     

转人乳铁蛋白基因大米的慢性毒性研究

目的 观察转人乳铁蛋白(hLF)基因大米是否具有慢性毒性作用.方法 初断乳的180只SD大鼠按性别、体重随机分为3组:转基因hLF基因大米组、亲本大米对照组和AIN-93对照组,分别饲喂相应饲料12个月.观察大鼠的体重、进食量、血常规、血生化情况,实验末期处死动物,称量脏器重量并对脏器进行病理学检查.结果 转hLF基因大米组血常规(LYM％、GRN％)、血生化(ALT、AST、GLU)在个别时间点上与亲本对照组或AIN-93对照组存在显著差异(P＜0.05);其他观察指标与两个对照组均无显著差异.结论 现有实验结果不能证实转hLF基因大米对大鼠有慢性毒性作用.     

Isolation of DNA from genetically modified oils by fast protein liquid chromatography

In this study, a novel method of fast protein liquid chromatography (FPLC) anion exchange chromatography was developed for isolation of DNA from processed genetically modified (GM) oils. Four kinds of different GM edible oil had been chosen as model sample. Salmon DNA was used as the control sample to determine the pH values and NaCl in mobile phase buffer. Applying pH 8 and NaCl gradient 0.5–2 m were chosen for the DNA isolation. The quality and purity of isolated DNA were tested with agarose gel electrophoresis, scanned with UV absorbance spectra and amplified by polymerase chain reaction (PCR). The result indicated that the quantity of DNA isolated by FPLC was suitable for further PCR analyses. Furthermore, it is more effective and less time‐consuming in comparison with cetyltrimethylammonium bromide method and High Pure GMO Sample Preparation Kit method.     

Analysis of caecal microbiota in rats fed with genetically modified rice by real-time quantitative PCR

The effect of genetically modified rice (GMR) on bacterial communities in caecal content was analyzed in a 90-d feeding rat model. A total of 12 groups of rats, which included male and female, were fed with the basal diets containing 30%, 50%, 70% GMR (B(1), B(2), B(3)) or 30%, 50%, 70% non-GMR (D(1), D(2), D(3)). The structure of intestinal microflora was estimated by real-time quantitative PCR (RQ-PCR) based on genus-specific 16s rDNA primers. SYBR Green was used for accurate detection and quantification of 6 kinds of major bacteria shared by humans and rats. According to RQ-PCR, the genome copies of Lactobacillus group from the cecum of male rats fed with 70% non-GMR was higher than those fed with 70% GMR and the relative abundance of Lactobacillus group also higher for group D. This result was in contrast with the E. coli subgroup, which was more numerous in proportion of group B, except D(2) and B(2) for male rats. The Clostridium perfringens subgroup was numerically more abundant in group D than group B of the same level, also except D(2) and B(2) for male rats. These results suggested that GMR had a complex effect on caecal microflora that may be related to the health of the host.     

基于近红外光谱和LSSVM方法的转基因大米鉴别研究

采用近红外漫反射光谱结合主成分分析(principal component analysis,PCA)和最小二乘支持向量机(least squares support vector machine,LSSVM)研究转基因大米的鉴别方法。采用PCA方法分析大米样品光谱空间分布;不同的光谱预处理方法:5点平滑、多元散射校正(multiplicative scatter correction,MSC)和标准正态变量变换(standard normal variate transformation,SNV)结合LSSVM用于定性判别模型的建立和优化;采用格点搜索方法对LSSVM模型的惩罚因子(c)和径向基核函数宽度(g)进行优化;正确识别率(correct recognition rate,CRR)用于判别模型的评价。结果表明:MSC结合LSSVM可用于转基因大米定性判别模型的建立,最优模型的CRR为97.50%。该方法有望成为转基因食品快速鉴别的一种辅助方法。      

变性淀粉在酶固定化中的应用

将酶固定在各种载体上,既可克服游离酶易变性失活的缺点,保持酶的生物催化特性,且易分离和重复利用,提高酶的利用效率。淀粉来源广泛,廉价易得,可通过改性满足不同酶固定化的需要,是一种较为理想的酶固定化载体。本文主要阐述了几种变性淀粉在酶固定化中的应用,并指出今后变性淀粉在酶固定化中的应用前景。