甘蓝自交不亲和决定因子的体外表达和相互作用的检测

 S位点受体激酶(SRK)和S位点富含半胱氨酸蛋白/S位点蛋白11(SCR/SP11)分别为甘蓝自交不亲和(self-incompatibility, SI)信号传导的雌雄决定因子。为了深入研究两者的作用机理和进行人工调控，本研究以结球甘蓝ZQ为材料，利用pET·NusA融合蛋白表达系统，将包含SRK胞外域和跨膜域的mSRK蛋白和SCR蛋白在大肠杆菌BL21中融合表达，经SDS-PAGE电泳检测表达出融合蛋白大小分别约为116 kD和74 kD。进一步将两融合蛋白进行体外相互作用的检测，结果表明mSRK与SCR蛋白能够相互结合形成稳定的复合体，这为下一步实现SRK-SCR复合体聚合与解离的人为调控提供了技术平台。     

小麦淀粉合酶基因I的克隆及反义和RNAi载体的构建

采用RT-PCR方法从小麦品种豫教2号发育的籽粒中克隆出淀粉合酶I基因(starch synthase I,SSI)部分cDNA片段(795 bp)(GenBank No.EF221762),同源性比较结果显示,它与GenBank上已报道的SSI基因有高度同源性。以p WM101质粒为基础,构建了由35S启动子调控的SS I基因的反义表达载体p WM101SSI;另外,还以pFGC5941质粒为基础,构建了SSI基因的RNAi载体pFGC5941SSIsa,这些载体的构建为研究此基因的功能奠定了基础。     

日本血吸虫GSK3蛋白编码cDNA的克隆、表达及其重组蛋白的免疫保护研究

本研究利用生物信息学分析了日本血吸虫(Schistosoma japonicum,sj)Glycogen synthase kinase 3(GSK3)蛋白,并对其中一个GSK3蛋白的编码cDNA进行了克隆和原核表达,制备了特异性的多克隆抗体.同时,还初步评估了重组蛋白的免疫保护效果.生物信息学分析表明在日本血吸虫数据库存在两种GSK3蛋白,且其中一个SjGSK3在日本血吸虫不同发育时期均有转录.Western blot结果表明本研究制备的抗体能特异性识别日本血吸虫SjGSK3重组蛋白,表明该重组蛋白具有良好的免疫原性.动物实验表明免疫SjGSK3重组蛋白的动物与佐剂对照组比较分别获得了平均10.6％减虫率和40.5％肝脏减卵率.     

凡纳滨对虾精氨酸激酶的分离纯化及性质研究

经过CM-纤维素批量层析、Separdex G-100柱层析、DEAE_纤维素柱层析等步骤,从凡纳滨对虾肌肉组织分离得到精氨酸激酶,经SDS-PAGE检测达到电泳纯,分子量约为40kDa.对该酶的性质进行分析结果表明,精氨酸激酶的最适作用温度为55℃,当温度高于65℃时,酶活力显著下降;pH 8时酶活力较高,低浓度的精氨酸对酶活力有促进作用,高浓度时表现抑制作用,而底物类似物精胺和氨基胍则对酶促反应表现出完全的抑制.NaCl,KCl对酶的活力具有促进作用,低浓度(10 mmol·L-1)MgCl2对酶活力表现出激活作用,而CuCl2与MnCl2则表现出完全抑制酶活力,CaCl2与ZnCl2在低浓度时对酶活力无明显影响,但是随着浓度升高,对酶具有抑制作用.     

Metabolic function of hepatic hexokinase in perch, Perca fluviatilis

Feeding previously fasted perch with a carbohydrate‐rich pelleted diet resulted in a rapid transient insulin response followed by a transient increase in the activity of the hepatic low‐affinity hexokinase (glucokinase, GK). The activity of the high‐affinity hexokinase (hexokinase, HK) increased more slowly and steadily to about seven times the fasting value on the 10th day after start of feeding, in parallel with an increase in the hepatic glycogen content. An insulin response was also observed when fasted perch were fed a diet consisting of boiled fillet of cod (a high‐protein, very low‐fat and practically carbohydrate‐free diet). In this case no GK activity was observed, whereas the glycogen content increased steadily from the start of feeding to about the same level as when fish were given the carbohydrate‐rich diet. There was a delayed increase in the activity of HK, starting on about the sixth day (after the glycogen stores were filled), concomitant with increased activities of lipogenic enzymes (glucose‐6‐phosphate dehydrogenase, ATP citrate lyase and malic enzyme). The results are in line with the hypothesis that the mitochondria‐bound hepatic HK has a particular anabolic function in glycogen and fat synthesis.     

宁夏枸杞八氢番茄红素合成酶基因的克隆与序列分析

类胡萝卜素是植物叶绿体光合作用的辅助色素,并能保护叶绿素免受高温强光的破坏(Bartley et al.,1995).现在越来越多的医学研究表明,类胡萝卜素与人类健康密切相关.约有10%的类胡萝卜素是维生素A的前体(Krinsky,1989).据统计,每年有70多个国家的3 000多万儿童因缺乏维生素A而造成致命性疾病(Cheryl et al.,1997).此外,类胡萝卜素在防癌抗癌、预防心血管疾病、增强人体免疫力等方面起着重要的作用(陶俊等,2002).通过对菲律宾、加拿大和中国的食道癌患者的调查已证明β-胡萝卜素能延缓疾病的进程(韩雅珊,1999).由于类胡萝卜素在许多植物中含量较低,并且难以用化学方法合成.因此,利用基因工程手段调控类胡萝卜素的代谢积累,进而大量生产类胡萝卜素已成为研究者们所共识的研究方向之一.目前通过基因工程手段已获得转类胡萝卜素合成酶基因的"金大米"和"金油菜"(陶俊等,2002).     

Molecular Cloning of a Cellulose Synthase Gene PtoCesA1 from Populus tomentosa and Its Genetic Transformation in Tobacco

A 3 125 bp cellulose synthase gene, PtoCesA1, which has a 98% identity to PtrCesA1 from Populus tremuloides, was cloned from cDNA prepared from secondary xylem of P tomentosa. Four anti-expression vectors with different fragments of PtoCesAl, named as pBIPF, pBICC1, pBIPR and pBIBR, were constructed. Some traits of transformed tobacco of pBICC1, pBIPR and pBIBR differed from wild types, such as small leaves, ＂dwarf＂ phenotype and thinner xylem and fiber cell walls than wild plants consistent with a loss of cellulose. It indicated that the growth of transgenic tobacco was restrained by the expression of anti-PtoCesA1. Transgenic tobacco was obtained and the contents of cellulose and lignin were analyzed as well as the width and length of fiber cells, and xylem thickness for both transgenic and control plants. Transformed tobacco showed a different phenotype from control plants and it implied that PtoCesA1 was essential for the cellulose biosynthesis in poplar stems.     

油茶查尔酮合酶和异构酶基因的cDNA克隆

查尔酮合酶和查尔酮异构酶是类黄酮代谢与色素苷代谢的关键酶.以油茶近成熟种子cDNA文库和EST文库为材料,通过分子克隆方法鉴定了1条查尔酮合酶基因全长cDNA和1条查尔酮异构酶全长cDNA.结果表明:油茶查尔酮合酶基因的cDNA含1479 bp,编码412个氨基酸,为目前最长的查尔酮合酶,与其它物种的查尔酮合酶具有极高的相似性,在进化上高度同源;油茶查尔酮异构酶基因的cDNA含899 bp,编码206个氨基酸,与茶的查尔酮异构酶基因高度同源,但与其它物种的相似性较低.     

Local pulmonary immune responses in domestic cats naturally infected with Cytauxzoon felis

Cytauxzoonosis is a hemoprotozoal disease of cats and wild felids in the South and Southeastern United States caused by Cytauxzoon felis. Although the causative agent has been recognized since the seventies, no study has examined the local immune response in affected organs, such as the lung, and compared them to the lungs of uninfected domestic cats. Previous studies have suggested that the histopathologic findings in the lungs of C. felis-infected cats are caused by the release of pro-inflammatory mediators, such as cytokines and increased production of inducible nitric oxide synthase (iNOS), by the infected macrophages. Our laboratory had previously found an upregulation of the adhesion molecule CD18, which can stimulate the release of these pro-inflammatory mediators. The objective of this study was to characterize local pulmonary immune responses in cats naturally infected with C. felis. Immunohistochemistry was performed to detect tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, iNOS, and major histocompatibility complex (MHC) II in 19 lungs from affected cats that died between 2005 and 2013. Results showed increased expression of all of these molecules when compared to lungs from uninfected, healthy cats. Furthermore, MHC II is expressed in the endothelium of C. felis naturally infected cats. These results support that there is a marked, local, pro-inflammatory immune response that can contribute to the pathogenesis of cytauxzoonosis in the lungs.     

Real-time PCR genotyping assay for feline erythrocyte pyruvate kinase deficiency and mutant allele frequency in purebred cats in Japan

Erythrocyte pyruvate kinase (PK) deficiency is an inherited glycolytic erythroenzymopathy caused by mutations of the PKLR gene. A causative mutation of the feline PKLR gene was originally identified in Abyssinian and Somali cats in the U.S.A. In the present study, a TaqMan probe-based real-time PCR genotyping assay was developed and evaluated for rapid genotyping and large-scale screening for this mutation. Furthermore, a genotyping survey was carried out in a population of four popular purebred cats in Japan to determine the current mutant allele frequency. The assay clearly displayed all genotypes of feline PK deficiency, indicating its suitability for large-scale survey as well as diagnosis. The survey demonstrated that the mutant allele frequency in Abyssinian and Somali cats was high enough to warrant measures to control and prevent the disease. The mutant allele frequency was relatively low in Bengal and American Shorthair cats; however, the testing should still be carried out to prevent the spread of the disease. In addition, PK deficiency should always be considered in the differential diagnosis of anemia in purebred cats in Japan as well as worldwide.