翅碱蓬CMO基因克隆、测序及植物表达载体的构建

从翅碱蓬叶片中提取总RNA,根据相关同源序列设计引物,使用反转录试剂盒进行RT-PCR,扩增得到翅碱蓬胆碱单加氧酶（CMO）cDNA,进行PCR产物测序;然后将CMOcDNAT-A克隆至pMD18-T-simple载体上,经测序正确后亚克隆至pBI121植物表达载体,进行酶切鉴定及克隆测序。     

HSV-1 UL30基因cDNA的克隆及筛选有效siRNA的融合载体构建

目的：克隆HSV-1 UL30 cDNA并测序,构建pEGFP-N1-Fi融合表达载体,为靶向UL30基因的siRNA的设计和筛选奠定基础。方法：从感染HSV-1 F株的病变VERO细胞提取总RNA,二次PCR扩增出UL30cDNA并克隆至pEGFP-N1质粒,测序鉴定序列;将UL30cDNA4个小片段亚克隆至pEGFP-N1-Fi融合表达质粒,转染VERO细胞,荧光显微镜观察融合蛋白表达情况。结果：成功克隆出UL30 cDNA,序列对比显示与基因库中的HSV-1株UL30序列同源性为99.4%;成功构建pEGFP-N1-Fi融合表达载体并实现Fi-EGFP融合蛋白的表达。结论：成功克隆出UL30cDNA,成功构建pEGFP-N1-Fi融合表达载体,为靶向UL30基因的siRNA的设计和筛选奠定基础。     

大鼠Mettl9基因的克隆及其慢病毒表达载体的构建

目的:克隆大鼠Mettl9基因,并构建慢病毒表达载体,为研究Mettl9基因功能奠定基础。方法:提取原代培养大鼠星形胶质细胞的总RNA,应用RT-PCR方法,扩增出Mettl9基因的cDNA,克隆至pGEM-T载体上并测序;再将该基因亚克隆至慢病毒载体pLenti6.3-MCS-IRES-EGFP,测序鉴定,利用脂质体lipofectamine 2000转染至293T细胞进行包装,感染U251细胞,荧光显微镜下观察其表达。结果:Mettl9 cDNA的RT-PCR扩增产物为954 bp的基因片段,连接到pGEM-T载体后测序结果向GenBank递交获得收录号HQ898855;构建的pLen-ti6.3-Mettl9-IRES-EGFP慢病毒载体在293T细胞完成包装,测得慢病毒滴度达1.825×108TU/mL;经感染U251细胞,荧光显微镜下可见绿色荧光。结论:成功克隆了SD大鼠Mettl9基因,构建了该基因的慢病毒表达载体pLenti6.3-Mettl9-IRES-EGFP。     

Annexin Ⅱ基因克隆及真核表达载体的构建

目的克隆人AnnexinⅡ全长cDNA序列,并构建其真核表达载体。方法根据Genbank中AnnexinⅡ基因的碱基序列,利用RT-PCR的方法从人肾脏组织中扩增编码AnnexinⅡ基因,将目的片段与pBS-T载体连接,利用亚克隆的方法将AnnexinⅡ的cDNA片段克隆到pcDNA3.1载体中,并进行序列测定。结果DNA测序证实该片段序列与Genbank完全一致。结论成功克隆人Annexin II全长cDNA序列,并构建出pcDNA3.1—Annexin II真核表达载体,为进一步研究其在肾脏疾病中的作用和机制奠定基础。     

人糖皮质激素β受体基因正、反义重组逆病毒载体构建

为研究人糖皮质激素受体hGRβ亚型的功能及其作用机制,用L-PCR方法从人胚脑表达文库内扩增hGRβ基因全长约2．4kb的cDNA片段,克隆至pGEM-Teasy载体;经限制性核酸内切酶谱证实后,进一步将其亚克隆至逆病毒载体pLXSN中,并经测序证实,为进一步的基因转移和基因治疗研究准备材料。     

盐度、氮、磷含量对翅碱蓬生长的影响

目的：探明盐度、氮、磷含量对翅碱蓬生长的影响。方法：采用盆栽方法,控制土壤的盐度、氮、磷浓度,设计正交试验,研究在不同水平的盐度、氮含量和磷含量条件下翅碱蓬的发芽和生长状况。使用三因素方差法对试验结果进行显著性分析。结果：盐度显著影响翅碱蓬种子萌发和苗重;盐度和氮含量显著影响翅碱蓬苗高、叶绿素积累及对氮磷的吸收;磷含量影响不显著。结论：盐度和氮含量是影响翅碱蓬生长的关键因子,低盐环境有利于翅碱蓬生长,氮含量增加促进翅碱蓬的生长。磷含量对翅碱蓬生长影响不显著。     

高亲和K+转运载体SsHKT1的抗体制备及表达分析

利用RT-PCR及RACE技术从盐生植物盐地碱蓬(Suaeda salsaL.)根中克隆了高亲和K 转运载体SsH-KT1的基因。以SsHKT1基因的cDNA为模板,扩增了cDNA中的亲水片段(403~612 bp),连接至原核表达载体pGEX-6P-3中并转化大肠杆菌BL21(DE3),IPTG诱导表达了融合蛋白。该蛋白主要以包涵体的形式存在,纯化后免疫新西兰兔,得到了特异性较高的SsHKT1多克隆抗体。利用此抗体进行了W estern b lot分析,表明SsHKT1可能主要存在于质膜上。进一步研究了K 饥饿及盐胁迫条件下SsHKT1蛋白表达量的变化,结果表明K 饥饿及盐胁迫条件下SsHKT1的表达量都明显增加,表明SsHKT1在盐地碱蓬K 吸收特别是高盐条件下K 营养中有重要作用。     

盐地碱蓬叶片液泡膜H^＋—ATPase B亚基的克隆及盐胁迫下表达分析

植物液泡膜H^ -ATPase在建立跨液泡膜质子梯度、促进液泡Na^ 区域化、提高植物耐盐性方面发挥着重要作用。本实验从盐生植物盐地碱蓬(Suaeda salsa L.)cDNA文库分离到碱蓬叶片液泡膜H^ -ATPase B亚基cDNA克隆。测序表明该基因长达1974bp,开放阅读框有1470bp编码489个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量约为54．29kD。Northern及Western印迹表明盐地碱蓬液泡膜H^ -ATPase B亚基表达明显受NaCl胁迫诱导,并且在NaCl胁迫下,B亚基在转录及翻译水平上与液泡膜H^ -ATPase c亚基存在协同作用。盐胁迫下,盐地碱蓬液泡H^ -ATPase B亚基与c亚基的协同表达增加了液泡H^ -ATPase的数量,从而提高了液泡H^ -ATPase活性,为碱蓬叶片液泡Na^ 区域化提供了动力,最终提高了碱蓬植株的耐盐性。     

盐地碱蓬叶片液泡膜H+-ATPase B亚基的克隆及盐胁迫下表达分析

植物液泡膜H -ATPase在建立跨液泡膜质子梯度、促进液泡Na 区域化、提高植物耐盐性方面发挥着重要作用.本实验从盐生植物盐地碱蓬(Suaeda salsa L.)cDNA文库分离到碱蓬叶片液泡膜H -ATPase B亚基cDNA克隆.测序表明该基因长达1 974 bp,开放阅读框有1 470 bp编码489个氨基酸,含有一个保守的ATP结合位点,其蛋白分子量约为54.29 kD.Northem及Western印迹表明盐地碱蓬液泡膜H -ATPase B亚基表达明显受NaCl胁迫诱导,并且在NaCl胁迫下,B亚基在转录及翻译水平上与液泡膜H -ATPase c亚基存在协同作用.盐胁迫下,盐地碱蓬液泡H -ATPase B亚基与c亚基的协同表达增加了液泡H -ATPase的数量,从而提高了液泡H -ATPase活性,为碱蓬叶片液泡Na 区域化提供了动力,最终提高了碱蓬植株的耐盐性.     

Investigation of the mechanism of temperature sensitivity of the electroreceptors of ampullae of lorenzini

In experiments on Black Sea skates (Raja clavata), the potential of the receptor epithelium of the ampullae of Lorenzini and spike activity of single nerve fibers connected to them were investigated during electrical and temperature stimulation. Usually the potential within the canal was between 0 and –2 mV, and the input resistance of the ampulla 250–400 k. Heating of the region of the receptor epithelium was accompanied by a negative wave of potential, an increase in input resistance, and inhibition of spike activity. With worsening of the animal's condition the transepithelial potential became positive (up to +10 mV) but the input resistance of the ampulla during stimulation with a positive current was nonlinear in some cases: a regenerative spike of positive polarity appeared in the channel. During heating, the spike response was sometimes reversed in sign. It is suggested that fluctuations of the transepithelial potential and spike responses to temperature stimulation reflect changes in the potential difference on the basal membrane of the receptor cells, which is described by a relationship of the Nernst's or Goldman's equation type.I. P. Pavlov Institute of Physiology, Academy of Sciences of the USSR, Leningrad. I. M. Sechenov, Institute of Evolutionary Physiology and Biochemistry, Academy of Sciences of the USSR, Leningrad. Pacific Institute of Oceanology, Far Eastern Scientific Center, Academy of Sciences of the USSR, Vladivostok. Translated from Neirofiziologiya, Vol. 12, No. 1, pp. 67–74, January–February, 1980.