恒河猴piwil1基因的表达

为了解人类近亲恒河猴piwil1基因的结构、表达和功能差异,利用RT-PCR方法克隆了恒河猴piwil1基因,并对其编码产物进行了相应的生物信息学分析,随后检测了piwil1 mRNA在成年恒河猴10种组织中的表达情况,最后利用免疫组化方法比较了PIWIL1蛋白在成人、成年恒河猴和幼年恒河猴睾丸组织中表达的异同.结果表明,恒河猴piwil1基因全长2 586 bp,编码861个氨基酸,氨基酸数目与人类piwil1基因相同.恒河猴PIWIL1蛋白与人的PIWIL1蛋白序列同源性达99%,均含有PAZ结构域和Piwi结构域.在成人和成年恒河猴的睾丸组织中,PIWIL1蛋白均在精母细胞和精细胞的胞浆中表达,而在幼年恒河猴睾丸组织中,PIWIL1表达与成年恒河猴相比存在差异,表达量相对较低,且主要表达于生精小管的细胞的细胞核上.上述结果提示,PIWIL1蛋白在成人和成年恒河猴可能具有相同作用,但是在不同的发育阶段中PIWIL1蛋白的功能不同.     

闽侯野生蕉LDOX基因的克隆及表达特性

为探讨闽侯野生蕉无色花青素双加氧酶基因(MiLDOX)的序列和表达特性,利用反转录PCR(RT-PCR)克隆MiLDOX基因cDNA序列,并对其进行系列生物信息学和不同组织器官中的表达特性分析.结果显示,MiLDOX基因包含1 083 bp完整的开放阅读框,可编码分子量(Mr)为40.73×10~3、包含360个氨基酸的蛋白质. MiLDOX基因属于α-酮戊二酸依赖性双加酶基因家族,其编码蛋白为不含信号肽和跨膜结构的亲水性蛋白,理论等电点为5.71.MiLDOX二级结构中不规则卷曲和α-螺旋占比较大.亚细胞定位预测结果显示MiLDOX可能定位于细胞质中.系统进化树显示MiLDOX与小果野蕉MaLDOX和阿比西尼亚红脉蕉EvLDOX亲缘关系最近.实时荧光定量PCR结果显示MiLDOX在闽侯野生蕉不同组织部位都有表达,在雄花中的表达量最高,且与类黄酮和花色素苷含量呈显著正相关.此外,通过研究MiLDOX基因在不同转色阶段果皮中的表达情况,发现它的表达随着果皮色素积累逐步升高.本研究表明MiLDOX基因可能分别参与闽侯野生蕉类黄酮和花色素苷的积累,并在果皮转色等过程发挥着重要作用.(图9表1参37)     

香蕉MaMPK1基因的克隆与表达模式

为研究香蕉MAPK1的序列特征及其在不同激素处理、逆境胁迫下的表达趋势,以‘天宝蕉’为材料,采用RTPCR技术克隆MaMPK1并对其进行生物信息学分析和不同处理下的表达模式分析.结果显示该基因编码区长为1182bp,可编码393个氨基酸.其编码蛋白具有STKc_TEY_MAPK结构域,属于MAPK基因家族TEY亚型A亚家族,是不稳定的脂溶性亲水酸性蛋白,无信号肽和跨膜结构,有多个磷酸化位点.亚细胞定位预测结果显示MaMPK1主要定位于细胞核.蛋白互作预测结果显示该蛋白与HSFA4A存在互作,暗示其可能在香蕉抗热反应过程中发挥作用.启动子顺式作用元件预测结果显示MaMPK1启动子包含多种激素和逆境胁迫相关作用元件.定量分析结果显示MaMPK1的表达受SA、45℃、低温和盐胁迫抑制,受茉莉酸甲酯(MeJA)和枯萎病菌侵染诱导上调,在脱落酸(ABA)处理后期极显著上调表达.本研究表明MaMPK1广泛参与香蕉逆境胁迫应答.(图9表1参35)     

文心兰‘南茜’谷氧还蛋白基因克隆定位及表达

为研究文心兰谷氧还蛋白超家族基因ROXY在花药发育中的功能,以文心兰‘南茜’(Oncidium Gower Ramsey)为试验材料,从‘南茜’的转录组网站选取一条与拟南芥At ROXY1/2相似度最高的基因序列(命名为OnROXY1),以该序列的cDNA为模版设计引物,用RT-PCR从‘南茜’花器官中扩增该基因,并对扩增产物进行测序和保守结构域分析.结果表明,OnROXY1基因编码区402 bp,编码133个氨基酸,与‘南茜’转录组网站中的预测序列只有一个碱基差异,但与该预测序列的氨基酸序列和保守结构域完全一致(GenBank登录号:MF696154).OnROXY1基因具有典型的谷氧还蛋白(Glutaredoxin,GRX)基因家族的结构域和CMCC活性位点,因此该基因属于GRX基因家族,并且属于亚类CC型的GRX基因.进化树分析表明,OnROXY1与水稻的Os ROXY1、Os ROXY2和拟南芥的At ROXY1、At ROXY2蛋白序列的一致性最高,进化距离最近.亚细胞定位结果显示,OnROXY1主要定位于细胞核中,少量定位在细胞质中.RT-PCR检测发现,在不同组织部位中,OnROXY1在假鳞茎中的表达量最高,唇瓣中的表达量最低;不同花期的定量结果显示,OnROXY1在脱落干枯期时的表达量最高,盛开期时的表达量最低.本研究推测谷氧还蛋白基因OnROXY1可能在文心兰假鳞茎与根的发育、消除植物体内过多的活性氧以及延缓文心兰花器官的衰老等方面具有重要作用.     

甜橙CsHAC1基因克隆及其在响应柑橘黄龙病侵染过程中的表达

为揭示甜橙组蛋白乙酰转移酶1基因(CsHAC1)在柑橘黄龙病(HLB)侵染过程中的响应机制,利用PCR和RTPCR分别克隆该基因gDNA和cDNA序列,并进行系列生物信息学分析.同时,还对CsHAC1互作蛋白进行预测并研究它们在感染HLB的柑橘中的表达情况.结果显示,该基因编码序列(CDS)全长为5 307 bp,预测可编码含有1 768个氨基酸、无信号肽和跨膜结构的蛋白质.亚细胞定位预测的结果显示CsHAC1主要定位在细胞核.CsHAC1含有ZnF_TAZ、PHD、HAT_KAT11、ZnF_ZZ等多个保守结构域,蛋白互作预测结果显示CsHAC1与ZC3H19L、SUMOs和HAM1L等蛋白存在互作关系.启动子顺式作用元件预测结果显示CsHAC1启动子除含有大量光响应元件外还含有一些逆境(如低温、防御和应激、厌氧等)和激素(如脱落酸、生长素、水杨酸等)相关元件.通过分析CsHAC1及其互作蛋白编码基因在感染黄龙病的柑橘根和叶片中的表达情况发现,CsHAC1在叶片中的表达受HLB诱导,且与HAM1L的表达呈显著负相关.本研究结果表明CsHAC1可能和它的互作蛋白基因一起通过表观遗传调控参与柑橘对HLB侵染的响应.(图9表3参48)     

一株受二噁英类化合物诱导表达绿色荧光蛋白的胃癌细胞系的建立

将人工合成的5个相连的XRE(Xeobiotic response element)和minimal CMV(Human cytomegalovirus)启动子,插入载体pEGFP-1,构建得到载体pXRE5-EGFP.以pXRE5-EGFP转染人胃癌细胞SGC-7901,经过筛选得到一株受2,3,7,8-四氯代二苯并二英(2,3,7,8-tetrachlorodibenzo-p-dioxin,TCDD)诱导表达绿色荧光蛋白的细胞系XRE5-SGC-7901.将XRE5-SGC-7901细胞株暴露于不同浓度TCDD(0、250、500pg·mL-1)中,发现TCDD暴露组XRE5-SGC-7901细胞中绿色荧光蛋白的表达量显著升高,且与TCDD暴露浓度呈正相关.新构建的细胞株具有二英类化合物诱导表达绿色荧光蛋白的功能.     

多花黄精几丁质诱导赤霉素应答基因（CIGR）克隆及其功能

为探讨多花黄精几丁质诱导赤霉素应答基因(chitin-inducible gibberellin-responsive,CIGR)在不同光处理下的的作用机制,采用全长验证的方法获得多花黄精CIGR基因的cDNA序列、gDNA序列,并对其进行生物信息学分析,再对CIGR蛋白进行亚细胞定位观察,同时对不同光质和光周期处理下CIGR基因的表达模式进行分析.结果显示,多花黄精CIGR基因cDNA序列和gDNA全长序列一致,完整的开放阅读框(ORF)长度为1 770 bp,共编码589个氨基酸,CIGR基因无内含子结构.生物信息学分析表明CIGR具有一个GRAS结构域,属于GRAS家族,是植物特有的转录因子,为碱性蛋白,无信号肽;Motif分析表明,CIGR蛋白在物种间比较保守,保守区主要位于中部至3′末端;氨基酸序列进化分析表明,多花黄精与石刁柏的CIGR亲缘关系最近,同源性达81%.进一步亚细胞定位显示CIGR蛋白定位于细胞核,与预测结果一致.实时荧光定量PCR(qPCR)结果显示,多花黄精CIGR基因具有器官表达特异性,在叶中表达量最高.在不同光质和光周期处理下,CIGR基因在蓝光下表达量较高,在远红光下表达量低;在光周期9 h/d的处理中出现峰值.本研究表明CIGR基因可能受蓝光及短光照周期调控从而影响多花黄精叶片的发育过程.(图9表1参49)     

甘蔗细胞色素P450还原酶基因的RT-PCR扩增与表达分析

细胞色素P450基因在电子传递链、次生代谢物质合成和对外源化学药物毒性降解中发挥着重要作用,为了深入了解该基因在甘蔗中的功能,通过RT-PCR扩增获得甘蔗细胞色素P450还原酶基因的cDNA全长序列,命名为ScCPR450(Gen Bank Accession Number:KR864841).该基因全长999 bp,含有744 bp的完整开放阅读框,编码247个氨基酸.亚细胞定位结果显示,ScCPR450蛋白分布于细胞质中,与生物信息学预测结果相符.q RT-PCR表达分析表明,该基因在甘蔗中组成型表达,但有组织特异性,芽中表达量最高,其次是叶,而皮中表达量最低.在脱落酸(ABA)、水杨酸(SA)、茉莉酸甲酯(Me JA)、聚乙二醇(PEG)和氯化铜(CuCl_2)胁迫诱导过程中,该基因的表达量呈现不同变化模式,其中SA胁迫6 h下,ScCPR450基因的表达量最高,约为对照的12.21倍;在PEG胁迫下,ScCPR450基因的表达量上调且表达量稳定,推测ScCPR450基因在甘蔗响应生物和非生物胁迫中发挥一定的作用.本研究可为该基因家族其它成员的克隆以及深入解析该基因的功能特性奠定基础,进而为基于基因工程技术对甘蔗品种进行定向改良提供基因资源.     

香蕉含VQ基序蛋白基因MaVQ1的克隆与表达

克隆香蕉含VQ基序蛋白基因MaVQ1,研究其序列特征及其在不同激素处理和逆境胁迫下的表达模式.采用RTPCR技术从‘天宝蕉’中克隆了该基因,对其进行生物信息学分析,并利用实时定量PCR技术(qRT-PCR)研究它在不同组织部位和不同激素、不同逆境胁迫处理下的表达情况.结果显示:MaVQ1编码序列(CDS)长为459 bp,可编码一个分子式为C_(732)H_(1164)N_(210)O_(207)S_3、分子量为16 314.76、等电点为10.19的不稳定亲水性蛋白.MaVQ1含有保守的VQ结构域,不含跨膜结构和信号肽,与小果野蕉VQ亲缘关系最近.亚细胞定位预测结果显示MaVQ1主要定位在细胞核.蛋白互作预测结果显示MaVQ1与其他香蕉VQ蛋白以及WRKY互作系数最高.启动子顺式作用元件预测结果显示MaVQ1启动子包含多种光响应元件、激素响应元件和逆境胁迫相关作用元件.转录因子结合位点分析结果显示其启动子上存在大量的ERF结合位点.qRT-PCR结果显示:MaVQ1在不同组织部位中的表达无显著差异,其表达受茉莉酸、脱落酸和低温显著诱导,受高温和干旱抑制.本研究表明,与其他植物VQ类似,MaVQ1的表达受多种激素和逆境影响,暗示其可能在香蕉抗逆防御反应过程中发挥着重要调节作用.(图6表2参33)     

基于转录组的‘三月李’及其红肉突变体ARF基因家族鉴定及分析

生长素反应因子(Auxin response factors,ARFs)是一类在植物生长发育过程中发挥着重要作用的转录调控因子.为鉴定ARF家族基因、了解成员特征及其在李果实成熟过程中表达模式,基于‘三月李’及其红肉突变体果实成熟过程的转录组数据,采用生物信息学分析方法进行PsARF家族基因鉴定,并对其蛋白质理化特性、亚细胞定位、系统进化树、蛋白质质保守结构域以及表达模式进行分析.共鉴定得到17个PsARF家族成员.生物信息学分析结果表明PsARF家族蛋白质长度在600-1 157 aa之间,分子量(Mr)在66.24×103-129.31×103之间,等电点介于5.44-8.31之间,均为位于细胞核的不稳定亲水蛋白质.系统进化树分析表明PsARF家族成员可分为4组. PsARF蛋白质均具有典型的B3和Auxin_resp结构域,且多数具有Aux/IAA结构域.保守基序分析表明,PsARF家族蛋白质包含15个保守基序,但非每个蛋白质均含有保守基序. 10个PsARF家族基因在‘三月李’及其红肉突变体果实成熟过程中差异表达,不同基因表达模式各不相同.其中,PsARF2和PsARF16在‘三月李’和红肉突变体中的表达模式相反;PsARF10在‘三月李’果肉中表达量显著高于红肉突变体果肉中的表达量.本研究表明PsARF家族成员可能在李果实成熟过程中具有不同的功能,结果可为深入研究PsARF家族基因在李果实成熟过程中的功能奠定基础.(图5表2参40)     

高赖氨酸蛋白基因在转基因生菜中的表达和遗传转化

以生菜(LactucasativaL.)为受体材料,将含有高赖氨酸蛋白基因的植物表达载体pLBI用农杆菌介导法进行遗传转化,获得47株转基因生菜植株.PCR和Southern-blotting检测证实目的片段在T0代的整合,RT-PCR检测表明目的基因的转录.T0代植株赖氨酸(Lys)含量的测定分析表明,赖氨酸有不同程度的提高,比对照提高最高的可达9.46%.T1代植株PCR检测证明,目的基因能够遗传.图5表1参25     

农杆菌介导将高赖氨酸蛋白基因导入谷秆两用水稻

采用农杆菌介导法将高赖氨酸蛋白基因导入到谷秆两用水稻中，GUS组织化学染色、PCR扩增、Southem blot分析表明，该基因已经整合到水稻基因组中，测定9株转基因水稻叶片赖氨酸含量，大部分植株有明显的提高，最高幅度达到了22．71％，图6参15     

Kinetics of osmoregulation in the crab Carcinus maenas

When shore crabs Carcinus maenas are transferred from 11 to 38 S at 11°C, new constant levels of hemolymph freezing points and of concentrations of Na, K, Ca, and Mg in the hemolymph are accomplished within 24h. From a decrease in serum protein and in serum free amino acids and an increase in the relative amounts of individual essential free amino acids in the body fluids of whole crabs, a participation of proteolytic activities and a transport of amino acids from hemolymph to cell is deduced; the stationary concentration of total intracellular free amino acids increases up to a nearly two-fold value, compared with the concentration in crabs remaining in diluted sea water. Also, the low molecular neutral sugars increase in whole crabs after high salinity stress, reaching values of more than two-fold initial concentrations within a period of 10 days. This increase is fully accounted for by a 6.7-fold increase in trehalose levels. The processes of increasing the concentrations of low molecular organic material seem to be slower than those of establishing new osmotic pressures in the hemolymph. The oxygen consumption decreases by 30 to 45% to new constant values within 8 to 12 h after the salinity change from 11 to 38, reflecting similar kinetics as the establishment of new osmotic pressures in the hemolymph.     

Determination of copper in embryos and very young specimens of Sepia officinalis

The total amount of copper in embryos and newly hatched young individuals of Sepia officinalis L. has been determined by microtechnique, using bathocuproine-sulfonate as complexing reagent. During embryonic life, the total amount of copper does not change; it remains at a level close to 3.8 g. The copper is found in the yolk sac of very early embryos; it is subsequently transferred into the embryo proper. After hatching, the copper content diminishes quickly in starved individuals. Fed S. officinalis also usually lose copper. The reason for this may be that the inner yolk sac of newly hatched individuals contains a great deal of the total copper, which is excreted with the yolk after the latter has become superfluous. Later on, copper must be taken up from the food. The mobilization of protein and copper from the yolk into the blood may account for the early appearance of embryonic hemocyanin in the blood.     

转基因烟草中赖氨酸含量的提高

采用叶盘法将高赖氨酸蛋白基因导入到烟草中.GUS组织化学染色、PCR扩增、Southernblot分析表明, 该基因已经整合到烟草基因组中.测定 10株转基因烟草叶片赖氨酸含量,大部分植株有着明显的提高,最高幅度达到了49. 89%. 图 6参 11     

纳豆激酶原核表达载体的构建及其活性鉴定

利用引物搭桥的方法,经PCR扩增,获得了纳豆激酶成熟肽基因(NK),从而构建了大肠杆菌表达质粒NK/pTWIN1,NK/PET32a及NK/PML-c2x,经分别转化宿主菌ER2566,BL21和ER2566,获得了转纳豆激酶基因重组菌.结果表明,NK/pTWIN1-ER2566和NK/PET32a-BL21的纳豆激酶蛋白表达量较高.本研究选用NK/pTWIN1-ER2566作为表达菌株,对其进行发酵表达.SDS-PAGE显示,目的蛋白约占菌体总蛋白的36%,该蛋白是以包涵体形式存在的.包涵体经收集、蛋白变性及复性,并经过SP Sepharose分离纯化,得到了纯化的纳豆激酶蛋白,琼脂糖-纤维蛋白平板法测出1mg纳豆激酶干粉的溶栓活性相当于600u尿激酶.本文从基因工程角度研究纳豆激酶基因的克隆、表达及纯化,为利用基因工程菌生产纳豆激酶奠定了基础.图5表1参11     

大凉疣螈脑源性神经营养因子(BDNF)基因的克隆与序列分析

大凉疣螈 (Ｔｙｌｏｔｏｔｒｉｔｏｎｔａｌｉａｎｇｅｎｓｉｓ)属于两栖纲有尾目蝾螈科 ,仅分布在我国四川省西南部 ,活动范围狭窄 ,数量有限 ,是我国二级保护动物[1] .两栖动物在生物进化中是承上启下的关键动物 ,它们开启了脊椎动物登上陆地的历史 ,而且是真正陆生脊椎动物—爬行动物的祖先 ,其生理、结构等方面与鱼类和爬行类都有着明显的差别 .脑源性神经营养因子 (ｂｒａｉｎ ｄｅｒｉｖｅｄｎｅｕｒｏｔｒｏｐｈｉｃｆａｃｔｏｒ,ＢＤＮＦ)是神经营养因子家族的一员 ,ＢＤＮＦ在脊椎动物胚胎神经系统的发育、分化及在成体神经系统维…     

Protein composition of white skeletal muscle from mesopelagic fishes having different water and protein contents

The consequences for white skeletal muscle of the whole body variation in water and protein content were examined in 11 mesopelagic fishes taken off the coast of Oregon, USA, in 1983. For such muscles, water content varied from 71 to 91% of muscle wet weight, and protein content ranged from 56 to 141 mg g^-1 muscle wet weight, depending on the species. Dilution by increased water content did not account for the decrease in protein content. Total muscle protein was partitioned into soluble (myogen or sarcoplasmic) and insoluble (myofibrillar) components. Both the myogen and myofibrillar components are reduced in muscle with decreased protein content. The activities (units g^-1 wet wt) of white muscle L-lactate dehydrogenase and L-malate dehydrogenase are higher in fishes undergoing diel vertical migration to surface waters than in fishes that either do not migrate or do not migrate to surface waters. The differences in enzyme activities are not due to a general dilution of muscle protein. The actin content of white skeletal muscle was maintained at a relatively constant level in all 11 species examined and was similar to actin levels observed previously in the white skeletal muscle of scombrids and demersal fishes. This conservation of actin content requires species with a reduced muscle protein content to maintain a significant fraction of their total protein as actin. The specific activities of the myofibrillar Mg²⁺–Ca²⁺-activated adenosine triphosphatases of the mesopelagic species are similar in all 11 species studied. Thus, the ratios of proteins in the isolated myofibrils are probably similar. These results suggest that, in species with decreased muscle protein, there is an increase in the non-myofibrillar form of actin.     

Combined effect of iron and zinc on micronutrient levels in wheat (Triticum aestivum L.)

A nutrient solution experiment was conducted to investigate the effect of Fe and Zn supply on Fe, Zn, Cu, and Mn concentrations in wheat plants. The experiment used a factorial combination of two Fe levels (0 and 5 mg l(-1)) and three Zn levels (0, 0.1 and 10 mg I(-1)). The supply of Fe (5 mg l(-1)) and Zn (0.1 mg l(-1)) increased plant dry weight and leaf chlorophyll content compared to the Fe or Zn deficient (0 mg 11) treatments. However, excess Zn supply (10 mg l(-1)) reduced plant dry weights and leaf chlorophyll content. Iron supply (5 mg l(-1)) reduced wheat Zn concentrations by 49%, Cu concentrations by 34%, and Mn by 56% respectively. Zinc supply (10 mg l(-1)) reduced wheat Fe concentrations by an average of 8%, but had no significant effect on Cu and Mn concentrations. Stepwise regression analyses indicated that Zn, Cu, and Mn concentrations were negatively correlated with root- and leaf-Fe concentrations, but positively correlated with stem-Fe concentrations. Leaf-Mn concentrations were negatively correlated with root-, stem- and leaf-Zn concentrations.     

A unique low molecular weight zinc-binding ligand in the kidney cytosol of the musselMytilus edulis,and its relationship to the inherent variability of zinc accumulation in this organism

Mussels,Mytilus edulis, are known to have a high degree of variability in their whole soft tissue zinc concentrations which cannot be explained by any known ecological or physiological factor. In the present study, 70 individual mussels collected from an uncontaminated site at Bellevue, Newfoundland, in Spring 1987 had kidney zinc concentrations ranging from 148 to 4 907 g g^-1 dry weight while 40 mussel exposed to 25 g l^-1 zinc for 18 d had kidney zinc concentrations ranging from 144 to 14 072 g g^-1. Pooled or individual kidneys were homogenized in 50 mM ammonium bicarbonate buffer (pH 8.0) and ultracentrifuged for 1 h at 105 000 g. On average, about 70% of the total kidney zinc load was found in the 105 000 g pellet. Cytosolic zinc was separated into two peaks on a column of Sephadex G-25. The first peak coincided with the void volume of the column representing molecules with molecular weights of at least 5 000 (exclusion limits of Sephadex G-25). This peak would include any metallothionein present since mussel metallothionein has a molecular weight of 10 000 to 20 000. Little variability was observed in this peak so it was concluded that zinc-thionein did not play a major role in the genesis of the inherent variability. The second peak represented zinc complexed to an unknown substance with an approximate molecular weight of 700 to 1 300. This very low molecular weight zinc showed an extremely high degree of inherent variability and a strong positive correlation with the whole kidney zinc concentration. It is concluded that this very low molecular weight zinc complex plays a major role with regard to kidney zinc variability. Some very low molecular weight zinc was also noted in the digestive gland and gills. It is speculated that this substance may aid in zinc transport as well as in the incorporation of zinc into granules.MSRL Contribution No. 720