珊瑚藻R-藻红蛋白γ亚基基因的克隆(英文)

根据珊瑚藻(Corallina afficinalis L.)R-藻红蛋白γ亚基N末端部分氨基酸序列(P83592)设计简并引物,结合RACE方法,扩增获得g亚基的全长cDNA序列。结果表明,序列全长为2308 bp(AY209894),5′非编码区长1203bp,3′非编码区长145 bp,编码区长960 bp,编码320个氨基酸组成的前体,包含71个氨基酸构成的信号肽和249个氨基酸组成的成熟蛋白。成熟蛋白序列内部存在重复序列与前人的报道一致。珊瑚藻亚基cDNA序列不同克隆子的测序结果表明,g亚基cDNA序列存在不同的3′末端,说明该基因可能存在多个拷贝或存在转录后加工。此外,扩增获得g亚基DNA序列(AY308999),比较表明编码区内部没有内含子存在。本文是对珊瑚藻R-藻红蛋白g亚基基因序列的首次报道。     

坛紫菜别藻蓝蛋白α亚基基因的原核表达与鉴定

通过PCR的方法从重组质粒pMD-apcAB中扩增坛紫菜别藻蓝蛋白α亚基基因(apcA),并将其克隆到高效表达外源基因的原核表达质粒pTO-T7。将构建好的质粒导入表达型大肠杆菌BL21(DE3),IPTG诱导表达,并对表达产物进行Western-blot和质谱鉴定。结果显示:apcA全长486bp,表达的α亚基(apcA)为带有原核表达载体T7g10的12个起始氨基酸的融合蛋白,其分子量约为19.7KD。0.5mmol/L的IPTG在37℃诱导6h时,apcA的表达量达到最大,达菌体总蛋白50%以上。Western-blot和质谱鉴定的结果表明获得的融合蛋白为重组坛紫菜别藻蓝蛋白α亚基。       

发菜固氮基因nifD和nifK克隆及其失水条件下的差异表达

nifD和nifK编码钼铁固氮酶中的钼铁蛋白。为了解发菜nifD和nifK分子信息及对水分胁迫的响应机制,该研究设计了简并性引物克隆发菜nifD和nifK全长,进行原核表达和生物信息学分析,并对不同失水状态下发菜nifD和nifK在转录水平的差异表达和固氮酶活性的变化进行分析。结果表明,发菜nifD和nifK全长分别为1 443 bp和1 536 bp (登陆号为分别为KU886164和KU886165);将nifD和nifK在大肠杆菌中表达,分别获得一个约57 kD和58 kD的外源蛋白;生物信息学分析表明,nifD和nifK核苷酸序列和推译的氨基酸序列均与点形念珠藻(Nostoc punctiforme PCC 73102)高度一致性;nifD和nifK的二级结构主要有α-螺旋、β-折叠、β-转角和随机卷曲。此外,随着藻体含水量的逐渐降低,发菜nifD和nifK在转录水平上的表达量逐渐增加,但固氮酶活性呈现先增加后下降的趋势。研究结果为深入全面研究发菜固氮酶基因结构及其响应水分胁迫的固氮机理及氮代谢途径提供了基础。     

珊瑚藻R-藻红蛋白γ亚基基因的克隆

根据珊瑚藻(Corallina afficinalis L.)R-藻红蛋白γ亚基N末端部分氨基酸序列(P83592)设计简并引物,结合RACE方法,扩增获得g亚基的全长cDNA序列.结果表明,序列全长为2 308 bp(AY209894),5'非编码区长1 203bp,3'非编码区长145 bp,编码区长960 bp,编码320个氨基酸组成的前体,包含71个氨基酸构成的信号肽和249个氨基酸组成的成熟蛋白.成熟蛋白序列内部存在重复序列与前人的报道一致.珊瑚藻亚基cDNA序列不同克隆子的测序结果表明,g亚基cDNA序列存在不同的3'末端,说明该基因可能存在多个拷贝或存在转录后加工.此外,扩增获得g亚基DNA序列(AY308999),比较表明编码区内部没有内含子存在.本文是对珊瑚藻R-藻红蛋白g亚基基因序列的首次报道.     

念珠藻葛仙米藻蓝蛋白α与β亚基基因克隆、表达及其单体结构模拟

天然抗氧化剂对于清除人体内自由基、保障健康具有重要作用。藻蓝蛋白(phycocyanin, PC)是蓝藻等藻类细胞内的一类特殊色素蛋白,具捕获光能的功能。体外研究发现,藻蓝蛋白具有较显著的抗氧化、抑癌细胞增殖等生物活性。试验利用食源性的拟球状念珠藻葛仙米(Nostoc sphaeroides)的基因组DNA为材料,借助PCR扩增克隆葛仙米藻蓝蛋白两个重要亚基α与β基因,Ns-PC-α和Ns-PC-β。结果表明,葛仙米基因组中,Ns-PC-α和Ns-PC-β基因编码框长度分别为492 bp和522 bp;Ns-PC-α和Ns-PC-β基因以双顺反子形式存在,Ns-PC-α位于Ns-PC-β下游。Ns-PC-α和Ns-PC-β分别编码由163与173个氨基酸组成的蛋白。Ns-PC-α和Ns-PC-β亚基蛋白多肽链中共含有3个半胱氨酸残基(Ns-PC-α的第85位与Ns-PC-β中的第83位氨基酸残基和第154位氨基酸残基),很可能是藻蓝胆素的结合位点。原核表达显示Ns-PC-α和Ns-PC-β分子量为近18 kD,Ns-PC-α比Ns-PC-β略小,与预测结果一致。模拟其高级结构,发现Ns-PC-α和Ns-PC-β单亚基均含有7个α-螺旋,除N端α-螺旋外,其它几个α-螺旋进一步折叠形成疏水核心区;并且,Ns-PC-α和Ns-PC-β结构上能够较好吻合,形成一稳定而封闭的单体,弧度近2π/3。Ns-PC-α和Ns-PC-β组成的这种单体结构是其进一步建构圆盘状三聚体Ns-PC-(α/β) 3的重要基础。本研究结果为食源性念珠藻葛仙米藻蓝蛋白抗氧化功能的进一步应用提供了重要依据。     

发菜G6PDH基因(GND)克隆及其干旱胁迫下的差异表达分析

由GND编码的葡萄糖-6-磷酸脱氢酶(G6PDH)是戊糖磷酸途径的关键酶。为了解发菜GND分子信息以及对干旱胁迫的响应机制,该研究设计了特异性引物克隆发菜GND全长,进行序列分析、原核表达和MALDITOF-TOF/MS鉴定,并对干旱胁迫下发菜GND的差异表达水平和G6PDH活性进行分析。结果表明:(1)发菜GND全长1 431bp(GenBank登录号为KX553955),与点形念珠藻(73102)的GND核苷酸序列相似性为96%,G6PDH氨基酸相似性为98%;氨基酸序列中第26和27位的Ile疏水性最强,在第302位的Arg亲水性最强,Thr有19个磷酸化位点,Ser有18个磷酸化位点,Tys有5个磷酸化位点,二级结构和三级结构主要由α螺旋、β折叠、β转角和随机卷曲构成。(2)将GND基因进行原核表达,获得47.23kD的外源表达蛋白G6PDH。(3)随干旱胁迫程度加剧,GND在转录水平的表达量和G6PDH活性均逐渐增加。研究结果为进一步探讨干旱胁迫条件下发菜GND表达调控奠定了基础。     

脊尾白虾血蓝蛋白大亚基基因的克隆及表达分析

应用RACE技术克隆脊尾白虾血蓝蛋白大亚基基因, 并通过攻毒实验揭示脊尾白虾血蓝蛋白基因的先天免疫防御作用, 为脊尾白虾(Exopalaemon carinicauda)的免疫防治研究提供依据和思路。研究成功克隆了脊尾白虾血蓝蛋白大亚基基因全长cDNA序列, 该大亚基cDNA全长 2192 bp, 开放式阅读框长 2034 bp, 5′非编码区长 21 bp, 3′非编码区长 137 bp, 将该基因命名为 EcHcL。EcHcL编码 667 个氨基酸, 前 21 个氨基酸组成信号肽, 推测成熟肽的分子量为 78.5 kD。Blast比对结果显示, 由脊尾白虾血蓝蛋白EcHcL序列推导的氨基酸序列与日本沼虾、凡纳滨对虾血蓝蛋白氨基酸序列的同源性分别达到 87%、73%, 其M结构域氨基酸序列与斑节对虾、日本对虾等物种同源性性高达 90% 左右, 由此推断该cDNA序列属于血蓝蛋白家族。组织表达分析结果显示, EcHcL基因在脊尾白虾鳃、卵巢、肝胰腺、心脏、肠、肌肉、胃、腹神经节、眼柄、血细胞中均有表达, 肝胰腺中相对表达量最高。Real-time PCR分析发现EcHcL基因在金黄色葡萄球菌、副溶血弧菌和对虾白斑综合征病毒(WSSV)感染后脊尾白虾肝胰腺和血细胞中的表达量显著增加, 并具有不同的时空表达模式, 推测脊尾白虾EcHcL基因在免疫防御中具有重要作用。     

条斑紫菜藻红、藻蓝蛋白α和β亚基基因序列测定及分析

为了获得江苏吕泗的条斑紫菜藻红蛋白α(Pea)和β(Peb)亚基、藻 蓝蛋白α(Pca)和β(Pcb)亚基基因的DNA序列,本文对其基因进行了克隆、 序列测定及分析.根据已发表的基因序列(DQ666487.1)设计引物,对提取自 条斑紫菜叶状体的DNA进行PCR和电泳鉴定,产物经测序证实获得藻红蛋白 序列1 357 bp (HM008263.1)和藻蓝蛋白序列1 335 bp (HM008262.1).上述 两段序列与已报道的条斑紫菜相关序列(DQ666487.1)同源性均为99％,与 其它几种紫菜相关序列同源性为88％～98％.两段序列均采用多顺反子转录 策略,排布顺序为5′Untranslated Regions(UTR)- Peb -间隔区- Pea -3 ′UTR 和 5′UTR- Pcb -间隔区- Pca -3′UTR.文中对基因翻译所得氨基 酸序列做了理化参数、功能位点及空间构型的预测.基于对序列开放阅读框 、启动子、Shine-Dalgarno (SD)序列的分析,本文对条斑紫菜分类地位进 行了讨论.     

美国王鸽脑组织中GABAA受体α1、β2和γ2亚基基因编码区的克隆与分析

目的:进行美国王鸽脑组织中GABAA受体α1亚基、β2亚基和γ2亚基的基因序列的克隆与分析。方法:从美国王鸽大脑中提取总RNA,应用通用引物逆转录获得基因组cDNA。设计GABAA受体三种亚基的特异引物,通过RT-PCR分别扩增出GABAA受体α1亚基、β2亚基和γ2亚基的基因序列,并对三段基因序列进行克隆、质粒鉴定及分析。结果:成功地测出GABAA受体α1亚基、β2亚基和γ2亚基的基因序列,长度依次为899bp、597bp和564bp,并得出其与Gallusgallus参考序列的同源率分别为94.99%、94.64%和96.28%。结论:鸽子脑组织中GABAA受体α1亚基、β2亚基和γ2亚基基因序列与已知原红鸡相应基因序列同源性较高,但也显示出一定的种属差别。     

干旱胁迫条件下发菜cphB差异表达与基因克隆

发菜是一种分布在干早、半干旱荒漠草原地区的陆生固氮蓝藻,对干旱具有极强的适应性。采用qRT-PCR技术,对干旱胁迫条件下发菜cphB的差异表达进行了分析,发现干旱胁迫条件下发菜cphB在转录水平上呈逐渐增加的趋势。根据发菜同源物种设计简并性引物克隆cphB基因,获得长度为864bp的DNA(GenBank登陆号为KX092456)。同源性比较发现发菜cphB基因具有较高的保守性,二级结构预测表明发菜CphB由α螺旋,β折叠和随机卷曲构成。三级结构预测表明CphB具有一个由α螺旋和β折叠组成的5股的结构域和由7个β折叠连成三明治结构的7股的结构域。将cphB基因在大肠杆菌中表达,获得符合预期的外源重组蛋白,CphB亚细胞定位于细胞质中。研究成果为进一步研究干旱胁迫条件下发菜cphB表达调控、藻蓝素的代谢机制及能量代谢机制方面奠定了基础。     

干旱胁迫条件下发菜Ferritin差异表达与基因克隆

发菜(Nostoc flagelliforme)是一种陆生固氮蓝藻,具有强烈的旱生生态适应性.运用双向电泳技术、凝胶图像分析、MALDI-TOF-TOF/MS质谱鉴定和数据库检索,发现发菜Ferritin在干旱胁迫条件下表达量逐渐降低.根据鉴定的Ferritin已知氨基酸序列设计简并性引物克隆该基因,获得了长度为540 bp的DNA,GenBank登陆号为HM854287.序列比较显示该基因具有较高的保守性,蛋白质二级结构主要由α螺旋和随机卷曲构成.RT-PCR分析表明,Ferritin mRNA在干旱胁迫条件下表达量逐渐降低,与Ferritin的表达趋势一致.将Ferritin基因在大肠杆菌中表达,获得符合预期的外源重组蛋白(22.4 kD).实验结果可为进一步研究发菜耐旱的分子机理及探讨发菜对极端干旱环境的适应和保护机制奠定基础.     

The structure of the large regulatory α subunit of phosphorylase kinase examined by modeling and hydrogen‐deuterium exchange

Phosphorylase kinase (PhK), a 1.3 MDa regulatory enzyme complex in the glycogenolysis cascade, has four copies each of four subunits, (αβγδ)₄, and 325 kDa of unique sequence (the mass of an αβγδ protomer). The α, β and δ subunits are regulatory, and contain allosteric activation sites that stimulate the activity of the catalytic γ subunit in response to diverse signaling molecules. Due to its size and complexity, no high resolution structures have been solved for the intact complex or its regulatory α and β subunits. Of PhK's four subunits, the least is known about the structure and function of its largest subunit, α. Here, we have modeled the full‐length α subunit, compared that structure against previously predicted domains within this subunit, and performed hydrogen‐deuterium exchange on the intact subunit within the PhK complex. Our modeling results show α to comprise two major domains: an N‐terminal glycoside hydrolase domain and a large C‐terminal importin α/β‐like domain. This structure is similar to our previously published model for the homologous β subunit, although clear structural differences are present. The overall highly helical structure with several intervening hinge regions is consistent with our hydrogen‐deuterium exchange results obtained for this subunit as part of the (αβγδ)₄ PhK complex. Several low exchanging regions predicted to lack ordered secondary structure are consistent with inter‐subunit contact sites for α in the quaternary structure of PhK; of particular interest is a low‐exchanging region in the C‐terminus of α that is known to bind the regulatory domain of the catalytic γ subunit.     

Enzyme induction in soybean infected by Phytophthora megasperma f.sp. glycinea

Laminin, the glycoprotein of basement membranes, consists of two subunits of 200,000 (α) and 400,000 (β) M_r on gel electrophoresis after reduction. We evaluated the relative proteolytic susceptibility of the two subunits using a variety of serine proteases. Human α-thrombin degraded the β subunit without altering the density or size of the α subunit. Chymotrypsin, plasmin, and cathepsin G all degraded both the β and α subunits producing limited digestion products. Chymotrypsin and cathepsin G both produced two major fragments of 160,000 and 130,000 M_r whereas plasmin produced two fragments of 180,000 and 140,000 M_r. Time course digestion studies demonstrated that the 400-kd β subunit was digested much more rapidly than the α subunit, and suggested that the major fragments (greater than 100,000 M_r) produced by chymotrypsin, plasmin, and cathepsin G were derived from the α subunit. The latter supposition was confirmed by first digesting laminin with thrombin to completely remove the β subunit, followed by digestion with chymotrypsin, cathepsin G, or plasmin. We conclude that the β subunit of laminin is highly protease labile. In contrast, the α subunit contains a large region resistant to serine proteases. Electron microscopic studies of the purified fragment of laminin derived from digestion with cathepsin G demonstrated that the protease resistant region of the α subunit contained three arms of similar appearance (32 nm) and included the intersection of the three short arms of the laminin molecule.     

The alpha and beta subunits of lamb kidney Na,K-ATPase are both glycoproteins

We have determined the carbohydrate compositions of the protein components of lamb kidney Na,K-ATPase. The α subunit contains a total of about 16 monosaccharide residues per mol of protein, while the β subunit contains about 36 residues per mol. The γ protein, a proteolipid associated with the Na,K-ATPase, contains only traces of carbohydrate. A comparison of our results with those of others shows considerable variability in the carbohydrate compositions of α and β subunits from different species.     

The reaction of tetranitromethane with human chorionic gonadotropin.

The reaction of tetranitromethane with human chorionic gonadotropin and its subunits has been investigated. The hormone consists of two subunits, α and β, containing four and three tyrosyl residues, respectively. Introduction of 1 nitrated tyrosine residue into the native hormone was accompanied by a 20% loss in immunological reactivity and a 50% loss in biological activity. This initial reaction occurred at α Tyr-88 and/or α Tyr-89. Exhaustive nitration of the hormone modified α tyrosines 65, 88, and 89 and resulted in 75% inactivation biologically and 50% immunologically. Either nitrated α subunit obtained by dissociation of the nitrated hormone recombined with the native β subunit to give a hormone whose activity was in reasonable agreement with that of the corresponding nitrated monomer. These results indicate involvement of α Tyr-88 and/or α Tyr 89 in binding of the hormone to its receptor. These residues are not required for binding to the β subunit, however. Tyr-65 of the α subunit is probably not involved in binding to either the β subunit or the hormone receptor. The β subunit obtained from the exhaustively nitrated hormone was unmodified and recombined with native α to give fully active hormone. About 25% of the protein was recovered as polymeric material following nitration; lesser amounts of crosslinked monomer were formed. Both were biologically inactive. The polymer products retained about 30% of the native immunological competence.Nitration of the isolated α subunit fully converted the remaining tyrosine (Tyr-37) to 3-nitrotyrosine in a two-step reaction. The fully nitrated α subunit did not recombine well with the native β subunit and the recombinant hormone has 10% or less of the native activity. Involvement of α Tyr-37 in binding to the β subunit is suggested by these data. However, exposure of this residue by a conformational change in the α subunit after dissociation of the native hormone, while it seems unlikely in view of the high disulfide content, is also consistent with the data. Reaction of the free β subunit with tetranitromethane resulted in complete nitration of Tyr-37, 85% nitration of Tyr-59, and 25% nitration of Tyr-82. The nitrated β subunit did not recombine well with native α but the isolated recombinant had two-thirds of the native activity. From these data we conclude that β Tyr-37 and/or β Tyr-59 are possibly involved in binding to the α subunit but do not have a role in the biological activity. Tyr-82 of β is apparently not involved in either subunit interactions or hormone-receptor binding.     

Molecular cloning of maltooligosyltrehalose trehalohydrolase gene from Nostoc flagelliforme and trehalose-related response to stresses

A genomic DNA fragment encoding a putative maltooligosyltrehalose trehalohydrolase (NfMTH) for trehalose biosynthesis was cloned by the degenerate primer- PCR from cyanobacterium Nostoc flagelliforme. The ORF of NfMTH is 1,848 bp in length and encodes 615 amino acid residues, constituting a 70 kDa protein. The deduced amino acid sequence of NfMTH contains 4 regions highly conserved for MTHs. By expression of NfMTH in E. coli, the function of this protein was demonstrated, where the recombinant protein catalyzed the hydrolysis of maltooligosyl trehalose to trehalose. The expressions of MTH and maltooligosyltrehalose synthase in the filaments of N. flagelliforme were upregulated significantly under dehydration stress, NaCl stress, and high temperature-drought stress. The accumulations of both trehalose and sucrose in the filaments of N. flagelliforme were also improved significantly under the above stresses. Furthermore, trehalose accumulated in smaller quantities than sucrose did when under NaCl stress, but accumulated in higher quantities than sucrose did when under temperature-drought stress, indicating that both trehalose and sucrose were involved in N. flagelliforme adapted to stresses and different strategies conducted in response to various stress conditions.