毛尖紫萼藓GpUCH基因克隆及表达分析

从毛尖紫萼藓干旱cDNA文库中筛选了一条与抗旱相关的泛素羧基末端水解酶基因的EST序列,采用RACE技术从毛尖紫萼藓中克隆出了该基因的cDNA全长序列,命名为白UCH。对该基因的序列特征、进化关系和表达谱进行了分析,为全面研究其功能奠定了基础。该基因cDNA全长951bp,开放阅读框711bp,共编码236个氨基酸。生物信息学分析显示,印UCH基因相对分子质量为25．7kD,等电点为4．67,为不稳定蛋白,属于跨膜蛋白但不存在信号肽。系统进化树分析表明,GpUCH与小立碗藓UCH蛋白一致性较高。实时荧光定量PCR结果显示,GpUCH基因在复水和干旱的条件下均能表达,但是在不同的条件下表达情况差异显著。表明该基因可能在毛尖紫萼藓干旱过程中发挥一定的作用。     

毛尖紫萼藓抗旱相关基因Gp-LEA的克隆与表达分析

以毛尖紫萼藓干旱cDNA文库中获得的一段与LEA基因同源性较高的EST序列为基础,采用RACE技术分离该基因cDNA全长序列,命名为Gp-LEA。Gp-LEA基因的cDNA全长814bp,开放阅读框456bp,编码含151个氨基酸蛋白质。生物信息学分析结果显示,Gp-LEA蛋白为稳定蛋白,分子质量为16.612kD,理论等电点（pI）为5.06,含有LEA₂功能结构域,不属于跨膜蛋白且不存在信号肽。系统发生分析表明,Gp-LEA基因编码蛋白与花旗松LEA蛋白亲缘关系最近。荧光定量PCR分析显示,Gp-LEA基因在复水和快速干旱模式下均能表达。推测Gp-LEA基因在毛尖紫萼藓的复水和干旱过程中起着重要作用。     

割手密醛脱氢酶ScALDH基因的克隆与表达分析

该研究从甘蔗细茎野生种(割手密Saccharum spontaneum)中克隆抗旱相关的基因Sc ALDH,并分析其在干旱处理条件下的表达情况和序列特征。利用RT-PCR技术克隆甘蔗的ALDH基因片段,并对其核苷酸和氨基酸序列进行分析。使用NCBI Blastx、ORF finder、Mega、NCBI Conserved Domain Search等程序对其分别进行不同物种氨基酸比较、开放阅读框(ORF)寻找、进化树及保守序列分析,并用Real Time-PCR分析所克隆基因在干旱胁迫前后的表达差异。结果表明:克隆出甘蔗的ALDH基因片段,总长度为1996bp,其中蛋白质编码区(CDS)全长1524bp,编码508个氨基酸;与其它物种ALDH类蛋白氨基酸序列有很高的同源性,有ALDH家族的保守序列,并含有完整的开放阅读框,系统进化树分析显示与玉米的蛋白质亲缘关系最近;Real timePCR数据表明,在干旱胁迫下,随着干旱时间的延长,该基因的表达量呈持续积累的表达模式。总体上来说,该基因对干旱胁迫显著表达。利用RT-PCR技术克隆的甘蔗ALDH基因,属于ALDH蛋白家族的一员,具有其典型的功能域,该基因在干旱胁迫过程中参与抗旱作用。该研究结果为野生种资源开发、优良抗旱亲本选择和培育抗旱性强甘蔗品种提供了参考依据。     

褪黑素改善认知作用研究进展

对褪黑素改善认知作用的研究进展进行综述,为褪黑素的进一步开发利用提供科学依据。     

小麦胁迫相关基因W1的克隆及表达模式分析

应用噬菌体原位杂交技术从干旱胁迫诱导的小麦cDNA文库中克隆到一个胁迫诱导的基因片段别。删全长cDNA为901bp,其中,编码区长498bp,编码166个氨基酸。Southern杂交表明,W1是一个低拷贝基因。RT—PCR结果表明,W1受干旱、低温的诱导,但不受高盐的诱导。氨基酸序列分析发现W1有一个USP保守区（pfam00582）。同源性分析发现W1与一个水稻胁迫诱导蛋白（NM_001061239）的同源性为83％,但该类蛋白的功能尚无报道。肼是小麦第1个被克隆的胁迫相关蛋白基因,该基因的克隆有助于阐明小麦的抗逆机制,并为今后培育抗逆性小麦品种提供候选基因。     

毛尖紫萼藓总RNA的提取方法研究

介绍一种提取苔藓植物毛尖紫萼藓(Grimmia pilifera P.Beauv)总RNA的方法。以新鲜的紫萼藓为材料,采用改良的SDS的方法,以氯化锂为沉淀试剂,成功地提取了该植物的总RNA。并与其他方法作了对比,结果发现该方法获得的RNA条带清晰、完整性好、纯度高、DNA污染小。可满足反转录及RT-PCR等实验要求。     

香蕉花叶病毒外壳蛋白基因克隆及表达载体的构建

从海南大田感染香蕉花叶病的香蕉叶片 ,获得香蕉花叶病毒 ,提纯其 RNA,在 AMV反转录酶作用下合成 c DNA第一链 ,经 PCR扩增 ,获得一约 70 0 bp的 DNA片段 ,测序结果显示所克隆的 DNA片段包含一完整的香蕉花叶病毒株系 ( CMV-BHI)外壳蛋白基因 ,长度为 6 5 7bp,然后将此 DNA片段 ,分别克隆到p BI1 2 1和 p KHG4质粒 ,构成两个含 Ca MV35 s启动子 ( 5 '-端 )、NOS终止子 ( 3'-端 )和分别含 NPT 标记基因和 NPT 及 HPT标记基因的植物表达载体 ( p TBB和 p TBK)。然后用 p AHC1 8中的 UBI promoter换下p BI1 2 1的 Ca MV35 s promoter,构成 p BIAH;再用 CMV-BHI外壳蛋白基因换下 p BIAH中 GUS基因 ,构成一含单子叶植物启动子 UBI和 NPT 标记基因的植物表达载体 ( p TBBU)。从而为 CMV-BHI外壳蛋白基因在香蕉中表达打下了基础     

野生种马铃薯SpPHYB基因的克隆与表达分析

采用RT-PCR技术从野生种马铃薯中克隆到一个光敏色素基因PHYB,其cDNA全长为3470bp。含有一个3393 bp的完整开放阅读框,编码一条长1130个氨基酸的蛋白,分子量为125kDa,等电点为5.6。该基因编码的蛋白序列与栽培种马铃薯、番茄和烟草同源基因编码的氨基酸序列一致性分别为98%、95%、92%,命名为SpPHYB.半定量PCR分析表明,根、茎、叶和芽中SpPHYB表达水平较高且相似,但在花和块茎成熟器官中表达量稍低.     

Molecular cloning and characterization of a tocopherol cyclase gene from Lactuca sativa (Asteraceae)

Tocopherol cyclase is a rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding tocopherol cyclase (designated as LsTC) was cloned from lettuce (Lactuca sativa) for the first time by rapid amplification of cDNA ends (RACE) and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTC was 1675 bp, with an open reading frame of 1521 bp, encoding a tocopherol cyclase protein of 506 amino acids, with a calculated molecular mass of 56.76 kD and an isoelectric point of 6.49. Comparative analysis revealed that LsTC has a close similarity with tocopherol cyclases from other plant species. Bioinformatic analysis indicated that LsTC shares a common evolutionary origin based on sequence and has the closest relationship to tocopherol cyclase from Helianthus annuus. Quantitative RT-PCR analysis suggested that expression of LsTC is induced and strengthened by oxidative stresses, such as strong light and drought. This cloning and characterization of LsTC will be helpful for further understanding of its role in the tocopherol biosynthesis pathway and provide a candidate gene for metabolic engineering of vitamin E.     

大蕉苯丙氨酸解氨酶基因的克隆、鉴定和表达分析

为了研究苯丙氨酸解氨酶基因与大蕉(Musa ABB cv. Dongguandajiao)抗枯萎病的关系,利用 RT-PCR 和 RACE技术克隆了大蕉苯丙氨酸解氨酶基因全长 cDNA。此 cDNA 长 1 300 bp,包含一个长为 1 191 bp,编码 397 个氨基酸的完整开放阅读框(ORF),推导的氨基酸序列与水稻 PAL 基因氨基酸序列同源性达 89%,将此基因命名为 M-PAL。Southern杂交结果表明大蕉中存在一个包含 4-5 个 PAL基因的基因家族,将此基因克隆到大肠杆菌表达载体 pET32(a )中,表达的蛋白质分子量大小与推导的相一致,并且表达的蛋白质表现出 PAL 酶活性。对接种香蕉枯萎病菌 4 号生理小种(Fusarium oxysporumf. sp. cubense (FOC) race 4 )后大蕉叶片中 M-PAL基因的转录谱进行研究表明,在接种枯萎病菌后,M-PAL基因在叶片中的转录水平提高,因此推测 M-PAL基因的表达可能与香蕉枯萎病抗性相关。     

水母雪莲查尔酮合酶基因的克隆、表达及酶活分析

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。用Ni-NTA预装柱对融合蛋白进行亲和纯化,对纯化蛋白进行酶活检测,结果表明融合蛋白具有查尔酮合酶活性,可催化底物4-香豆酰辅酶A和丙二酰辅酶A缩合生成产物柚皮素查尔酮。     

长春花Crlea基因的克隆及原核表达初步分析

晚期胚胎丰富（Late Embryogenesis Abundant, LEA）蛋白是植物在干旱胁迫下响应并被描述为具有潜在的抗旱功能的一类重要的抗旱蛋白。通过建立干旱胁迫下长春花（Catharanthus roseus）的cDNA文库并进行测序筛选分析,首次分离得到Crlea（Crlea for Catharanthus roseus late embryogenesis abundant）全长基因。该基因具有492 bp的开放读码框,编码163个氨基酸,其中偏性氨基酸含量占总蛋白的55.9%。同源性分析表明该假定蛋白与胡萝卜（Daucus carota）LEA DC3 的同源性达69%。亲水性分析表明具有极强的亲水性。为进一步验证CrLEA蛋白的功能,构建了Crlea基因的原核表达载体并在大肠杆菌中对其表达进行了分析。结果表明,原核载体成功的表达了CrLEA蛋白,亲水性实验及热稳定性实验表明CrLEA蛋白具有极强的亲水性和热稳定性。     

Cloning and expression analysis of a water stress-induced gene from Brassica oleracea.

Plant growth and productivity are greatly affected by water stress, such as drought and salinity. Here we report on the cloning and expression analysis of a water stress-induced gene from Brassica oleracea (designated as BoWS, GenBank accession number AY571333) by rapid amplification of cDNA ends (RACE). The full-length cDNA of BoWS consisted of 594 bp and contained a 285 bp open reading frame (ORF) encoding a 95-amino-acid protein. The deduced protein had a calculated molecular mass of 10.53 kDa and an isoelectric point of 6.93. The sequence similarity and comparative analysis showed that BoWS was 84% identical to Arabidopsis thaliana putative water stress-induced protein (GenBank accession number AAM67282). Southern blot analysis indicated that BoWS was a low-copy gene. Northern blot analysis revealed that the expression of BoWS was upregulated by abscisic acid (ABA), mannitol, NaCl, drought, salicylic acid (SA) and hydrogen peroxide (H(2)O(2)). Our results indicate that BoWS is extremely related to the water-deficit stress in B. oleracea.     

Molecular cloning of a dehydration-responsive protein gene (MRD22) from mulberry,and determination of abiotic stress patterns of MRD22 gene expression

A full-length cDNA sequence coding for dehydration-responsive protein gene of mulberry tree, which we designated was MRD22 (GenBank accession number: JQ804833) was cloned based on mulberry expressed sequence tags (ESTs). MRD22 is 1503 bp long, contains a 334 bp 5′-UTR (untranslated region) and a 563 bp 3′-UTR, encodes 201 amino acids with a predicted molecular weight of 54.28 kDa and an isoelectric point of 9.35. Phylogenetic analysis based on MRD22 sequences from different species showed that mulberry has close relationship with Populus trichocarpa, Ricinus communis, Camellia sinensis, Gossypium hirsutum, Gossypium barbadense and so on. The expression level of the MRD22 gene under conditions of drought, low temperature and salt stresses was quantified by qRT-PCR. The results show that the expression level changed significantly under the stress conditions compared to the normal growth environment. It helps us to get a better understanding of the molecular basis for signal transduction mechanisms underlying the stress response in mulberry.     

金黄色葡萄球菌特异性PCR检测靶点的自动化筛选

从水母雪莲Saussurea medusa Maxim. cDNA文库中得到一段查尔酮合酶基因 (SmCHS) 片段,然后通过RT-PCR得到完整的查尔酮合酶基因cDNA。序列分析表明SmCHS全长1 313 bp,其开放阅读框为1 170 bp,编码389个氨基酸,预测表达蛋白的分子量为43 kDa。构建原核表达质粒pET28a(+)-SmCHS,重组质粒转化大肠杆菌BL21(DE3),获得表达菌株。经IPTG诱导表达后,对表达产物进行SDS-PAGE分析,结果显示,表达的融合蛋白以部分可溶的形式存在。     

齐口裂腹鱼和鲤鱼H-FABP基因的克隆及其表达谱

心型脂肪酸结合蛋白（heart fatty acid binding protein, H-FABP）的水平与影响肉质性状的肌内脂肪含量有关,鱼类H-FABP的表达水平对其肌内脂肪含量是否相关仍未见报道.本研究获得齐口裂腹鱼和鲤鱼心脏型脂肪酸结合蛋白基因序列,利用半定量RT-PCR分析其表达特性并测定肌内脂肪含量,比较H-FABP基因在不同生活环境的2种鲤科鱼肌内脂肪沉积中的作用.结果显示,齐口裂腹鱼和鲤鱼H-FABP基因的ORF为402 bp,编码133个氨基酸,它们的氨基酸序列相同,与人、猪、小鼠、斑马鱼、大西洋鲑、虹鳟等的同源性为71.3%~ 90%;H-FABP基因在2种鲤科鱼的心、肌肉、脂肪、肝、脑、脾、肾和鳃等组织中均有表达,肝中的表达量显著高于其它组织（P<0.05）,H-FABP基因的肌肉表达谱在齐口裂腹鱼和鲤鱼中存在明显差异：齐口裂腹鱼中的表达随生长发育呈上升趋势,在大体重鱼（500 g）中的表达显著高于小体重鱼（P<0.05),其表达与肌内脂肪含量呈显著正相关（R=0.370,P<0.05）;H-FABP基因在鲤鱼生长发育中呈下降趋势,而小体重鱼（50~60 g）中的表达显著高于其它大体重鱼(P<0.05),其表达与肌内脂肪含量呈显著负相关（R=-7.083,P<0.01）.据此推测,齐口裂腹鱼和鲤鱼肌肉组织H-FABP基因表达与肌内脂肪关联性的差异可能与2种鱼的生活环境不同有关.     

Molecular cloning and characterization of gene coding for γ-tocopherol methyltransferase from lettuce (Lactuca sativa)

γ-tocopherol methyltransferase is an important rate-limiting enzyme involved in tocopherol biosynthesis. The full-length cDNA encoding γ-tocopherol methyltransferase (designated as LsTMT) was cloned from Lactuca sativa for the first time by rapid amplification of cDNA ends and characterized by means of quantitative RT-PCR. The full-length cDNA of LsTMT was 1131 bp, with an open reading frame of 897 bp encoding a γ-tocopherol methyltransferase protein of 298 amino acids, with a calculated molecular mass of 33.06 kDa and an isoelectric point of 5.86. Comparative analysis revealed that LsTMT has a close similarity with γ-TMTs from other plant species. Bioinformatic analysis indicated that LsTMT shares a common evolutionary origin based on sequence similarity and has the closest relationship to γ-TMT from the sunflower, Helianthus annuus. Based on quantitative RT-PCR analysis, we found that expression of LsTMT is induced and strengthened by oxidative stresses such as strong light and drought. The cloning and characterization of LsTMT will be helpful to further understanding its role in the tocopherol biosynthesis pathway. We consider it to be a candidate gene for metabolic engineering of vitamin E in vegetable crops.     

中国水仙水杨酸甲酯合成酶基因克隆与表达分析

为了解中国水仙(Narcissus tazetta var. chinensis)花香气形成的分子机理,以花发育形成相关基因的SSH文库获得的cDNA片段为基础,利用RACE技术从中国水仙花朵中克隆了水杨酸甲酯合成酶基因,命名为NtSAMT1 (GeneBank No. JX273470),其cDNA全长1323 bp,包含1个1131 bp完整阅读框架,编码376个氨基酸,具有Methyltransf_7 superfamily蛋白保守区,与仙女扇(Clarkia breweri) SAMT蛋白(1m6eX)的三维结构相似。系统进化树分析表明,中国水仙与粳稻的SAMT蛋白亲缘关系最近。原核表达结果表明NtSAMT1在大肠杆菌中能高效表达。半定量RT-PCR分析表明,NtSAMT1在花蕾期就有表达,第1天完全开放时各部位均有表达,以雄蕊与雌蕊的表达量最高,第8天已检测不到表达。