马尾松纤维素合成酶基因PmCesA2的克隆及其植物表达载体的构建

为了探究马尾松木材形成的分子机制,以马尾松嫩枝的总RNA反转录得到的cDNA为模板,利用反转录PCR和RACE技术克隆得到马尾松CesA2基因的全长cDNA序列(命名为PmCesA2),序列长度为3 500 bp,包含一个长为3 174 bp的开放阅读框(open reading frame,ORF),编码1 057个氨基酸.序列分析表明:PmCesA2序列与已报道的火炬松CesA2基因(AY789651.1)的相似性达98％.将全长基因克隆到载体pBI121质粒的SnaBI,Sacl双酶切位点之间,构建正义表达栽体pBI121-PmCesA2.     

刚毛柽柳 FeSOD 基因的克隆及耐盐应答分析磁

植物响应盐胁迫逆境,涉及一系列的保护酶类基因的表达和调控, SOD酶是其中重要的一类。本研究通过对盐胁迫后7个柽柳转录组分析和进一步测序验证,获得了2个柽柳FeSOD基因全长cDNA序列,它们编码蛋白的氨基酸残基数分别为256、308,分子量为63.8、75.4 kDa,理论等电点为5.0和5.1。实时荧光定量PCR结果显示这2个FeSOD基因在盐胁迫后柽柳根和叶中呈现不同的表达模式。在根中,第9天时, ThFeSOD1和ThFeSOD2基因的表达均明显上调。在叶中, ThFeSOD1和ThFeSOD2在胁迫3天受明显的诱导,而5天表达受明显的抑制。     

盐胁迫对5个柳树无性系幼苗根系生长发育的影响

在水培条件下,研究了盐(NaCl)6种质量分数处理对5个柳树杂种无性系(J172,J795,J799,J842和JW9-6)根系生长发育的影响。结果表明:盐处理对柳树无性系根系的生长发育影响显著,低盐处理对柳树无性系幼苗根系的生长(根数、最长根长和根鲜重)有一定的刺激作用,高盐处理对根系生长则有明显的抑制作用。J799的根数和根长在NaCl质量分数为0.10%时与对照相比,分别增加了24%和50%,在0.40%时减少了57%和69%。JW9-6根鲜重在NaCl质量分数为0.10%时与对照相比,增加了94%,在0.30%时减少了53%。盐胁迫对5个无性系根系生长的抑制程度不同,其中无性系J842受抑制度最小(在NaCl质量分数为0.30%时,根数、根长和根鲜重抑制度分别为7%,-34%和7%),J799受抑制度最大(在NaCl质量分数为0.30%时,根数、根长和根鲜重抑制度分别为19%,50%和100%)。     

百合MDHAR基因的克隆与表达分析

抗坏血酸(ascorbic acid,AsA)作为植物细胞内重要的自由基清除剂,是通过参与一系列的氧化还原反应而发挥抗氧化剂的功能.单脱氢抗坏血酸还原酶(monodehydroascorbate reductase,MDHAR)在抗坏血酸-谷胱甘肽循环中,可以将氧化型抗坏血酸(MDHA)还原为还原型抗坏血酸(AsA),在维持植物体内抗坏血酸的正常代谢和植物体氧化还原平衡方面起着重要作用(Chen et al.,2003;2005).     

馥郁滇丁香LgFKF1基因的克隆及节律表达分析

[目的]研究馥郁滇丁香LgFKF1基因的结构特点及其在特异组织中的节律表达特征,探索其在成花调控过程中的作用。[方法]采用RACE技术克隆获得馥郁滇丁香LgFKF1基因的cDNA全长序列,利用生物信息学方法对获得的基因核苷酸序列及编码蛋白质序列进行分析,利用qRT-PCR技术对特异组织的节律表达模式进行分析。[结果]序列分析表明:LgFKF1基因cDNA全长为2 271 bp,开放阅读框为1 917 bp,编码一个638个氨基酸的蛋白质;推导的氨基酸序列与扁果树、软枝黄蝉、纳塔尔倒吊笔、马利筋的FKF1蛋白具有较高同源性,达92.59%;预测LgFKF1蛋白属于不稳定的亲水性蛋白,定位在细胞核,无信号肽和跨膜区;结构主要由无规则卷曲结构、α螺旋结构、扩展长链和β-转角构成;遗传进化关系与扁果树最亲近。qRT-PCR分析表明:LgFKF1在诱导光周期下处理7 d时较非诱导光周期下的表达量高,而诱导光周期下处理10 d后其表达量则低于非诱导光周期的表达。一天中,LgFKF1在各组织中均有表达,且以叶片中的相对表达量较高。LgFKF1因组织不同在不同的时间呈现出单峰和双峰表达,其中,根、芽及...     

转BpTCP10基因抑制表达白桦的耐盐性分析

[目的]TCP家族基因是植物特有的一类转录因子,广泛参与了植物的各类生命进程,起到至关重要的调控作用.白桦BpTCP10基因是属于TCP家族的一个重要基因,探究BpTCP10基因在白桦生长发育及非生物胁迫过程中的作用,能为揭示该基因的功能、培育白桦耐盐良种提供理论依据.[方法]以白桦为试材,在克隆BpTCP10基因的基...     

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).     

盐胁迫对柳树无性系SH31离子含量及光合作用的影响

以柳树无性系SH31为试材,研究了NaCl溶液半抑制浓度胁迫下根、茎、叶中Na⁺、K⁺、Ca²⁺ 的分布规律及叶片光合特性的变化。结果表明:在NaCl溶液半抑制浓度(0.6%)下,SH31耐盐性较强;在0.6% NaCl溶液胁迫下,根部在前16 d积累大量Na⁺并拦截部分Na⁺向地上部运输;盐胁迫降低了根、茎部对 K⁺ 的吸收,而叶部却保持较高的K⁺ 含量来维持离子平衡;Ca²⁺含量在根、茎、叶中与对照的相当。在NaCl溶液半抑制浓度盐胁迫下,SH31 的净光合速率前7 d降低之后保持稳定,而气孔导度、蒸腾速率、胞间CO₂先降后升,表明前7 d的蒸腾速率下降的主要原因在于气孔限制。SH31根部对Na⁺ 选择性吸收并维持K⁺、Ca²⁺的高含量及维持较高光合作用是其重要的耐盐机制。     

毛白杨油酸去饱和酶基因PtFAD2的克隆与表达分析

以毛白杨为材料,结合生物信息学和分子生物学技术,利用现有的杨树基因组EST序列库资源,通过同源序列搜索,经过多次拼接合并,获得了理论的杨树油酸去饱和酶基因PtFAD2 cDNA序列;首次利用RT-PCR方法从杨树这个物种中成功克隆得到毛白杨PtFAD2全长编码序列cDNA(GenBank注册号:DQ316788),该cDNA长1 276bp,开放阅读框编码388个氨基酸.RT-PCR半定量研究表明:PtFAD2在叶片、茎、根不同组织中的表达量基本一致,并且在4℃低温处理过程中转录表达量没有变化,说明短时间内该基因表达不受低温诱导.     

Transcriptome analysis reveals dynamic changes in the salt stress response in Salix

Soil salinization is a serious ecological problem worldwide and information regarding the salt tolerance mechanisms of Salix is scarce. To elucidate the dynamic changes in the molecular mechanisms of Salix under salt stress, we generated gene expression profiles and examined changes in the expression of those genes. RNA-Seq was used to produce six c DNA libraries constructed from the leaves of Salix 9 jiangsuensis CL ‘J2345' treated with Na Cl for 0, 2, 6, 12, 24 and 48 h. In total, 249 million clean reads were assembled into 12,739 unigenes, all of which were clustered into 10 profiles based on their temporal expression patterns. KEGG analysis revealed that as an early defense response, the biosynthesis pathways of cutin,suberin and wax, which are involved in cell wall structure,were activated beginning at 2 h. The expression of secondary metabolism genes, including those involved in the phenylpropanoid, flavonoid, stilbenoid, diarylheptanoidand gingerol pathways, peaked at 6 h and 24 h; the upregulated genes were mainly involved in plant hormone pathways and beta-alanine, galactose and betalain metabolism. We identified roles of key phytohormones and found ETH to be the major signaling molecule activating TFs at 12 h; ETH, ABA, IAA and SA were the key molecules at 24 h. Moreover, we found that the upregulated genes were associated with elevated levels of amino acids, sucrose, inositol, stress proteins and ROS-scavenging enzymes, contributing to the maintenance of water balance. This research constitutes the first detailed analysis of salt stress-related mechanisms in Salix and identifies potential targets for genetic manipulation to improve yields.     

Full length cDNA cloning and expression analysis of annexinA2 gene from deer antler tissue

ANXA2（AnnexinA2）, a calcium-dependent phospholipid bind- ing protein, is involved in various Ca2＋-related biological activities. In the present study, full-length cDNA of ANXA2 was isolated from the velvet antler tip tissue of sika deer （Cervus nippon hortulomm）; the amino acid sequence and gene expression was analyzed by using bioinformatics and real-time reverse transcdptase polymerase chain reaction （RT-PCR） techniques. Nucleotide sequence analysis reveals that the full-length cDNA of the ANXA2 gene was 1372 bp, of which 1020 bp was in the opan-reading frame （OR.F） encoding 339 amino acids; its relative mo- lecular weight was 38.3 kDa; and isoelectrie point was 6.72. Sequence analysis indicates that the protein includes four conserved tan- dem-duplication ANX domains. The gene-aceession nucleotide sequence number in GenBank is JX315571. Expression analysis by RT-PCR re- veals that ANXA2 gene expression has a significant positive correlation with the antler-tissue mineralization process, indicating that this gene may play an important role in the regulation of antler-tissue mineraliza- tion.     

Molecular cloning and analysis of the partial sequence ofRhinopithecus roxellanae growth hormone gene

Growth hormone gene (GH) ofRhinopithecus roxellanae was amplified by PCR based on the sequences of the reported mammalian growth hormone gene for the first time. The amplified fragment was about 1.8 kb. It was cloned and its upper stream was sequenced. This sequencing region consists of a 5′flanking regulatory region, exon I and part of exon II, intron I of growth hormone gene. Comparing the corresponding sequences of growth hormone gene betweenRhinopithecus roxellanae and the porcine, we concluded that the homology reached 81% in the region, and there was high conservation in the 5′flanking sequence. The kinds of amino acids of exon I and exon II for about 90% were the same to those in pig. Many mutations occurred in the degenerate site of the triplet code. In the nucleotides of intron I, there were only 72% homologies with those in pig. It means that introns and 3′flanking sequence maybe play an important part in growth hormone gene regulation of the different animals. The project is supported by National Nature Science Foundation of china (No. 39970103). Xu Laixiang, male, born in March 1960, professor, Department of Biology, Qufu Normal University, Qufu 273165, P.R. China. Responsible editor: Zhu Hong     

Molecular cloning and analysis of the partial sequence of Rhinopithecus roxellanae growth hormone gene

IntroductionGrowth hormone gene (GH) of the vertebrate, a 22kDa pfot6in hormone synthesized in anterior pituitarygland, was r6quired for preadult groWth (Franchimontand Burger 1975i Barinaga et al. 1985). The genesenCOding rat, human, bovine, porcine, ovine, turkeyand duck GH have been well studied at both proteinand gene levels (Page et al. 1981, DeNotO et al. 1981,WOychik et aL 1982; Wze et aL 1987i Byrne et al.1987; FOSter et aL 1990). GroWth hormone gene isalso a member Of a mul…     

马尾松苯丙氨酸解氨酶基因的克隆与序列分析

苯丙氨酸解氨酶(Phenylalanine ammonia lyase,PAL)是苯丙烷类代谢的关键酶,催化苯丙氨酸转化为肉桂酸,促进黄酮、木质素等次生代谢产物的合成。根据其他植物PAL基因的DNA保守序列设计的简并引物,经过PCR扩增,得到1条864 bp的特异性片段,编码288个氨基酸。通过分析其序列和编码的氨基酸序列,发现其与其他物种的PAL基因高度同源,证实为马尾松PAL基因的核心序列。所得序列已经登陆在NCBI的Genbank中,序列号为GQ142010。     

Molecular cloning and expression analysis of a gene encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) from Jatropha curcas

A cDNA clone encoding a putative beta-ketoacyl-acyl carrier protein (ACP) synthase III (KAS III) was isolated from Jatropha curcas L., a woody oil plant. The cDNA clone (named JcKAS III) contained a 1203-bp open reading frame coding for 400 amino acids with a predicted molecular mass of about 42 kDa. The deduced amino acid sequence of the cDNA clone shares about 80% identity to KAS III from other plants, and contains a conserved Cys(176) in the active site and the amino acid motif G(355)NTSAAS(361) which is responsible for binding regulatory acyl-ACPs. Southern blotting analysis indicated that JcKAS III is a single copy gene in the J. curcas genome. Quantitative real-time PCR analysis showed that JcKAS III was expressed in all tissues examined with highest expression in roots, and that expression of JcKAS III increased as seeds developed.     

盐胁迫对盐蒿种子萌发及保护酶活性的影响

为更好地开发盐蒿资源,对不同浓度的NaCl和Na2SO4胁迫下盐蒿种子萌发过程中发芽率、酶活性(SOD、POD)、MDA含量的变化进行了研究。结果表明:3种盐处理均抑制种子萌发,NaCl、Na2SO4提高了SOD酶活性,降低了POD的酶活性。