青蒿过氧化物酶的克隆及其在体外对青蒿素生物合成的影响

用RACE方法从青蒿(Artemisia annua L.)高产株系001中克隆了一个过氧化物酶.将此基因在大肠杆菌BL21(DE3)pLysS细胞中进行原核表达得到重组蛋白(APOD1),表达的蛋白分别以抗坏血酸、愈创木酚为底物进行过氧化反应,结果显示,APOD1催化愈创木酚的活力是抗坏血酸的1.8倍左右,由此表明,克隆的APOD1类属于植物经典过氧化物酶(第三大类过氧化物酶).经与其他植物过氧化物酶同源性比较分析,推测APOD1的氨基酸序列与白羽扇豆(Lupinus albus)、辣根菜(Armoracia rusticana)、小麦(Triticum aestivum)、烟草(Nicotiana tabacum)和蕃茄(Lycopersicon esculentum)的一致性分别为42.0%、36.2%、38.9%、33.6%和32.8%.Northern杂交分析表明,此基因在青蒿的根、茎和叶中均有表达.加入APOD1至青蒿细胞提取液有利于青蒿酸向青蒿素的生物转化,但APOD1并不能直接以青蒿酸作为氧化底物.     

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.     

西府海棠(Malus micromalus)MaMAPK基因的克隆及表达特性

依据高等植物MAPK基因的保守区设计简并引物,用 RT-PCR方法,首次从西府海棠幼苗叶片中克隆了促分裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)基因(MaMAPK)的cDNA全序列.Southern杂交结果表明在西府海棠中存在一个小的MAPK基因家族.20%PEG处理西府海棠幼苗不同时间后的Northern杂交分析表明,该基因在根系和叶片中均有表达,随着胁迫时间延长表达量增加,说明西府海棠MaMAPK基因在转录水平上受水分胁迫诱导表达.     

Molecular Cloning of a HMG-CoA Reductase Gene from Eucommia ulmoides Oliver

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate, which is the first committed step in the pathway for isoprenoid biosynthesis in plants. A full-length cDNA encoding HMGR (designated as EuHMGR, GenBank Accession No. AY796343) was isolated from Eucommia ulmoides by rapid amplification of cDNA ends (RACE). The full-length cDNA of EuHMGR comprises 2281 bp with a 1770-bp open reading frame (ORF) encoding a 590-amino-acid polypeptide with two trans-membrane domains revealed by bioinformatic analysis. Molecular modeling showed that EuHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. The deduced protein has an isoelectric point (pI) of 6.89 and a calculated molecular weight of about 63 kDa. Sequence comparison analysis showed that EuHMGR had highest homology to HMGR from Hevea brasiliensis. As expected, phylogenetic tree analysis indicated that EuHMGR belongs to plant HMGR group. Tissue expression pattern analysis showed that EuHMGR is strongly expressed in the leaves and stems whereas it is only poorly expressed in the roots, which implies that EuHMGR may be a constitutively expressing gene. Functional complementation of EuHMGR in HMGR-deficient mutant yeast JRY2394 demonstrated that EuHMGR mediates the mevalonate biosynthesis in yeast.     

南京椴HMGR基因的克隆和功能验证

3-羟基-3-甲基戊二酰辅酶A还原酶（3-hydroxy-3-methylglutaryl-CoA reductase,HMGR）是植物萜类代谢中甲羟戊酸途径的关键酶,本研究运用cDNA末端快速扩增（RACE）技术,首次从珍稀植物南京椴中克隆出HMGR的全长基因TmiHMGR,其长度为2 160 bp,包含一个1 758 bp的开放阅读框,其推导蛋白TmiHMGR编码585个氨基酸残基,相对分子量为62.9 kD,pI为6.11。将TmiHMGR与其他植物HMGR氨基酸序列构建进化树,结果显示TmiHMGR与苹果的HMGR聚为一枝。采用半定量RT-PCR分析TmiHMGR在根、茎和叶中的表达情况,结果表明该基因在茎中的表达量最高,根和叶中的表达量相对较弱。验证功能的颜色互补实验结果显示,TmiHMGR能够使代谢流明显朝类胡萝卜素合成的方向进行,说明TmiHMGR在萜类产物生物合成中是一个重要因子。     

百脉根液泡膜H+-PPase基因的cDNA克隆与分析

采用EST电子克隆和RACE技术从豆科模式植物百脉根中克隆到一个液泡膜H -PPase基因的cDNA,命名为LcVP1。该cDNA长为2962bp,含2304bp的完整开放阅读框,编码767个氨基酸,其推测的氨基酸序列与绿豆、拟南芥等I类液泡膜H -PPase的氨基酸序列同源性在80%以上,且有很高的功能区段保守性。该cDNA序列已提交GenBank,登录号为EF440187。半定量RT-PCR表明,LcVP1在根、茎、叶中的表达不同,叶中表达最多,茎中最少。     

镉在旱柳中亚细胞分布及存在的化学形态

以2个旱柳无性系幼苗为材料,通过营养液培养并结合差速离心与化学试剂提取法,分析了不同浓度Cd2+胁迫下旱柳叶和根中Cd的亚细胞分布及其存在的化学形态.结果显示,(1)随着培养介质Cd2+浓度升高,旱柳无性系幼苗叶和根中各亚细胞组分Cd含量随之增加.叶片的Cd主要富集于细胞壁、叶绿体和可溶性部分,它们的含量分别占65%～69%、14%～22%、6.8%～7.7%,仅少量Cd发现于膜部分;而根中Cd主要积累于细胞壁和可溶性部分,其中含量分别占59%～66%和14%～25%,Cd在根亚细胞组分中积累量依次为细胞壁>可溶性部分>质体>膜部分.(2)旱柳体内Cd以不同的化学形态存在,大部分为HCl(FHCl)、NaCl(FNaCl)、醋酸(HAC,FHAC)提取态,极少部分为乙醇(EtOH,FEtOH)和水提取态(Fwater),叶和根中5种Cd提取态含量依次为FHCl>FNaCl>FHAC>Fwater>FEtOH,而叶和根中HCl和NaCl提取态Cd占有比例大于30%以上.研究表明,旱柳无性系中Cd主要与蛋白质和有机酸螯合或以金属磷酸盐沉淀的形态存在,其根、叶的细胞壁和液泡在Cd忍耐与解毒中起到重要作用.     

侵染落葵的黄瓜花叶病毒分离物CP基因序列分析

通过间接酶联免疫法(ID-ELISA)检测到染病落葵病样中存在黄瓜花叶病毒（Cucumber Mosaic Virus,CMV）。从病叶中提取总RNA,用RT-PCR方法扩增得到657bp的CMV CP基因片断,将扩增产物与T载体连接并进行测序。用DNA MAN将得到的CP基因序列与GenBank收录的黄瓜花叶病毒两亚组部分株系或分离物的CP基因序列进行比较,结果表明该CP基因与CMV亚组Ⅰ、亚组Ⅱ之间的核苷酸序列同源性分别为91.17~95.43%和75.30~75.76%,推导氨基酸序列同源性分别为95.41~97.71%和81.28~81.74%,表明CMV-Ba与亚组Ⅰ同源关系密切。     

菜心和水稻绿叶中不同等电点的乙醇酸氧化酶

The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%～20% native\|PAGE under a pH8.3 buffer system.GOⅢ's p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ's p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ's p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.     

Cloning and sequence analysis of a cDNA encoding ferric leghemoglobin reductase from soybean nodules.

A cDNA encoding soybean (Glycine max [L.] Merr) ferric leghemoglobin reductase (FLbR), an enzyme that is postulated to play an important role in maintaining leghemoglobin in its functional ferrous state, has been cloned and characterized. A group of highly degenerate oligonucleotides deduced from the N-terminal amino acid sequence of FLbR was used to prime the polymerase chain reaction (PCR) on soybean nodule mRNA and cDNA. A full-length clone of FLbR cDNA was isolated by screening a lambda gt11 soybean nodule cDNA library using the specific PCR-amplified FLbR cDNA fragment as a probe. The cDNA contained about 1.8 kb and had a coding sequence for 523 amino acids with a predicted molecular mass of 55,729 D, which included a putative 30-residue signal peptide and a 493-residue mature protein. Computer-aided analysis of the deduced FLbR amino acid sequence showed considerable homology (varied from 20-50% with enzymes and species) to dihydrolipoamide dehydrogenase (EC 1.8.1.4), glutathione reductase (EC 1.6.4.2), mercuric reductase (EC 1.16.1.1), and trypanothione reductase (EC 1.6.4.8) in a superfamily of pyridine nucleotide-disulfide oxidoreductases from various organisms. Northern blot analysis using FLbR cDNA as a probe showed that the FLbR gene was expressed in soybean nodules, leaves, roots, and stems, with a greater level of expression in nodules and leaves than in roots and stems. Southern blot analysis of the genomic DNA showed the presence of two homologous FLbR genes in the soybean genome.     

An Apomictic Autotriploid Line TAR Identified in Oryza sativa

Since apomixis has a close correlation with polyploidy and sterility, a number of autotriploids with no sexual reproductivity were induced and apomictic germplasm were screened in Oryza sativa L. As a result, an autotriploid line, named TAR, was cytoembryologically identified which possessed apomictic property, with an average seed-set rate of 10% per panicle. Karyotype analysis proved that all the progeny seeds of TAR carried 36 chromosomes in the generations tested. Priliminary cytological observations revealed that all the ovaries of TAR had embryo sac differentiation, 33% of which developed into normal megagametophyte, 9% with previous embryogenesis prior to anthesis, and about 58% differentiated abnormally, i.e. disordered polarization, absent female generative unit and more than 2 polar nuclei. In TAR, the frequencies of chromosome configuration of 12 Ⅲ, 11 Ⅱ + 1 Ⅱ +1 Ⅰ. L0Ⅲ +2Ⅱ +2 Ⅰ, 9Ⅲ+3Ⅱ +3 Ⅰ, 8Ⅲ+4Ⅱ +4 Ⅰ and 7Ⅲ+5 Ⅱ +5 Ⅰ were ll%, 17%, 15%, 26%, 20% and 11% respectively at metaphase Ⅰ . While in the check line T-15 of autotriploid only 7 % of the ovaries observed had embryo sac development, and the progenies of this triploid line were aneuploids with chromosome number of 25～27. In T-15, the frequencies of chromosome configuration of 12 Ⅲ, 11Ⅲ +1 Ⅱ +1 Ⅰ, 10 Ⅲ +2 Ⅱ +2 Ⅰ , 9Ⅲ+3 Ⅱ +3 Ⅰ and 8 Ⅲ+4 Ⅱ +4 Ⅰ were 24%, 16%, 36%, 17% and 7% respectively at metaphse Ⅰ . The above observations indicated that some megaspore mother cell in TAR might undergo apomeiosis and where it gave rise to unreduced embryo sac, the unreduced eggs or synergids developed into embryos without fertilization and polar nuclei produced endosperm by pseudogamy.     

野生茄子（Solanum torvum）抗黄萎病相关基因Sto Vel的克隆与分析

采用RT—PCR和RAcE技术从野生茄子中扩增克隆到一个抗黄萎病相关基因,命名为StoVel,其cDNA全长3400bp,含有3153bp的完整开放阅读框,编码1051个氨基酸,该基因编码的蛋白序列与刚果野茄、类番茄和番茄Vel编码的氨基酸序列同源性分别为82％、81％和80％,且有很高的功能区段保守性。将该cDNA全长序列提交Gen Bank,登陆号为DQ020574。半定量PCR表明该基因为组成型表达,在根中表达最多,叶中最少。     

烟草黄酮醇合成酶基因的克隆及其序列分析

根据已知的黄酮醇合成酶cDNA保守序列设计引物,用RT-PCR技术从烟草叶片中扩增获得黄酮醇合成酶cDNA片段,再用RACE方法得到其两端序列。根据获得的序列,设计引物分离得到完整的1188bp的黄酮醇合成酶基因,其开放阅读框编码346个氨基酸。序列分析显示,烟草黄酮醇合成酶与高杯花、矮牵牛和马铃薯的同源性分别为87％、86％和84％,与其它物种中的同源性也在80％左右,表明不同物种中黄酮醇合成酶基因具有高度同源性。此外,在氨基酸水平上,该酶与其它依赖于2-酮戊二酸的双加氧酶及其相关酶也具有同源性。     

兜兰DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的克隆及表达分析

以‘同色兜兰’品种为材料,采用RT-PCR和RACE技术获得了DEFICIENS(DEF)-和GLOBOSA(GLO)-like基因的cDNA全长,命名为PcDEF和PcGLO,并用半定量RT-PCR和实时PCR研究了PcDEF和PcGLO在花芽发育过程和不同组织部位的表达特性。结果表明,PcDEF和PcGLO的全长cDNA分别为1 039bp和934bp,分别编码224和210个氨基酸;蛋白比对表明,PcDEF和PcGLO蛋白都具有典型MADS-box蛋白的MADS和K结构域;蛋白同源性分析显示,PcDEF和PcGLO与已登录的其它兰科植物的DEF/AP3和GLO/PI蛋白的相似性分别在75%～96%和87%～98%;系统进化树分析表明,PcDEF和PcGLO分别属于B类MADS-box蛋白家族的AP3和PI亚家族。表达分析显示,PcDEF和PcGLO在花芽发育中均有表达,PcDEF在成熟花、唇瓣和花瓣中的表达量高,在蕊柱、萼片、苞叶和根中次之,在花茎和叶中较低,在子房中几乎不表达;PcGLO在各组织中均有不同丰度的表达。     

矮化苹果氮肥施用技术

以8年生烟富3/M26/平邑甜茶为试材,采用¹⁵N同位素示踪技术,研究不同施氮方式(Ⅰ:春季1次性施氮,Ⅱ:分2次施氮,Ⅲ:集约技术施氮,即氮肥减量和分次施氮)对烟富3/M26/平邑甜茶¹⁵N-尿素吸收、利用、损失和果实品质的影响.结果表明: 处理Ⅲ植株叶片的叶面积、叶绿素含量(SPAD)、光合速率(P_n)、叶片全氮含量和生物量(果实除外)显著高于处理Ⅰ和处理Ⅱ,植株根冠比也显著增加.处理Ⅲ显著提高了叶片的保护酶活性(超氧化物歧化酶、过氧化物酶和过氧化氢酶),降低了叶片丙二醛(MDA)含量.3个处理各器官从肥料中吸收分配到的¹⁵N量对该器官全氮量的贡献率(Ndff)表现一致,果实的Ndff值最大,其次是一年生枝、叶片和根,且各器官的Ndff值均以处理Ⅲ最大.在果实成熟期,处理Ⅲ的单株总氮含量为93.0 mg·kg^-1,显著高于处理Ⅰ(70.2 mg·kg^-1)和处理Ⅱ(81.9 mg·kg^-1);处理Ⅲ的¹⁵N肥料利用率为33.6%,显著高于处理Ⅰ(20.4%)和处理Ⅱ(26.0%);而 ¹⁵N损失率为46.9%,显著低于处理Ⅰ(56.5%)和处理Ⅱ(52.9%).不同施氮方式下植株的平均单果质量、可溶性固形物、硬度、可溶性糖、可滴定酸、糖酸比均存在显著差异,且均以处理Ⅲ最高,其次是处理Ⅱ,处理Ⅰ最低.     

矮化苹果氮肥施用技术

以8年生烟富3/M26/平邑甜茶为试材,采用¹⁵N同位素示踪技术,研究不同施氮方式(Ⅰ:春季1次性施氮,Ⅱ:分2次施氮,Ⅲ:集约技术施氮,即氮肥减量和分次施氮)对烟富3/M26/平邑甜茶¹⁵N-尿素吸收、利用、损失和果实品质的影响.结果表明:处理Ⅲ植株叶片的叶面积、叶绿素含量(SPAD)、光合速率(P_n)、叶片全氮含量和生物量(果实除外)显著高于处理Ⅰ和处理Ⅱ,植株根冠比也显著增加.处理Ⅲ显著提高了叶片的保护酶活性(超氧化物歧化酶、过氧化物酶和过氧化氢酶),降低了叶片丙二醛(MDA)含量.3个处理各器官从肥料中吸收分配到的¹⁵N量对该器官全氮量的贡献率(Ndff)表现一致,果实的Ndff值最大,其次是一年生枝、叶片和根,且各器官的Ndff值均以处理Ⅲ最大.在果实成熟期,处理Ⅲ的单株总氮含量为93.0 mg·kg^-1,显著高于处理Ⅰ(70.2 mg·kg^-1)和处理Ⅱ(81.9 mg·kg^-1);处理Ⅲ的¹⁵N肥料利用率为33.6%,显著高于处理Ⅰ(20.4%)和处理Ⅱ(26.0%);而¹⁵N损失率为46.9%,显著低于处理Ⅰ(56.5%)和处理Ⅱ(52.9%).不同施氮方式下植株的平均单果质量、可溶性固形物、硬度、可溶性糖、可滴定酸、糖酸比均存在显著差异,且均以处理Ⅲ最高,其次是处理Ⅱ,处理Ⅰ最低.     

Molecular cloning and functional analysis of the gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from hazel (Corylus avellana L. Gasaway)

The enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR; EC1.1.1.34) catalyzes the first committed step of isoprenoids biosynthesis in MVA pathway. Here we report for the first time the cloning and characterization of a full-length cDNA encoding HMGR (designated as CgHMGR, GenBank accession number EF206343) from hazel (Corylus avellana L. Gasaway), a taxol-producing plant species. The full-length cDNA of CgHMGR was 2064 bp containing a 1704-bp ORF encoding 567 amino acids. Bioinformatic analyses revealed that the deduced CgHMGR had extensive homology with other plant HMGRs and contained two transmembrane domains and a catalytic domain. The predicted 3-D model of CgHMGR had a typical spatial structure of HMGRs. Southern blot analysis indicated that CgHMGR belonged to a small gene family. Expression analysis revealed that CgHMGR expressed high in roots, and low in leaves and stems, and the expression of CgHMGR could be up-regulated by methyl jasmonate (MeJA). The functional color assay in Escherichia coli showed that CgHMGR could accelerate the biosynthesis of beta-carotene, indicating that CgHMGR encoded a functional protein. The cloning, characterization and functional analysis of CgHMGR gene will enable us to further understand the role of CgHMGR involved in taxol biosynthetic pathway in C. avellana at molecular level.     

Molecular characterization of tobacco mitochondrial L-galactono-gamma-lactone dehydrogenase and its expression in Escherichia coli

A cDNA clone encoding L-galactono-gamma-lactone (GAL) dehydrogenase (EC 1.3.2.3) was isolated from tobacco leaves. The cDNA clone contained an open reading frame encoding the protein of 501 amino acids with a calculated molecular mass of 56,926 Da, preceded by a putative mitochondrial targeting signal consisting of 86 amino acid residues. In fact, GAL dehydrogenase was localized in the mitochondria of tobacco cells. The deduced amino acid sequence of the cDNA showed 77 and 82% homology to cauliflower and sweet potato GAL dehydrogenases, respectively. Southern blot analysis showed that tobacco contains one copy of the gene for the enzyme. Northern blot analysis showed that GAL dehydrogenase mRNA (2.0 kb) is expressed in the leaves, stems, and roots in almost equal quantities. We introduced the cDNA clone encoding tobacco GAL dehydrogenase into a pET expression vector to overexpress this protein in Escherichia coli. The partially purified recombinant enzyme was used for comparative studies on the native enzymes from tobacco and other sources; its enzymatic properties were similar to those of other GAL dehydrogenases.     

培养料配方对姬松茸子实体产量和氨基酸、有害物含量的影响

采用3种培养料配方,研究其对姬松茸(N2)产量和氨基酸含量有害物含量的影响。结果表明:姬松茸子实体中镉含量比培养料中高184～215倍,姬松茸有富集镉的特性。配方1、配方2、配方3子实体镉含量分别为13,10,9.6mg/kg,产量分别为9.5,9.4,8.8kg/m2,氨基酸总量分别为27.59%,25.80%,20.58%。配方2子实体中镉含量显著低于配方1,其产量、氨基酸总量与配方1相近;配方3的产量、氨基酸总量最低,与配方1、配方2差异显著,表明采用KH2PO4代换过磷酸钙,可以降低子实体镉含量,而不宜用化学氮代换牛粪;配方1子实体砷含量最高,与配方2、配方3差异显著。3种配方子实体中六六六、滴滴涕、敌敌畏、四环素等有害物含量基本相同。