大豆类钙调磷酸酶B亚基GmCBL1互作候选蛋白的筛选

Ca2+是非生物胁迫信号转导途径中的重要信号分子,植物类钙调磷酸酶B亚基蛋白(CBL,calcineurin B-like proteins)是一类重要的钙信号受体蛋白,主要通过与其他蛋白的特异结合传递信号,使植物形成对非生物胁迫的响应。本实验室已经获得大豆Gm CBL1基因,功能鉴定显示Gm CBL1增强了转基因拟南芥对非生物胁迫的耐性。为了进一步研究Gm CBL1的作用机理,本研究构建诱饵载体p GBKT7::Gm CBL1,利用酵母双杂交技术筛选大豆Gm CBL1的互作蛋白。通过对筛选获得的106个蛋白基因测序和Blast比对分析,并根据其可能的生理功能对这些候选蛋白归类,整理得到4类蛋白:能量代谢相关蛋白、修饰蛋白、防御蛋白、钙信号转导相关蛋白。筛选得到候选蛋白的功能预测初步表明,大豆Gm CBL1参与多条信号途径,为进一步研究探索大豆CBL介导的抗逆信号转导途径奠定了基础。     

Isolation and Analysis of Differential Expressed ESTs from Stem Trichomes of Lycopersicon esculentum (Solanaceae)

Glandular trichomes are special organs involved in plant defense response and synthesis of volatile secondary metabolites, analyzing trichome specific expressed sequence tags will help us further understand the specific function of plant trichomes. In this paper, suppression subtractive hybridization（SSH） based on magnetic beads technology was used to isolate differential expressed genes of the glandular trichomes in Lycopersicon esculentum. The differential expressing cDNA library was constructed using the glandular trichomes cDNA as tester and the cDNA from the stem without glandular trichomes as driver. After randomly sequencing 108 differential ESTs, Blast2go program was used to do blastx, functional annotation and metabolism analysis. The results show that most ESTs are related to substance metabolism, response to stress, biotic or abiotic stimulus, and have binding and catalytic function. These differential genes lay the foundation for further research on defense mechanism of the tomato trichomes.     

Characterization of nodule-specific cDNA clones from Sesbania rostrata and expression of the corresponding genes during the initial stages of stem nodules and root nodules formation

We have constructed a Sesbania rostrata stem nodule-specific cDNA library. By screening with heterologous probes from pea and soybean, we have isolated several nodulin cDNA clones. On the basis of nucleotide and amino acid sequence homology, two nearly full-length cDNA clones coding for two different leghemoglobin-like proteins have been identified. The inserts of two other clones reveal a high degree of amino acid sequence homology (81% and 72%) to the early nodulin Enod2 from soybean; the characteristic heptapeptide repeat units PPHEKPP and PPYEKPP of the soybean Enod2 are conserved in the proteins encoded by these Sesbania cDNA clones. The time course of Enod2 and leghemoglobin mRNA appearance during the formation of stem nodules and root nodules on S. rostrata was analyzed by northern blot hybridization. Significant differences were found for the initiation of mRNA accumulation of these nodulins between S. rostrata and soybean.     

Identification of protein fold and catalytic residues of gamma-hexachlorocyclohexane dehydrochlorinase LinA.

gamma-Hexachlorocyclohexane dehydrochlorinase (LinA) is a unique dehydrochlorinase that has no homologous sequence at the amino acid-sequence level and for which the evolutionary origin is unknown. We here propose that LinA is a member of a novel structural superfamily of proteins containing scytalone dehydratase, 3-oxo-Delta(5)-steroid isomerase, nuclear transport factor 2, and the beta-subunit of naphthalene dioxygenase-all known structures with different functions. The catalytic and the active site residues of LinA are predicted on the basis of its homology model. Nine mutants that carry substitutions of the proposed catalytic residues were constructed by site-directed mutagenesis. In addition to these, eight mutants that have a potential to make contact with the substrate were prepared by site-directed mutagenesis. These mutants were expressed in Escherichia coli, and their activities in crude extract were evaluated. Most of the features of the LinA mutants could be explained on the basis of the present LinA model, indicating its validity. We conclude that LinA catalyzes the proton abstraction via the catalytic dyad H73-D25 by a similar mechanism as described for scytalone dehydratase. The results suggest that LinA and scytalone dehydratase evolved from a common ancestor. LinA may have evolved from an enzyme showing a dehydratase activity.     

2-Hydroxyisoflavanone dehydratase is a critical determinant of isoflavone productivity in hairy root cultures of Lotus japonicus

Hairy root cultures of a model legume, Lotus japonicus, were established to characterize two heterologous cDNAs encoding enzymes involved in isoflavone biosynthesis, i.e. licorice 2-hydroxyisoflavanone synthase (IFS) and soybean 2-hydroxyisoflavanone dehydratase (HID) catalyzing sequential reactions to yield isoflavones. While the control and the IFS overexpressor did not accumulate detectable isoflavones, the HID overexpressors did accumulate daidzein and genistein, showing that HID is a critical determinant of isoflavone productivity. Production of coumestrol in all the genotypes and isoliquiritigenin/liquiritigenin in IFS + HID-overexpressing lines was also noted. These results provide insight into the regulatory mechanism that controls isoflavonoid biosynthesis.     

Induced plant responses to pathogen attack. Analysis and heterologous expression of the key enzyme in the biosynthesis of phytoalexins in soybean (Glycine max L. Merr. cv. Harosoy 63).

In soybean (Glycine max L.), pathogen attack induces the formation of glyceollin-type phytoalexins. The biosynthetic key enzyme is a reductase which synthesizes 4,2', 4'-trihydroxychalcone in co-action with chalcone synthase. Screening of a soybean cDNA library from elicitor-induced RNA in lambda gt11 yielded two classes of reductase-specific clones. The deduced proteins match to 100% and 95%, respectively, with 229 amino acids sequenced in the purified plant protein. Four clones of class A were expressed in Escherichia coli, and the proteins were tested for enzyme activity in extracts supplemented with chalcone synthase. All were active in 4,2',4'-trihydroxychalcone formation, and the quantification showed that shorter lengths of the cDNAs at the 5' end correlated with progressively decreasing enzyme activities. Genomic blots with DNA from plants capable of 4,2',4'-trihydroxychalcone synthesis revealed related sequences in bean (Phaseolus vulgaris L.) and peanut (Arachis hypogaea L.), but not in pea (Pisum sativum L.). No hybridization was observed with parsley (Petroselinum crispum) and carrot (Daucus carota) which synthesize other phytoalexins. The reductase protein contains a leucine-zipper motif and reveals a marked similarity with other oxidoreductases most of which are involved in carbohydrate metabolism.     

Calmodulin-binding protein BP-10, a probable new member of plant nonspecific lipid transfer protein superfamily.

CaMBP-10 is a novel plant endogenous calmodulin-binding protein with important physiological functions. The partial cDNA sequence of this protein was cloned using RT-PCR. The deduced peptide (designated PCBP10) is composed of 74 amino acid residues containing a basic amphiphilic alpha-helix typical for calmodulin-binding proteins. PCBP10 shows very high amino acid sequence homology with plant nonspecific lipid-transfer proteins (nsLTPs). Sequence analysis also reveals that PCBP10 has similar amino acid composition to plant nsLTPs, and seven of the eight conserved cysteine residues are found in PCBP10. Furthermore, the secondary structure features of PCBP10 are very similar to those of plant nsLTPs. In addition, there are striking resemblances between CaMBP-10 and plant nsLTPs in their biochemical and physical properties. Our results suggest that CaMBP-10 is a novel member of the plant and nsLTP gene family, and the Ca(2+)/CaM regulative system may also play roles in lipid metabolism, defense reactions, and the adaptation of plants to natural environment.     

Primary structure of rat liver serine dehydratase deduced from the cDNA sequence

The nucleotide sequence of serine dehydratase mRNA of rat liver has been determined from a recombinant cDNA clone, previously cloned in this laboratory, and from a recombinant cDNA clone screened from a primer-extended cDNA library. The sequence of 1322 nucleotides includes the entire protein coding region and noncoding regions on the 3'- and 5'-sides. The deduced polypeptide consists of 327 amino acid residues with a calculated molecular mass of 34,462 Da. Comparison of the amino acid sequences of the serine dehydratase polypeptide with those of biosynthetic threonine dehydratase of yeast and biodegradative threonine dehydratase of E. coli revealed various extents of homology. A heptapeptide sequence, Gly-Ser-Phe-Lys-Ile-Arg-Gly, which is the pyridoxal-binding site in the yeast and E. coli threonine dehydratases was found as a highly conserved sequence.     

Aminolevulinic acid dehydratase in pea (Pisum sativum L.). Identification of an unusual metal-binding domain in the plant enzyme

Aminolevulinic acid dehydratase (ALA dehydratase) catalyzes the second step of tetrapyrrole synthesis leading to the formation of heme and chlorophyll in higher plant cells. Antibodies elicited against spinach leaf ALA dehydratase were used to immunoscreen lambda gt11 cDNA libraries constructed from etiolated pea (Pisum sativum L.) leaf poly(A)+ RNAs. A set of overlapping cDNAs was characterized that encode the pea enzyme. The predicted amino acid sequence of the pea ALA dehydratase is similar to those reported for other eukaryotic and prokaryotic ALA dehydratases. The pea enzyme has an active site domain centered on lysine that is highly conserved in comparison to other known ALA dehydratases. Consistent with the previously reported requirement of Mg2+ for catalytic activity by plant ALA dehydratases, the pea enzyme lacks the characteristic Zn(2+)-binding domain present in other eukaryotic ALA dehydratases, but contains a distinctive metal ligand-binding domain based upon aspartate. Northern blot analyses demonstrated that ALA dehydratase mRNA is present in leaves, stems, and to a lesser extent in roots. Steady state levels of mRNA encoding ALA dehydratase exhibit little or no change during light-induced greening.     

The sng2 mutant of Arabidopsis is defective in the gene encoding the serine carboxypeptidase-like protein sinapoylglucose:choline sinapoyltransferase

Serine carboxypeptidase-like (SCPL) proteins have traditionally been assigned roles in the hydrolytic processing of proteins; however, several SCPL proteins have recently been identified as catalysts in transacylation reactions of plant secondary metabolism. The novel functions of these enzymes suggest a catalytic diversity for plant SCPL proteins that extends beyond simple hydrolysis reactions. Characterization of the Arabidopsis sng2 (sinapoylglucose accumulator 2) mutant has identified another SCPL protein involved in plant secondary metabolism. The sng2 mutant was isolated by screening seed extracts for altered levels of sinapate esters, a group of phenylpropanoid compounds found in Arabidopsis and some other members of the Brassicaceae. Homozygous sng2 seeds accumulate sinapoylglucose instead of sinapoylcholine, and have increased levels of choline and decreased activity of the enzyme sinapoylglucose:choline sinapoyltransferase (SCT). Cloning of the SNG2 gene by a combination of map-based and candidate gene approaches demonstrates that SCT is another member of the growing class of SCPL acyltransferases involved in plant secondary metabolism.     

Plant delta-aminolevulinic acid dehydratase. Expression in soybean root nodules and evidence for a bacterial lineage of the Alad gene.

We isolated a soybean (Glycine max) cDNA encoding the heme and chlorophyll synthesis enzyme delta-aminolevulinic acid (ALA) dehydratase by functional complementation of an Escherichia coli hemB mutant, and we designated the gene Alad. ALA dehydratase was strongly expressed in nodules but not in uninfected roots, although Alad mRNA was only 2- to 3-fold greater in the symbiotic tissue. Light was not essential for expression of Alad in leaves of dark-grown etiolated plantlets as discerned by mRNA, protein, and enzyme activity levels; hence, its expression in subterranean nodules was not unique in that regard. The data show that soybean can metabolize the ALA it synthesizes in nodules, which argues in favor of tetrapyrrole formation by the plant host in that organ. Molecular phylogenetic analysis of ALA dehydratases from 11 organisms indicated that plant and bacterial enzymes have a common lineage not shared by animals and yeast. We suggest that plant ALA dehydratase is descended from the bacterial endosymbiont ancestor of chloroplasts and that the Alad gene was transferred to the nucleus during plant evolution.     

The P34 syringolide elicitor receptor interacts with a soybean photorespiration enzyme,NADH-dependent hydroxypyruvate reductase

The syringolide receptor P34 mediates avrD-Rpg4 gene-for-gene complementarity in soybean. However, the mechanism underlying P34 signal transmission after syringolide binding is unknown. In an effort to identify a second messenger for P34, soybean leaf proteins were run though a P34-affinity column. A 42-kDa protein which specifically bound to the column was identified as a putative plant NADH-dependent hydroxypyruvate reductase (HPR) by N-terminal peptide sequencing. HPR is an important enzyme involved in the plant photorespiration system. Screening of a soybean cDNA library yielded two distinct HPR clones that encoded proteins with 97% identity (P42-1 and P42-2). Surprisingly, only P42-2 displayed good binding with P34 in a yeast two-hybrid assay, indicating that P42-2, but not P42-1, is a potential second messenger for P34. Glycerate and its analogs, which are utilized in the photorespiration system, were tested for their inhibitory effect on syringolide-induced hypersensitive response (HR) to evaluate the biological significance of P42-2. Interestingly, the downstream products of HPR (glycerate and 3-phosphoglycerate) inhibited HR but the upstream compounds (hydroxypyruvate or serine) did not have a significant effect on HR. These results suggest that P42-2 is a primary target for a P34/syringolide complex and that P42-2 binding with the complex probably induces HR by inhibiting one or more HPR functions in soybean.     

高山植物黄管秦艽cDNA 文库构建与表达序列标签(EST) 分析

黄管秦艽( Gentiana officinalis) 是一种重要的藏药高山植物, 本研究构建了该物种开花期的cDNA 文库。经检测达到中等cDNA 文库水平, 文库滴度为1 . 2×107 pfu􊄯ml , 重组率95.9% , 插入片段平均长度大于500 bp。对343 个随机挑选的重组克隆进行部分测序, 获得的ESTs 经编辑后共有181 条有效序列。经生物信息学方法分析181 条表达序列标签(EST) 代表144 个单克隆序列, 其中55 个与已鉴定的基因同源, 35 个序列与未鉴定的EST 匹配, 54 个未找到同源序列; 后两者共有89 个EST 序列未发现功能相似的蛋白。对已鉴定的EST进行功能分析发现, 相关基因主要编码以下蛋白: 与蛋白表达相关的占35%; 光合作用相关的占22%; 新陈代谢相关的占18%; 抗性相关的占11%; 质膜运输和细胞分裂相关的分别占5% ; 染色体变化和细胞信号转导的分别占2%。根据有效EST 序列设计引物, 通过RT-PCR 进一验证了所得EST 的准确性。这些研究结果为将来研究黄管秦艽的功能基因以及该物种与相关物种的群体遗传学、进化生物学等方面提供了基础。     

Kinetic, mutagenic, and structural homology analysis of L-serine dehydratase from Legionella pneumophila

A structural database search has revealed that the same fold found in the allosteric substrate binding (ASB) domain of Mycobacterium tuberculosis D-3-phosphoglycerate dehydrogenase (PGDH) is found in l-serine dehydratase from Legionella pneumophila. The M. tuberculosis PGDH ASB domain functions in the control of catalytic activity. Bacterial l-serine dehydratases are 4Fe-4S proteins that convert l-serine to pyruvate and ammonia. Sequence homology reveals two types depending on whether their α and β domains are on the same (Type 2) or separate (Type 1) polypeptides. The α domains contain the catalytic iron-sulfur center while the β domains do not yet have a described function, but the structural homology with PGDH suggests a regulatory role. Type 1 β domains also contain additional sequence homologous to PGDH ACT domains. A continuous assay for l-serine dehydratase is used to demonstrate homotropic cooperativity, a broad pH range, and essential irreversibility. Product inhibition analysis reveals a Uni-Bi ordered mechanism with ammonia dissociating before pyruvate. l-Threonine is a poor substrate and l-cysteine and d-serine are competitive inhibitors with K(i) values that differ by almost 10-fold from those reported for Escherichia colil-serine dehydratase. Mutagenesis identifies the three cysteine residues at the active site that anchor the iron-sulfur complex.     

Glycinin A3B4 mRNA. Cloning and sequencing of double-stranded cDNA complementary to a soybean storage protein

The cDNA clones encoding the precursor form of glycinin A3B4 subunit have been identified from a library of soybean cotyledonary cDNA clones in the plasmid pBR322 by a combination of differential colony hybridizations, and then by immunoprecipitation of hybrid-selected translation product with A3-mono-specific antiserum. A recombinant plasmid, designated pGA3B41425, from one of six clones covering codons for the NH2-terminal region of the subunit was sequenced, and the amino acid sequence was inferred from the nucleotide sequence, which showed that the mRNA codes for a precursor protein of 516 amino acids. Analysis of this cDNA also showed that it contained 1786 nucleotides of mRNA sequence with a 5'-terminal nontranslated region of 46 nucleotides, a signal peptide region corresponding to 24 amino acids, an A3 acidic subunit region corresponding to 320 amino acids followed by a B4 basic subunit region corresponding to 172 amino acids, and a 3'-terminal nontranslated region of 192 nucleotides, which contained two characteristic AAUAAA sequences that ended 110 nucleotides and 26 nucleotides from a 3'-terminal poly(A) segment, respectively. Our results confirm that glycinin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs via disulfide bonds. The inferred amino acid sequence of the mature basic subunit, B4, was compared to that of the basic subunit of pea legumin, Leg Beta, which contained 185 amino acids. Using an alignment that permitted a maximum homology of amino acids, it was found that overall 42% of the amino acid positions are identical in both proteins. These results led us to conclude that both storage proteins have a common ancestor.     

Leucine aminopeptidase from Arabidopsis thaliana. Molecular evidence for a phylogenetically conserved enzyme of protein turnover in higher plants.

Leucine aminopeptidases are exopeptidases which are presumably involved in the processing and regular turnover of intracellular proteins; however, their precise function in cellular metabolism remains to be established. Towards this goal, a full-length complementary DNA encoding a plant leucine aminopeptidase was isolated from a cDNA library of Arabidopsis thaliana and sequenced. The nucleotide sequence showed 49.5% identity to the Escherichia coli xerB-encoded leucine aminopeptidase. Sequence analysis revealed that the cDNA encodes a polypeptide of 520 amino acids with a calculated molecular mass of 54,506 Da. The C-terminal part (amino acids 200-520) of the deduced amino acid sequence showed 43.8% sequence identity to the xerB-encoded leucine aminopeptidase and 42.6% sequence identity to the amino acid sequence of bovine lens leucine aminopeptidase (EC 3.4.11.1). No sequence similarity (not even over short sequence elements) was observed with any other known peptidase or proteinase sequence. The cDNA was expressed as a fusion protein from the lacZ promoter in E. coli. Enzymatic analysis proved that the cloned cDNA encoded an active leucine aminopeptidase. The properties of this enzyme, including metal requirements, inhibitor sensitivity, pH optimum and the remarkable temperature stability, are very similar to those reported for leucine aminopeptidases from other tissues. Amino acids involved in metal and substrate binding in bovine lens aminopeptidase are completely conserved in the plant enzyme as well as in the XerB protein. Our results show that leucine aminopeptidases form a superfamily of highly conserved enzymes, spanning the evolutionary period from the bacteria to animals and higher plants. This is the first aminopeptidase cloned from a plant.