PO149, a new member of pollen pectate lyase-like gene family from alfalfa

PO149 is a low-copy-number gene expressed in the late stages of pollen development. The promoter region contains no similarities in DNA sequence to those of other pollen-specific genes, except for a tobacco sequence (AAATGA), which occurs four times in this alfalfa gene and much further upstream than in tobacco. Four distinct TATA boxes were detected in the promoter with the distal and proximal TATA boxes being separated by a spacer of 269 nucleotides. Hairpin loop structures were found in the 5-and 3-untranslated regions of PO149 mRNA. The coding region of PO149 is interrupted by two introns and encodes a putative prepeptide of 450 amino acids with homology to pollen pectate lyase-like proteins and pollen allergens. The coding region also contains sequences characteristic of both a signal peptide and a nuclear localization signal.     

Cloning and nucleotide sequence of a novel cry gene from Bacillus thuringiensis

A cry1Ab-type gene was cloned from a new isolate of Bacillus thuringiensis by PCR. When restriction pattern was compared with that of known genes it was found to have additional restriction site for ClaI. Nucleotide sequencing and homology search revealed that the toxin shared 95% homology with the known Cry1Ab proteins as compared to more than 98% homology among the other reported Cry1Ab toxins. The gene encoded a sequence of 1,177 amino acids compared to 1,155 amino acids encoded by all the other 16 cry1Ab genes reported so far. An additional stretch of 22 amino acids after the amino acid G793 in the new toxin sequence showed 100% homology with several other cry genes within cry1 family. Homology search indicated that the new cry1Ab-type gene might have resulted by nucleotide rearrangement between cry1Ab and cry1Aa/cry1Ac genes.     

A proline-rich chitinase from Beta vulgaris

A gene (Chl) encoding a novel type of chitinase was isolated from Beta vulgaris. The Ch1 protein consists of an N-terminal hydrophobic prepeptide of 25 amino acids followed by a hevein-like domain of 22 amino acid residues, an unusually long proline-rich domain of 131 amino acid residues with 90 prolines, and finally a catalytic domain of 261 amino acid residues. Proteins with similar proline-rich domains are present in some other plants. The Chl gene shows a transient expression in response to fungal infection.     

10kD玉米醇溶蛋白基因的克隆与植物表达载体构建

由于紫花苜蓿中的含硫氨基酸水平很低,采用基因工程手段将富硫蛋白基因-10kD玉米醇溶蛋白(zein)基因转入苜蓿中以提高其水平。根据该基因的保守序列设计引物,并在下游引物终止密码子前引入内质网驻留蛋白信号序列(KDEL),通过PCR技术从玉米(Zea mays L.)黄化幼苗基因组中扩增目的基因的开放阅读框(ORF)。序列测序得到全长为465bp的目的片断,该片段编码154个氨基酸,其中甲硫氨酸(M)31个占20.13%,半胱氨酸(C)6个占3.90%,含硫氨基酸共占24.03%。该基因与报道的10kD玉米醇溶蛋基因具有很高的同源性。将该基因构建到植物表达载体pCAMBIA13011上,以转化紫花苜蓿提高其含硫氨基酸的水平。     

Protein content and amino acids composition of bee-pollens from major floral sources in Al-Ahsa,eastern Saudi Arabia

Protein content and amino acids composition of bee-pollens from major pollen floral sources in Al-Ahsa, Saudi Arabia were determined to investigate the nutritive value of pollen protein relative to requirements of honeybees and adult humans. The major pollen sources were alfalfa, date palm, rape, summer squash, and sunflower. Bee-pollens from alfalfa and date palm showed high content of crude protein and amino acid concentrations. Bee-pollen from sunflower had low content of those components. Eighteen amino acids were found in bee-pollens from the five major floral sources. The highest concentrations of individual amino acids valine, leucine, isoleucine, phenylalanine and proline were obtained from alfalfa bee-pollen; lysine, arginine, cysteine, tryptophan and tyrosine from date palm; methionine, histidine, glycine and alanine from summer squash; threonine, serine and glutamic acid from sunflower; and aspartic acid from rape bee-pollen. The amino acid composition obtained from sunflower bee-pollen showed the lowest concentrations of the essential amino acids: isoleucine, leucine, methionine, phenylalanine and valine. Apart from methionine, arginine and isoleucine, the essential amino acids of bee-pollen from alfalfa, date palm, summer squash and rape exceeded the honeybees’ requirements. Methionine was the limiting amino acid in bee-pollens from the five selected sources. Concentrations of essential amino acids in the tested bee-pollens were variable and significantly correlated to their botanical origin of pollen. Bee-pollens from alfalfa, date palm and summer squash was found to be rich source of protein and amino acids for bees and for humans.     

Dodder infection induces the expression of a pathogenesis-related gene of the family PR-10 in alfalfa

A full-length cDNA, PPRG2, representing a gene highly expressed in dodder (Cuscuta trifolii Bab et. Gibs)-infected alfalfa (Medicago sativa L.) stems was isolated by differential screening. The predicted protein contains 157 amino acids and belongs to the PR-10 family of the pathogenesis-related genes with putative ribonuclease activities. Northern hybridizations showed that PPRG2 is transcribed in root and crops of uninfected alfalfa and is induced not only by dodder attack but also by bacterial infections and a large variety of environmental stresses.     

斑地锦RT-qPCR内参基因的筛选

实时荧光定量PCR(RT-qPCR)的前提条件之一是具有合适的内参基因。为筛选斑地锦(Euphorbia maculata)合适的RT-qPCR内参基因,该文利用同源克隆法克隆斑地锦GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP等基因片段,RT-qPCR检测7个候选内参基因在斑地锦不同生长期根、茎、叶和果实中的表达情况,并用geNorm、NormFinder和BestKeeper等生物学软件对各候选基因表达稳定性进行评价。结果表明:(1)克隆的GAPDH、EF-1α、act、UBQ、TUB-α、eIF-4A、CYP基因片段为729、808、753、422、233、656、313 bp,分别编码242、269、250、140、77、218、103个氨基酸,与其他植物相应氨基酸序列的最高同源性均在85%以上。(2)综合3个分析软件分析内参基因表达稳定性得出,表达稳定性排名为UBQ>EF-1α>TUB-α>eIF-4A>GAPDH>CYP>act。因此,可以选取UBQ作为斑地锦RT-qPCR分析的内参基因,用于不同生长期基因组织特异性表达研究。     

Molecular characterization of NADH-dependent glutamate synthase from alfalfa nodules.

Alfalfa NADH-dependent glutamate synthase (NADH-GOGAT), together with glutamine synthetase, plays a central role in the assimilation of symbiotically fixed nitrogen into amino acids in root nodules. Antibodies previously raised against purified NADH-GOGAT were employed to screen a cDNA library prepared using RNA isolated from nodules of 20-day-old alfalfa plants. A 7.2-kb cDNA clone was obtained that contained the entire protein coding region of NADH-GOGAT. Analysis of this cDNA and determination of the amino-terminal amino acids of the purified protein revealed that NADH-GOGAT is synthesized as a 2194-amino acid protein that includes a 101-amino acid presequence. The deduced amino acid sequence shares significant identity with maize ferredoxin-dependent GOGAT, and with both large and small subunits of Escherichia coli NADPH-GOGAT. DNA gel blot analysis of alfalfa genomic DNA suggests the presence of a single NADH-GOGAT gene or a small gene family. The expression of NADH-GOGAT mRNA, enzyme protein, and enzyme activity was developmentally regulated in root nodules. A dramatic increase in gene expression occurred coincidentally with the onset of nitrogen fixation in the bacteroid, and was absent in both ineffective plants that were nodulated with effective Rhizobium meliloti and effective plants that had been nodulated with ineffective R. meliloti strains. Maximum NADH-GOGAT expression, therefore, appears to require an effective, nitrogen-fixing symbiosis.     

Production of a truncated human c-myc protein which binds to DNA

Two kinds of truncated human c-myc proteins were produced in Escherichia coli. The human c-myc gene is composed of three exons, exons 2 and 3 having coding capacity for a protein of 439 amino acids. 252 N-terminal amino acids are encoded by exon 2, the C-terminal 187 amino acids being encoded by exon 3. One of the proteins (p42) produced in E. coli corresponds to 342 amino acids from the 98th Gln to the C-terminus, plus 21 amino acids derived from the H-ras gene at the N-terminus. The other (p23) corresponds to 155 amino acids from the 98th Gln to the 252nd Ser, plus five amino acids (Gly-Gly-Thr-Arg-Arg) at the C-terminus, plus 21 amino acids from the H-ras gene at the N-terminus. The p23 protein was produced by using cDNA in which a frame shift occurred at the boundary between exons 2 and 3. We investigated the DNA-binding activity in p42 and p23 proteins. DNA-cellulose column chromatography showed that p42 binds to DNA, whereas p23 does not. This DNA-binding activity of p42 was inhibited by antiserum prepared against p42 but not by antiserum against p23. This indicates that the DNA-binding activity of c-myc protein is localized in the portion encoded by exon 3.     

Role of rhizobial biosynthetic pathways of amino acids,nucleotide bases and vitamins in symbiosis

Rhizobia require the availability of 20 amino acids for the establishment of effective symbiosis with legumes. Some of these amino acids are synthesized by rhizobium, whereas the remaining are supplied by the host plant. The supply from plant appears to be plant-type specific. Alfalfa provides arginine, cysteine, isoleucine, valine and tryptophan, and cowpea and soybean provide histidine. The production of ornithine and anthranilic acid, the intermediates in the biosynthetic pathways of arginine and tryptophan, respectively, seems to be essential for effective symbiosis of Sinorhizobium meliloti with alfalfa. The expression of ilvC gene of S. meliloti is required for induction of nodules on the roots of alfalfa plants. An undiminished metabolic flow through the rhizobial pathways for the synthesis of purines and pyrimidines and the synthesis of biotin, nicotinic acid, riboflavin and thiamine by rhizobium appear to be requirements for normal symbiosis. To the best of our knowledge, this is the first review article on the role of rhizobial biosynthetic pathways of amino acids, nucleotide bases and vitamins in rhizobium-legume symbiosis. The scientific developments of about 35 years in this field have been reviewed.     

The fission yeast rad22 gene, having a function in mating-type switching and repair of DNA damages, encodes a protein homolog to Rad52 of Saccharomyces cerevisiae.

The gene rad22 of the fission yeast Schizosaccharomyces pombe has a function in DNA repair and mating-type switching. We have cloned the rad22 gene from a genomic gene bank by functional complementation of the switching defect. An open reading frame coding for a putative protein of 469 amino acids was found by sequence analyses. The rad22 gene contains no intron. A region of 126 amino acids in the N-terminal half of the Rad22 protein has significant homologies (56% identity and 36% similarity) to the Rad52 protein of Saccharomyces cerevisiae. A rad22 disruption strain was constructed which seems to be inviable in a homothallic background. Southern blot analyses have shown that the rad22-67 mutant frequently gives rise to deletions in the mating-type region. These data indicate that the Rad22 protein has a function in the repair of DNA double-strand breaks.     

Domain structures of chlorophyllide<Emphasis Type="Italic"> a</Emphasis> oxygenase of green plants and<Emphasis Type="Italic"> Prochlorothrix hollandica</Emphasis> in relation to catalytic functions

Chlorophyll b is a photosynthetic antenna pigment found in prochlorophytes and chlorophytes. In chlorophytes, its biosynthesis regulates the photosynthetic antenna size. Chlorophyll b is synthesized from chlorophyll a in a two-step oxygenation reaction by chlorophyllide a oxygenase (CAO). In this study, we first identified the entire sequence of a prochlorophyte CAO gene from Prochlorothrix hollandica to compare it with those from chlorophytes, and we examined the catalytic activity of the gene product. Southern blot analysis showed that the CAO gene is presented in one copy in the P. hollandica genome. The P. hollandica CAO gene (PhCAO) has a coding capacity for 367 amino acids, which is much smaller than that of Arabidopsis thaliana (537 amino acids) and Oryza sativa (542 amino acids) CAO genes. In spite of the small size, PhCAO catalyzed the formation of chlorophyll b. By comparing these sequences, we classified the land-plant sequences into four parts: the N-terminal sequence predicted to be a transit peptide, the successive conserved sequence unique in land plants (A-domain, 134 amino acids), a less-conserved sequence (B-domain, 30 amino acids) and the C-terminal conserved sequence common in chlorophytes and prochlorophytes (C-domain, 337 to 344 amino acids). We demonstrated that the C-domain is sufficient for catalytic activity by transforming the cyanobacterium Synechocystis sp. PCC6803 with the C-domain from A. thaliana. In this paper, the role of the A-domain is discussed in relation to the formation of light-harvesting chlorophyll a/b–protein complexes in land plants.Abbreviations CAO Chlorophyllide a oxygenase - CP Chlorophyll protein - HPLC High-performance liquid chromatography - LHC Light-harvesting complex - PCR Polymerase chain reaction - PS Photosystem     

Physiology of behavioral mutants of Rhizobium meliloti: evidence for a dual chemotaxis pathway

Wild-type and nonchemotactic mutant strains of Rhizobium meliloti were tested for attraction to localized sites on alfalfa roots and for attraction to numerous small molecules, including sugars, amino acids, and two fractions derived from alfalfa root extracts. Four strains (carrying mutations che-6, che-11, che-12, and che-26) lost all responses and were classified as generally nonchemotactic mutants. One strain (carrying mutation che-7) lost responses to a group of structurally unrelated amino acids but retained all other responses and was classified as a putative sensory transducer mutant. Two strains (carrying mutations che-1 and che-3) lost responses to all the amino acids and sugars tested but retained normal responses to localized sites on roots and to the root fractions. These two mutant strains could not be classified according to the generally accepted model for a sensory pathway, derived from studies of enteric bacteria, and provided evidence for a dual chemotaxis pathway in R. meliloti.     

Enhanced levels of free and protein-bound threonine in transgenic alfalfa (Medicago sativa L.) expressing a bacterial feedback-insensitive aspartate kinase gene

Threonine, lysine, methionine, and tryptophan are essential amino acids for humans and monogastric animals. Many of the commonly used diet formulations, particularly for pigs and poultry, contain limiting amounts of these amino acids. One approach for raising the level of essential amino acids is based on altering the regulation of their biosynthetic pathways in transgenic plants. Here we describe the first production of a transgenic forage plant, alfalfa (Medicago sativa L.) with modified regulation of the aspartate-family amino acid biosynthetic pathway. This was achieved by over-expressing the Escherichia coli feedback-insensitive aspartate kinase (AK) in transgenic plants. These plants showed enhanced levels of both free and protein-bound threonine. In many transgenic plants the rise in free threonine was accompanied by a significant reduction both in aspartate and in glutamate. Our data suggest that in alfalfa, AK might not be the only limiting factor for threonine biosynthesis, and that the free threonine pool in this plant limits its incorporation into plant proteins.