Cloning of Soybean 24 kDa Oleosin Gene and Its Transient Expression as a Carrier for Foreign Protein

The genomic DNA sequence encoding soybean 24 kDa oleosin and its promoter were cloned andanalyzed for investigation of the potentials of the oleosin acted as a carrier forproduction of recombinant proteins in plant. The -300 box, GA-rich, G-box, SEF-3, SEF-4, RY box, ABA box, CAn and TATA box were found in the upstream region of the soybeanoleosin gene, which shows the functional oleosin promoter available. Homology comparisonreveals that the soybean 24 kDa oleosin shares the highest identity with the soybeanoleosin isoform A (U09118, GenBank), reaching to 98.4% in nucleotide. A soybean oleosin-hirudin fusion gene driven by the oleosin promoter was constructed and inserted intoplant binary expression vector. The intact tobacco plantlets were transformed by meansof vacuum infiltration approach, with the Agrobacterium tumefaciens harboring the abovevector. The transient correct expression of oleosin-hirudin fusion gene was identifiedby SDS/PAGE, western blotting and enterokinase treatment.

Frequency and Distribution of Microsatellites in the Genome of Filamentous Fungus,Neurospora crassa

A total of 38.0 Mb of publicly available DNA sequence in Neurospora crassa was researched for mono- to hexanucleotide simple sequence repeats (SSR or microsatellite) to determine the type, size and frequency. A total of 14 788 SSRs were observed in the whole genomic DNA sequence, about one every 2.57 kb, with the criteria of SSR length >15 bp and 80% matches. The most abundant microsatellite was trinucleotide repeat, the number was 4 729, followed by hexanucleotide and mononucleotide repeats, the numbers were 2 940 and 2 489 respectively, and the least abundance was dinucleotide repeat, only 691 were found. Among the 10 082 ORFs, 4 094 SSRs were harbored in 2 373 ORF (no intron) of the organism. One thousand and fifty six ORFs harbored only one SSR. Similar with other organisms, tri- and hexanucleotide repeats were predominant in ORFs, 54.1 and 48.8% of tri- and hexanucleotide repeats were distributed in ORF region. The density of these two motifs was overpresented in coding regions, because ORF region and coding region constitutes only 46 and 38.3% of genomic sequence, respectively. Upstream and downstream 300 bp of regulatory regions were high density regions of SSRs, particularly density of pentanucleotide SSR in upstream region was as high as five times of average density in genomic DNA, density of di- and tetranucleotide SSR was also more than two times of average density. The density of penta-, tetra-, di- and mononucleotide SSRs was relatively higher than average density. There were 47 SSRs in mitochondria 64 840 bp DNA sequence, their distribution is similar with genomic DNA sequence. These results suggested that SSRs were clustered in regulatory regions of genomic DNA.

Cloning and Sequence Analysis of Disease Resistance Gene Analogues from Three Wild Rice Species in Yunnan

Two sets of degenerate oligonucleotide primers were designed according to amino acid conserved regions of reported plant disease resistance genes which encode proteins that contain nucleotide-binding site and leucine-rich repeats(NBS-LRR), and the plant disease resistance genes which encode serine/threonine protein kinase(STK). By polymerase chain reaction(PCR), disease resistance gene analogues have been amplified from three wild rice species in Yunnan Province, China. The DIN A fragments from amplification have been cloned into the pGEM-T vector respectively. Sequencing of the DNA fragments indicated that 7 classes, 2 classes and 6 classes NBS-LRR disease resistance gene analogues from Oryza rufipogon Griff. , Oryza officinalis Wall. , and Oryza meyeriana Baill. were obtained respectively. The two representative fragments of TO12 from Oryza officinalis Wall, and TR19 from Oryza rufipogon Griff, belong to the same class and homology of their sequences are 100%. The result shows that the sequences of the same class disease resistance gene analogues have no difference among different species of wild rice. 5 classes STK disease resistance gene analogues were also obtained among which 4 classes from Oryza rufipogon Griff. , 1 class from Oryza officinalis Wall. By comparison analysis of amino acid sequences. we found that the obtained disease resistance gene analogues have very low identity(low to 25%) with the reported disease resistance gene L6, N, Bs2, Prf, Pto, Lr10 and Xa21 etc. The finding suggests that the obtained disease resistance gene analogues are analogues of putative disease resistance genes that have not been isolated so far.

Overexpression of glutamine synthetases confers transgenic rice herbicide resistance

Glutamine synthetase (GS, E.C.6.3.1.2) is a key enzyme involved in the assimilation of inorganic nitrogen in higher plants and gram-negative microorganisms. GS is the targeting enzyme of a herbicide phosphinothricin (PPT) or Basta. In order to generate PPT-resistant transgenic rice via overexpression of GS, we constructed a plant expression vector p2GS harboring two different isoenzymes GS1 and GS2 cDNAs under the control of constitutive promoters of rice Act1 and maize Ubiquitin(Ubi) genes. The p2GS was introduced into rice genome by Agrobacterium-mediated transformation and confirmed by PCR and Southern blot hybridization. GS-transgene expression was first detected by Northern blot analyses. Results from Basta test indicated that GS-transgenic plants can tolerate as high as 0.3% Basta solution. In addition, our results also demonstrated that GS overexpression conferred transformed rice calli PPT resistance. Thus, GS cassette can serve as a selective marker gene instead of bar cassette for selection of PPT transformants.

Detection of RAPD Markers Linked to Gene lx_1 in Soybeans

Near-isogenic lines of soybean lipoxygenase, which contain genes Lox, lx1, lx2, lx3, lx1.3, lx2.3,respectively, were used for polymorphic analysis by RAPD technique.520 10-mer-oligonucleotideprimers were screened, and thirteen primers showed polymorphism among near-isogenic lines.There were six primers showed special polymorphic bands among lines lx1 and lx1.3. Especially,primer S352 presented the stable results in which a 900 bp band was found in the lines lx1 andlx1.3, and primer S352900 was detected with F2 generation of cross 96P11×Century-1, indicatedprimer S352900 could be identified as a RAPD marker linked to gene lx1 in soybeans, the distanceof linkage was 7.6 cM.

Cloning of Bt cry Genes by Rapid Screening of DNA Libraries with PCR-RFLP

Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, crylCa insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR I fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR I complete and 5au3A I partial digestion. On the basis of every 50 transformants pooled together from 5-10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa , and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletropora-tion. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality.

Isolation, purification and characterization of a new organphosphorus hydrolase OPHC2

A bacterium with the capability of degrading organphosphorus, identified as Pseudomonas pseudoaicaligenes, is isolated from OP-treated soil. The organphosphorus hydrolase OPHC2 from this bacterium has been purified and characterized. OPHC2 has optimum activity for the reaction at 65℃ and pH 9.0 with methyl parathion as a substrate, it also shows good thermal and pH stability. Most metal ions and chemicals have no effect on the activity of OPHC2. The analyses of nucleotide sequence encoding OPHC2 and amino acid sequence of OPHC2 show that there are lower homologies with those of organphosphorus hydrolase reported in GenBank.

Cloning and expression of ophc2,a new organphosphorus hydrolase gene

The amino acid sequences of N-terminal and internal peptide of OPHC2,purified from Pseudomonas pseudoalcaligenes strain C2-1 in our lab,are determined.The full-length organphosphorus hydrolase gene ophc2 is cloned by PCR using the degenerate primers designed according to the sequences and future inverse PCR.The ophc2 gene is 975 bp long with G+C content of 63%,comprising one open reading frame encoding a polypeptide of 324 amino acids with a molecular weight of 36 kD.The nucleotide sequence of ophc2 shows low homolo- gies with those organphosphorus hydrolase genes deposited in Gen- Bank,one of which exhibits the highest homology of 46.4% with ophc2.The organphosphorus hydrolase protein expressed in E.coli bears normal bioactivity.

Evaluation and characterization of an endosperm-specific sbeⅡa promoter in wheat

Starch, the main component of the wheat grain, is the product of a complex biochemical pathway. The sbeⅡa gene plays a key role in controlling the synthesis of starch, in particular, the biosynthesis of amylopectin, in maturing wheat grain. To investigate its regulatory mechanisms and endosperm-specific expression pattern, the sbeⅡa promoter (3094 bp in length) was cloned using APCR and sequenced. The effect of a series of deletions was studied using a GUS transient assay system. Results showed that the 3094 bp se-quence (sbe.g construct) exhibited full stable promoting ac-tivity and that the activities of 5′ or 3′ deletions reduced levels of GUS expression. Some constructs with internal dele-tions showed only weak activity, however, sbe.e, with a dele-tion from -1579—-1210 bp resulted in higher levels of ex-pression than the full-length promoter sequence, sbe.g. This indicates that motifs such as the -300 bp element, G-box and/or P-box act as positive elements and are necessary in determining the promoters endosperm-specific pattern and that negative repressor elements or motifs may also be pre-sent within the -1579—-1210 bp sequence. The age of wheat endosperm tissue used in the GUS-transient assay system is shown to be of significant importance.

Effect of GbKTN1 from Gossypium barbadense on cell elongation of fission yeast (Schizosaccharomyces pombe)

The GbKTN1 gene was isolated from 10 DPA fiber cells of Gossypium barbadense using 5′RACE/3′RACE.Full-length cDNA of this gene is 2006 bp, including a 113 bp of 5′untranslated region, a 1563 bp of an open reading frame(ORF), and a 327 bp of 3′untranslated region (excluding the stop codon TAA). The ORF of GbKTN1 encodes a 521-amino acid protein with a predicted size of 55 kD. Near C-terminal of the deduced protein there is a putative ATP binding site between amino acid residues from 233 to 414. Southern blot analysis indicated that the GbKTN1 was a single copy gene in G barbadense. Combining semi-quantitative RT-PCR with Southern blot hybridization revealed that GbKTN1 expressed in all the organs detected such as roots, stems, leaves and fibers. However, the mRNA of GbKTN1 was the most abundant in fiber cells, while it was the lowest in leaves. The GbKTN1 cDNA was transformed into S. pombe to verify its function on cell elongation. Results showed that most yeast cells over expressing GbKTN1 gene were elongated dramatically with an average length increase of 2.18 times than that of the non-induced cells. Even the morphology of some yeast cells appeared irregularly. To the best of our knowledge this is the first evidence that KTN1 is correlated with cell elongation in vivo.