Cloning and identification of an angiostatic molecule IP-10/crg-2

AIM To obtain human and murine cDNAsencoding IFN--y inducible protein 10 (lP--10) andcytokine responsive gene--2 (Crg-2).METHODS The encoding genes of lP-10 and Crg2 were amplified by RT--PCR from Cultured humanfibroblast cells and Balb/ c mouse liver treatedby IFN-y and TNF-a, respectively, and clonedinto plasmids of PUC19 and PGEM3Zf(+ ).RESULTS The nucleotide sequences ot theamplified DNA were confirmed by endonucleasesdigestion and sequencing.CONCLUSION Recombinant lP-10/ crg--2 geneclones with 306 hp and 314 hp inserts wereestablished for further research on biologicalactivities and ligands of hiP-10/mCrg--2.

Identification and Cloning of Resistance Gene Analogues (RGAs) Encoding NBS-LRR Proteins from Gossypium arboreum L.

Plants have developed a complicated defense mechanism during evolution to resist the harmful pathogens they encountered.The mechanism involves the interaction of the plant resistance(R)

Genotypic Analysis, Construction of the Expression System and Immunological Identification of the Recombinant Proteins of the LipL32 Gene in the Dominant Serogroups of Leptospira interrogans in China

To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Tort. ＆ Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction （PCR） assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Identification and Cloning of Differentially Expressed Genes Involved in the Interaction Between Potato and Phytophthora infestans using a Subtractive Hybridization and cDNA-AFLP Combinational Approach

Using a subtractive hybridization （SH）/cDNA-AFLP combinational approach, differentially expressed genes involved in the potato-Phytophthora infestans interaction were identified. These included genes potentially controlling pathogenesis or avr genes in P. infestans as well as those potentially involved in potato resistance or susceptibility to this pathogen. Forty-one differentially expressed transcript, derived fragments （TDFs）, resulting from the interaction, were cloned and sequenced. Two TDFs, suggested as potential pathogenicity factors, have sequence similarity to N-succinyl diaminopimelate aminotransferase and a transcriptional regulator, TetR family gene, respectively. Two other TDFs, suggested as potential avr genes, have sequence similarity to an EST sequence from Avr41Cf.41Avr91Cf- 9 and a P. infestans avirulence-associated gene, respectively. Genes＇ expression and origin were confirmed using Southern blots, Northern blots and qRT-PCR, he., potential resistance gene DL81 was induced at 12 hpi in the moderately resistant cultivar, whereas it was down-regulated as early as 6 hpi in the susceptible cultivar. On the other hand, DL21 was induced at 6 hpi （3.38-fold） in response to the highly aggressive isolate （US8） and strongly up-regulated thereafter （25.13-fold at 120 hpi.）, whereas it was only slightly up-regulated in response to the weakly aggressive isolate US11 （3.82-fold at 96 hpi）, suggesting its potential involvement as a susceptibility gene.

Development of two microsatellite multiplex PCR systems for high throughput genotyping in Populus euphratica

Eighteen microsatellite primer pairs previously developed at Oak Ridge National Laboratory for Populus tremuloides Michx. and Populus trichocarpa Torr. & Gray were screened for amplification in Euphrates poplar, Populus euphratica Oliv. Thirteen loci were found to express polymorphisms ranging from two to 17 alleles. The eight most variable loci were selected to set up and optimize two multiplex polymerase chain reaction (PCR) assays. Three populations containing altogether 436 trees were used to characterize the selected loci and ascertain their applicability for parentage analysis and genotyping studies. Through cross-checking of clonal identity against sex of the genotyped trees we estimated the maximum error rate for merging genotypes to be less than 0.045.

Cloning of fatty acid elongase1 gene and molecular identification of A and C genome in Brassica species

The fatty acid elongase 1 (FAE1) genes of Brassic napus were cloned from two cultivars, i.e. Zhong- shuan No. 9 with low erucic acid content, and Zhongyou 821 with high erucic acid content, using the degenerate PCR primers. The sequence analysis showed that there was no intron within the FAE1 genes. The FAE1 genes from Zhongyou 821 contained a coding sequence of 1521 nucleotides, and those cloned from Zhongshuan No. 9 contained a 1517 bp coding sequence. Alignment of the FAE1 sequences from Brassica rapa, B. oleracea and B. napus detected 31 single nucleotide polymorphic sites (2.03%), which resulted in 7 amino-acid substitutions. Further analysis indicated that 19 SNPs were genome-specific, of which, 95% were synonymous mutations. The nucleotide substitution at po- sition 1217 in the FAE1 genes led to a specific site of restricted cleavage. An AvrII cleavage site was present only in the C genome genes and absent in the A genome FAE1 genes. Digestion profile of the FAE1 sequences from B. rapa, B. oleracea and B. napus produced with AvrII confirmed that the FAE1 genes of B. oleracea origin was recognized and digested, while that of B. rapa origin could not. The results indicated that by AvrII cleavage it was possible to distinguish B. rapa from B. oleracea and be- tween the A and C genome of B. napus. In addition, the FAE1 genes could be used as marker genes to detect the pollen flow of B. napus, thus providing an alternative method for risk assessment of gene flow.