MILDEW RESISTANCE LOCUS O GENE CLONING, CHARACTERIZATION, AND EXPRESSION PATTERN IN MULBERRY AND ITS PROKARYOTIC EXPRESSION IN E. coli, "Биоорганическая химия"

Abstract Background Downy mildew, the most devastating disease of spinach (Spinacia oleracea L.), is caused by the oomycete Peronospora effusa [=P. farinosa f. sp. spinaciae]. The P. effusa shows race specificities to the resistant host and comprises 19 reported races and many novel isolates. Sixteen new P. effusa races were identified during the past three decades, and the new pathogen races are continually overcoming the genetic resistances used in commercial cultivars. A spinach breeding population derived from the cross between cultivars Whale and Lazio was inoculated with P. effusa race 16 in an environment-controlled facility; disease response was recorded and genotyped using genotyping by sequencing (GBS). The main objective of this study was to identify resistance-associated single nucleotide polymorphism (SNP) markers from the cultivar Whale against the P. effusa race 16. Results Association analysis conducted using GBS markers identified six significant SNPs (S3_658,306, S3_692697, S3_1050601, S3_1227787, S3_1227802, S3_1231197). The downy mildew resistance locus from cultivar Whale was mapped to a 0.57 Mb region on chromosome 3, including four disease resistance candidate genes (Spo12736, Spo12784, Spo12908, and Spo12821) within 2.69–11.28 Kb of the peak SNP. Conclusions Genomewide association analysis approach was used to map the P. effusa race 16 resistance loci and identify associated SNP markers and the candidate genes. The results from this study could be valuable in understanding the genetic basis of downy mildew resistance, and the SNP marker will be useful in spinach breeding to select resistant lines.

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Cloning and abiotic stress expression analysis of galactose-binding lectin (GBL) gene from mulberry and its prokaryotic expression in E. coli

The Journal of Horticultural Science and Biotechnology ◽

10.1080/14620316.2020.1769507 ◽

2020 ◽

Vol 96 (1) ◽

pp. 24-33

Author(s):

Yang Li ◽

Jian Zhang ◽

Fei Hu ◽

Acheampong Adolf ◽

Michael Ackah ◽

...

Keyword(s):

Abiotic Stress ◽

Expression Analysis ◽

Prokaryotic Expression ◽

E Coli ◽

Binding Lectin ◽

Galactose Binding Lectin

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Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

Applied and Environmental Microbiology ◽

10.1128/aem.02511-06 ◽

2007 ◽

Vol 73 (7) ◽

pp. 2199-2206 ◽

Cited By ~ 38

Author(s):

Stuart A. Thompson ◽

Elizabeth V. Maani ◽

Angela H. Lindell ◽

Catherine J. King ◽

J. Vaun McArthur

Keyword(s):

Serratia Marcescens ◽

Efflux Pump ◽

Tetracycline Resistance ◽

Amino Acid Identity ◽

Resistance Determinant ◽

Mercury Resistance ◽

Resistance Locus ◽

Environmental Strain ◽

Apparent Lack ◽

E Coli

ABSTRACT Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.

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Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

Biochemical Journal ◽

10.1042/bj2730587 ◽

1991 ◽

Vol 273 (3) ◽

pp. 587-592 ◽

Cited By ~ 22

Author(s):

K M LeVan ◽

E Goldberg

Keyword(s):

Escherichia Coli ◽

Lactate Dehydrogenase ◽

Prokaryotic Expression ◽

Specific Activity ◽

Human Testis ◽

Reading Frame ◽

E Coli ◽

Heat Stable ◽

Prokaryotic Expression Vector ◽

Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

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