Identification and evaluation of new reference genes in Gossypium hirsutum for accurate normalization of real-time quantitative RT-PCR data

<p>Abstract</p> <p>Background</p> <p>Normalizing through reference genes, or housekeeping genes, can make more accurate and reliable results from reverse transcription real-time quantitative polymerase chain reaction (qPCR). Recent studies have shown that no single housekeeping gene is universal for all experiments. Thus, suitable reference genes should be the first step of any qPCR analysis. Only a few studies on the identification of housekeeping gene have been carried on plants. Therefore qPCR studies on important crops such as cotton has been hampered by the lack of suitable reference genes.</p> <p>Results</p> <p>By the use of two distinct algorithms, implemented by <it>geNorm </it>and <it>NormFinder</it>, we have assessed the gene expression of nine candidate reference genes in cotton: <it>GhACT4, GhEF1α5, GhFBX6, GhPP2A1, GhMZA, GhPTB, GhGAPC2, GhβTUB3 </it>and <it>GhUBQ14</it>. The candidate reference genes were evaluated in 23 experimental samples consisting of six distinct plant organs, eight stages of flower development, four stages of fruit development and in flower verticils. The expression of <it>GhPP2A1 </it>and <it>GhUBQ14 </it>genes were the most stable across all samples and also when distinct plants organs are examined. <it>GhACT4 </it>and <it>GhUBQ14 </it>present more stable expression during flower development, <it>GhACT4 </it>and <it>GhFBX6 </it>in the floral verticils and <it>GhMZA </it>and <it>GhPTB </it>during fruit development. Our analysis provided the most suitable combination of reference genes for each experimental set tested as internal control for reliable qPCR data normalization. In addition, to illustrate the use of cotton reference genes we checked the expression of two cotton MADS-box genes in distinct plant and floral organs and also during flower development.</p> <p>Conclusion</p> <p>We have tested the expression stabilities of nine candidate genes in a set of 23 tissue samples from cotton plants divided into five different experimental sets. As a result of this evaluation, we recommend the use of <it>GhUBQ14 </it>and <it>GhPP2A1 </it>housekeeping genes as superior references for normalization of gene expression measures in different cotton plant organs; <it>GhACT4 </it>and <it>GhUBQ14 </it>for flower development, <it>GhACT4 </it>and <it>GhFBX6 </it>for the floral organs and <it>GhMZA </it>and <it>GhPTB </it>for fruit development. We also provide the primer sequences whose performance in qPCR experiments is demonstrated. These genes will enable more accurate and reliable normalization of qPCR results for gene expression studies in this important crop, the major source of natural fiber and also an important source of edible oil. The use of bona fide reference genes allowed a detailed and accurate characterization of the temporal and spatial expression pattern of two MADS-box genes in cotton.</p

Meloidogyne incognita: molecular cloning and characterization of a cDNA encoding a cathepsin D-like aspartic proteinase

Herein we describe the cloning and characterization of a cDNA encoding an aspartic proteinase from the root-knot nematode Meloidogyne incognita. Using PCR techniques, a 1471-bp cDNA fragment encoding a cathepsin D-like (Mi-asp1) transcript was isolated from second- stage larvae mRNA. Its predicted amino acid sequence comprises a pro-region of 71 amino acid residues and a mature protease of 378 amino acid residues with a predicted molecular mass of 41.502 kDa. Protein sequence comparisons of Mi-asp1 with GenBank ™ (DQ360827) sequences showed 59–71% identity with nematode- specific cathepsin D-like aspartic proteinases. Southern blot analysis, RT-PCR amplification and EST mining suggest the existence of a developmentally expressed gene family encoding aspartic proteinases in M. incognita. Mi-asp1 may represent a potential target for molecular intervention for the purposes of plant–parasitic nematode control

Characterization of novel Brazilian Bacillus thuringiensis strains active against Spodoptera frugiperda and other insect pests

Abstract: Brazilian strains of Bacillus thuringiensis, namely S701, S764 and S1265 were analysed regarding their cry gene and protein contents, crystal type, and activity against larvae of the lepidopteran fall armyworm (Spodoptera frugiperda Smith), the velvet caterpillar (Anticarsia gemmatalis), the dipterans (Culex quinquefasciatus and Aedes aegypti) and the coleopteran (Tenebrio molitor). The LC50 of the strains against second instar larvae of S. frugiperda or A. gemmatalis revealed a high potency against those insect species. The spore–crystal mixtures of the isolates were analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and showed similar protein pattern as the B. thuringiensis subsp. kurstaki strain HD-1 (proteins approximately 130 and 65 kDa) for isolates S701 and S764, respectively, and only one major protein of approximately 130 kDa for isolate S1265. The polymerase chain reaction (PCR) using total DNA of the isolates and general and specific primers showed the presence of cry1Aa, cry1Ac, cry1Ia and cry2Ab genes in the two isolates serotyped as B. thuringiensis kurstaki (S701 and S764) and the presence of cry1D and cry2Ad in B. thuringiensis morrisoni S1265 strain. Scanning electron microscopy of strains S701 and S764, showed the presence of bipyramidal, cuboidal and round crystals, like in strain HD-1 and bipyramidal and round crystals like in strain S1265