RESUMO
Parkia pendula (Willd.) Walp. (Fabaceae) is a neotropical species of the genus Parkia more abundantly distributed in Central to South America. From the seeds of P. pendula a glucose/mannose specific lectin (PpeL) was isolated that has been characterised and used as a biotechnological tool but until now this is the first manuscript to analyse P. pendula mRNA expression in seedlings. For this porpoise a Differential display reverse transcription polimerase chain reaction (DDRT-PCR) was used to evaluate the expression of P. pendula lectin mRNAs in non-rooted seedlings. No bands were observed in the agarose gel, indicating the absence of mRNA of PpeL seedlings. our findings confirm that lectins mRNAs are differently regulated among species even if they are grouped in the same class.
Parkia pendula (Willd.) Walp. (Fabaceae) é a espécie neotropical do gênero Parkia mais abundantemente distribuída na América Central a do Sul. Das sementes de P. pendula foi isolada uma lectina glicose/manose específica (Ppel) que foi caracterizada e usada como ferramenta biotecnológica, porém até o momento esse é o primeiro artigo a analisar a expressão do mRNA nas plântulas de P. pendula. Para esse propósito uma reação de PCR diferencial de transcriptase reversa (DDRT-PCR) foi utilizada para avaliar a expressão do mRNA da lectina de P. pendula em plântulas não enraizadas. Nenhuma banda foi observada no gel de agarose, indicando a ausência de mRNA das plântulas de PpeL. Nossos achados confirmam que os mRNAs de lectinas são regulados de forma diferentes entre as espécies, mesmo que sejam agrupadas na mesma classe.
Assuntos
Fabaceae/genética , Lectinas de Plantas/genética , RNA Mensageiro/análise , RNA de Plantas/análise , Fabaceae/química , Lectinas de Plantas/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , PlântulaRESUMO
Chickpea is the world's third most important pulse crop and India produces 75% of the world's supply. Chickpea seeds are attacked by Callosobruchus maculatus and C. chinensis which cause extensive damage. The alpha-amylase inhibitor gene isolated from Phaseolus vulgaris seeds was introduced into chickpea cultivar K850 through Agrobacterium-mediated transformation. A total of 288 kanamycin resistant plants were regenerated. Only 0.3% of these were true transformants. Polymerase chain reaction (PCR) analysis and Southern hybridization confirmed the presence of 4.9 kb alpha-amylase inhibitor gene in the transformed plants. Western blot confirmed the presence of alpha-amylase inhibitor protein. The results of bioassay study revealed a significant reduction in the survival rate of bruchid weevil C. maculatus reared on transgenic chickpea seeds. All the transgenic plants exhibited a segregation ratio of 3:1.
Assuntos
Cicer/anatomia & histologia , Imunidade Inata/genética , Fito-Hemaglutininas/genética , Lectinas de Plantas/genética , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Engenharia de Proteínas , Rhizobium/genética , Sementes/genética , Transformação Genética , Gorgulhos/crescimento & desenvolvimento , alfa-Amilases/antagonistas & inibidoresRESUMO
During its biosynthesis in developing Canavalia brasiliensis seeds, the lectin ConBr undergoes a form of protein splicing in which the order of the N- and C-domains of the protein is reversed. To investigate whether these events can occur in other eukaryotic organisms, an expression system based on Pichia pastoris cells was established. A DNA fragment encoding prepro-ConBr was cloned into the vector pPICZB, and the recombinant plasmid was transformed in P. pastoris strain GS115. Ten clones were screened for effective recombinant protein production. Based on Western blot analysis of the two clones with the highest level of protein expression: 1) diffuse high-molecular mass immunoreactive bands were produced as early as 24 h after induction; 2) a single-, high-molecular mass protein was secreted into the medium, and 3) a significant fraction of the recombinant polypeptides that cross-reacted with anti-ConBr antibodies comprised a band of approximately 34.5 kDa. Diffuse protein bands with high molecular masses are attributed to hyperglycosylation at the single potential N-glycosylation site located in the linker peptide of prepro-ConBr. In contrast, native ConBr is made up of three polypeptides, the intact alpha chain (aa 1-237) and the fragments beta (aa 1-118) and gamma (aa 119-237), which have apparent molecular masses of 30, 16 and 12 kDa, respectively. Apparently, the yeast P. pastoris is not able to carry out all the complex post-translational proteolytic processing necessary for the biosynthesis of ConBr.
Assuntos
Canavalia/química , Lectinas de Plantas/genética , Modelos Genéticos , Pichia/metabolismo , Processamento de Proteína/genética , Regulação da Expressão Gênica de Plantas/genética , Lectinas de Plantas/biossíntese , Lectinas de Plantas/química , Reação em Cadeia da Polimerase , Vetores Genéticos , Western BlottingRESUMO
In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the gna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.