Isolation Of Early Nodulin Genes From Pisum Sativum And Introduction Of Psl Gene Into Nicotiana Tobacum

Legume root nodule formation and symbiotic nitrogen fixtion are induced by rhizobia, which inoculate legumes specifically. The successive steps in root nodule development involve a lot of host genes and rhizobium genes that express in this process. Former studies have showed that the leguminous lectins play an important role in the recognition of rhizobia. Some legumes could recognise the rhizobium that hadn't been recognized after the relevant lectin gene being introduced. However, when lectin genes were introduced into non-legumes using manual promoter the anticipant result was not obtained. So, it is necessary to simulate the original mechanism of lectin genes' expression, research on the interaction of transgenic non-legumes and rhizobia.The successive steps in root nodule development involve the induction of host genes that are not expressed in uninoculated roots. Of these genes, the genes that are not expressed in any other plant organ are named nodulin genes. Nodulin genes are very important for the formation legume root nodule , maintaining the function of nitrogen fixtion and the metabolism of products. According to the expression time, nodulin genes were divided as early nodulin genes and late nodulin genes. Early nodulin genes were induced at the early stage of legume root nodule formation. It played an important role in the information communication between plants and rhizobia, the process of infection and the formation of root nodule. So far several key early nodulin genes have been found, and some function of them were explained to some extent. Whether these genes would bring the same function after being introduced into non-legumes need to be made sure.In this study PsENOD12A, PsSYM10 and PsENOD40 gene, which are nodulin genes, and psl gene were obtained from pea (Pisum sativum L.) by PCR amplification. The expression vector of psl gene was constructed, and then, we introduced psl gene into tobacco (Nicotiana tobacum L.cv.Wisconsin38) via Agrobacterium tumefaciens mediated method. The main results show as follows:1. Cloning of psl gene , construction of its plant expression vector and Introduction of psl into tobaccoThe psl gene fragment was amplified by meas of PCR using pea genomic DNA as template and a pair of specific oligonucleotides at 5'- and 3'- end as primer. Sequence analysis showed that the fragment consisted of 1948 bps, which contained an open reading frame of 828 bps, and encoded a polypeptide of 275 amino acids. Comparison with previously published sequence (location in GenBank as X66368) showed 99.4% homologies in nucleotide sequence and 100% in amino acid sequence respectively. The plant expression vector pVCT-PsL was constructed, and then, we introduced psl gene into tobacco via Agrobacterium tumefaciens mediated method. Ten kanamycin-resistant tobacco plants were obtained and all of these plants were alive. PCR detection showed that the psl gene has been integrated into their genome.2. Cloning of PsENOD12A geneThe PsENOD12A gene fragment was amplified by meas of PCR using pea genomic DNA as template and a pair of specific oligonucleotides at 5'- and 3'- end as primer. Sequence analysis showed that the fragment consisted of 1282 bps, which contained an open reading frame of 333 bps, and encoded a polypeptide of 110 amino acids. Comparison with previously published sequence (location in GenBank as X81366) showed 99.9% homologies in nucleotide sequence and 100% in amino acid sequence respectively.3. Cloning of PsSYM10 geneThe PsSYM10 gene fragment was amplified by meas of PCR using pea genomic DNA as template and a pair of specific oligonucleotides at 5'- and 3'- end as primer. Sequence analysis showed that the fragment consisted of 3310 bps, which contains contained an open reading frame of 1785 bps, and encoded a polypeptide of 594 amino acids. Comparison with previously published sequence (location in GenBank as AJ575250) showed 98.8% homologies in nucleotide sequence and 100% in amino acid sequence respectively.4. Cloning of PsENOD40 geneThe PsENOD40 gene fragment was amplified by meas of PCR using pea genomic DNA as template and a pair of specific oligonucleotides at 5'- and 3'- end as primer. Sequence analysis showed that the fragment consisted of 459 bps, which contains contained an open reading frame of 228 bps, and encoded a polypeptide of 75 amino acids. The iniation codon of this gene was not ATG but TTA. Comparison with previously published sequence (location in GenBank as X81064) showed 100% homologies in nucleotide sequence and 100% in amino acid sequence respectively.