Isolation Of Early Nodulin Genes From Pisum Sativum And Introduction Of PsENOD12A And PsLectin Into Nicotiana Tabacum

Symbiotic nitrogen fixtion is the result of interaction between Legume and Rhizobium.The successive steps from the Rhizobium come into contact with root hair to form mature nodulin,in fact, is the program of gene expression of the Rhizobium and its host.This include molecule communication,recognition reaction,information dissemination,network-manipulation of gene expression.If we can make clear the sections of this process,we can not only make use of the symbiotic nitrogen fixtion in Legume,but also introduce it into non-legumes,especially agricultural crop.Through this,we can improve the gross production;reduce the presure of short-resources and environmental pollution.It's a green revolution that brings benefit to mankind.Former studies have showed that the leguminous lectins play an important role in the recognition of rhizobia.Some legumes could recognize the rhizobinm that hadn't been recognized after the relevant lectin gene being introduced.In various ecological environment,one host can interact with several Rhizobium,this indicate the diversity of symbiosis between Legume and Rhizobium.It is modification and developments to traditional concept of specify of Rhizobium-host and co-inoculate of plants.Edwards et al(1991) and H(?)fte et al(1991) had introduced the leguminous lectins into potato and tobacco;YIQUN WANG,YUXIANG JIN(1999) had constructed the plant-expression vector of leguminous lectins and leghemoglobin genes,then introduced it into tobacco and rice.They had studied the expression mechanism and Cell biology orientation.The main aim is to introduce leguminous lectins into non-legumes,and let the non-legumes distinguish from rhizobia,ultimately,extend the host of rhizobia.In the system of symbiotic nitrogen,there are suchlike genes in the host that express in the program of nodulation.It's called Nodulin Gene.Including early nodulin gene(ENOD) and late nodulin gene(NOD).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,more and more early nodulin genes were differentiated,cloned and sequenced.But the mechanism of their function was not well-informed yet.In this study,PsENOD12A,PsSym7 and PsSym35 gene,which are early nodulin genes,were obtained from pea(Pisum sativum L.) by PCR amplification.The expression vector of PsENOD12A and PsLetin gene was constructed,and then,we introduced the expression vector into tobacco (Nicotiana tabacum Lcv.Wisconsin38) via Agrobacterium tumefaciens mediated method.The main results show as follows:1.Cloned early nodulin Genes -PsENOD12A gene from Pisum sativumThe PsENOD12A gene fragment was amplified by means 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 bp,which contained an open reading frame of 333 bp, 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.2.Cloned early nodulin Genes -PsSym7 gene from Pisum sativumThe PsSym7 gene fragment was amplified by means of PCR using pea genomic DNA as template and a pair of annex ollgonucleotides as primer.The sequence length is 743bp,Using Geneious Pro tril 4.5.4 analyze it on net,we found that 99.1%homologies in nucleotide sequence Pisum sativum Sym7 gene for GRAS family protein(located in AJ832139).It encoded a polypeptide of 248 amino acids,99.8%in amino acid sequence respectively with Pisum sativum GRAS family protein(CAH55769).3.Cloned early nodulin Genes -PsSym35 gene from Pisum sativumThe PsSym35 gene fragment was amplified by means of PCR using pea genomic DNA as template and a pair of specific oligonucleotides as primer.Because the gene bigger than 5 kb,so we cloned it by two sequences,and then connected to one.The fragment of PsSym35up(promoter region) consisted of 838 bp,PsSym35down(CDS region) consisted of 3199 bp.Analyzing it with Geneious Pro tril 4.5.4,the sequence has three axons.Contrasted with PsNIN(located in GenBank AJ493063),homologies in nucleotide sequence is 99.9%.And 98.7%in amino acid sequence respectively with nodule inception protein of Pisum sativum(CAD37946).4.Introduced PsENOD12A and PsLectin into tobacco's genomeThe PsENOD12A gene was first linked in mid-vector of pVCT-2020,joined with the plant expression of pVCT-PsL which was constructed beforetime,we built the expression vector of pVCT2120,and then,we introduced the two genes into tobacco via Agrobacterium tumefaciens mediated method.Eight kanamycin-resistant tobacco plants were obtained and all of these plants were alive.PCR detection showed that the PsENOD12,4 and PsLetin gene have been integrated into tobacco's genome.