Studies On Construction Of Seed-specific Expression Vectors And Optimizing The Techno-System Of Genetic Transformation Mediated By Agrobacterium Tumefaciens In Peanut(Arachis Hypogara L.)

Peanut (Arachis hypogaea L.), the world's fourth largest oilseed crop, is the oil and economic crop grown worldwide, also is the oil crop that having highest single yield,total yield and export income in our country. So improving peanut trait by genetic transformation is the important field of molecular breeding. Two seed-specific promoters were isolated from genome DNA of Fenghua3 by homology cloning, then seed-specific expression vectors were constructed; the effect of genotype,medium for the inducement of buds and medium for the elongation of buds to regeneration frequency were researched, regeneration system was Optimized in three factors; Genetic transformation frequencies of 21 genotypes were compared; Genetic transformation seeding were obtained, and identified by PCR. All these establish basics for molecular breeding of improving peanut quality. The main results were as follows:1 Cloning of the Oleosin promotor from peanut and constructing of its seed- specific expression vectorThe primers were designed according to the published sequence of Oleosin17.8 gene on GenBank (EF695400). Extracting genome DNA from peanut Fenghua3, the fragment of promotor OL1584 was obtained by PCR. The OL1584 was cloned into pGM-T easy vector for sequencing and named pT-OL1584. Sequencing analysis indicated that the inserted fragment was showing 99.37% homology to the reported sequence Oleosin17.8 .And the accession number of sequence OL1584 in GeneBank is EU518466. TATA,CAAT,two AT-Rich elemengts,two RY elements,five CATG boxes,five E-Boxes,six GATA-Boxes two TACACAT elements and fourteen AAAG boxes are existed in OL1584. The recombinant plasmid pT-OL1584 and pBI121 were digested by HindⅢand BamHⅠ, the aimed 1584bp fragment and the longer of pBI121 resected 35S gene were recycled, and ligated by T4 DNA Ligase.The result proved that OL1584 was ligated into the normal vector of pBI121. And the seed-specific expression vector pB-OL1584 was constructed. Then the OL1584 5'deletion plasmids pT-OL755 and pT-OL375 were constructed by PCR with pT-OL1584 for template and different up-primers.2 Cloning of the Ara h1 promotor from peanut and constructing of its seed-specific expression vectorThe primers were designed according to the published sequence of Ara h1 gene (WANG jing, 2005; Olga M, 2003). Extracting genome DNA from peanut Fenghua3, the fragment of promotor Ah1968 was obtained by PCR. Then Ah1968 was cloned into pGM-T easy vector for sequencing and named pT-Ah1968. Sequencing analysis indicated that the inserted fragment was showing 99.14% homology to the reported sequence by WANG Jing(2005), 98.27% homology to the reported sequence by Olga M(2003).And the accession number of sequence Ah1968 in GeneBank is EU526898.TATA,CAAT,three AT enhancers,two RY elements,three CATG boxes,two E-Boxes,ten GATA-Boxes two TACACAT elements and thirteen AAAG boxes are existed in Ah1968. The recombinant plasmid pT-Ah1968 and pBI121 were digested by HindⅢand BamHⅠ, the aimed 1716bp fragment and the longer of pBI121 resected 35S gene were recycled and ligated by T4 DNA Ligase.The result proved that Ah1716 was inserted into the normal vector of pBI121. Then the seed-specific expression vector pB-Ah1716 was constructed. The Ah1968 5'deletion plasmids pT-Ah1300,pT-Ah1000 and pT-Ah500 were constructed by PCR with pT-Ah1968 for template and different up-primers .3 Optimization of the Regeneration Technique from independent Leaflet of Peanut(1)Study on the suitable medium for the inducement of budsThe experimental results indicated that the suitable medium for the inducement of buds were MSB + 3.0mg/L BA + 0.7mg/LNAA for Fenghua2, frequencies of buds inducement and regeneration are 81.49% and 94.39% respectively. MSB + 4.5mg/L BA + 0.2mg/L NAA is suitable for Fenghua3, frequencies of buds inducement and regeneration are 72.33% and 10.70% respectively.(2)Study on the suitable medium for the elongation of budsThe experimental results indicated that the best media for the elongation of buds were MSB+ 2.5mg/L BA for fenghua2, the rate of seeding in buds and regeneration were 172.00% and 122.10% respectively. And MSB+ 5.0mg/L KIN is suitable for Fenghua3, the rate of seeding in buds and regeneration were 33.33% and 4.03% respectively.Conclusion:There are significant differences on bud differentiation and plant regeneration between the two genotypes. Genotype is the main element of regeneration difference. The lesser change of Plant hormone deepness is not enough to offset the regeneration difference of genotypes. The best combination of three factors for higher regeneration frequency: Fenghua2, buds inducement media is MSB + 3.0mg/L BA + 0.7mg/L NAA, buds elongation media is MSB+ 2.5mg/L BA. These could be as accepter system for peanut genetic transformation.4 Comparing on transformation efficiency of different genotypes mediated by Agrobacterium tumefaciensThe experiment conducted genetic transformation research on peanut using vector harboringγ-tmt and bar genes, and leaflet of 21 genotypes as explants. The results indicated that the ten genotypes which have higher regeneration rate are Fenghua2,02P181,Fenghua3,Bujieliuxi,Fenghua1,Baisha1016,Fenghua6,05D651,Haihua1,PengLaiyiwohou;Five genotypes with higher transformation efficiency are Fenghua2,Fenghua1,Fenghua3,Baisha1016,02P181;Fenghua2 is the highest one for 2.67%;PCR positive plants were obtained from the five genotypes.