Construction And Genetic Transformation Of Pollen-specific Vector Of α-amylase Gene In Maize

Maize is the first food crop in our country.And it is one of the crops that produced hybrids seeds with CMS lines for the first. GMS can not be used widely for the production of hybrid seeds in maize before the coming of SPT technology. SPT bring us the hope for using GMS for seeds production. In this study, combined the SOE-PCR with digestion and connection technology together were used to construct the expression vector.Then transformed the vector into the maize inbred line 18-599R via agrobacterium mediation. The results are as follows:1.Pollen specific expression promoter Pg（X666920）, amyloplast signal peptide bt1（NM₀01112419）, and terminator In-2-1 of substituted benzenesulfona mides were cloned from the B73 genomic DNA with high-fidelity DNA polymerase KOD Fx specificly amplify. Gene of α-amylase（NM₀01156806） was cloned from the RNA of the germinated seed of 18-599 red. Compared the sequences of clone genes with known sequences listed in the NCBI, the sequence similarity are 99%, 100%,100%å'Œ 95% respectively.2. Touch-down overlapping extension PCR （TD-SOE-PCR） were employed to connect the four target fragments in the specific order. Four group of primers were designed with reverse complementary end. high-fidelity DNA polymerase KOD Fx was used to amplify the four target fragments which have reverse complementary end. At the same time, the enzyme loci of Hind â…¢ and EcoR â…  of Pg’s 5’and In2-1’s 3’ were introduced. Employing TD-SOE-PCR and high-fidelity DNA polymerase reconstruct the four fragments which have reverse complementary end in order. Transform the α-amylase gene expression cassette into E. coli, and select single clone then conduct microbial propagation. Extracting and sequencing the target plasmid, the result are consistent with the DNA sequencing.3. Ligating the α-amylase gene expression cassette to plant expression vector CPB.Using the endonu-clease Hindâ…¢ and EcoR â…  digest the plasmid which connect the a-amylase gene expression cassette, and then recovery the objective genes cassette.At the same time digestion the expression vector CPB, recovery the linearized vector. Ligating the objective genes cassette to linearized vector with the T4 ligase.Transform the target vector into E. coli, with kana to filter the positive plasmid.Digesting the positive plastid overnight under 37℃, and then agarose gel electrophoresis was conducted. The result shows that the vetor sequence constructed in this study is correct.4. Transform the target vector to embryonic callus of 18-199R via agrobacterium-mediated method. After resistance selection with different levels of basta（1.5mg/L,2.5mg/L,3mg/L）,17 regeneration plants were obtained. But PCR amplification results show the target vetor’s T-DNA region did not integrated into the genome of the 18-599Red. The result indicates that the regeneration plants may be false positive.