Method for processing plant biomass
A biomass, plant technology, applied in biochemical equipment and methods, animal feed, animal feed, etc., can solve problems such as adverse phenotypic effects
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Embodiment 1-p
[0107] Example 1 - pSB11
[0108] see figure 1 , the vector in one embodiment of the present invention can be based on the pSB11 intermediate plasmid (a derivative of pBR322). pSB11 was obtained from Japan Tobacco. The pSB11 plasmid is suitable for cloning and is easily maintained in E. coli. The two vectors are coupled and maintained in LB4404 Agrobacterium by homologous recombination using cos and ori sites present in both pSB11 and pSB1 "super binary" acceptor vectors (non-oncogenic Ti plasmids) within the strain. The integrated product represents a hybrid vector that can be used for subsequent plant transformation. pSB1 includes virulence genes such as virB, virC and virG that are essential for T-DNA processing and delivery to plant cells. pSB11 has multiple cloning sites including unique restriction enzyme recognition sites for cloning expression cassettes with gene sequences of interest.
Embodiment 2
[0109] Example 2 - pAG1000
[0110] see Figure 2A , pAG1000 was formed by modifying pSB11 to accept several gene expression cassettes. First, the original expression cassette was cloned from the pNOV2819 plasmid (Syngenta Biotechnology) and cloned into pSB11 as a HindIII-KpnI fragment to form pAG1000. Phosphomannose Isomerase (PMI) Driven by C.
Embodiment 3
[0111] Example 3 - pAG1001, pAG1002 and pAG1003
[0112] pAG1000 was further modified by removing the EcoRI site (nucleotide position #7) to form pAG1001 ( Figure 2B ), and then remove the KpnI site (nucleotide position #1) to form pAG1002 ( Figure 2C ). These modifications made the EcoRI and KpnI sites available for subsequent cloning of the expression cassette with the gene of interest (GOI). see Figure 3A , the following new multiple cloning site (MCS) sequence, including PacI, XhoI, SnaBI, NcoI, KpnI, XmaI, AvrII, EcoRI sites, was synthesized as a 249bp PmeI-HindIII fragment by PCR, and was cloned into pAG1002 In the PmeI-HindIII site, thus providing the pAG1003 vector.
[0113] >MCS
[0114] GTTTAAACTGAAGGCGGGAAACGACAACCTGATCATGAGCGGAGAATTAAGGGAGTCACGTTATGACCCCCGCCGATGACGCGGGACAAGCCGTTTTACGTTTGGAACTGACAGAACCGCAACGTTGAAGGAGCCACTCAGCTTAATTAAGTCTAACTCGAGTTACTGGTACCAAATCCATGGAATCAAGIDACCATCAATCCCGGGTATCATGAAATGT7
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