中科院植物基因工程课件转化系统1 pCAMBIA载体

As intellectual property restrictions become more onerous, it is clear the ability to remove segments of DNA which are not readily licensed, or for which freedom-tooperate cannot be obtained, is of great importance for commercial applications. Therefore, developing a ‘modular’ structure that encompasses an upgrade path is of the highest priority. Thus, there is still a pressing need to have a single set of vectors which can be used for routine and efficient transformation of most crops, both dicot and monocot, with numerous genetic selections, including routes to simple novel selection upgrades. These vectors should also facilitate advanced construction, modification and analysis of transgenes and variants.
CAMBIA has constructed a new set of vectors to meet these requirements for its own in-house use, and has now made these available to the research community. The starting material for all these vectors was the excellent backbone developed by Peter Hajdukiewicz, Zora Svab and Pal Maliga, and generously provided to CAMBIA by Pal Maliga (Hajdukiewicz et al., 1994). These vectors, the pPZP series, have a wide-host-range origin of replication from the Pseudomonas plasmid pVS1, which is extremely stable in the absence of selection; the pBR322 ORIGIN (pMB9-type) to allow high-yielding DNA preparations in E. coli; the T-DNA borders, including overdrive; and a CaMV35S cassette. While pPZPs served as the backbones for the pCAMBIA series, they have been very extensively modified for particular uses.
2.1General Structure of pCAMBIA vectors
2.2 Design Features of the pCAMBIA Modular format:
1. Consensus sequences for both prokaryotic (Shine/Dalgarno) and eukaryotic (Kozak) translational initiation, to allow strong ribosome binding and efficient translation of reporter or other genes incorporated within the modular structure. A stop codon upstream from the S/D and initiator ensures no read-through translational fusion. Choice of 5’-cloning sites, Nco I provides the start codon and second base of Kozak consensus. 5’-SpeI and 3’-Nhe I have compatible sticky overhangs, allowing easy gene rearrangement or reorientation. These sites have been engineered to provide the same reading frame relative to their sticky ends.
2. Outline and Nomenclature of pCAMBIA Vector Structure
A few points about our strategy:
The pUC18 polylinker was used in some vectors, but pUC8 and pUC9 poБайду номын сангаасylinkers were also used to: simplify the choice of cloning enzyme – in the age of PCR, it is no longer necessary to have a large number of cloning sites; and
These substandard features of most vectors include: • low-copy origin of replication resulting in low yield DNA preps; • unstable replicons, causing variable loss of the plasmid during propagation; • large size; • lack of convenient restriction sites for manipulation; • limited choice of selectable markers for both bacteria and plants; • lack of simple ways to construct reporter gene fusions; • incompletely sequenced regions; • no path for ‘upgrades’.
T0 rice plants by southern hybridization confirmed integration of the
selectable marker gene - hygromycin with copy numbers ranging from 1-3; more than 50% of the transgenic rice lines produced with Agrobacterium
a.
b.
eliminate potential conflicts with sites such as Sph I (which has an ATG) or Xba I (which has a TAG). This makes other sites in the vector more useful (such as the Sph I site outside of the Right TDNA Border, or the Sac II site outside of the Left T-DNA Border).
pCAMBIA vectors. Transient and stable expression of the GUS reporter
gene indicating successful transformation was observed by X-GlcA staining in transformed rice calli and tobacco shoots. Molecular analysis of
pCAMBIA vectors
•国际农业分子生物学应用中心 •- CAMBIA是一个独立的、非营利的国际研 究组织，注册在澳洲的首都。主要是对农业 分子生物学的研究。
1.Introduction
1.1Why a new set of vectors?
Plant transformation is now routine in hundreds of laboratories worldwide, using both Agrobacterium or direct methods, notably particle bombardment. Recently it has become clear that even the majority of monocots are susceptible to efficient Agrobacterium-mediated transformation (for rice, see Hiei et al. 1997). However, the vectors used vary widely between laboratories making comparisons of results between groups difficult, and most are ‘historical’ relics with many substandard features that make advanced DNA constructions awkward or cumbersome.