Cosmid vectors: (1) Possessing the characteristics of λ phage (cos site): After being packaged, they can be efficiently transformed into E. coli. (2) Possessing the characteristics of plasmid vectors: autonomous replication, with resistance markers, and easy selection of positive clones. (3) High-capacity cloning ability: can carry up to 45kb of foreign DNA. (4) Because it does not contain the λ gene, it will not lyse. (5) Easy to clone in a targeted manner.
What is an artificial chromosome vector?
An artificially constructed vector system containing the basic functional units of natural chromosomes.
Yeast artificial chromosomes (YACs)
① There is a high proportion of chimeras, that is, a YAC clone contains two independent fragments that are not originally connected;
Some clones are unstable and may undergo deletion or rearrangement during subculture;
Difficult to distinguish from yeast chromosomes because YACs have similar structures to yeast chromosomes;
Mechanical cutting of chromosomes is likely to occur during operation;
The conversion efficiency is low.
Bacterial artificial chromosomes (BACs)
Easy to transform E. coli by electroporation (transformation efficiency is 10-100 times higher than that of yeast);
Supercoiled circular vector, easy to operate;
The genes carried by the F' plasmid itself control the replication of the plasmid;
Rearrangements rarely occur in vivo.
(3) Other types of artificial chromosomes (PAC and TAC)
- P1 Artificial Chromosome (PAC)
- Plant genetically modified artificial chromosomes
What are the commonly used gene cloning methods? What are their characteristics? What are the requirements for practical applications?
Gene cloning methods based on known sequences
Gene cloning method based on molecular marker linkage map
Gene cloning methods based on artificial mutants
Gene cloning methods based on expression differences
A gene cloning method that utilizes the interactions between biological macromolecules.
5. What is a gene library? What are the two types of gene libraries?
6. What are the two types of biological vectors used for plant genetic transformation?
(1) Viral vectors:
?Single-stranded RNA plant viruses
?Single-stranded DNA plant viruses
?Double-stranded DNA plant viruses
(2) Plasmid vector:
Ti plasmid (Agrobacterium tumefaciens)
?Ri plasmid (Agrobacterium rhizogenes)
7. Why can Agrobacterium tumefaciens induce the formation of crown galls in plants?
8. The main structural regions and functions of Ti plasmid.
(1) Toxicity zone (vir zone): activates the transfer of TT-DNA
(2) T-DNA region: When infecting plants, it is cut from the Ti plasmid and transferred to plant cells, carrying the genes involved in tumor formation.
Related genes
(3) Plasmid conjugative transfer region (Con region): contains genes related to conjugation between bacteria
(4) Plasmid replication origin region (Ori region): ensures self-replication of Ti plasmid.
(5) Ozone decomposition site
9. The structure and function of T-DNA.
Structure: (1) Generally around 23kb, containing a core region of about 8kb (2) Both the 5' and 3' ends of the T-DNA contain eukaryotic expression signals, such as TATA-box, AATAA-box, and poly(A)n (3) No introns (4) Contains tumorigenic genes (genes related to hormone synthesis) (5) Contains genes for opine synthesis and metabolism (6) Left and right border (RB and LB) sequences (importance for T-DNA transfer: the right border is more conserved)
Function: When infecting plants, it is cut from the Ti plasmid and transferred into plant cells, carrying the tumor-forming
Related genes
The main structure and function of the Vir region.
The total length is approximately 35 kb and consists of 7 complementary groups, named VirA, VirB, VirC, VirD, VirE, VirG and VirH.
VirA
Structure: Single gene, 2.8 kb, encoding a 92 kDa polypeptide, a membrane-bound receptor protein (periplasmic domain, linker domain, kinase domain, receiver domain); the conserved region at the C-terminus of the VirA protein undergoes ATP-specific spontaneous phosphorylation (phosphorylation site is at histidine residue 474, forming stable 1-phosphohistidine and 3-phosphohistidine); phosphorylated VirA protein activates VirG protein (by transferring its phosphate group to an aspartic acid residue of the VirG protein)
Function: Helps plant cells receive plant signal molecules and initiate the expression of toxic regions
VirG
Structure: 1.2kb, single copy gene, encoding DNA binding activation protein; the aspartic acid at position 52 at the N-terminus of VirG protein can be phosphorylated to form acyl phosphate (VirG protein must be in the presence of VirA protein to be able to specifically
phosphorylation)
Function: Transcription factor. It can specifically bind to the 5' non-transcribed region of VirB, C, D, E, G and H operons to activate transcription.
VirB
Structure: Encodes at least 11 open reading frames, encoding membrane transport proteins.
The N-terminus of the protein carries a signal sequence that helps transport the protein across the membrane, allowing it to cross the inner membrane independently or with the help of molecular chaperone receptors.
Function: It may change the structure of bacterial cell membrane to create a membrane-penetrating channel, allowing T-DNA to be transferred outside the bacterial cell; it may also serve as a component of the T-DNA complex.
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