Gene bank refers to the collection of all genes of a certain biological type. This collection is in the form of reorganization. The DNA fragment population of the organism is recombined with the carrier molecule, and then the host cell is transformed. A single colony (or plaque) (or living cell) in which transformed cells grow on selective medium is a clone of DNA fragment. The aggregate of all DNA fragment clones is the gene library of the organism. The significance of constructing gene library is not only to store biological genetic information in a stable recombinant form, but more importantly, it is the main way to isolate and clone the target gene. For complex chromosomal DNA molecules, the proportion of a single gene is very small. In order to isolate from a huge genome, it is generally necessary to amplify it first, so it is necessary to construct a gene library. In many cases, the separation of the target gene can not be separated from the gene library. In addition, gene library is also an important basis for complex genome mapping. The construction of gene library includes the following basic procedures: ① DNA extraction and fragmentation, or cDNA synthesis. ② Selection and preparation of carrier. ③ DNA fragment or cDNA is connected with the vector. ④ The recombinant transformed the host cell. ⑤ Screening of transformed cells. When the host cell containing the recombinant was obtained, the gene cloning was completed. Gene cloning is only the basis of gene separation. After gene cloning, the cloned gene should be separated, that is, the target gene should be separated from the library by various means. In order to isolate the target gene, it is necessary to detect and analyze it, such as sequencing, transcription and translation in vitro, functional complementarity experiment and so on. Through these experiments, the structure and function of the gene were determined. Only in this way can the target gene be isolated. Therefore, gene cloning, isolation of cloned genes and identification of isolated genes are the main contents of gene library technology to isolate target genes. I. Types of gene banks 1. Genome library and cDNA library According to gene types, gene libraries can be divided into genome library and cDNA library. Genome library refers to the collection formed by cutting all the genome DNA of an organism into DNA fragments with a certain length and cloning them into a certain carrier. CDNA library refers to the collection of clones formed by connecting cDNA fragments of mRNA transcribed in a certain development period with vectors. Genome library is divided into nuclear genome library, chloroplast genome library and mitochondrial genome library according to the source of DNA. The difference between genome library and cDNA library is that cDNA library is time-sensitive. The information donor in library construction is the total mRNA of cells in a certain time and space, which reflects the gene expression of an organism in a certain development period and certain environmental conditions at the transcription level, and cannot contain all the genes of the organism. In a sense, it can express the functional information of genome. In addition, the cDNA library only reflects the molecular structure of mRNA. CDNA does not contain spacer sequences and regulatory regions of eukaryotic genes, so it is not a real gene. When the genome library is constructed, the donor of genetic information is genomic DNA, so there is no specificity of development period and tissues and organs. A complete genome library contains all clones of coding region and non-coding region of genomic DNA. Every gene in the organism has its clone in the library, and the cloned gene fragments contain spacer sequences, so the genome library can truly display all the structural information of the genome. At present, these two kinds of gene libraries have been effectively applied in genetic engineering. Which one to choose depends mainly on the purpose of the experiment. When isolating RNA virus genes, studying functional protein sequences, and isolating genes specifically expressed in specific development stages or specific tissues, it is necessary to construct CDNA libraries. It is necessary to construct a nuclear genome library when studying the sequences that do not exist in mRNA molecules and mapping the genome. 2. Cloning library and expression library can be divided into cloning library and expression library according to the function of gene library. Cloning library is constructed by cloning vector. There are replicons, multiple cloning sites and selection markers in the vector, and the cloned fragments can be propagated by bacterial culture. Construction of expression library with expression vector. In addition to the above elements, the vector also contains sequences that control gene expression (such as promoter, SD sequence, ATG, terminator, etc.). ), which can express the coding products of cloned fragments in host cells. Expression vectors can be divided into fusion protein expression vectors and natural protein expression vectors. Nucleic acid probes are mainly used to separate target clones from the clone bank, which can be oligonucleotide probes synthesized according to protein sequence, or probes with the same or the same homologous sequence. When the target clone is isolated from the expression library, because the expression product protein of the clone fragment has antigenicity and biological activity, besides nucleic acid probes, immunological probes and biological functions can also be used for screening. The expression library is suitable for isolating the target gene which is unknown to the amino acid sequence of protein and cannot be screened by nucleic acid probe. 3. Gene libraries with different vectors At present, the vectors used to construct gene libraries mainly include plasmids, phages, cosmids and artificial chromosomes. There are many different operators in each category. Different vectors are suitable for constructing different gene libraries. (1). Plasmid library Plasmids are the earliest vectors used for gene cloning. There are various commercial plasmids such as cloning, expression and sequencing, which are suitable for different tasks. However, in the construction of gene library, because the plasmid is relatively small, it can only accommodate smaller fragments than itself, so it can not be used to construct nuclear genome library, and usually it is only used to construct short sequence clone library. For example, chloroplast DNA molecules are very small, and plasmids can be used to construct chloroplast DNA libraries. Plasmid vector can be used to construct biological cDNA library. But it is only suitable for high abundance mRNA. (2) Phage Library At present, there are many phage vectors and their derivative vectors used for gene cloning, such as single-stranded M 13 phage vector, λ phage vector, P 1 phage vector, phage particles (phage particles or phasmid) and so on. Among them, phage is the most commonly used. λ-DNA is a double-stranded structure with a length of 49kb. There is a sticky end of 12 nucleotides at both ends of a linear molecule, which is called cos site. There is an unnecessary gene region of about 1.5 kb in the molecule, which is also called "filling region". The sequences on both sides of the "filling region" contain all the genes necessary for its proliferation, which are called left arm and right arm. The "filling region" can be replaced by foreign DNA to form a recombinant, which is the structural basis for it to become a cloning vector. Due to the limitation of the packaging capacity of phage head, the molecular size of recombinant λ-DNA can only be between 39 and 52 KB. (3) The cosmid, also known as cosmid plasmid, is a special kind of plasmid vector consisting of the COS sequence of λ phage, the replicon sequence of plasmid and the antibiotic resistance gene sequence. COS sequence is necessary for DNA packaging into phage particles. Replicators usually use ColEl or pMBl as replication initiation sites. This cosmid has some characteristics of λ phage. After cloning foreign DNA fragments with appropriate size in vitro and packaging them into phage particles, Escherichia coli host cells sensitive to phage entry can be transduced efficiently. It is cyclized by λ phage in the host cell, but it can't pass the bacteriolysis cycle and can't form offspring phage particles (because the molecule doesn't enter all the necessary genes of phage). It also has the main properties of plasmid vector, and can replicate in host cells like other plasmids, just like relaxed plasmids. Appropriate amount of chloramphenicol can promote amplification. Due to the antibiotic gene, the recombinant can be screened by antibiotic resistance. When constructing cosmid vector, multiple cloning sites were added to insert inactivated genes. The molecule of cosmid carrier is relatively small (2.8-24 KB), but its cloning ability is very high. The length of exogenous DNA is required to be 30-45 KB, and the upper limit is almost twice that of phage vector (23 kb). Therefore, cosmid vector has considerable advantages in constructing nuclear genome library, and can clone complete plant genes including 3,5' regulatory regions. (4) Artificial chromosome library An artificial chromosome vector is an artificial vector that uses functional elements of eukaryotic chromosomes or prokaryotic genomes to clone DNA fragments larger than 50 KB. Some of these vectors can be used for cloning and direct transformation, and are good vectors for gene function research. The artificial chromosome libraries developed in recent years include YAC library, BAC library, BIBAC library, PAC library and TAC library. Second, the construction of nuclear genome library The construction of nuclear genome library mainly uses λ phage replacement vector or cosmid vector. 1. The number of clones in a random library means that the number of moles of DNA representing all parts of the genome is equal. For random library: n = ln (1-p); Ln( 1-x/y) N: the number of clones p: the set probability value (for example, 0.99, indicating that the probability of finding any sequence from the library is not less than 0.99 when the fragments are randomly distributed) x: the average size of inserted fragments (15 ~ 20kb) y: including 51x. When the fragments are randomly distributed, the probability of finding any sequence from the library is not less than 0.99. Random fragments can be produced by mechanical cutting or restriction enzyme digestion. A uniform random fragment can be obtained by mechanical cutting, but the fragment can not be directly used for cloning, and it needs terminal modification, methylation, ligation and restriction enzyme digestion to produce sticky ends. Although restriction enzyme digestion can directly produce sticky ends, the randomness of fragments is poor, so when using the latter method, the number of clones in the library should be greater than the calculated value. 3. Construction of c DNA Library by PCR The initial information material of cDNA library construction is mRNA. Therefore, the first problem to be considered in constructing cDNA library is the content and quality of mRNA. The mRNA content in biological cells is very low. Usually, the construction of cDNA library needs ug-level mRNA. For low abundance mRNA (