PCR amplification cloning method is a method to clone gene sequences according to known plant gene sequences. Firstly, the related gene sequences were found from the gene bank, and then a pair of oligonucleotide primers were synthesized. Chromosome DNA or RNA was extracted from plants and amplified by PCR. The amplified fragment was purified and inserted into a suitable plasmid vector. Restriction restriction enzyme analysis and sequence analysis were carried out on the recombinant, and the target gene was obtained by comparing with the known gene sequence.
2. Functional cloning method
Functional cloning is carried out according to the basic biochemical characteristics of traits after determining the functions of known genes. This method constructs a cDNA library or a genome library after purifying the corresponding encoded protein. Then, two methods were used to screen genes from the library. One is to sequence the amino acids of the purified protein, and then synthesize oligonucleotide probes to screen the coding genes from cDNA library or genome library. Secondly, protein was made into corresponding antibody probes, and the corresponding clones were screened from the cDNA vector expression library. The key step of functional cloning is to isolate high-purity protein. This method can't clone genes whose products are unknown.
3. Location (map location) cloning method
Localization cloning is based on the position of the target gene on the chromosome, and then cloning by chromosome walking. It is necessary to establish a suitable genetic segregation population (near isogenic line or segregation population) with the target gene in advance, accurately locate the target gene in a specific position on the chromosome, screen the genome library with molecular markers (RFLP markers) closely linked on both sides of the target gene as probes, then use the ends of positive clones as probes to obtain large-segment clones containing the target gene, and then use these new DNA as probes to obtain DNA fragments closer to the target gene, thus narrowing the screening scope continuously.
4. Transposon labeling method
Transposon is a DNA fragment that can be transferred from one gene position to another, but the original DNA fragment (transposon) has not disappeared, and only a copy of transposon is transferred. Gene transposition can cause insertion mutation, inactivate the gene at the insertion position and induce mutation. The location of the mutant gene can be detected by marking the gene, and then the mutant gene can be cloned. In this way, the transposon tag is called transposon tag, and the gene is cloned by transposon insertion and mosaic on chromosome. Para & gt5. Subtractive hybridization
This method clones plant genes according to the difference of plant phenotype or gene expression in different tissues or organs. Specifically expressed genes have different mRNA expressions. MRNA was extracted from plant tissues expressing specific genes and tissues not expressing specific genes, and then reverse transcribed into cDNA, and the two were hybridized. Gene products expressed in two tissues form hybrid molecules, while cDNA transcribed by specific mRNA remains single-stranded. Isolation of this single-stranded cDNA is a differentially expressed gene. This strategy is called subtractive hybridization.
6.mRNA differential display method
This method can effectively identify and clone differentially expressed genes. After different mRNA is extracted, it is reverse transcribed into cDNA with the same primer, and differentially expressed cDNA sequences are obtained by PCR amplification, which can be used as probes to screen genes in cDNA library or genome library and perform functional analysis. This method can directly screen differentially expressed genes, which is more convenient and faster than subtractive hybridization, requires less total RNA and has high efficiency, and can complete cDNA amplification, identification and cloning in a short time.