Deployment of new biotechnologies in plant breeding Maria Lusser1,3, Claudia Parisi1,3, Damien Plan2 Emilio Rodríguez-Cerezo Nature biotechnology， volume 30 number 3 march 2012 原文下载： www.nature.com/nbt/journal/v30/n3/full/nbt.2142.html The seven techniques we focused on are described below. • ZFN technology. ZFNs are synthetic restriction endonucleases, custom designed to cut DNA at specific sequences. They consist of a zinc-finger domain that recognizes specific DNA sequences and a nuclease domain. Genes encoding the ZFNs are delivered to plant cells in an expression plasmid. Depending on the method, the expression plasmid may additionally contain a short template sequence or a stretch of DNA to be inserted. The ZFNs create a double-strand break (DSB) at a specific site in the DNA. The double-strand break stimulates the cell’s repair mechanism, the process of homologous recombination and the insertion of DNA. Essentially three methods are in development: ZFN-1, ZFN genes are delivered to plant cells without a repair template. The ZFN binds to a specific DNA sequence and generates a site-specific DSB. Gene repair mechanisms of the plant cell intervene to repair the break and generate site-specific mutations, which consist of changes of single or few base pairs, short deletions or insertions. ZFN-2, ZFN genes are delivered to plant cells along with a short repair template, consisting of a DNA sequence homologous to the targeted area with the exception of a point mutation. The ZFN binds to a specific DNA sequence and generates a site-specific DSB. Gene repair mechanisms of the plant cell intervene to repair the break and generate site-specific point mutations by copying the repair template. ZFN-3, ZFN genes are delivered to plant cells along with a large stretch of DNA (e.g., a gene of interest). The ZFN binds to a specific DNA sequence and generates a site-specific DSB. The ends of the DNA stretch are homologous to the sites flanking the DSB; therefore, the DNA stretch is site-specifically inserted into the plant genome. The rationale of ZFN technology is to create site-specific mutations or gene inactivation leading to the desired phenotype, like herbicide resistance. The ZFN-3 approach can be used for targeted addition of genes of interest, gene replacement and trait stacking. Specific gene targeting can prevent so-called ‘position effects’ caused by random insertion of genes in the genome. • Oligonucleotide directed mutagenesis (ODM). Also known as targeted gene repair,oligonucleotide-directed gene targeting, genoplasty and chimeraplasty. Oligonucleotides are chemically synthesized to share homology with a target sequence, with the exception of a few nucleotides. Oligonucleotides induce site-specific mutation at the target sequence. The genetic changes include the introduction of a new mutation (replacement of one or a few base pairs), the reversal of an existing mutation or the induction of short deletions. • Cisgenesis and intragenesis. Cisgenic and intragenic plants are produced by the same transformation techniques as transgenic plants, but the DNA transferred belongs to the same species of the transformed plant, or to a cross-compatible species. In cisgenesis, the DNA sequence includes the gene of interest flanked by its own promoter and terminator. In intragenesis, the gene of interest can be combined with regulatory elements from the species itself or from a cross-compatible species. Both approaches aim to confer a new property to the modified plant. By definition only cisgenics could achieve results also possible by traditional breeding methods, whereas intragenesis offers more options for modifying gene expression and trait development. Intragenesis can also include the use of silencing approaches, for example, RNA interference, by introducing inverted DNA repeats. • RNA-dependent DNA methylation (RdDM). RdDM induces transcriptional gene silencing by methylation of promoter sequences. Genes encoding RNAs homologous to promoter regions are delivered to the plant cells. These genes give rise to the formation of small double-stranded RNAs that induce methylation and silencing of the homologous sequences. RdDM allows breeders to produce plants that do not contain foreign DNA sequences and in which no changes or mutations are made in the nucleotide sequence but in which gene expression is modified epigenetically. • Grafting. A chimeric plant is produced by grafting a nongenetically modified scion on a genetically modified rootstock. Consequently, the fruits of the plant do not contain the inserted DNA sequence. The rootstock can be modified to improve its rooting capacity or resistance to soil-borne diseases, resulting in a substantial increase in the yield of harvestable components. The rootstock can also be modified for obtaining gene silencing through the technique of RNA interference. In grafted plants, the small RNAs can also move through the graft so that the silencing signal can affect gene expression in the scion. • Reverse breeding. Homozygous parental lines of a selected heterozygous plant are reproduced. The genes involved in the meiotic recombination process are silenced through transgenesis. Consequently, nonrecombined haploid lines are obtained from the heterozygous plant and their chromosomes are doubled through the double-haploid technique. The doubled haploids obtained are screened to find a pair that, would reconstitute the original heterozygous plants. Only nontransgenic plants are selected, thus the offspring of the selected parental lines would not carry any additional genomic change. • Agro-infiltration. Three types of agro-infiltration can be distinguished: ‘Sensu stricto,’ nongermline tissues, mostly leaves, are infiltrated with a liquid suspension of Agrobacterium carrying a gene of interest. The gene is locally expressed at a high level, without being integrated into the plant genome; Agro-infection, nongermline tissues, typically leaves, are infiltrated with a full-length virus vector containing a gene of interest. Through the virus vector, the expression of the gene of interest is spread in the entire plant; The floral dip technique involves immersion of germline tissues, typically flowers, into a suspension of Agrobacterium carrying a gene of interest so as to obtain stable transformation. Transformed embryos are then selected at the germination state. Agro-infiltration can be used to screen for plants with valuable phenotypes that can then be used in breeding programs, for instance, with specific genes from pathogens to evaluate plant resistance. The technique has also been developed as a production platform for high-value recombinant proteins. However, the technique is mostly used in a research context, for example, to study plant-pathogen interaction in living tissues (leaves) or to test the functionality of regulatory elements in gene constructs.
标签: 基因工程
