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1、Chapter 15 Genetically Modified BreedingSection 1 Development and Application of Transgenic Crop BreedingSection 2 Integration Mechanism and Genetic Characteristics of ExogenousSection 3 Cultivation of Genetically Modified CropsSection 4 Biosafety of Genetically Modified CropsSection 3 Basic procedu
2、res of crop transgenic breedingCropsmain breeding targetThe acquisition of the target geneThe breeding using of transgenic material Obtaining and identification of transgenic plantsGenetic transformation of target geneChoice of receptor materialConstruction of plant expression vectorsBasic procedure
3、s of crop transgenic breeding1.Targeting of breeding of genetically modified crops2.The acquisition of the target gene3.The construction of plant gene expression carrier4.The choice of receptor material5.Methods of genetic transformation6.Acquisition and method of genetically modified plants7.Breedi
4、ng and utilization of genetically modified materialsObtaining the gene of the destinationCarrier building遗传转化遗传转化RegenerationIdentificationUsing1.The formulation of the breeding target sedatiby targets for genetically modified cropsa.Develop breeding plans for major problems in crop productionb.Dete
5、rmine specific traits based on the factors that affect breeding objectivesc.Determining forward-looking breeding targets based on social development needsd.Taking full account of the biosecurity of genetically modified cropse.Consider the reasonable matching of varieties when setting breeding target
6、sTarget genesGenes used for gene recombination,altering receptor crop traits,and obtaining expression products.Target genes can be divided into functional genes(encoding specific functional proteins)and regulatory genes(encoding transcription factors and small RNAs,etc.).I.The acquisition of the tar
7、get geneHomology based candidate gene method is a rapid cloning method of unknown members of gene families based on the characteristics of conserved amino acid sequences in the protein structure encoded by gene family members.1.Homology sequence methodExpressed sequence tag(EST)refers to a partial s
8、equence that can specifically label a gene,and usually contains enough structural information area of the gene so that it can be distinguished from other genes.Screening of EST database,acquisition and integration of EST contigs,sequence analysis,acquisition of target genes,and sequencing identifica
9、tion of cloned genes.2.Expressed sequence tagging methodThe full-length target gene can also be obtained by nested PCR,5or 3RACE,or cDNA library screening.Steps：The way in which different genes occur in different stages of an organisms individual development or in different tissues and cells are seq
10、uentially expressed in time and space is called gene differential expression.3.Gene cloning based on differential expressionDifferential expression(1)Analysis and cloning of the target gene by gene chip hybridizationUsing in-situ synthesis or microscopic printing,tens of thousands of nucleic acid pr
11、obes are cured on a special glass or silicon chip to produce a DNA probe array.Gene chip is also called DNA microarray Using DNA chip technology,high-throughput analysis of gene expression profiles of plants at specific developmental stages,specific tissues,and different environmental conditions can
12、 be performed.Gene chip cloning steps1）Ordering or making gene chips;2）Extraction and labeling of mRNA for specific samples(treatment and control);3）Chip hybridization and data analysis;4）Obtaining differentially expressed gene information；5）Cloning and identification of target genes.(2)mRNA differe
13、ntial display 1）Isolate the total mRNA from a pair of cell populations at different developmental stages(or different genotypes)and reverse transcribe the first with the selected 3-end anchor primers(12 kinds of 5-TTTTTTTTTTTTTMN3)Strand cDNA2）Using random primers and anchored primer pairs,adding ra
14、dioisotope labeled dNTP,using the first step reverse transcript as a template for PCR amplification.3）Amplified sample DNA sequencing gel electrophoresis.(3)Suppression subtractive hybridizationprincipleHigh-abundance single-stranded cDNA produces homologous hybridization faster than low-abundance s
15、ingle-stranded cDNA during annealing,and utilizes the advantage of intra-strand annealing over inter-strand annealing to selectively inhibit the amplification of non-target fragments.The samples are divided into two groups,with different adaptors connected to the 5end.Sample and control double-stran
16、ded cDNA were synthesized and digested to blunt ends.1）Prepare2）Adaptor Connection4）Second crossSTEPS:The sample was added with excess control,then denatured and annealed.The two groups of samples were directly mixed without denaturation.3）First cross(subtractive cross)First PCRSecond PCR6）Two PCR r
17、eactionsThe 5 end of the adapter is a primer.The 3 end of the adapter is a primer.5）Fill ends4.Cloning the target gene by screening genomic library or cDNA libraryA probe is synthesized based on a DNA sequence homologous to the target gene,and then the probe is hybridized with the library to screen
18、clones with the target gene.Nucleic acid hybridization screening5.Map cloning（1）Identify DNA markers linked to target genes(preliminary mapping)Frary et al.(2000),Science 289:85-88（2）Construction of high-density genetic maps(fine mapping)The separated population should be large enough(more than 1,00
19、0 plants).Identify recombinant individuals close to the target gene,CornScreen all DNA fragments of the genomic library with a specific marker as a probe.Chromosome walking1）Unsequenced crops30%2）Sequenced crops6.Insertion inactivation technique(1)T-DNA labeling methodT-DNA tagging is a method of ta
20、rget gene isolation based on transgenic technology.Generally,an exogenous reporter gene is inserted into T-DNA,and plant cells are genetically transformed,and then a target trait mutant caused by T-DNA insertion is selected from the transgenic population.The gene of interest was cloned from the geno
21、me of the mutant based on the inserted sequence.(2)Transposon labeling methodThe transposon tagging method uses the movement of transposons to create mutants,and then screens the mutant genome library with specific probes to isolate and clone the gene of interest.7.Comparison of cloned target genes
22、through proteome differencesProteomicsRefers to the collection of all proteins expressed under specific space-time conditions in an organism.Find the differentially expressed target protein by two-dimensional electrophoresis,and then design PCR primers or probes according to the amino acid sequence
23、of the protein to clone the target gene.I.The acquisition of the target gene1.Homology sequence method7.Differential expressed Protein method6.Insertion inactivation technique5.Map-based cloning4.Cloning the target gene by screening genomic library or cDNA library3.Differential expression2.Expressed
24、 sequence tagging.Construction of plant expression vectors1.Selection and optimization of gene expression regulatory elements(1)Main elements affecting the transcription of the target gene5promoter,3termination sequenceConstitutive promoterA strong promoter that is not restricted by spatiotemporal c
25、haracteristics.35S，Ubiquitin IInducible promoterA promoter that drives the expression of a gene of interest induced by signals inside or outside the plant or by chemical substances.Plant hormone-induced promoter,stress-induced factor-induced promoter,chemical substance-induced promoter.Tissue-specif
26、ic promoterA promoter that specifically initiates expression in a specific tissue or organ.Anther-specific type,TA29Leaf-specific type,corn PEPCPhloem-specific type，Rice sucrose synthase gene RSs1Fiber-specific type，cotton E6(2)Main elements affecting the translation of the target geneThe target gen
27、e must include the start codon,stop codon and reading frame；In order to improve the translation efficiency of the target gene,the surrounding sequence of the start codon of the target gene also needs to be considered.For the target gene from the source prokaryote,codon optimization is required,prefe
28、rably including the 5and 3untranslated regions of the eukaryote gene.5UTRStart codonSTOP codonCoding sequence(reading frame)3UTR2.Construction of the target gene expression cassetteGene expression cassetteA DNA vector framework containing expression regulatory elements such as the gene of interest,p
29、romoter and termination.The gene expression cassette of interest is stored in an intermediate cloning vector(such as pUC).1)Overexpression vectorGhADFThe target gene is positively connected downstream of the 35S promoter.2)Antisense suppression vectorThe target gene is reversely linked downstream of
30、 the 35S promoter。3）RNAi vectorThe forward and reverse fragments of the target gene are connected in series downstream of the 35S promoter,and an intron is inserted between the forward and reverse fragments.4）CRISPR-Cas9 gene editing vector（Clustered regularly interspaced short palindromic repeats，C
31、RISPR)(CRISPR-associated,Cas)Techniques for specific DNA modification of targeted genes.Cas9 first combines with crRNA and tracrRNA,and then combines and invades DNA through the PAM sequence to form an RNA-DNA composite structure,and then cleaves the target DNA double strand to break the DNA double
32、strand.Gene editing crop quantitative trait loci,Create a lot of variation,Improve and increase crop yieldGene editing and haploid breedingZmPLA1Gene editing and apomixis3.Recombinant vector(plant expression vector)systemThe target gene expression cassette is cut out from the cloning vector and inse
33、rted into an appropriate position of the plant expression vector.Unary carrier systemThe intermediate vector containing the target gene expression cassette and the modified Ti plasmid are a composite vector produced by homologous recombination,that is,a co-integration vector.Because the T-DNA and Ti
34、 plasmids are connected to the Vir region,they are also called cis vectors.Binary carrier systemOne Ti plasmid carries the Vir gene,and the other Ti plasmid carries two border repeat sequences of T-NDA.Recipient material should have the following conditions:1）efficient and stable regeneration abilit
35、y2)high genetic stability3）stable source of explants4）sensitive to screening agents.Receptor materialReceptor material refers to cells,tissues or organs used in genetic transformation of plants and receiving exogenous DNA.Choice of receptor material(1)Callus regeneration system(2)Direct differentiat
36、ion regeneration system(3)Protoplast regeneration system(4)Embryoid body regeneration system(5)Germ cell regeneration systemtypes of commonly used receptor materials.Genetic transformation of target geneGentic transformationGenetic transformation is the process of introducing a exogenous gene into a
37、 recipient cell and integrating it into the nuclear or plastid genome.(1)Agrobacterium-mediated methodAgrobacterium Ti plasmidAgrobacterium rhizogenes Ri PlasmidThe target gene is inserted into T-DNA,the target gene is introduced into the genome of the recipient plant cell by means of Agrobacterium,
38、and a transgenic plant is obtained.The gene gun method is a method of injecting metal particles that have absorbed exogenous DNA into a recipient cell or tissue by means of pressure generated by gunpowder,high-pressure discharge,or high-pressure gas,and integrating exogenous genes into the recipient
39、 cell genome.(2)Gene gun methodApply the exogenous DNA to the stigma of pollination,use the pollen tube formed by pollen germination after the plant is pollinated,or inject the exogenous DNA into the ovary with a micro syringe to allow the exogenous DNA to enter the embryo sac and transform the zygo
40、tes that do not have normal cell walls.（3）Pollen tube channel(ovary injection)method（3）Nano Magnetic Bead ConversionPlasmid carrier DNA adsorbed on nano magnetic beadsNano magnetic beads into pollenPollen pollinationSeed collectionScreening and identification.Obtaining and identification of transgen
41、ic plantsNPT-/Kan resistance1.Screening of plant transformantsEPSPS/Glyphosate resistanceThe selectable marker gene and the target gene are introduced into the recipient cell at the same time,and the expression of the marker gene makes the transformed cell have specific resistance.plant transformant
42、A plant transformant is a plant cell or plant that directs the introduction of a exogenous gene,that is,a transgenic cell or plant.GUS(-gluconase)geneGUS gene product can decompose 4-methylumbelliferone-glucuronide and produce fluorescent substance 4-methylumbelliferone.LUC(luciferase)geneluciferase
43、+ATPAdenylate fluorescein acylation complexLUCOxidative decarboxylationOxyfluorescein+AMP+lightGFP(Green fluorescent protein)geneGreen fluorescent protein will emit green fluorescence when excited by light in the blue wavelength range.Based on the sequences at both ends of the target gene,a pair of
44、specific primers for the target gene are designed.1)PCR analysis2.Identification of transformants(1)Identification in DNA level2)Dot hybridization and southern blottingWhen the nucleotides of two single-stranded DNA molecules from different sources are complementary,double-stranded DNA can be formed
45、 under renaturation conditions.The total DNA of the transformed plants was extracted,cut with restriction enzymes,and then gel electrophoresed,transferred to the hybridization membrane,and hybridized with probes.Southern blotting DNA marked with32P,biotin,digoxigeninprobe(2)Identification of transcr
46、iption levelRNA can form a double strand with the template DNA strand that transcribes the RNA.Northernblotting The DNA probe of the target gene is denatured and hybridized with the total RNA of the transformed plant.mRNA reverse transcription PCR RT-PCRqRT-PCRreal time quantitative PCR1)Gel electro
47、phoresis identification(3)Identification of translation level2)Western blottingThe total protein of the transformants is extracted,subjected to SDS-polyacrylamide gel electrophoresis,blotted and transferred to the membrane,and then reacted and developed with an antibody of the target protein(primary antibody)and a secondary antibody with specific label that can bind to the primary antibody.(4)Phenotypic identification