CN108531506A - It is a kind of regulation and control crop plant type structure method and obtain the compact crop of plant type method - Google Patents

It is a kind of regulation and control crop plant type structure method and obtain the compact crop of plant type method Download PDF

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CN108531506A
CN108531506A CN201810365360.XA CN201810365360A CN108531506A CN 108531506 A CN108531506 A CN 108531506A CN 201810365360 A CN201810365360 A CN 201810365360A CN 108531506 A CN108531506 A CN 108531506A
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plant type
crop
crops
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control
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CN108531506B (en
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张晓辉
李锡香
宋江萍
邱杨
王海平
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Abstract

The present invention provides a kind of methods of regulation and control crop plant type structure, belong to plant genetic engineering field.The present invention realizes the regulation and control to crop plant type by a kind of method of the regulation and control a variety of economical characters of crop, this method by the expression of regulation and control SBP Box genes;And then obtain plant type it is compact, suitable for dense planting, facilitate harvesting, crop resistant to lodging;By the way that the plant type structure of crops in genetic transformation to crops, will be optimized, the lodging resistance of crops is improved, the planting density of crops, convenient artificial and mechanical harvesting are improved;With preferable actual application value.

Description

It is a kind of regulation and control crop plant type structure method and obtain the compact crop of plant type method
Technical field
The present invention relates to plant genetic engineering field, a kind of method in particular to regulation and control crop plant type structure and The method for obtaining the compact crop of plant type.
Background technology
In agricultural production, the economical character of crops has larger impact to the yield of crop, in agricultural research, reason Think that the research of plant type constantly carries out, researcher is by constantly adjusting the plant type of crop plant, to improve the production of crop Amount, cultivation, management, harvesting performance.
Traditional genetic breeding is the path of a comparative maturity, but the time of genetic breeding is longer, time and effort consuming, also Differ and surely obtains ideal plant type;It, can be greatly by the means of molecular biology but with the development of molecular biology Shorten breeding time, and the probability for obtaining target plant can also be improved, is a preferable means.
Plant morphological structure is a major traits for crops, at present in the rose family and Solanaceae, about grinding for plant morphological structure Study carefully not enough;Research especially in apple and tomato need further to reinforce.
Invention content
The first object of the present invention is to provide a kind of method of regulation and control crop plant type structure, by the strain for regulating and controlling crops Type character, correlation (derivative) economical character and improvement planting type of further regulation and control crops;These related (derivative) agricultures Skill character includes:Lodging resistance;Amendatory planting type includes:Suitable for dense planting, convenient artificial and mechanical harvesting.
The second object of the present invention is to provide a kind of method obtaining the compact crop of plant type, by optimizing plant type structure, Can improve crop it is resistant to lodging, suitable for dense planting, facilitate the ability of harvesting;Plant type compact genetically modified plants are obtained, are had anti-fall Volt, suitable dense planting, easy recovery characteristic provide to increase the yield of crops, improving plant exterior quality and reducing labour's expenditure Important guarantee.
In order to realize the above-mentioned purpose of the present invention, using following technical scheme:
A method of regulation and control crop plant type structure, by regulating and controlling crop SBP-Box gene expressions, to regulate and control the agriculture of crop Skill character.
A method of the compact crop of plant type is obtained, is included the following steps:
SBP-Box genes are connected to expression vector, obtain conversion carrier;
Conversion carrier is transformed into crops, plant type compact, resistant to lodging, suitable dense planting, the transgenosis easily harvested are obtained Crop.
Compared with prior art, beneficial effects of the present invention are:The present invention passes through a kind of side of regulation and control crop plant type structure Method, this method realize the regulation and control to crop economical character by the expression of regulation and control SBP-Box genes;And then to the outer of crops It sees quality and planting type has an impact;Obtain plant type compact, resistant to lodging, suitable dense planting, easy harvesting crop method, pass through by In genetic transformation to crops, plant type structure, lodging resistance, space availability ratio, the artificial and mechanical harvesting of crops are improved Energy;With preferable actual application value.
Description of the drawings
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the transgenic line PCR testing result figures that the embodiment of the present invention 1 provides;
Fig. 2 is that the transgenic line plant type that the embodiment of the present invention 1 provides compares figure;
Fig. 3 is that the transgenic line plant height that the embodiment of the present invention 1 provides compares figure;
Fig. 4 is that the transgenic line set section length that the embodiment of the present invention 1 provides compares figure;
Fig. 5 is that the transgenic line joint number that experimental example 1 of the present invention provides compares figure;
Fig. 6 is that the transgenic line blade length that experimental example 1 of the present invention provides compares figure;
Fig. 7 is that the transgenic line that provides of experimental example 1 of the present invention section of bearing fruit compares figure;
Fig. 8 is that the transgenic line L22 plant heights that experimental example 1 of the present invention provides compare figure;
Fig. 9, which is the CK1 and CK2 that the embodiment of the present invention 2 provides, has expression correlation fitted figure;
Figure 10, which is the CK1 and CK3 that the embodiment of the present invention 2 provides, has expression correlation fitted figure;
Figure 11, which is the CK2 and CK3 that the embodiment of the present invention 2 provides, has expression correlation fitted figure;
Figure 12, which is the L16 and L22 that the embodiment of the present invention 2 provides, has expression correlation fitted figure;
Figure 13, which is the L4 and L16 that the embodiment of the present invention 2 provides, has expression correlation fitted figure;
Figure 14, which is the L4 and L22 that the embodiment of the present invention 2 provides, has expression correlation fitted figure.
Specific implementation mode
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is The conventional products that can be obtained by commercially available purchase.
Method to a kind of regulation and control crop plant type structure of the embodiment of the present invention and the method for obtaining genetically modified crops below It is specifically described.
A method of regulation and control crop plant type structure, by regulating and controlling crop SBP-Box gene expressions, to regulate and control the agriculture of crop Skill character.
Further, in preferred embodiments of the present invention, the base sequence such as SEQ ID NO.1 institutes of SBP-Box genes Show.
Further, in preferred embodiments of the present invention, economical character includes plant type.
A method of genetically modified crops are obtained, are included the following steps:
SBP-Box genes are connected to expression vector, obtain conversion carrier;
Conversion carrier is transformed into crops, plant type compact, resistant to lodging, suitable dense planting, the transgenosis easily harvested are obtained Crop.
Further, in preferred embodiments of the present invention, further include that conversion carrier is transformed into host strain, converted Host strain obtains genetically modified crops with conversion host strain conversion crop.
Further, in preferred embodiments of the present invention, host strain is Agrobacterium.
Further, in preferred embodiments of the present invention, crops are rose family crop or solanaceous crops.
Further, in preferred embodiments of the present invention, rose family crop is apple, and solanaceous crops are tomato.
The feature and performance of the present invention are described in further detail with reference to embodiments.
Embodiment 1
The present embodiment provides a kind of methods of regulation and control crop plant type structure, the i.e. table by regulating and controlling crop SBP-Box genes It reaches, to achieve the purpose that regulate and control crop plant type structure;The base sequence of SBP-Box genes is as shown in SEQ ID NO.1.
The present embodiment provides a kind of methods of regulation and control crop plant type structure, include the following steps:
Restriction enzyme site is designed on primer by 1.1 two suitable restriction enzyme sites of selection by design of primers, passes through PCR Restriction enzyme site is introduced into the both ends of SBP-Box genes by amplification or artificial synthesized method;
1.2 double digestions carry restriction enzyme site SBP-Box genetic fragments and pBI121 vector plasmids, and gel electrophoresis is recycled;
1.3 electrophoresis recycle, and obtain SBP-Box segments and pBI121 carrier segments;
1.4 are connected SBP-Box segments with pBI121 carrier segments with ligase, obtain pBI121-SBP-Box recombination matter Grain.
1.5, by pBI121-SBP-Box recombinant plasmid transformeds to Escherichia coli, identify positive colony, select positive colony survey Whether sequence, verification SBP-Box sequences are correct.
Further include the conversion of Agrobacterium and the identification of positive colony, includes the following steps:
2.1 take 100 μ L Agrobacterium competence, and 1-2 μ L Plasmid DNA is added, and 1800V electricity turns, and 400 μ L YEP cultures are added Base;
2.2 at 28 DEG C, under conditions of 100rpm, renewal cultivation 1h;It is coated on the LB tablets containing corresponding antibiotic, 28 DEG C, cultivate 36-48h;
2.3 picking single bacteriums are fallen in the LB culture mediums (containing corresponding antibiotic) of 1mL, 28 DEG C, after 200rpm cultivates 48h, into Row PCR verifications;It verifies correct monoclonal and carries out guarantor bacterium, be used for subsequent experiment.
The present embodiment also provides the positive Agrobacterium-mediated Transformation tomato containing pBI121-SBP-Box recombinant plasmids obtained, obtains Obtain the experiment of transgene tomato.The step of experimentation, is as follows:
3.1 take tomato seeds, and with distilled water flushing one time, 70% ethyl alcohol is washed 45 seconds, 1% hypochlorite disinfectant 5-8 minutes, Aseptic water washing 5 times, is seeded in 1/2MS culture mediums and sprouts;
3.2 cut the aseptic seedling cotyledon sprouted, and blade is paraxial downwards, is seeded on precultivation medium, pre- to train It supports 1-3 days;
The 3.3 single bacterium colony Agrobacterium tumefaciems of picking one are inoculated into YEB the or LB culture solutions that 20mL adds corresponding antibiotic In, when culture on 28 DEG C of constant-temperature tables to OD values is 0.2-0.5, is centrifuged 5 minutes and received in 3000rpm with 50mL sterile centrifugation tubes Collect thalline, can be used to convert with liquid MS medium resuspension after abandoning supernatant;
3.4 on superclean bench, and bacterium solution is poured into sterile petri dish, and the explant of preculture is taken out from culture dish, It is put into bacterium solution and steeps 1-5 minutes, take out the bacterium solution that explant sucks attachment on aseptic filter paper, be then seeded in differentiation culture On base, 28 DEG C of light cultures 2-4 days;
3.5 will be transferred to by the explant co-cultured on the differential medium added with selection pressure.Under conditions of illumination, 25 DEG C carry out selection culture;
After 3.6 selection cultures 2-4 weeks, the transformed cells of explant will differentiate resistance adventitious bud, or generate resistance and be cured Injured tissue;These resistant materials are transferred on corresponding subculture Selective agar medium, or is transferred to the growth of additional selection pressure and divides Change in culture medium, growth and induction is enabled to break up;
3.7 when adventitious bud grows to 1cm or more, cuts and is inserted into progress root training on the root media containing selection pressure It supports, grows adventitious root within two weeks or so.
By Agrobacterium-mediated genetic transformation, 31 resistant plants are obtained altogether, and the card with T1 for strain is checked by PCR Mycin screening is drawn, 15 transgenic lines are obtained.
PCR inspection results are as shown in Figure 1, it can be seen that obtain 15 transgenic lines from 31 resistant plants, 15 A strain is L1, L3, L4, L6, L7, L10, L12, L15, L16, L22, L23, L25, L27, L28 and L30 respectively.
The plant type of transfer-gen plant as shown in Fig. 2, the plant and control group (CK) of 15 transgenic lines obtained ratio T1 compared with, transfer-gen plant shows plant type short and smallization for plant since Seedling Stage, the phenotypic character of shortened internodes, especially Transgenic line L22 performances are more apparent.
The plant height of transfer-gen plant is as shown in Figure 3, section is long as shown in Figure 4, joint number is as shown in Figure 5, blade is grown such as Fig. 6 institutes Show, section of bearing fruit it is as shown in Figure 7;As can be seen that in the transfer-gen plant maturity period, the plant height of transfer-gen plant, internode length with it is right Photograph is than significantly reducing, and length of blade, plant joint number with compare no significant difference, the first tire fruit section of bearing fruit is increased.
As can be seen that the transfer-gen plant of SBP-Box genes can regulate and control plant height, plant type, the high economical character of section.
As shown in figure 8, by transfer-gen plant L22 strains compared with control group memory, the plant type of transgenic line L22 is found Obviously become smaller.
To sum up, SBP-Box genes are a plant type structure controlling genes, and overexpressing the gene in right amount, can to generate plant type tight The plant gathered.
Therefore, the expression vector of SBP-Box genes is built by above-mentioned method, and is transformed into plant, can be obtained Transfer-gen plant and the economical character for regulating and controlling crop.Certainly also can by the rose families such as SBP-Box genetic transformation to apple or with Tomato is, by regulating and controlling the expression of SBP-Box genes, to regulate and control the crops such as tomato, apple in other crops of the Solanaceae of representative Economical character.
Since the compact crop of plant type has preferable resistant to lodging, suitable dense planting, easily harvests performance, passes through and regulate and control SBP- Box genes can optimize crop plant type, improve resistant to lodging, suitable dense planting, easily harvest performance.
Embodiment 2
The present embodiment is to obtaining 3 transgenic lines (L4, L6 and L22) of transgene tomato and other 3 and non-turning base Because strain (CK1, CK2 and CK3) carries out transcriptome analysis.
Respectively to obtaining 3 transgenic lines (L4, L6 and L22) and other 3 and non-transgenic strain of transgene tomato It is that (CK1, CK2 and CK3) carries out illumina transcript profile sequencings, sequencing reading length is 100bp, and each sample is obtained more than 4G Clean data data.The data of acquisition are as shown in table 1.
The sequencing of table 1 obtains Clean data data statistics
The distributions of reads in the genome are consistent with the distribution of gene on chromosome, show that the sequencing result is Unbiased.
By using short reads compare software SOAPaligner/SOAP2 by the clean data of each sample respectively with Reference gene and reference gene group compare, and 5 base mispairings, the reads of 87.30%-88.95% is at most allowed to compare to ginseng It examines on gene, it can be seen that most genes can compare, and illustrate that sequencing result is still more reliable and accurate;Than To it is as shown in table 2.
The statistical result that 2 sample of table is compared with reference gene
Correlation between transgenosis and respective 3 strains of control is evaluated, the correlation between sample and quasi- For collaboration number as shown in Fig. 9 to Figure 14, related coefficient is more than 0.97, shows between these strains there is good biology to repeat Property.
Expression analysis shows that 3 transgenic lines compared with the control, there is 1091,655,1187 gene upregulations respectively Expression, 1196,584,842 gene deregulation expression, the results are shown in Table 3 for expression analysis.
3 sample difference expression gene number of table
It repeats to treat using 3 strains as biology, uses DESeq (http://www.bioconductor.org/ Packages/release/bioc/html/DESeq.html) sample does variance analysis the group that software repeats biology, sieves It is FDR≤0.001AND to select condition | log2Ratio | >=1, it is found that and 333 genes have notable table between transgenosis and control group Up to difference, wherein 248 gene upregulation expression, 85 gene deregulation expression.The results are shown in Table 4.
4 group difference expressing gene number of table
Group Total gene number Up-regulation It lowers
Transgenosis group VS is non-to turn group 333 248 85
To the expression pattern clustering of these differential genes, transgenic line is clustered respectively with non-transgenic strain to 2 In a branch, show that these differences are mainly caused by institute's transgenosis.Classify to the GO functional annotations of difference expression gene, sends out Cellular process and metabolic process entries in existing biological process classification, cellular Cell and cell part entries in component classification, the catalytic in molecular function classification Activity and binding entries are the entries containing most difference expression genes.
Gene is compared into KEGG databases using blast softwares, obtains the pathway annotation informations of gene.Then According to the difference expression gene list obtained, the pathway for doing difference expression gene is examined to be enriched with analysis using hypergeometry. Metabolic pathways, biosynthesis of secondary metabolites and plant hormone Signal transduction are notable impacted metabolic pathways.Show that turned SBP-box genes are by influencing plant Hormone signal conducts, and then influences plant metabolism and secondary metabolism, to regulate and control plant plant type structure and other growth courses.
In conclusion the method for regulation and control crop plant type structure provided by the invention, is explained and is confirmed in molecular level, Operating process through the invention can obtain the good genetically modified crops of performance.
Embodiments described above is a part of the embodiment of the present invention, instead of all the embodiments.The reality of the present invention The detailed description for applying example is not intended to limit the range of claimed invention, but is merely representative of the selected implementation of the present invention Example.Based on the embodiments of the present invention, those of ordinary skill in the art are obtained without creative efforts Every other embodiment, shall fall within the protection scope of the present invention.
SEQUENCE LISTING
<110>Vegetable & Flower Inst., Chinese Academy of Agriculture Science
<120>It is a kind of regulation and control crop plant type structure method and obtain the compact crop of plant type method
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 489
<212> DNA
<213> Apple
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atggagggga tgagtaaatt ggacttggac aagcagatga aggagaagcc gcatgtggtg 60
gtggtggtga agaaggagga ggaggaattt gatgatgagc tgcaagtgga caggatgaag 120
aaaggtaaga gatcgttgtc gtcatcatca ggaggaggag gaggtgcaat gagacggtgt 180
caggcagaca ggtgcacagc tgatctgagc gatgaaaaga aatatcacag aaagcataag 240
gtttgtgacc ttcattccaa gtctcaggtt gtgcttgtct ctggcctcca ccaaaggttt 300
tgccagcaat gcagcagatt tcatgagcta tcagaatttg acgacaccaa acggagctgt 360
cgtagccgtt tgtcaggaca caatgaacga cgaaggaaga atccagccga gtctcatgct 420
gtggaaggct caagccgcaa tgttggtaca aggactcagt ccactcacaa gcacttccaa 480
atcaaataa 489

Claims (10)

1. a kind of method of regulation and control crop plant type structure, which is characterized in that by regulating and controlling crop SBP-Box gene expressions, to adjust Control the economical character of crop.
2. the method for regulation and control crop plant type structure according to claim 1, which is characterized in that the SBP-Box genes Base sequence is as shown in SEQ ID NO.1.
3. the method for regulation and control crop plant type structure according to claim 1, which is characterized in that the economical character includes strain Type.
4. the method for regulation and control crop plant type structure according to claim 3, which is characterized in that the plant type includes resistant to lodging Plant type, suitable dense planting plant type easily harvest plant type.
5. a kind of method obtaining the compact crop of plant type, which is characterized in that include the following steps:
SBP-Box genes are connected to expression vector, obtain conversion carrier;
The conversion carrier is transformed into crops, obtain plant type compact, resistant to lodging, suitable dense planting, easily harvest described turn Gene crops.
6. the method according to claim 5 for obtaining genetically modified crops, which is characterized in that further include by the conversion carrier It is transformed into host strain, obtains conversion host strain, genetically modified crops are obtained with conversion host strain conversion crop.
7. the method according to claim 6 for obtaining genetically modified crops, which is characterized in that the host strain is Agrobacterium.
8. the method according to claim 6 for obtaining genetically modified crops, which is characterized in that the crops make for the rose family Object or solanaceous crops.
9. the method according to claim 8 for obtaining genetically modified crops, which is characterized in that the rose family crop is apple Fruit.
10. the method according to claim 8 for obtaining genetically modified crops, which is characterized in that the solanaceous crops are tomato.
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CN111072761A (en) * 2020-01-09 2020-04-28 西南大学 EjSPL5 gene for promoting loquat flowering conversion and encoding protein and application thereof
CN112695041A (en) * 2019-10-22 2021-04-23 华南农业大学 Application of ZmSBP28 gene in regulation of corn plant type

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110835367A (en) * 2018-08-14 2020-02-25 南京农业大学 Pear flowering regulating transcription factor PbrSPL15 and application thereof
CN110835367B (en) * 2018-08-14 2022-04-29 南京农业大学 Pear flowering regulating transcription factor PbrSPL15 and application thereof
CN112695041A (en) * 2019-10-22 2021-04-23 华南农业大学 Application of ZmSBP28 gene in regulation of corn plant type
CN112695041B (en) * 2019-10-22 2024-03-26 华南农业大学 Application of ZmSBP28 gene in regulation of corn plant type
CN111072761A (en) * 2020-01-09 2020-04-28 西南大学 EjSPL5 gene for promoting loquat flowering conversion and encoding protein and application thereof

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