CN106591357A - Rape genetic transformation method - Google Patents
Rape genetic transformation method Download PDFInfo
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- CN106591357A CN106591357A CN201710015224.3A CN201710015224A CN106591357A CN 106591357 A CN106591357 A CN 106591357A CN 201710015224 A CN201710015224 A CN 201710015224A CN 106591357 A CN106591357 A CN 106591357A
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- brassica campestris
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- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 1
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- 101800000135 N-terminal protein Proteins 0.000 description 1
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- ARJASMXQBRNAGI-YESZJQIVSA-N Tyr-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)N ARJASMXQBRNAGI-YESZJQIVSA-N 0.000 description 1
- QHFQQRKNGCXTHL-AUTRQRHGSA-N Val-Gln-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QHFQQRKNGCXTHL-AUTRQRHGSA-N 0.000 description 1
- XBJKAZATRJBDCU-GUBZILKMSA-N Val-Pro-Ala Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O XBJKAZATRJBDCU-GUBZILKMSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
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- 230000003321 amplification Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 229910052796 boron Inorganic materials 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- ODZHLDRQCZXQFQ-UHFFFAOYSA-N chlorferron Chemical compound C1=C(O)C=CC2=C1OC(=O)C(Cl)=C2C ODZHLDRQCZXQFQ-UHFFFAOYSA-N 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010028295 histidylhistidine Proteins 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
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- 239000002689 soil Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention discloses a rape genetic transformation method. The method comprises (1) introducing a recombinant expression vector containing a target gene into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens, (2) taking the recombinant Agrobacterium tumefaciens and infecting an explant of rape through the recombinant Agrobacterium tumefaciens, (3) taking the explant and co-culturing the explant in a co-culture medium, (4) taking the explant, placing the explant in a selection medium and carrying out culture, (5) taking the explant, placing the explant in a differentiated medium and carrying out culture to obtain a differentiated bud, (6) taking the differentiated bud, placing the differentiated bud in a rooting medium and carrying out culture to obtain transgenic rape. Experiment results show that the rape genetic transformation method has high transformation efficiency of 12.0-12.5% and short time consumption. The method provides a strong tool for rape transgenosis and has a very important application value.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of method of Brassica campestris L genetic transformation.
Background technology
Brassica campestris L is important oil crop.Before 20 century 70s, the genetic improvement of Brassica campestris L is all by hybridization, mutation etc.
Traditional breeding way is realizing.From 1985 first using agrobacterium tumefaciens-mediated transformation obtain transgenic brassica napus with
Come, the transgenic research of Brassica campestris L is developed rapidly, particularly gene recombinaton in recent years and the appearance of transgenic technology, is even more Brassica campestris L
Genetic improvement open new path.However, extensively also having set up ideal without a kind of in the rape variety of cultivation
Transformation system.Brassica campestris L genetic transformation efficiency is low to have become restriction Brassica campestris L functional genomicses research and Brassica campestris L transgenic research
Bottleneck problem.At present conventional rape variety is Europe class Brassica campestris L pattern kind Westar, and it obtains transfer-gen plant needs 6-8
Week, transformation efficiency is about 0.8%.
The content of the invention
The technical problem to be solved is to provide the higher Brassica campestris L genetic transforming method of transformation efficiency.
To solve above-mentioned technical problem, present invention firstly provides a kind of Brassica campestris L genetic transforming method.
Brassica campestris L genetic transforming method provided by the present invention, may include successively following steps：
(1) recombinant expression carrier containing genes of interest is imported into Agrobacterium, obtains recombinational agrobacterium；
(2) recombinational agrobacterium that step (1) is obtained is taken, the explant of Brassica campestris L is infected；
(3) after completing step (2), the explant is taken, is placed in co-cultivation culture medium, co-cultured；
(4) after completing step (3), the explant is taken, is placed in Selective agar medium, cultivated；
(5) after completing step (4), the explant is taken, is placed in division culture medium and is cultivated, obtain Bud Differentiation；
(6) after completing step (5), the Bud Differentiation is taken, is placed in root media and is cultivated, obtain transgene rape.
In said method, in the co-cultivation culture medium 1.0-1.5mg/L 1- naphthalene acetic acids, 4.0-6.0mg/L 6- are contained
Benzyl aminoadenine and 4.0-6.0mg/L AgNO3.Contain 0.8-1.2mg/L 1- naphthalene acetic acids, 2.0- in the Selective agar medium
4.0mg/L 6- benzyls aminoadenines and 80-120mg/L cefalotin.Contain 0.1-0.3mg/L 1- in the division culture medium
Naphthalene acetic acid, 1.0-3.0mg/L 6- benzyl aminoadenines, 30-50mg/L kanamycin and 80-120mg/L cefalotin.It is described
Containing 80-120mg/L kanamycin and 80-120mg/L cefalotin in root media.
The co-cultivation culture medium concretely contains 1.25mg/L 1- naphthalene acetic acids, 5.0mg/L 6- benzyl aminoadenines
With 5.0mg/L AgNO3MS0Culture medium.The Selective agar medium concretely contains 1.0mg/L 1- naphthalene acetic acids, 3.0mg/L
The MS of 6- benzyls aminoadenine and 100mg/L cefalotin0Culture medium.The division culture medium concretely contains 0.2mg/L
The MS of 1- naphthalene acetic acids, 2.0mg/L 6- benzyl aminoadenines, 40mg/L kanamycin and 100mg/L cefalotin0Culture medium.Institute
State the root media concretely MS containing 100mg/L kanamycin and 100mg/L cefalotin0Culture medium.
In said method, in the step (2), the explant of the Brassica campestris L can be the cotyledon of the Brassica campestris L.
In said method, the acquisition modes of the cotyledon of the Brassica campestris L can be：Aseptic Semen Brassicae campestris are inoculated in into culture medium,
Alternation of light and darkness culture 5-12d (such as 5d, 7d or 12d), then takes cotyledon part.The culture medium can be MS0Culture medium.The son
Leaf portion point need to be close proximity to growing point.
In said method, in the step (2), can be as follows the step of described " infecting "：The cotyledon of the Brassica campestris L is placed in
Agrobacterium infects 8-15min in liquid；It is to be suspended in the recombinational agrobacterium to infect culture medium and obtain that the Agrobacterium infects liquid
's.Described infecting contain in culture medium 80-120mg/L acetosyringones.
In said method, in the step (2), specifically can be as follows the step of described " infecting "：By the cotyledon of the Brassica campestris L
It is placed in Agrobacterium and infects 10min in liquid；It is to be suspended in the recombinational agrobacterium to infect culture medium and obtain that the Agrobacterium infects liquid
Arrive.It is described to infect culture medium concretely containing the MS of 100mg/L acetosyringones0Culture medium.
In said method, the Agrobacterium infects the OD of liquid600nmValue can be 0.15～0.25.The Agrobacterium infects liquid
OD600nmValue concretely 0.20.
In said method, in the step (3), the condition of the co-cultivation is 23-27 DEG C, light culture 3-5 days.The step
Suddenly in (4), the condition of the culture is 23-27 DEG C, alternation of light and darkness culture 3-5 days.In the step (5), the bar of the culture
Part is 23-27 DEG C, alternation of light and darkness culture 3-5 is all.In the step (6), the condition of the culture is 23-27 DEG C.
In said method, in the step (3), concretely 25 DEG C of the condition of the co-cultivation, light culture 4 days.It is described
In step (4), concretely 25 DEG C of the condition of the culture, alternation of light and darkness culture 4 days.In the step (5), the culture
Concretely 25 DEG C of condition, alternation of light and darkness culture 4 weeks.In the step (6), concretely 25 DEG C of the condition of the culture.
In said method, the cycle concretely illumination cultivation/10 hour dark training in 14 hours of the alternation of light and darkness culture
Support.Intensity of illumination during the alternation of light and darkness culture is 13000-17000Lx.Intensity of illumination tool during the alternation of light and darkness culture
Body can be 15000Lx.
In any of the above-described described method, the Brassica campestris L is concretely double No. 4 in rape variety.
Any of the above-described " recombinant expression carrier containing genes of interest " can be the polyclone to carrier pCAMBIA2301
Insert genes of interest, the recombiant plasmid for obtaining in site.
Any of the above-described genes of interest concretely encodes the nucleic acid molecules of the precursor of salmon calcitonin see calcimar.
The nucleic acid molecules of precursor of the coding salmon calcitonin see calcimar can be following b1) or b2) or b3) or b4) shown in DNA
Molecule：
B1) there is DNA molecular of the sequence 1 from 5 ' ends shown in the 22nd to 120 nucleotide in sequence table；
B2) nucleotide sequence is DNA molecular of the sequence 1 from 5 ' ends shown in the 22nd to 120 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 85% or more than 85% homogeneity, and encodes salmon drop calcium
The DNA molecular of the precursor of element；
B4) under strict conditions with b1) or the b2) nucleotide sequence hybridization that limits, and encode the salmon calcitonin see calcimar
The DNA molecular of precursor；
The precursor of the salmon calcitonin see calcimar can be sequence 5 in aminoacid sequence such as sequence table the 158th to 190 from N-terminal
Protein shown in position.
Any of the above-described " recombinant expression carrier containing genes of interest " can be the recombiant plasmid containing expression cassette.It is described
Expression cassette may include successively following element from upstream to downstream：The promoter of vegetable oils protein gene, fusion gene and termination
Sequence；
Can be containing section first and section second in the fusion gene；The section first coded plant oil body protein；The area
Section second encodes the precursor of salmon calcitonin see calcimar；The precursor of the salmon calcitonin see calcimar can for aminoacid sequence as in sequence table sequence 5 from N
Play the protein shown in the 158th to 190 in end.
In the fusion gene, 5 ' ends are start codon, and 3 ' ends are termination codon, and centre is continuous coding
Area.
The precursor of the salmon calcitonin see calcimar can spontaneously form salmon calcitonin see calcimar in plant body.
The section second can be following b1) or b2) or b3) or b4) shown in DNA molecular：
B1) there is DNA molecular of the sequence 1 from 5 ' ends shown in the 22nd to 120 nucleotide in sequence table；
B2) nucleotide sequence is DNA molecular of the sequence 1 from 5 ' ends shown in the 22nd to 120 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 85% or more than 85% homogeneity, and encodes the salmon
The DNA molecular of the precursor of calcitonin；
B4) under strict conditions with b1) or the b2) nucleotide sequence hybridization that limits, and encode the salmon calcitonin see calcimar
The DNA molecular of precursor.
The fusion gene may also include more than one section third；The section the third encoding proteins label.The albumen mark
Sign concretely 6 × His labels.
The fusion gene may also include section fourth；The section fourth can be the restriction endonuclease recognition sequence of protease.
The protease can be the protease for meeting following condition：The protease is produced for the expression of the fusion gene
The enzyme action position of thing is between particular amino acid residue and its previous amino acids residue；The particular amino acid residue is described
First amino acid residue of the precursor of salmon calcitonin see calcimar.
The protease concretely enterokinase.
The fusion gene may include successively such as lower curtate from upstream to downstream：It is the section third, the section first, described
Section fourth and the section second.
The promoter of the vegetable oils protein gene can be Brassica campestris L oil body protein (No. GenBank is X94225.1) gene
Promoter.The nucleotide sequence of the promoter of Brassica campestris L oil body protein (No. GenBank the is X94225.1) gene specifically may be used
As shown in sequence 4 the 1st to 875 from 5 ' ends in sequence table.
The vegetable oils albumen in " the section first coded plant oil body protein " can be Semen Sesami oil body protein
(No. GenBank is AAB58402.1).When the section first encodes Semen Sesami oil body protein (No. GenBank is AAB58402.1)
When, the nucleotide sequence of the section first specifically can be as shown in sequence 4 the 913rd to 1341 from 5 ' ends in sequence table.
The terminator sequence can be Nos polyA terminators.The nucleotide sequence of the Nos polyA terminators specifically may be used
As shown in sequence 4 the 1474th to 1721 from 5 ' ends in sequence table.
The nucleotide sequence of the expression cassette specifically can be as shown in sequence 4 in sequence table.
Any of the above-described " recombinant expression carrier containing genes of interest " concretely recombiant plasmid pBO-sCT2301.
The recombiant plasmid pBO-sCT2301 is by the restricted enzyme Hind III and EcoRI recognition sequences of carrier pCAMBIA2301
Between small fragment replace with DNA molecular in sequence table shown in sequence 4, the recombiant plasmid for obtaining.The recombiant plasmid pBO-
Protein in sCT2301 expressed sequence tables shown in sequence 5.
Any of the above-described recombinational agrobacterium concretely LBA4404/pBO-sCT2301.The LBA4404/pBO-
SCT2301 is that recombiant plasmid pBO-sCT2301 is imported into agrobacterium tumefaciens lba4404 using electric shocking method, obtains recombinational agrobacterium.
To solve above-mentioned technical problem, present invention also offers for the test kit of Brassica campestris L genetic transformation.
Test kit for Brassica campestris L genetic transformation provided by the present invention contain it is any of the above-described it is described co-cultivation culture medium and/
Or any of the above-described Selective agar medium and/or any of the above-described division culture medium and/or any of the above-described root culture
Base.
In mentioned reagent box, the Brassica campestris L is concretely double No. 4 in rape variety.
Any of the above-described MS0The preparation method of culture medium is：By NH4NO3 1650mg、KNO3 1900mg、KH2PO4
170mg、MgSO4·7H2O 370mg、CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、Na2EDTA 37.30mg、
MnSO4·4H2O 22.30mg、ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、Na2MOO4·2H2O
0.25mg、CuSO4·5H2O 0.025mg、COCl2·6H2O 0.025mg, inositol 100.00mg, VB10.1mg、VB60.5mg、
Nicotinic acid 0.5mg, glycine 2.0mg and agar 7g are dissolved in 1L distilled water, adjust pH value to 5.8.
It is demonstrated experimentally that the method provided using the present invention carries out Brassica campestris L genetic transformation, transformation efficiency height (up to 12.0%-
12.5) it is, time-consuming short, powerful instrument is provided for Brassica campestris L transgenic, with very important using value.
Description of the drawings
Fig. 1 is the experimental result of the step 6 of embodiment 2.
Fig. 2 is the experimental result of the step 7 of embodiment 2.
Fig. 3 is the experimental result of the step 7 of embodiment 2.
Fig. 4 is the experimental result of the step 7 of embodiment 2.
Fig. 5 is the experimental result of (1) in the step 8 of embodiment 2.
Fig. 6 is the experimental result of (2) in the step 8 of embodiment 2.
Specific embodiment
The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining
The bright present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiments, if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, commercially obtain.
Rape variety Qingyou14 is recorded in following document：Su Youzhi. Opius dimidiatus Ashmead new varieties-Qingyou14
[J]. Chinese agricultural technology spread, 03 phase in 1997. hereinafter, rape variety Qingyou14 abbreviation Qingyou14.
Rape variety Zhejiang oil 758 is recorded in following document：Zhejiang Academy of Agricultural Science crop institute Brassica campestris L group. high yield and high quality
New rape variety Zhejiang oil 758 [J]. Zhejiang Agriculture science, 1998 (4)：Hereinafter, rape variety Zhejiang oil 758 is simple for 164-166.
Claim Zhejiang oil 758.
Rape variety Zhejiang oil 19 is recorded in following document：Rape variety Zhejiang oil 19 [J]. Zhejiang Agriculture science, 2012,1
(8)：1218-1218. hereinafter, rape variety Zhejiang oil 19 abbreviation Zhejiang oil 19.
Rape variety Zhejiang double -72 is recorded in following document：Geng Yuhua. double -72 kind brief introductions [J] in Zhejiang. Ningbo agricultural
Science and technology, 2002 (1):5-5. hereinafter, double -72 abbreviation Zhejiang, rape variety Zhejiang double -72.
Excellent oily No. 1 of rape variety Zhejiang is recorded in following document：Chen Manling, Zhao Jianyi, Zhu Yizhang. excellent oily No. 1, No. 2, Zhejiang
Main Agronomic, quality trait and yield [J]. Chinese oil crop journal, 1996 (1):64-66. hereinafter, rape variety
Excellent oily No. 1 abbreviation Zhejiang, Zhejiang is excellent oily No. 1.
Double No. 4 are recorded in following document in rape variety：Luo Xiaohui, Ye Minghai. " in double No. 4 " culturing and transplanting seedlings high yield is planted
Training [J]. Shanghai Agricultural science and technology, 05 phase in 1999. it is hereinafter, double No. 4 in double No. 4 abbreviations in rape variety.
Oil 821 is recorded in following document in rape variety：Tong Tingfeng. middle oily 821 high-yield culture technique [J]. Anhui agriculture
Industry, 09 phase in 1996. hereinafter, oil 821 in the abbreviation of oil 821 in rape variety.
The high oil 9 of rape variety is recorded in following document：Academy of agricultural sciences of Shanxi Province High-cold regions crop research institute. high oily 9. mountain
Western agricultural sciences, the 3rd phase in 2007. hereinafter, the referred to as high oil 9 of the high oil 9 of rape variety.
The blue or green oil 10 of rape variety is recorded in following document：Nian Fuzhao etc. periodically except boron stress is blue or green to cabbage type rape
Oily No. 10 yield, the impacts [C] of quality. Crops In China association Annual Conference collection of thesis .2006 in 2006. hereinafter, oil
The referred to as blue or green oil 10 of the blue or green oil 10 of vegetable kind.
Rape variety cloud oil 22 is recorded in following document：Wu Jianhua. cabbage type rape " double high, double low " kind cloud oil
No. 22 [J]. Crop Diseases in Yunnan science and technology, 2001 (5):38-38. hereinafter, rape variety cloud oil 22 abbreviation cloud oil 22.
Rape variety Henan oil 5 is recorded in following document：Zhang Shufen etc. quality rape cenospecies Henan No. 5 [J] of oil. river
Southern agricultural sciences, 09 phase in 1998. hereinafter, No. 5 abbreviation Henan oil 5 of rape variety Henan oil.
Rape variety H165 is recorded in following document：Wang Zhanyu, Hou Guicai. new rape variety H165 high-yield culturing skills
Art. Hebei Agriculture science and technology, 04 phase in 1997. hereinafter, rape variety H165 abbreviation H165.
Miscellaneous No. 1 of oil is recorded in following document in rape variety：High-quality double low, high yield, cross-bred rape new varieties-middle oil
Miscellaneous No. 1 [J]. Agriculture of Anhui, 07 phase in 2000. hereinafter, oil is miscellaneous No. 1 in miscellaneous No. 1 abbreviation of oil in rape variety.
Rape variety 92-7-58 is recorded in following document：What pleasant virtue, Hu Yunfeng, Su Hua, Xu Changwang. new rape variety
92-7-58 characteristics and Cultural technique [J]. Agriculture of Anhui, 09 phase in 1998. hereinafter, rape variety 92-7-58 abbreviation 92-
7-58。
Double No. 6 are recorded in following document in rape variety：Flat China of Huai etc. in double No. 6 rape high yield cultivation technologies [J].
Seed science and technology, 2003,21 (6):365-365. hereinafter, in rape variety double 6 in double No. 6 abbreviations.
Double No. 3 of rape variety Zhejiang is recorded in following document：Zhang Yaofeng, Zhang Dongqing. double-low rapeseed Zhe pair 3
Selection-breeding [J]. Zhejiang Agriculture science, 2001,1 (6):303-304. hereinafter, double No. 3 abbreviation Zhejiang double 3, rape variety Zhejiang.
Oil 119 is recorded in following document in rape variety：Zheng Changmin. 119 [J] of oil in quality rape new varieties. Sichuan
Agricultural science and technology, 04 phase in 1994. hereinafter, oil 119 in the abbreviation of oil 119 in rape variety Zhejiang.
Double No. 3 of rape variety China oil is recorded in following document：Double-low rapeseed routine new varieties：Magnificent oil is double No. 3 [J]. agriculture
Village's practical technique and information, 09 phase in 2000. hereinafter, the double No. 3 referred to as magnificent oil double 3 of rape variety China oil.
The blue or green oil nine of rape variety is recorded in following document：Liu Ziyu. new rape variety-green grass or young crops No. nine [J] of oil. Xinjiang
Agricultural science and technology, the 01st phase in 1987. hereinafter, the referred to as blue or green oil 9 of the blue or green oil nine of rape variety.
Rape variety Henan oil 2 is recorded in following document：Song Wenguang, Wen Yancheng. the selection-breeding of new rape variety Henan oil 2
[J]. Henan Agricultural Sciences, 05 phase in 1991. hereinafter, No. 2 abbreviation Henan oil 2 of rape variety Henan oil.
The condition of alternation of light and darkness culture in following embodiments (i.e. illumination cultivation and light culture replace) is：25℃.Illumination
Intensity of illumination during culture is 15000Lx.The cycle of alternation of light and darkness culture is specially：14h illumination cultivation/10h dark culturing.
MS0Culture medium：By NH4NO3 1650mg、KNO3 1900mg、KH2PO4 170mg、MgSO4·7H2O 370mg、
CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、Na2EDTA 37.30mg、MnSO4·4H2O 22.30mg、
ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、Na2MOO4·2H2O 0.25mg、CuSO4·5H2O
0.025mg、COCl2·6H2O 0.025mg, inositol 100.00mg, VB10.1mg、VB60.5mg, nicotinic acid 0.5mg, glycine
2.0mg and agar 7g are dissolved in 1L distilled water, adjust pH value to 5.8.
Henan sesame No. 4 is recorded in following document：Cao Yuping. Henan sesame No. 4. agricultural science and technology is communicated, 11 phases in 1991.
No. GenBank of Semen Sesami oil body protein is AAB58402.1.No. GenBank of Brassica campestris L oil body protein be
X94225.1。
Carrier pUC57 is the product of Biomics Bioisystech Co., Ltd, and catalog number is BK0033.Carrier
PEASY-T1 is the product of Beijing Quanshijin Biotechnology Co., Ltd, and catalog number is CT101.Carrier pCAMBIA2301
For the product of ocean biology (Beijing) Science and Technology Ltd. of CHMC, catalog number is VECT0310.λDNA/EcoRI+HindⅢ
Marker is the product of Beijing Kang Run bio tech ltd, and catalog number is M125-01.
Carrier pT Ω 4a：Small fragment between the restricted enzyme Hind III and EcoRI recognition sequences of carrier pUC19 is replaced
It is changed to the recombiant plasmid that the DNA molecular in sequence table shown in sequence 6 is obtained.Carrier pUC19 for TaKaRa companies product, article No.
For D3219.
The impact that embodiment 1, different explants, rape variety and hormone concentration break up to bud
First, the impact that different explants break up to bud
1st, the acquisition of explant
The seed of Qingyou14 is taken, first 2min is soaked with the ethanol water of 75% (v/v), it is then water-soluble with sodium hypochlorite
Liquid (being mixed by 1 parts by volume liquor natrii hypochloritises (effective chlorine >=10.0) and 4 parts by volume water) soaks 10min, finally with nothing
Bacterium water is rinsed repeatedly, and MS is inoculated in after draining0In culture medium, alternation of light and darkness culture 5d obtains the material of seedling age 5d.Seedling taking age 5d
Material, clip cotyledon (close proximity to growing point) obtains the cotyledon of seedling age 5d；Clip hypocotyls, obtain the lower embryo of seedling age 5d
Axle.
According to the method described above, " alternation of light and darkness culture 5d " is replaced with into " alternation of light and darkness culture 7d ", other steps are constant,
Obtain the cotyledon of seedling age 7d and the hypocotyls of seedling age 7d.
According to the method described above, " alternation of light and darkness culture 5d " is replaced with into " alternation of light and darkness culture 12d ", other steps are not
Become, obtain the cotyledon of seedling age 12d and the hypocotyls of seedling age 12d.
2nd, the impact that different explants break up to bud
After completing step one, by explant (cotyledon of seedling age 5d, the hypocotyls of seedling age 5d, the cotyledon of seedling age 7d, seedling age 7d
Hypocotyls, the cotyledon of seedling age 12d or seedling age 12d hypocotyls) be inoculated in containing 1.0～3.0mg/L 6- benzyl aminoadenines
(6-Benzylaminopurine, 6-BA), 0.0～1.0mg/L 1- naphthalene acetic acids (1-Naphthaleneacetic acid,
) and 5.0mg/L AgNO NAA3MS0In culture medium (cotyledon needs face-up), alternation of light and darkness culture 4-6 weeks are (every during culture
New culture medium was changed once every 2 weeks), obtain Bud Differentiation (including from bud and simple bud).Statistics inductivity (inductivity=can divide
Dissolve the explant number/explant number of bud × 100%) and average inductivity.
Experimental result is shown in Tables 1 and 2.
The inductivity (%) of the explant of table 1
Note："/" represented and do not exist.
The average inductivity (%) of the explant of table 2
Experimental result is as follows：Cotyledon and hypocotylar inductivity, can be used as Brassica campestris L genetic transformations without significant difference
Explant is used；The inductivity of the cotyledon of the cotyledon, the cotyledon of seedling age 7d and seedling age 12d of seedling age 5d is without significant difference；Seedling age
The hypocotylar inductivity of the hypocotyls, the hypocotyls of seedling age 7d and seedling age 12d of 5d is without significant difference；When explant inoculation
When the content of 6-BA is between 1.0～3.0mg/L in culture medium, the bud point of NAA explants in the range of 0.0～1.0mg/L
Rate is without regularity；When the content of 6-BA in the culture medium of explant inoculation is 3.0mg/L, and NAA is 0.2 or 0.5mg/L,
The growth conditions of regeneration plant are normal, and bar-shaped deformity or vitrification are presented when NAA is other contents, regeneration plant more；Work as explant
When the content of 6-BA is more than 0.2mg/L for 1.0mg/L or 2.0mg/L, NAA in the culture medium of body inoculation, explant is easier to life
Root.
2nd, the impact that different rape varieties break up to bud
1st, the acquisition of explant
The seed of rape variety to be selected is taken, first 2min is soaked with the ethanol water of 75% (v/v), sodium hypochlorite is then used
Aqueous solution (being mixed by 1 parts by volume liquor natrii hypochloritises (effective chlorine >=10.0) and 4 parts by volume water) soaks 10min, finally
Rinsed repeatedly with sterilized water, MS is inoculated in after draining0In culture medium, alternation of light and darkness culture 5d obtains the material of rape variety to be selected
Material.
The material of rape variety to be selected is taken, clip cotyledon (close proximity to growing point) obtains the cotyledon of rape variety to be selected.
The material of rape variety to be selected is taken, clip hypocotyls obtain the hypocotyls of rape variety to be selected.
Rape variety to be selected is that Qingyou14, Zhejiang oil 758, Zhejiang oil 19, Zhejiang are double -72, Zhejiang is excellent oily No. 1, in double No. 4, middle oil
821st, high oil 9, blue or green oil 10, cloud oil 22, Henan oil 5, H165, miscellaneous No. 1 middle oil, 92-7-58, in double 6, Zhejiang pair 3, middle oily 119,
Magnificent oil double 3, blue or green oily 9 or Henan oil 2.
2nd, the structure of recombiant plasmid pBO-sCT2301
(1) double chain DNA molecule in artificial synthesized sequence table shown in sequence 1.In sequence table in sequence 1, from 5 ' ends
1st to 6 recognition sequence for restricted enzyme BamH I, the 7th to 21 recognition sequence for enterokinase, the 22nd to 120
For the encoding gene (hereinafter referred to as sCT genes) of the precursor of salmon calcitonin see calcimar, the 121st to 126 is two termination codoies, the
127 to 132 recognition sequences for restricted enzyme Xho I.
(2) double chain DNA molecule that step (1) synthesizes is connected to into carrier pUC57, obtains recombiant plasmid psCT.
(3) total serum IgE of the Henan sesame No. 4 Post flowerings seed of 5 weeks is extracted, then the total serum IgE goes out the first chain with M-MLV reverse transcriptions
CDNA, obtains the cDNA of Semen Sesami.CDNA with Semen Sesami as template, using primer Olem5：5′-CTGCAGATGCATCATCACCATCATCATGCGGACGAACC-3 ' (underscore is the recognition sequence of restricted enzyme PstI)
With primer Olem3：5′-ATGGGATCC(underscore is restricted enzyme BamHI to CGGTCTTATTACATCTTGGCTTC-3 '
Recognition sequence) enter performing PCR amplification, obtain pcr amplification product.Reaction condition is：94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 1min, 35
Individual circulation；72 DEG C of extension 10min.
Jing is sequenced, and the nucleotide sequence of the pcr amplification product is as shown in the sequence 2 in sequence table.Sequence 2 in sequence table
In, the 1st to 6 recognition sequence for restricted enzyme PstI from 5 ' ends, the 7th to 9 is that start codon ATG (is compiled
Code methionine), the 10th to 27 nucleotide sequence for coding 6 × His labels, the 28th to 456 is coding Semen Sesami oil body
Albumen nucleotide sequence of the 2nd to 144 from N-terminal, the 457th to the 462 identification sequence for restricted enzyme BamHI
Row.
(4) pcr amplification product that step (3) is obtained is connected to into carrier pEASY-T1, obtains recombiant plasmid first.
(5) with restricted enzyme PstI and BamHI double digestion recombiant plasmid first, the DNA fragmentation first of 465bp is reclaimed.
(6) with restricted enzyme PstI and BamHI double digestion carrier pT Ω 4a, the carrier framework first of about 2.7kb is reclaimed.
(7) DNA fragmentation first and carrier framework first are attached, obtain intermediate carrier first.
(8) with the restricted enzyme BamH I and double digestion recombiant plasmid psCT of Xho I, the DNA fragmentation second of 132bp is reclaimed.
(9) with restricted enzyme BamH I and the double digestion intermediate carrier first of Xho I, the carrier framework second of about 3.0kb is reclaimed.
(10) DNA fragmentation second and carrier framework second are attached, obtain intermediate carrier second.
(11) with blue or green oily 14 genomic DNA of rape variety as template, using primer NP5：
5′-ATTCAAGCTT(underscore is the knowledge of restricted enzyme Hind III to GTCGGATCATGACGTTTCCAG-3 '
Other sequence) and primer NP3：5′-GCTTCTGCAG(underscore is restricted enzyme to AATTGAGAGAGATCGAAGAG-3 '
The recognition sequence of PstI) enter performing PCR amplification, obtain pcr amplification product.Reaction condition is：94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C
1min, 35 circulations；72 DEG C of extension 10min.
Jing is sequenced, and the nucleotide sequence of the pcr amplification product is as shown in the sequence 3 in sequence table.Sequence 3 in sequence table
In, the 5th to 10 recognition sequence for restricted enzyme Hind III from 5 ' ends, the 11st to 885 is Brassica campestris L oil body egg
The nucleotide sequence of white promoter, the 896th to 901 recognition sequence for restricted enzyme PstI.
(12) pcr amplification product obtained with restricted enzyme Hind III and PstI double digestion steps (11), reclaims
The DNA fragmentation third of 905bp.
(13) with restricted enzyme Hind III and PstI double digestion intermediate carrier second, the carrier framework of about 3.1kb is reclaimed
Third.
(14) DNA fragmentation third and carrier framework third are attached, obtain intermediate carrier third.
(15) with restricted enzyme Hind III and EcoRI double digestions intermediate carrier third, the DNA fragmentation of about 2.0kb is reclaimed
Fourth.
(16) with restricted enzyme Hind III and EcoRI double digestion carrier pCAMBIA2301, the load of about 11.6kb is reclaimed
Body skeleton fourth.
(17) DNA fragmentation fourth and carrier framework fourth are attached, obtain recombiant plasmid pBO-sCT2301.
According to sequencing result, structure is carried out to recombiant plasmid pBO-sCT2301 and is described as follows：By carrier pCAMBIA2301
Restricted enzyme Hind III and EcoRI recognition sequences between small fragment replace with DNA in sequence table shown in sequence 4 point
Son.Fusion protein first in recombiant plasmid pBO-sCT2301 expressed sequence tables shown in sequence 5.
In sequence table in sequence 4, the 1st to 875 nucleotides sequence for the promoter of Brassica campestris L oil body protein from 5 ' ends
Row, the 886th to 891 recognition sequence for restricted enzyme PstI, the 892nd to 894 is start codon ATG (codings
Methionine), the 895th to 912 nucleotide sequence for coding 6 × His labels, the 913rd to 1341 is coding Oleum sesami
Body protein nucleotide sequence of the 2nd to 144 from N-terminal, the 1342nd to 1347 identification for restricted enzyme BamHI
Sequence, the 1348th to 1362 recognition sequence for enterokinase, the 1363rd to 1461 coding for the precursor of salmon calcitonin see calcimar
Gene, the 1462nd to 1467 is two termination codoies, the 1468th to the 1473 identification sequence for restricted enzyme Xho I
Row, the 1474th to 1721 nucleotide sequence for Nos polyA terminators.
In sequence table in sequence 5, the 1st is methionine (initial amino acid) from N-terminal, and the 2nd to 7 is 6 × His
Label, the 8th to 150 be Semen Sesami oil body protein the 2nd to 144 from N-terminal, the 153rd to the 157 identification position for enterokinase
Point, the 158th to 190 precursor for salmon calcitonin see calcimar.
3rd, Agrobacterium infects the preparation of liquid
(1) recombiant plasmid pBO-sCT2301 is imported by agrobacterium tumefaciens lba4404 using electric shocking method, obtains agriculture bar of recombinating
Bacterium, is named as LBA4404/pBO-sCT2301.
(2) by the monoclonal of LBA4404/pBO-sCT2301 be seeded to 5mL kanamycin containing 100mg/L (Kanamycin,
Kan) and 50mg/L rifampicin (Rifampicin, Rif) YEB fluid mediums, 28 DEG C, 200rpm shaken cultivation 12h are obtained
Culture bacterium solution 1.
(3) 1mL cultures bacterium solution 1 is seeded to into 50mL kanamycin containing 100mg/L (Kanamycin, Kan) and 50mg/L is sharp
The YEB fluid mediums of good fortune flat (Rifampicin, Rif), 28 DEG C, 200rpm shaken cultivation to OD600nmIt is worth for 0.3～0.4, obtains
To culture bacterium solution 2.
(4) 2,4 DEG C of culture bacterium solution, 5000rpm centrifugation 5min that step (3) is obtained, collects thalline 1 are taken.
(5) thalline 1 of step (4) collection is taken, with the MS containing 1.0mg/L 6-BA0Culture medium is resuspended, then 4 DEG C,
5000rpm is centrifuged 5min, collects thalline 2.
(6) thalline 2 of step (5) collection is taken, with the MS of acetosyringone containing 100mg/L (Acetosyringone, As)0
Culture medium is resuspended, obtains Agrobacterium and infects liquid.With the MS containing 100mg/L As0Culture medium is reference, and adjustment Agrobacterium infects liquid
OD600nmIt is worth to 0.15～0.25.
4th, infect
Take step 1 acquisition explant (hypocotyls of the cotyledon of rape variety to be selected or rape variety to be selected), be placed in from
Heart pipe, is subsequently adding Agrobacterium and infects liquid (OD600nmIt is worth 0.2) to soak 10min.
5th, co-culture
After completing step 4, the explant is transferred to into (cotyledon needs face-up) in co-cultivation culture medium and co-cultures 4d.
Co-culture culture medium：NAA containing 1.25mg/L, 5.0mg/L 6-BA and 5.0mg/L AgNO3MS0Culture medium.
Co-cultivation condition：25 DEG C, light culture.
6th, culture is selected
After completing step 5, the explant is transferred on Selective agar medium (cotyledon needs face-up), alternation of light and darkness training
Foster 4d.
Selective agar medium：NAA containing 1.0mg/L, 3.0mg/L 6-BA and 100mg/L cefalotin (cefalotin, Cf)
MS0Culture medium.
7th, differentiation culture
After completing step 6, the explant is transferred on division culture medium (cotyledon needs face-up), alternation of light and darkness training
Support 8 weeks, obtain Bud Differentiation (including clump bud and simple bud).
Division culture medium：NAA containing 0.2mg/L, 2.0mg/L 6-BA, 40mg/L Kan, the MS of 100mg/L Cf0Culture
Base.
Experimental result is as follows：Using the cotyledon of rape variety to be selected, used as explant, (cotyledon of each rape variety to be selected is not
Less than 40), only in the cotyledon of double No. 4 differentiate 2 Bud Differentiations, the cotyledon (totally 868) of remaining rape variety to be selected is not
Differentiate Bud Differentiation；Using the hypocotyls of rape variety to be selected, used as explant, (hypocotyls of each rape variety to be selected are no less than
40), the hypocotyls (totally 871) of rape variety to be selected it is undifferentiated go out Bud Differentiation.
The above results show, different rape varieties have a certain impact to bud differentiation, in double No. 4 cotyledon be Brassica campestris L heredity
The ideal material of conversion.
3rd, the impact that hormon concentration is broken up to bud
1st, double No. 4 seed in taking, first soaks 2min with the ethanol water of 75% (v/v), then uses javelwater
Solution (being mixed by 1 parts by volume liquor natrii hypochloritises (effective chlorine >=10.0) and 4 parts by volume water) soaks 10min, finally uses
Sterilized water is rinsed repeatedly, and MS is inoculated in after draining0In culture medium, alternation of light and darkness culture 5d, double No. 4 material in obtaining.In taking
The material of double No. 4, clip cotyledon (close proximity to growing point), double No. 4 cotyledon in obtaining.
2nd, complete after step 1, double No. 4 cotyledons is inoculated in containing 1.0～3.0mg/L 6-BA, 0.0～0.5mg/L by
NAA and 5.0mg/L AgNO3MS0In culture medium (cotyledon need face-up), alternation of light and darkness culture 4 weeks is (every 2 during culture
Change once new culture medium week), obtain Bud Differentiation (including clump bud and simple bud).Statistics inductivity (inductivity=can break up
Explant number/explant the number for sprouting × 100%).
Experimental result is shown in Table 3：In double No. 4 there is very high hormone-sensitive, inductivity is up to more than 40%.As a result
Show, hormon concentration has a certain impact to bud differentiation；In the differentiation capability of double No. 4 it is very strong, be Brassica campestris L genetic transformation
Ideal material.
Table 3
The establishment of embodiment 2, Brassica campestris L genetic conversion system
First, Agrobacterium infects the preparation of liquid
With in the step 2 of embodiment 13.
The building process of the recombiant plasmid pBO-sCT2301 is with the step 2 of embodiment 12.
2nd, the acquisition of double No. 4 cotyledons in
Double No. 4 seed in taking, first soaks 2min with the ethanol water of 75% (v/v), then water-soluble with sodium hypochlorite
Liquid (being mixed by 1 parts by volume liquor natrii hypochloritises (effective chlorine >=10.0) and 4 parts by volume water) soaks 10min, finally with nothing
Bacterium water is rinsed repeatedly, and MS is inoculated in after draining0In culture medium, alternation of light and darkness culture 5d, double No. 4 material in obtaining.
Double No. 4 material in taking, clip cotyledon (close proximity to growing point), double No. 4 cotyledon in obtaining.
3rd, infect
Cotyledons double No. 4 during step 2 is obtained are taken, centrifuge tube is placed in, Agrobacterium is subsequently adding and is infected liquid (OD600nmIt is worth and is
0.2) 10min is soaked.
4th, co-culture
After completing step 3, will be described in the cotyledon of double No. 4 be transferred to (cotyledon needs face-up) in co-cultivation culture medium
Co-culture 4d.
Co-culture culture medium：NAA containing 1.25mg/L, 5.0mg/L 6-BA and 5.0mg/L AgNO3MS0Culture medium.
Co-cultivation condition：25 DEG C, light culture.
5th, culture is selected
After completing step 4, will be described in the cotyledon of double No. 4 be transferred on Selective agar medium (cotyledon needs face-up), light
Dark alternate culture 4d.
Selective agar medium：NAA containing 1.0mg/L, 3.0mg/L 6-BA and 100mg/L cefalotin (cefalotin, Cf)
MS0Culture medium.
6th, differentiation culture
After completing step 5, will be described in the cotyledon of double No. 4 be transferred on division culture medium (cotyledon needs face-up), light
Dark alternate culture 4 weeks, obtains being about the Bud Differentiation (see Fig. 1) of 1cm.
Division culture medium：NAA containing 0.2mg/L, 2.0mg/L 6-BA, 40mg/L Kan, the MS of 100mg/L Cf0Culture
Base.
7th, root culture
After completing step 6, the Bud Differentiation is transferred on root media, alternation of light and darkness culture 2 weeks, obtains and intend turning
SCT vector for transgenic rape (see Fig. 2 and Fig. 3), then will intend turning sCT vector for transgenic rape and transplant to Nutrition Soil normally to cultivate (see Fig. 4).
Root media：The MS of Kan containing 100mg/L and 100mg/L Cf0Culture medium.
Random selection 6 plants of plans turn sCT vector for transgenic rape, be named as successively Siv1, Siv3, Siv4, Siv7, Siv11 and
Siv12。
8th, Molecular Detection
(1) PCR detections
Respectively obtain 6 plants of extraction step seven are intended turning the genomic DNAs of sCT vector for transgenic rape blades and as template, with
CT5：
5'-GCCGGTCTCATTCTTACC-3' and CT3：5'-CTCAGCCTCGAGTTACTATCC-3' is carried out for primer
PCR is expanded, and obtains pcr amplification product.Replace plan to turn the genomic DNA of the blade of sCT vector for transgenic rape with equal-volume ultra-pure water, make
For negative control.The genome of the blade for being replaced turning sCT vector for transgenic rape with the DNA of equal-volume recombiant plasmid pBO-sCT2301
DNA, as positive control.
PCR response procedures are：94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 1min, 72 DEG C of extension 1min, 35
Individual circulation；72 DEG C of extension 10min, 4 DEG C of holdings.
After the completion of pcr amplification reaction, taking 10 μ L pcr amplification products carries out 1% agarose gel electrophoresiies.Experimental result is shown in
Fig. 5 (swimming lane 1 be the Marker of λ DNA/EcoRI+Hind III, swimming lane 2 be Siv1, swimming lane 3 be Siv3, swimming lane 4 be Siv4, swimming lane 5
For Siv7, swimming lane 6 is Siv11, and swimming lane 7 is Siv12, and swimming lane 8 is positive control, and swimming lane 9 is negative control).As a result show, such as
Fruit can amplify the band of about 433bp with the genomic DNA of blade intending turning sCT vector for transgenic rape as template, then the plan turns
SCT vector for transgenic rape is and turns sCT vector for transgenic rape；Negative control can not obtain the band of 433bp；Positive control can be obtained
The band of 433bp.
(2) RT-PCR detections
Coding Oleosin albumen gene (hereinafter referred to as Oleosin genes) be the specifically expressing in seed gene, one
As start transcription in the seed in Post flowering 4-6 weeks, the content of mRNA reaches top level in the seed in 9-11 weeks, seed into
The mRNA contents in ripe later stage are drastically reduced.
A () extracts the seed in Brassica campestris L (Siv1, Siv3, Siv4, Siv7, Siv11 or Siv12) Post flowering 9-11 weeks to be measured
Total serum IgE, then total serum IgE M-MLV reverse transcriptions go out the first chain cDNA, obtain the cDNA of Brassica campestris L to be measured.The cDNA of Brassica campestris L to be measured
In, DNA concentration is 500ng/ μ L.
The cDNA of b Brassica campestris L to be measured that () is obtained with step (a) as template, with CT5：5'-GCCGGTCTCATTCTTACC-3'
And CT3：5'-CTCAGCCTCGAGTTACTATCC-3' carries out RT-PCR amplifications for primer, obtains pcr amplification product.With grade body
Product ultra-pure water replaces the cDNA of Brassica campestris L to be measured, used as negative control.
PCR response procedures are：94 DEG C of denaturations 5min；94 DEG C of degeneration 30s, 55 DEG C of annealing 1min, 72 DEG C of extension 1min, 35
Individual circulation；72 DEG C of extension 10min, 4 DEG C of holdings.
After the completion of pcr amplification reaction, taking 10 μ L pcr amplification products carries out 1% agarose gel electrophoresiies.Experimental result is shown in
Fig. 6 (swimming lane 1 be the Marker of λ DNA/EcoRI+Hind III, swimming lane 2 be Siv1, swimming lane 3 be Siv3, swimming lane 4 be Siv4, swimming lane 5
For negative control).As a result show, if with intend turn sCT vector for transgenic rape cDNA as template, the bar of about 433bp can be amplified
Band, then the plan turn sCT vector for transgenic rape and be to turn sCT vector for transgenic rape, illustrate sCT genes be incorporated in the genome of pairs No. 4,
And transcriptional expression；Negative control can not obtain the band of 433bp.
PCR is detected and the result of RT-PCR detections is completely the same.Therefore, Siv1, Siv3, Siv4, Siv7, Siv11 and
Siv12 is and turns sCT vector for transgenic rape.
Transformation efficiency is counted (in transformation efficiency (%)=positive plant strain number/infect according to the testing result of step 8
The cotyledon sum of double No. 4 × 100%).As a result show, the transformation efficiency of the method for the Brassica campestris L genetic transformation that the present invention is provided reaches
12.0%.
The above results show that not only transformation efficiency is high for the method for the Brassica campestris L genetic transformation that the present invention is provided, and time-consuming short.
The method provides powerful instrument for Brassica campestris L transgenic, with very important using value.
According to the method described above, " the alternation of light and darkness culture 5d " in step 2 is replaced with into " alternation of light and darkness culture 7d ", other
Step is constant, obtains turning sCT vector for transgenic rape.The transformation efficiency of the method is 12.1%, with the transformation efficiency of said method without
Significant difference.
According to the method described above, " the alternation of light and darkness culture 5d " in step 2 is replaced with into " alternation of light and darkness culture 12d ", other
Step is constant, obtains turning sCT vector for transgenic rape.The transformation efficiency of the method is 12.5%, with the transformation efficiency of said method without
Significant difference.
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>A kind of method of Brassica campestris L genetic transformation
<160> 6
<170> PatentInversion3.5
<210>1
<211>132
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>1
ggatccgatg atgacgataa gtgctcaaac ttgtctacct gtgttcttgg taagttgtct 60
caggaacttc acaagttgca aacctaccca aggactaaca ctggttctgg aaccccagga 120
tagtaactcg ag 132
<210>2
<211>465
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>2
ctgcagatgc atcatcacca tcatcatgcg gacgaacccc acgaccagcg ccccaccgac 60
gtcatcaaga gctacctccc cgaaaagggt ccctccacct ctcaagtcct cgccgtcgtg 120
accctcttcc ccctcggcgc cgtcctcctc tgcctagccg gtctcattct taccgggacc 180
atcatcggcc tcgccgtcgc caccccgctc ttcgtcatct tcagccccat cttggtcccc 240
gccgccctaa ccatcgccct agccgtcacc ggtttcttga cctccggagc tttcggcatc 300
accgccctgt cctcgatttc gtggttgctg aactacgtta ggcgaatgcg ggggagcttg 360
ccagagcagc tggatcatgc acggcggcgc gtgcaggaga cggtgggcca gaagacaagg 420
gaggcggggc agagaagcca agatgtaata agaccgggat cccat 465
<210>3
<211>905
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>3
attcaagctt gtcggatcat gacgtttcca gaaaacatcg agcaagctct caaagctgac 60
ctctttcgga tcgtactgaa cccgaacaat ctcgttatgc cccgtcgtct ccgaacagac 120
atcctcgtaa gtaggattgt cgacgaatcc atggctatac ccaacctccg tcttcgtcac 180
gcctggaacc ctctggtaag ccaattccgc tccccagaag caaccggcgc cgaattgcgc 240
gaattgctga ccaggcgaag gaacatcgtc gtcgggtcct tgtgcgattg cggaggaagc 300
cgccgggtcg ggttggggac gagacccgaa tccgagcctg gtgaataggt tgttcatcgg 360
agatttatag acggagatgg atcgagcggt tttggggaaa ggggaagtgg gtttggctct 420
tttggataga gagagtgcag ctttggcgag agactggaga ggtttagaga gagacacggc 480
ggagattacc ggaggagagg cgacgataga tagcattatc gaagggacgg gagaaagagt 540
gacgtggaga aataagaaac cgttaagagt cggatattta ttatattaaa agcccactgg 600
gcctaaaccc atttaaacaa gacaagataa atgggccgtg tgtgagtgtt aacgttcggt 660
ttcaaatgcc aacgccattg gaacaaaaca aacgtgtcct caagtaaacc cctgtcgttt 720
acacctcaat ggctgcatgg tgaagccatt aacacgtggc gtagaatgca tgacgacgcc 780
attgacacct gactctcttt ccttctcttc atatatctct aatcaattca acactcattg 840
tcatagctat tcggaaaata tatacacatc cttttctctt cgatctctct caattctgca 900
gaagc 905
<210>4
<211>1721
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>4
gtcggatcat gacgtttcca gaaaacatcg agcaagctct caaagctgac ctctttcgga 60
tcgtactgaa cccgaacaat ctcgttatgc cccgtcgtct ccgaacagac atcctcgtaa 120
gtaggattgt cgacgaatcc atggctatac ccaacctccg tcttcgtcac gcctggaacc 180
ctctggtaag ccaattccgc tccccagaag caaccggcgc cgaattgcgc gaattgctga 240
ccaggcgaag gaacatcgtc gtcgggtcct tgtgcgattg cggaggaagc cgccgggtcg 300
ggttggggac gagacccgaa tccgagcctg gtgaataggt tgttcatcgg agatttatag 360
acggagatgg atcgagcggt tttggggaaa ggggaagtgg gtttggctct tttggataga 420
gagagtgcag ctttggcgag agactggaga ggtttagaga gagacacggc ggagattacc 480
ggaggagagg cgacgataga tagcattatc gaagggacgg gagaaagagt gacgtggaga 540
aataagaaac cgttaagagt cggatattta ttatattaaa agcccactgg gcctaaaccc 600
atttaaacaa gacaagataa atgggccgtg tgtgagtgtt aacgttcggt ttcaaatgcc 660
aacgccattg gaacaaaaca aacgtgtcct caagtaaacc cctgtcgttt acacctcaat 720
ggctgcatgg tgaagccatt aacacgtggc gtagaatgca tgacgacgcc attgacacct 780
gactctcttt ccttctcttc atatatctct aatcaattca acactcattg tcatagctat 840
tcggaaaata tatacacatc cttttctctt cgatctctct caattctgca gatgcatcat 900
caccatcatc atgcggacga accccacgac cagcgcccca ccgacgtcat caagagctac 960
ctccccgaaa agggtccctc cacctctcaa gtcctcgccg tcgtgaccct cttccccctc 1020
ggcgccgtcc tcctctgcct agccggtctc attcttaccg ggaccatcat cggcctcgcc 1080
gtcgccaccc cgctcttcgt catcttcagc cccatcttgg tccccgccgc cctaaccatc 1140
gccctagccg tcaccggttt cttgacctcc ggagctttcg gcatcaccgc cctgtcctcg 1200
atttcgtggt tgctgaacta cgttaggcga atgcggggga gcttgccaga gcagctggat 1260
catgcacggc ggcgcgtgca ggagacggtg ggccagaaga caagggaggc ggggcagaga 1320
agccaagatg taataagacc gggatccgat gatgacgata agtgctcaaa cttgtctacc 1380
tgtgttcttg gtaagttgtc tcaggaactt cacaagttgc aaacctaccc aaggactaac 1440
actggttctg gaaccccagg atagtaactc gaggctgagt aaggttaact ttgagtatta 1500
tggcattgga aaagccattg ttctgcttgt aatttactgt gttctttcag ttttgttttc 1560
ggacatcaag ttaaccaaaa aaaaaaaaaa aaaaaaaaat tttaccaaaa aaaaaaaaaa 1620
aaaaaaaaaa atttccccaa aaaaaaaaaa aaaaaaaaaa atttaaagag ctcgaatttc 1680
cccgatcgtt caaacatttg gcaataaagt ttcttaagat t 1721
<210>5
<211>190
<212>PRT
<213>Artificial sequence
<220>
<223>
<400>5
Met His His His His His His Ala Asp Glu Pro His Asp Gln Arg Pro
1 5 10 15
Thr Asp Val Ile Lys Ser Tyr Leu Pro Glu Lys Gly Pro Ser Thr Ser
20 25 30
Gln Val Leu Ala Val Val Thr Leu Phe Pro Leu Gly Ala Val Leu Leu
35 40 45
Cys Leu Ala Gly Leu Ile Leu Thr Gly Thr Ile Ile Gly Leu Ala Val
50 55 60
Ala Thr Pro Leu Phe Val Ile Phe Ser Pro Ile Leu Val Pro Ala Ala
65 70 75 80
Leu Thr Ile Ala Leu Ala Val Thr Gly Phe Leu Thr Ser Gly Ala Phe
85 90 95
Gly Ile Thr Ala Leu Ser Ser Ile Ser Trp Leu Leu Asn Tyr Val Arg
100 105 110
Arg Met Arg Gly Ser Leu Pro Glu Gln Leu Asp His Ala Arg Arg Arg
115 120 125
Val Gln Glu Thr Val Gly Gln Lys Thr Arg Glu Ala Gly Gln Arg Ser
130 135 140
Gln Asp Val Ile Arg Pro Gly Ser Asp Asp Asp Asp Lys Cys Ser Asn
145 150 155 160
Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu His Lys Leu
165 170 175
Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro Gly
180 185 190
<210>6
<211>1383
<212>DNA
<213>Artificial sequence
<220>
<223>
<400>6
aagcttcata cagagctcaa tgacaagaag aaaatcttcg tcaacatggt ggagctctct 60
tacgaacgac acgattgtct actccaaaaa tatcaaagat acagtctcag aagaccaaag 120
ggcaattgag acttttcaac aaagggtaat atccggaaac ctcctcggat tccattgccc 180
agctatctgt cactttattg tgaagatagt ggaaaaggaa ggtggctcct tacaatgcca 240
tcattgcgat aaaggaaagg ccatcgttga agatgcctct gccgacagtg gtcccaaaga 300
tggaccccca cccacgagga gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa 360
gcaagtggat tgatgtgata ctccaaaaat atcaaagata cagtctcaga agaccaaagg 420
gcaattgaga cttttcaaca aagggtaata tccggaaacc tcctcggatt ccattgccca 480
gctatctgtc actttattgt gaagatagtg gaaaaggaag gtggctcctt acaatgccat 540
cattgcgata aaggaaaggc catcgttgaa gatgcctctg ccgacagtgg tcccaaagat 600
ggacccccac ccacgaggag catcgtggaa aaagaagacg ttccaaccac gtcttcaaag 660
caagtggatt gatgtgatat ctccactgac gtaagggatg acgcacaatc ccactatcct 720
tcgcaagacc cttcctctat ataaggaagt tcatttcatt tggagaggac acgctgaaat 780
cacctctaga ggatccatcc tatttttaca acaattacca acaacaacaa acaacaaaca 840
acattacaat tactatttac aaataacaat ggactgcagg tcgactctag aggatcccct 900
actcgaggct gagtaaggtt aactttgagt attatggcat tggaaaagcc attgttctgc 960
ttgtaattta ctgtgttctt tcagttttgt tttcggacat caagttaacc aaaaaaaaaa 1020
aaaaaaaaaa aaattttacc aaaaaaaaaa aaaaaaaaaa aaaaatttcc ccaaaaaaaa 1080
aaaaaaaaaa aaaaatttaa agagctcgaa tttccccgat cgttcaaaca tttggcaata 1140
aagtttctta agattgaatc ctgttgccgg tcttgcgatg attatcatat aatttctgtt 1200
gaattacgtt aagcatgtaa taattaacat gtaatgcatg acgttattta tgagatgggt 1260
ttttatgatt agagtcccgc aattatacat ttaatacgcg acgcgataga aaacaaaata 1320
tagcgcgcaa actaggataa attatcgcgc gcggtgtcat ctatgttact agatcgggaa 1380
ttc 1383
Claims (10)
1. a kind of Brassica campestris L genetic transforming method, in turn includes the following steps：
(1) recombinant expression carrier containing genes of interest is imported into Agrobacterium, obtains recombinational agrobacterium；
(2) recombinational agrobacterium that step (1) is obtained is taken, the explant of Brassica campestris L is infected；
(3) after completing step (2), the explant is taken, is placed in co-cultivation culture medium, co-cultured；
(4) after completing step (3), the explant is taken, is placed in Selective agar medium, cultivated；
(5) after completing step (4), the explant is taken, is placed in division culture medium and is cultivated, obtain Bud Differentiation；
(6) after completing step (5), the Bud Differentiation is taken, is placed in root media and is cultivated, obtain transgene rape.
2. the method for claim 1, it is characterised in that：
In the co-cultivation culture medium containing 1.0-1.5mg/L 1- naphthalene acetic acids, 4.0-6.0mg/L 6- benzyls aminoadenines and
4.0-6.0mg/L AgNO3；
Contain 0.8-1.2mg/L 1- naphthalene acetic acids, 2.0-4.0mg/L 6- benzyls aminoadenines and 80- in the Selective agar medium
120mg/L cefalotin；
Contain 0.1-0.3mg/L 1- naphthalene acetic acids, 1.0-3.0mg/L 6- benzyl aminoadenines, 30- in the division culture medium
50mg/L kanamycin and 80-120mg/L cefalotin；
Containing 80-120mg/L kanamycin and 80-120mg/L cefalotin in the root media.
3. method as claimed in claim 1 or 2, it is characterised in that：In the step (2), the explant of the Brassica campestris L is oil
The cotyledon of dish.
4. method as claimed in claim 3, it is characterised in that：The acquisition modes of the cotyledon of the Brassica campestris L are：By aseptic oil
Colza is inoculated in culture medium, and then alternation of light and darkness culture 5-12d takes cotyledon part.
5. the method as described in Claims 1-4 is arbitrary, it is characterised in that：In the step (2), the step of described " infecting "
It is as follows：The cotyledon of the Brassica campestris L is placed in into Agrobacterium and infects 8-15min in liquid；It is by the restructuring agriculture that the Agrobacterium infects liquid
Bacillus is suspended in infects what culture medium was obtained；Described infecting contain in culture medium 80-120mg/L acetosyringones.
6. method as claimed in claim 5, it is characterised in that：The Agrobacterium infects the OD of liquid600nmIt is worth for 0.15～0.25.
7. the method as described in claim 1 to 6 is arbitrary, it is characterised in that：
In the step (3), the condition of the co-cultivation is 23-27 DEG C, light culture 3-5 days；
In the step (4), the condition of the culture is 23-27 DEG C, alternation of light and darkness culture 3-5 days；
In the step (5), the condition of the culture is 23-27 DEG C, alternation of light and darkness culture 3-5 is all；
In the step (6), the condition of the culture is 23-27 DEG C.
8. the method as described in claim 1 to 7 is arbitrary, it is characterised in that：It is described " containing genes of interest in the step (1)
Recombinant expression carrier " be the recombiant plasmid containing expression cassette；The expression cassette may include successively following unit from upstream to downstream
Part：The promoter of vegetable oils protein gene, fusion gene and terminator sequence；
Can be containing section first and section second in the fusion gene；The section first coded plant oil body protein；The section second
The precursor of coding salmon calcitonin see calcimar；The precursor of the salmon calcitonin see calcimar can be sequence 5 in aminoacid sequence such as sequence table from N-terminal
Play the protein shown in the 158th to 190.
9. the method as described in claim 1 to 8 is arbitrary, it is characterised in that：The Brassica campestris L is double No. 4 in rape variety.
10. the test kit of Brassica campestris L genetic transformation is used for；The test kit contains and co-culture described in claim 1 or 2 culture medium
And/or the Selective agar medium and/or the division culture medium and/or the root media.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107475174A (en) * | 2017-08-16 | 2017-12-15 | 北京大北农生物技术有限公司 | The method for converting rape |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101911915A (en) * | 2010-09-08 | 2010-12-15 | 安徽工程大学 | Special culture medium for agrobacterium-mediated transformation of rape hypocotyls |
| CN103966258A (en) * | 2014-04-29 | 2014-08-06 | 浙江大学 | Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101911915A (en) * | 2010-09-08 | 2010-12-15 | 安徽工程大学 | Special culture medium for agrobacterium-mediated transformation of rape hypocotyls |
| CN103966258A (en) * | 2014-04-29 | 2014-08-06 | 浙江大学 | Agrobacterium tumefaciens mediated cabbage type oilseed rape genetic transformation method |
Non-Patent Citations (1)
| Title |
|---|
| 刘昱辉: "植物油体表达体系的建立及Profilin2维管束特异表达启动子的区段缺失分析", 《中国优秀博硕士学位论文全文数据库 (博士)基础科学辑》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107475174A (en) * | 2017-08-16 | 2017-12-15 | 北京大北农生物技术有限公司 | The method for converting rape |
| CN107475174B (en) * | 2017-08-16 | 2020-08-28 | 北京大北农生物技术有限公司 | Method for transforming rape |
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