CN106591355A - Method for breeding high glucoraphanin content Brassica rapa - Google Patents
Method for breeding high glucoraphanin content Brassica rapa Download PDFInfo
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Abstract
The invention relates to a method for breeding high glucoraphanin content Brassica rapa. The method comprises A: introducing a gene which is lost in function in a donor material into a receptor material to be cultured so that a polymerized single plant is obtained, and B: systematically breeding the polymerized single plant to obtain high glucoraphanin content Brassica rapa, wherein the gene losing functions comprises at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3. The high glucoraphanin content Brassica rapa contains the protein losing functions and the protein losing functions comprises at least two of BrAOP2.1, BrAOP2.2 and BrAOP2.3. The method utilizes a molecular marker-assisted selection technique, saves a breeding cost, shortens breeding time and improves breeding efficiency.
Description
Technical field
The invention belongs to breeding technical field, and in particular to a kind of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group
Selection.
Background technology
Chinese cabbage group (Brassica rapa, L.syn.Brassica campestris) belongs to Cruciferae rape
Category, comprising many important vegetables, feeding and oil crop is used.China is the origin and evolution center of Chinese cabbage group.Cabbage
Crop gradually develops including Chinese cabbage, pakchoi, turnip, cabbage heart, tender flower stalk in existing more than the 2000 years cultivation history of China
Deng polytype, become China's distribution most extensively, one of maximum greengrocery crop of cultivated area occupies act foot in agricultural production
The status of weight.
Moisture (abbreviation sulphur glucoside) is a kind of distinctive secondary metabolite, this metabolism in crucifer
Product combines to form specific catabolite with extracellular myrosin immediately after plant cell is destroyed, these drops
Solution product not only facilitates plant defense pest and disease damage, and is also the important original of the special taste and flavor of brassicaceous vegetable generation
Cause.In recent years, to the research discovery of sulphur glucoside catabolite, the particularly catabolite of some sulphur glucosides, 4- methyl sulphur oxygen-butyl sulphur
The catabolite sulforaphen of glucoside (GRA) by blocking the cell cycle and can promote apoptotic mode to suppress tumour
Growth.Increasing research finds that sulforaphen can effectively suppress intestinal cancer, cancer of the stomach, lung cancer, prostate cancer, liver cancer, colon
The growth of the kinds of tumor cells such as cancer.In brassicaceous vegetable, detect in the cabbage vegetable thing such as broccoli, cauliflower
The GRA of high level, however, in most of Chinese cabbage groups, is only able to detect that micro, or even can't detect GRA's
Content.
The enzyme of AOP2 gene codes is the key factor that the beneficial sulphur glucoside GRA of catalysis is converted into NAP (gluconapin).Though
So cabbage and brassica crop all there occurs full-length genome duplicate event, all there are 3 AOP2 genes, but in Chinese cabbage group
In 3 AOP2 genes it is all active, and have in wild cabbage 2 AOP2 genes due to occur base deletion, so as to cause to shift to an earlier date
Terminate so as to lose function.This is probably the reason for causing brassica crop to contain higher GRA.Further, since AOP2 genes
The presence of redundancy phenomena, may cause the forfeiture of a copy function can not significantly affect the accumulation of its internal sulphur glucoside GRA.Cause
This, establishment carries the Chinese cabbage group of the BrAOP2 genes of two or three function forfeitures to cultivating the beneficial sulphur glucoside of high-load
The new varieties of all kinds of Chinese cabbage crops of GRA are significant.
The content of the invention
The technical problem to be solved is for the deficiencies in the prior art, there is provided a kind of high-load 4- methyl sulphur oxygen
The selection of butyl sulphur glucoside Chinese cabbage group, the method is selected by convergent cross and Molecular Marker Assisted Selection Technology
The Chinese cabbage group that two or three BrAOP2 gene functions are lost is carried simultaneously, so as to quick breeding goes out the beneficial sulphur of high-load
The cabbage material of glucoside GRA.
For this purpose, the invention provides a kind of selection of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group, its
Including：
Step A, the channel genes of loss of function in donor material are treated in the acceptor material of breeding, obtain polymerization individual plant；
Step B, to being polymerized, individual plant carries out systematic breeding, obtains high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups；
Wherein, the gene of the loss of function includes at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3.
In some enforcements of the present invention, the gene containing loss of function in the polymerization individual plant, the loss of function
Gene includes at least two in BrAOP2.1, BrAOP2.2 and BrAOP2.3.
In other embodiments of invention, containing funeral in the high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group
The albumen of function is lost, the albumen of the loss of function includes in BrAOP2.1, BrAOP2.2 and BrAOP2.3 at least two.
In some embodiments of invention, the donor material is at least one plant, when the donor material is more than two plants
When, the gene of loss of function between donor material is different.
In other embodiments of the present invention, the gene of loss of function passes through natural mutation, EMS in the donor material
Mutagenesis, RNAi interference knock out method acquisition using CRISPR/Cas9.
In some embodiments of the invention, by convergent cross the channel genes of loss of function in donor material are waited to educate
In the acceptor material planted, and by molecular marker assisted selection method screening polymerization individual plant.
In other embodiments of invention, systematic breeding is carried out to the individual plant that is polymerized by molecular marker assisted selection method.
In some embodiments of the invention, the algebraically of the systematic breeding is 2-4 generations.
According to the present invention, the donor material is Chinese cabbage group with the acceptor material for treating breeding.
According to the present invention, the 4- methyl sulphur oxygen-butyl sulphur in the high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group
The content of glucoside is 5-10 times of the 4- methyl sulphur oxygen-butyl glucosinolate contents in common white cole crop.
Beneficial effects of the present invention are：Selection of the present invention is by convergent cross and molecular marker assisted selection skill
Art, selects while carry the Chinese cabbage group that two or three BrAOP2 gene functions are lost, so as to quick breeding goes out height
The cabbage material of the beneficial sulphur glucoside GRA of content, and the cabbage material of the beneficial sulphur glucoside GRA of high-load for selecting can use directly
To prepare the combination of the Chinese cabbage containing high beneficial sulphur glucoside GRA.Meanwhile, the selection passes through Molecular Marker Assisted Selection Technology,
Avoid and go to determine the GRA contents of every part of material by instrument, greatly save breeding cost, shorten breeding time, improve
Breeding efficiency.
Description of the drawings
Below in conjunction with the accompanying drawings illustrating the present invention.
Fig. 1 is the flow chart of high-load GRA Chinese cabbage group selections in embodiment 1；Wherein, the digital table in bracket
Show the strain number of plantation.
Fig. 2 is the enzymatic characterization schematic diagram of BrAOP2.1, BrAOP2.2 and BrAOP2.3 in donor material R-O-18.
Fig. 3 is the genotyping result schematic diagram of BrAOP2.2 marks；Wherein, FAM represents the plant with FAM fluorescence；Hex generations
Table has the plant of Hex fluorescence；Both represents the heterozygous plant with two kinds of fluorescence of FAM and Hex；NTC represents negative control.
Fig. 4 is the Molecular Detection result schematic diagram of BrAOP2.3 marks.
Fig. 5 be braop2.2braop2.3 individual plants, BrAOP2.2BrAOP2.3 individual plants, braop2.2BrAOP2.3 individual plants,
BrAOP2.2braop2.3 and L58, R-O-18, F1The measurement result schematic diagram of middle GRA glucosinolate contents.
Specific embodiment
To make the present invention easy to understand, the present invention is described more detail below.
As it was previously stated, the cabbage material for creating the BrAOP2 genes for carrying two or three function forfeitures is high to cultivating
The new varieties of all kinds of Chinese cabbage crops of the beneficial sulphur glucoside GRA of content are significant, and it is new not have the Chinese cabbage group at present
The quick breeding method of kind.
Forefathers carry out RNAi interference to the AOP2 genes of rape, reduce the activity of AOP2, so as to significantly improve rape
The activity of the content of middle GRA, i.e. AOP2 is with the content of GRA in negative correlation；In addition, three BrAOP2 genes in Chinese cabbage group
It is distributed on three chromosomes (A02, A03, A09), is appropriate to the mutation that convergent cross will be dispersed in different cultivars
BrAOP2 channel genes are in same breeds of Chinese cabbage.The present invention is based on what the above was made.
For this purpose, a kind of selection of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group involved in the present invention,
It includes：
(1) disturbed by natural mutation, EMS mutagenesis, RNAi first or knock out method using CRISPR/Cas9 and obtained and may take
The donor material of the gene with loss of function；Gone out by gene sequencing, prokaryotic expression and external enzyme activity experiment screening
Carry the donor material of the gene of loss of function；The gene of the loss of function include BrAOP2.1, BrAOP2.2 and
At least one of BrAOP2.3；
(2) gene of loss of function in donor material is imported the acceptor material for treating breeding by way of convergent cross
In, and successful positive plant is imported using the screening of molecular marker assisted selection method, obtain polymerization individual plant；
Gene containing loss of function in the polymerization individual plant of acquisition, the gene of the loss of function include BrAOP2.1,
In BrAOP2.2 and BrAOP2.3 at least two, and the gene of loss of function is heterozygote form, i.e., the albumen that it is encoded is still
With function；
The donor material is at least one plant；When the donor material is more than two plants, work(is lost between donor material
The gene of energy is different；The gene of the loss of function includes at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3；
It is using the concrete mode of molecular marker assisted selection method screening polymerization individual plant：
(for example, the sequence difference lost according to function first between gene and function normal gene designs molecular labeling
KASP is marked and InDel marks), this is marked at function and loses in the plant of gene and the plant of function normal gene has significantly
Difference (as amplified fragments are of different sizes, fluorescence signal is different)；
Then the DNA of plant to be sieved is extracted, using the molecular labeling of design performing PCR amplification is entered, detect pcr amplification product；
If the mark that the mark in pcr amplification product loses gene with function meets, show in plant to be sieved containing target gene (funeral
Lose the gene of function), selecting the plant containing respective objects gene can both obtain polymerization individual plant.
(3) systematic breeding is carried out to the individual plant that is polymerized, obtains high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups；
Albumen containing loss of function in the high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group, the forfeiture work(
The albumen of energy includes at least two in BrAOP2.1, BrAOP2.2 and BrAOP2.3；The height obtained i.e. Jing after systematic breeding contains
The gene of the loss of function in amount 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups is homozygote shape；
Binding molecule marker assisted selection method during systematic breeding, carry out it is excellent in select excellent, obtain stable with higher
The Chinese cabbage group of content GRA；The algebraically of the systematic breeding is 2-4 generations；The algebraically of General System seed selection can shorten to 2
Generation；
The concrete mode for carrying out systematic breeding using molecular marker assisted selection method is：
(for example, the sequence difference lost according to function first between gene and function normal gene designs molecular labeling
KASP is marked and InDel marks)；
The DNA of seed selection plant is treated in extraction, and using the molecular labeling of design performing PCR amplification is entered, and detects pcr amplification product；If
The mark that mark in pcr amplification product loses material with gene function meets, then show to treat to contain target base in seed selection plant
Because of (gene of loss of function), select the plant containing respective objects gene and both may be used.
According to the present invention, the donor material is Chinese cabbage group with the acceptor material for treating breeding.
Inventor is by detecting discovery, 4- in the high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups of seed selection of the present invention
Methyl sulphur oxygen-butyl glucosinolate content is 0.999 ± 0.150umol/g of DW, and the 4- methyl sulphur oxygen in common white cole crop
Butyl glucosinolate content is 0.109 ± 0.040umol/g of DW；High-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage of the present invention
The content of the 4- methyl sulphur oxygen-butyl sulphur glucosides in class crop is the 4- methyl sulphur oxygen-butyl glucosinolate contents in common white cole crop
5-10 times.
The implication of term " loss of function " of the present invention is " not with function or miopragia ".
Term " braop2.2 " of the present invention represents the BrAOP2.2 genes of loss of function；" braop2.3 " is represented and lost
The BrAOP2.3 genes of function；" braop2.2braop2.3 individual plants " represents that genotype is
Braop2.2braop2.2braop2.3braop2.3 individual plants；" BrAOP2.2BrAOP2.3 individual plants " represents that genotype is
BrAOP2.2BrAOP2.2BrAOP2.3BrAOP2.3 individual plants；" braop2.2BrAOP2.3 individual plants " represents that genotype is
Braop2.2braop2.2BrAOP2.3BrAOP2.3 individual plants；" BrAOP2.2braop2.3 individual plants " represents that genotype is
BrAOP2.2BrAOP2.2braop2.3braop2.3 individual plants.
Embodiment
To make the present invention easier to understand, the present invention is further described below in conjunction with drawings and Examples,
These embodiments only play illustrative effect, it is not limited to the range of application of the present invention.Raw material or group used in the present invention
If divide can be obtained by commercial sources or conventional method without specified otherwise.
Embodiment 1：The selection of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups
The flow chart of the selection is as shown in figure 1, specific operating procedure is as follows：
(1) select donor material and treat the acceptor material of breeding
By gene sequencing, select and carry the R-O-18 materials that BrAOP2.2 and BrAOP2.3 functions may be lost
(one kind in Yellow Sarson types)；Tested by prokaryotic expression and external enzyme activity, in R-O-18 materials
The enzyme activity of BrAOP2.1, BrAOP2.2 and BrAOP2.3 detected, as a result as shown in Figure 2；As can be seen from Figure 2 in R-O-18 materials
BrAOP2.1 has catalysis activity, and BrAOP2.2 and BrAOP2.3 lose catalysis activity, therefore is made from R-O-18 materials
For donor material；
BrAOP2.1, BrAOP2.2 and BrAOP2.3 is selected to be respectively provided with the L58 materials (four or nine cabbage heart) of activity as breeding
Acceptor material；
(2) acquisition of polymerization individual plant
Separately designed with the difference of BrAOP2.2 and BrAOP2.3 gene orders in R-O-18 materials according to parent L58 materials
KASP marks and InDel mark；
By the way that R-O-18 materials are hybridized with L58 materials, F is obtained1(first-filial generation)；Plantation F1, with parent's L58 materials
Material is returned, and obtains BC1(first backcross generation) seed；
Plantation BC1, obtain 424 plants of BC1, in BC1Seedling stage extracts the DNA of each strain using modified CTAB method, using above-mentioned two
Mark is to BC1Individual plant is screened；Wherein, gene BrAOP2.2 specificities mark KASP mark detections, are drawn using KASP marks
Thing enters performing PCR amplification, and KASP labeled primers are respectively：
Hex fluorescent primers：5’-GAAGGTCGGAGTCAACGGATTGTGGTGCTGAATACTTCATCAGTCA-3’(SEQ
ID NO:1)；
FAM fluorescent primers：5’-GAAGGTGACCAAGTTCATGCTGGTGCTGAATACTTCATCAGTCG-3’(SEQ ID
NO:2)；
Universal primer：5’-AGAACCTCAAGTCAATGAATTACCGTCTA-3’(SEQ ID NO:3)；
The product Jing qPCR instrument (model of the instrument for being used：ABI 7900HT-fast、ABI Quantstudio
12K or Roche Lightcycler II 480 etc.) detected, testing result is as shown in figure 3, select have FAM and Hex double
Fluorescently-labeled BC1Individual plant.
The mark InDel mark detections of gene BrAOP2.3 specificities, using InDel labeled primers performing PCR amplification is entered,
InDel labeled primers are respectively：
Upstream primer F：5’-GTGCTGGTGATGGTGCTAATGATG-3’(SEQ ID NO:4)；
InDel marks R：5’-CAACACCAGCACTACTGAGAGTAC-3’(SEQ ID NO:5)；
Product Jing Ago-Gel is detected that testing result is as shown in figure 4, select with 268bp and 476bp
BC1Individual plant.
The testing result of two marks of comprehensive analysis, in BC1Filtering out 112 plants of marker genetypes in big colony (424) is
These polymerization individual plants are carried out context marker selection by the polymerization individual plant of BrAOP2.2braop2.2BrAOP2.3braop2.3, are obtained
Background is obtained closest to the polymerization individual plant BC of L58 materials1- 018 (individual plant in first backcross generation)；Wherein context marker selects to make
Method referring to：Meng Lin, the QTLs analyses of Chinese cabbage group nitrogen use efficiency, Yunnan Prov Agriculture University's master thesis,
Page 2012,38-39.
(3) systematic breeding obtains high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups
Plantation BC1- 018, carry out backcrossing with parent L58 materials and obtain BC2(second backcross generation) seed；
Plantation BC2, obtain 224 plants of BC2, seedling stage is marked using KASP and InDel is marked at BC264 are filtered out in big colony
Strain marker genetype is the individual plant of BrAOP2.2braop2.2BrAOP2.3braop2.3, and to these individual plants context marker choosing is carried out
Select, obtain background closest to the BC of L58 materials2Material BC2- 227 (individual plants in second backcross generation).
BC2- 227 carry out selfing obtains BC2S1(a selfing generation for second backcross generation) seed, plants BC2S1, obtain 400 plants
BC2S1, seedling stage is marked using KASP and InDel is marked at BC2S1Filtering out marker genetype in big colony is
Braop2.2braop2.2braop2.3braop2.3 homozygosis individual plants, the as cabbage of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside
Crop.
Braop2.2braop2.3 individual plants and L58, R-O-18, F to acquisition1, BrAOP2.2BrAOP2.3 individual plants,
Braop2.2BrAOP2.3 individual plants, BrAOP2.2braop2.3 individual plants carry out the measure of GRA sulphur glucosides, and measurement result is as shown in Figure 5.
As can be seen from Figure 5, genotype is 1.02 μm of ol/g DW for the GRA average contents in braop2.2braop2.3 individual plants, hence it is evident that be higher than
The GRA contents of parent L58 (0.13 μm of ol/g DW).
It should be noted that embodiment described above is only used for explaining the present invention, do not constitute to any of the present invention
Limit.By referring to exemplary embodiments, invention has been described, it should be appreciated that word wherein used is descriptive
With explanatory vocabulary, rather than limited vocabulary.By regulation the present invention can be made within the scope of the claims
Modification, and the present invention is revised in without departing substantially from scope and spirit of the present invention.Although the present invention described in it is related to
And specific method, material and embodiment, it is not intended that the present invention is limited to wherein disclosed particular case, conversely, this
It is bright to can be extended to other all methods and applications with identical function.
SEQUENCE LISTING
<110>Vegetable ＆ Flower Inst., Chinese Academy of Agriculture Science
<120>A kind of selection of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group
<130> 2016
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 46
<212> DNA
<213>The Hex fluorescent primers of KASP marks
<400> 1
gaaggtcgga gtcaacggat tgtggtgctg aatacttcat cagtca 46
<210> 2
<211> 44
<212> DNA
<213>The FAM fluorescent primers of KASP marks
<400> 2
gaaggtgacc aagttcatgc tggtgctgaa tacttcatca gtcg 44
<210> 3
<211> 29
<212> DNA
<213>The universal primer of KASP marks
<400> 3
agaacctcaa gtcaatgaat taccgtcta 29
<210> 4
<211> 24
<212> DNA
<213>The upstream primer of InDel marks
<400> 4
gtgctggtga tggtgctaat gatg 24
<210> 5
<211> 24
<212> DNA
<213>The downstream primer of InDel marks
<400> 5
caacaccagc actactgaga gtac 24
Claims (10)
1. a kind of selection of high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage group, it includes：
Step A, the channel genes of loss of function in donor material are treated in the acceptor material of breeding, obtain polymerization individual plant；
Step B, to being polymerized, individual plant carries out systematic breeding, obtains high-load 4- methyl sulphur oxygen-butyl sulphur glucoside Chinese cabbage groups；
Wherein, the gene of the loss of function includes at least one of BrAOP2.1, BrAOP2.2 and BrAOP2.3.
2. selection according to claim 1, it is characterised in that the base containing loss of function in the polymerization individual plant
Cause, the gene of the loss of function includes in BrAOP2.1, BrAOP2.2 and BrAOP2.3 at least two.
3. selection according to claim 1 and 2, it is characterised in that the high-load 4- methyl sulphur oxygen-butyl sulphur glucoside
Albumen containing loss of function in Chinese cabbage group, the albumen of the loss of function include BrAOP2.1, BrAOP2.2 and
In BrAOP2.3 at least two.
4. the selection according to any one of claim 1-3, it is characterised in that the donor material is at least a plant；
When the donor material is more than two plants, the gene of loss of function between donor material is different.
5. the selection according to any one of claim 1-4, it is characterised in that loss of function in the donor material
Gene is disturbed by natural mutation, EMS mutagenesis, RNAi or knocks out method using CRISPR/Cas9 and obtained.
6. the selection according to any one of claim 1-5, it is characterised in that by convergent cross by donor material
The channel genes of loss of function are treated in the acceptor material of breeding, and screen polymerization individual plant by molecular marker assisted selection method.
7. the selection according to any one of claim 1-6, it is characterised in that by molecular marker assisted selection method pair
Polymerization individual plant carries out systematic breeding.
8. the selection according to any one of claim 1-7, it is characterised in that the algebraically of the systematic breeding is 2-4
Generation.
9. the selection according to any one of claim 1-8, it is characterised in that the donor material and treat receiving for breeding
Body material is Chinese cabbage group.
10. the selection according to any one of claim 1-9, it is characterised in that the high-load 4- methyl sulphur oxygen fourth
The content of the 4- methyl sulphur oxygen-butyl sulphur glucosides in base sulphur glucoside Chinese cabbage group is the 4- methyl sulphur oxygen fourths in common white cole crop
5-10 times of base glucosinolate content.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114931148A (en) * | 2022-04-07 | 2022-08-23 | 中国农业科学院蔬菜花卉研究所 | Application of nanocarbon sol in improvement of content of aliphatic glucosinolates in broccoli curd |
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| CN101631865A (en) * | 2006-08-22 | 2010-01-20 | 哥本哈根大学 | Polypeptides and nucleic acids involved in glucosinolate biosynthesis |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114931148A (en) * | 2022-04-07 | 2022-08-23 | 中国农业科学院蔬菜花卉研究所 | Application of nanocarbon sol in improvement of content of aliphatic glucosinolates in broccoli curd |
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