CN108935077A - A kind of breeding method of blast resisting, anti-snout moth's larva transgenic paddy rice sterile line - Google Patents
A kind of breeding method of blast resisting, anti-snout moth's larva transgenic paddy rice sterile line Download PDFInfo
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- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
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- 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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
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Abstract
A kind of blast resisting, the breeding method of anti-snout moth's larva transgenic paddy rice sterile line, this method is to carry the sterile line E agriculture 1S of the endogenous blast resistant gene Pi-1 of rice and Pi-2 gene as female parent, rice homozygous lines mfb-MH86 with marker-free transform insect-resistant gene Cry1Ab is male parent, by first hybridizing, the breeding mode being successively returned three times again, it is detected in conjunction with Cry1Ab albumen test strips, Molecular Detection, Fertility identification and economical character are investigated, not only it is low to have obtained fertility origin temp for screening, stable fertility, it polymerize blast resisting, the new rice variety of anti-snout moth's larva, and it ensure that the high accuracy and high confidence level of selection result, improve the working efficiency of transgenic pest-resistant rice sterile line pyramiding breeding.
Description
Technical field
The invention belongs to field of plant variety breeding technology, and in particular to a kind of blast resisting, anti-snout moth's larva transgenic paddy rice infertility
The breeding method of system.
Background technique
Rice is China the first generalized grain crop, rice year sown area at 30,000,000 hectares or so, total yield is about
The 40% of China's total output of grain is accounted for, Rice Production is the mainstay for guaranteeing national food security.It is applied to the rice of production at present
Variety Disease-resistance worm ability is poor, vulnerable to pest and disease damage harm and cause rice the underproduction and quality performance it is unstable, therefore,
Breeding simultaneously have disease-resistant, insect resistance capacity rice varieties be improve paddy disease-resistant, pest-resistant and yield integration capability it is important
Measure.
Gene pyramiding refers to that will be dispersed in the target gene in different cultivars is aggregated in a kind, passes through this side
Method, it is possible to cultivate integrate high yield, excellent paddy rice cross breeding rice parent that is high-quality, more resisting or product in a relatively short period of time
Kind.Gene pyramiding can increase the persistence of Rice Resistance disease pest ability, different Resistant Genes can also be aggregated to one
In crop varieties, the technical way of current rice pyramiding breeding be using hybridization backcrossing method by disease-resistant gene with it is pest-resistant
Gene pyramiding is into a germplasm.
For a long time, the main diseases and insect pests for endangering China's rice have rice blast, snout moth's larva, brown paddy plant hopper etc..Rice blast is water
One of big Major Diseases of rice three can cause the rice significantly underproduction, underproduction 40%-50% when serious, or even total crop failure.Rice blast exists
The major rice region in China has different degrees of generation, occurs in recent years gradually in rising trend.It cultivates and promotes anti-rice blast rice
Kind is that rice blast is most economical, most efficient method for prevention and treatment.Rice lepidoptera pest such as striped rice borer, yellow rice borer, rice leaf roller
Etc. being primary pest in China's Rice Production, hazard area is up to 50,000,000 mu of mus or more, and brown paddy plant hopper is to hinder the strain of rice rice
Therefore internal water and nutrient transporting, and the entomophila of the propagation various diseases of rice are cultivated and promote while having anti-rice blast
Disease, brown planthopper resistant, anti-snout moth's larva ability the new rice varieties of multiple resistance target polymerizations be the main target to solve the above problems.
Summary of the invention
The purpose of the present invention is overcome the problems, such as that rice disease, insect pest restrict production capacity of rice in the prior art, provide
A kind of multiple target character pyramiding breeding method of transgenic paddy rice sterile line.
In order to achieve the above object, the present invention provides following technical schemes:
A kind of breeding method of blast resisting, anti-snout moth's larva transgenic paddy rice sterile line, successively the following steps are included:
One, elder generation is female parent, with no choosing with the sterile line E agriculture 1S for carrying the endogenous blast resistant gene Pi-1 and Pi-2 of rice
The rice homozygous lines mfb-MH86 for selecting label transform insect-resistant gene Cry1Ab is male parent, and the two hybridizes to obtain F1Seed, then plant F1
It is returned after seed with female parent and obtains BC1F1Seed；
Two, BC is first planted1F1Seed is to obtain BC1F1Group, then in BC1F1Anti insect gene Cry1Ab is selected to obtain in group
BC is obtained to the plant of expression and maternal backcrossing2F1Seed；
Three, BC is first planted2F1Seed is to obtain BC2F1Group, then in BC2F1Anti insect gene Cry1Ab is selected to obtain in group
BC is obtained to the plant of expression and maternal backcrossing3F1Seed；
Four, BC is first planted3F1Seed is to obtain BC3F1Group, then in BC3F1More strain molecule testing results are selected in group
For positive and excellent economical character plant, plantation obtains BC after single plant sowing3F2Strain；
Five, BC is first planted3F2Seed is to obtain BC3F2Strain, then in BC3F2Select Fertility identification qualified, anti-in strain
More plants of the plant that worm gene C ry1Ab is expressed and economical character is excellent, single plant sowing obtains BC3F3Seed；
Six, BC is first planted3F3Seed obtains BC3F3Strain, then in seedling stage to BC3F3Strain carries out Cry1Ab gene by strain
Molecular Detection, and examined by artificial water cooling pond processing selection Fertility al-teration origin temp lower than 23.5 DEG C, Cry1Ab gene molecule
More plants of strain that result is homozygous are surveyed, economical character and the smallest strain of maternal difference is filtered out wherein, harvests the strain kind
Son continues plantation and obtains BC3F4Seed；
Seven, BC is first planted3F4Seed is to obtain BC3F4Then group harvests multiple single plants and obtains BC3F5Seed, breeding choosing
The selfed seed that middle strain obtains as cultivates the Rice New Material of obtained blast resisting, anti-snout moth's larva, at this point, completing breeding.
It is described Step 2: in three, five, the method whether detection anti insect gene Cry1Ab is expressed is that Cry1Ab albumen tries
Paper slip detection.
The Cry1Ab albumen test strips detection are as follows: first take 1 ﹣ 2cm rice seedling or Mature Leaf to be placed in EP pipe, add
Enter sterile water and be ground into homogenate, stood after centrifugation, then Bt Cry1Ab protein immunization chromatograph test strip is taken to be put into EP pipe, makes EP
The liquid level of pipe is no more than the detection line of test strips, test strips is observed after 5min, if control band and These positive bands all go out in test strips
Now then judge that anti insect gene Cry1Ab is expressed, and judges anti insect gene Cry1Ab if only occurring control band in test strips
Do not expressed.
In the step 4, Molecular Detection is blast resistant gene Pi-1's and Pi-2 and anti-snout moth's larva gene C ry1Ab
PCR detection；
In the step 5, Fertility identification 1%I2- KI solution stained pollen method；
In the step 6, Cry1Ab gene molecule is detected as the PCR detection of anti-snout moth's larva gene C ry1Ab.
The total volume of the PCR reaction system of the PCR detection is 50 μ L, including buffer, two final concentrations are 1pM/L
Primer, the dNTP of final concentration of 200 μM/L, Taq enzyme, sterile distilled water, Plasmid DNA template and the sample mould that vigor is 2U
Plate, the volume of the buffer are 5 μ L, and the volume of described two primers is respectively that the volume of 1 μ L, the dNTP is 1 μ L, described
The volume of Taq enzyme is 1 μ L, and the volume of the distilled water is 41 μ L.
Primer used by the PCR of the blast resistant gene Pi-1 is detected are as follows:
L:5'-ATTGCTGCAAAGTGGGAGAC-3'
R:3'-AAGTGGAGGCAGTTCACCAC-5'；
Primer used by the PCR of the blast resistant gene Pi-2 is detected are as follows:
L:5'-GTGCATGAGTCCAGCTCAAA-3'
R:3'-GTGTACTCCCATGGCTGCTC-5'；
Primer used by the PCR of the anti-snout moth's larva gene C ry1Ab is detected are as follows:
L:5'-AAGTTCCTCTCTTGTCCGTGTACG-3'
R:3'-GATGAATCCATGAGAACATAGGAGC-5'。
In the step 5,1%I2- KI solution stained pollen method refers to: first using 1%I2- KI solution contaminates pollen
Color, then the single plant for selecting pollen sterile rate to be not less than 99.5% by continuous microscopy 3 days observation pollen staining situations and shape,
In, when microscopy, taken on every fringe each three grain husks in upper, middle and lower spend in anther be placed on a glass slide and dye, every 2 ﹣ of each selection
Pollen stainings and shape are observed to judge pollen fertility, pollen sterile rate=pollen sterile number/total pollen number * in 3 visuals field
100%.
In the step 6, the processing of artificial water cooling pond refers to: first breaking up for the 4th~7 phase for plant from crop field in rice young panicle
It digs out potting and is placed on nature down toward the plant root and stem of certain plants living, then the single plant of the potting root and stem of certain plants living is put into water cooling pond, pour at cold water
Reason is moved back to growing under natural conditions for 6~8 days, then selects sword-like leave and two leaf pulvinus are away from single fringe in ± 2cm, wait ear
Microscopy afterwards, wherein water temperature is arranged during the processing are as follows: 23.1 DEG C of turn-on temperature, 22.8 DEG C of shutdown temperature.
Compared with prior art, the invention has the benefit that
A kind of blast resisting of the present invention, anti-snout moth's larva transgenic paddy rice sterile line breeding method using mfb-MH86 as male parent,
Be female parent with E agriculture 1S, by the breeding mode for once hybridizing, being returned three times, by Cry1Ab anti insect gene be aggregated to brown planthopper resistant,
In the good rice varieties of blast resisting, and combine the detection of Cry1Ab albumen test strips, Molecular Detection, Fertility identification, field agriculture
Skill character observation and the processing of artificial water cooling pond realize rice blast resistant gene, brown planthopper resistant gene and anti-snout moth's larva gene
Effectively polymerization and homozygous ensure that critical temperature of sterility is low, stable fertility, improve the working efficiency of pyramiding breeding.Therefore, originally
Invention has not only cultivated not only blast resisting but also the polymerization resistant transgenic new rice variety of brown planthopper resistant and snout moth's larva, but also improves
Working efficiency.
Specific embodiment
The invention will be further described With reference to embodiment.
A kind of breeding method of blast resisting, anti-snout moth's larva transgenic paddy rice sterile line, successively the following steps are included:
One, elder generation is female parent, with no choosing with the sterile line E agriculture 1S for carrying the endogenous blast resistant gene Pi-1 and Pi-2 of rice
The rice homozygous lines mfb-MH86 for selecting label transform insect-resistant gene Cry1Ab is male parent, and the two hybridizes to obtain F1Seed, then plant F1
It is returned after seed with female parent and obtains BC1F1Seed；
Two, BC is first planted1F1Seed is to obtain BC1F1Group, then in BC1F1Anti insect gene Cry1Ab is selected to obtain in group
BC is obtained to the plant of expression and maternal backcrossing2F1Seed；
Three, BC is first planted2F1Seed is to obtain BC2F1Group, then in BC2F1Anti insect gene Cry1Ab is selected to obtain in group
BC is obtained to the plant of expression and maternal backcrossing3F1Seed；
Four, BC is first planted3F1Seed is to obtain BC3F1Group, then in BC3F1More strain molecule testing results are selected in group
For positive and excellent economical character plant, plantation obtains BC after single plant sowing3F2Strain；
Five, BC is first planted3F2Seed is to obtain BC3F2Strain, then in BC3F2Select Fertility identification qualified, anti-in strain
More plants of the plant that worm gene C ry1Ab is expressed and economical character is excellent, single plant sowing obtains BC3F3Seed；
Six, BC is first planted3F3Seed obtains BC3F3Strain, then in seedling stage to BC3F3Strain carries out Cry1Ab gene by strain
Molecular Detection, and examined by artificial water cooling pond processing selection Fertility al-teration origin temp lower than 23.5 DEG C, Cry1Ab gene molecule
More plants of strain that result is homozygous are surveyed, economical character and the smallest strain of maternal difference is filtered out wherein, harvests the strain kind
Son continues plantation and obtains BC3F4Seed；
Seven, BC is first planted3F4Seed is to obtain BC3F4Then group harvests multiple single plants and obtains BC3F5Seed, breeding choosing
The selfed seed that middle strain obtains as cultivates the Rice New Material of obtained blast resisting, anti-snout moth's larva, at this point, completing breeding.
It is described Step 2: in three, five, the method whether detection anti insect gene Cry1Ab is expressed is that Cry1Ab albumen tries
Paper slip detection.
The Cry1Ab albumen test strips detection are as follows: first take 1 ﹣ 2cm rice seedling or Mature Leaf to be placed in EP pipe, add
Enter sterile water and be ground into homogenate, stood after centrifugation, then Bt Cry1Ab protein immunization chromatograph test strip is taken to be put into EP pipe, makes EP
The liquid level of pipe is no more than the detection line of test strips, test strips is observed after 5min, if control band and These positive bands all go out in test strips
Now then judge that anti insect gene Cry1Ab is expressed, and judges anti insect gene Cry1Ab if only occurring control band in test strips
Do not expressed.
In the step 4, Molecular Detection is blast resistant gene Pi-1's and Pi-2 and anti-snout moth's larva gene C ry1Ab
PCR detection；
In the step 5, Fertility identification 1%I2- KI solution stained pollen method；
In the step 6, Cry1Ab gene molecule is detected as the PCR detection of anti-snout moth's larva gene C ry1Ab.
The total volume of the PCR reaction system of the PCR detection is 50 μ L, including buffer, two final concentrations are 1pM/L
Primer, the dNTP of final concentration of 200 μM/L, Taq enzyme, sterile distilled water, Plasmid DNA template and the sample mould that vigor is 2U
Plate, the volume of the buffer are 5 μ L, and the volume of described two primers is respectively that the volume of 1 μ L, the dNTP is 1 μ L, described
The volume of Taq enzyme is 1 μ L, and the volume of the distilled water is 41 μ L.
Primer used by the PCR of the blast resistant gene Pi-1 is detected are as follows:
L:5'-ATTGCTGCAAAGTGGGAGAC-3'
R:3'-AAGTGGAGGCAGTTCACCAC-5'；
Primer used by the PCR of the blast resistant gene Pi-2 is detected are as follows:
L:5'-GTGCATGAGTCCAGCTCAAA-3'
R:3'-GTGTACTCCCATGGCTGCTC-5'；
Primer used by the PCR of the anti-snout moth's larva gene C ry1Ab is detected are as follows:
L:5'-AAGTTCCTCTCTTGTCCGTGTACG-3'
R:3'-GATGAATCCATGAGAACATAGGAGC-5'。
In the step 5,1%I2- KI solution stained pollen method refers to: first using 1%I2- KI solution contaminates pollen
Color, then the single plant for selecting pollen sterile rate to be not less than 99.5% by continuous microscopy 3 days observation pollen staining situations and shape,
In, when microscopy, taken on every fringe each three grain husks in upper, middle and lower spend in anther be placed on a glass slide and dye, every 2 ﹣ of each selection
Pollen stainings and shape are observed to judge pollen fertility, pollen sterile rate=pollen sterile number/total pollen number * in 3 visuals field
100%.
In the step 6, the processing of artificial water cooling pond refers to: first breaking up for the 4th~7 phase for plant from crop field in rice young panicle
It digs out potting and is placed on nature down toward the plant root and stem of certain plants living, then the single plant of the potting root and stem of certain plants living is put into water cooling pond, pour at cold water
Reason is moved back to growing under natural conditions for 6~8 days, then selects sword-like leave and two leaf pulvinus are away from single fringe in ± 2cm, wait ear
Microscopy afterwards, wherein water temperature is arranged during the processing are as follows: 23.1 DEG C of turn-on temperature, 22.8 DEG C of shutdown temperature.
The principle of the present invention is described as follows:
Mfb-MH86 of the present invention is Inst. of Genetics and Development Biology, CAS and Fujian Agricultural
The rice homozygous lines of the marker-free transform insect-resistant gene cry1Ab of academy of sciences's biotechnology research institute breeding, E agriculture 1S is lake
Indica type the two of northern Shanxi Academy of Agricultural Sciences cereal crops research institute cultivation are line with genic sterile, carry the endogenous blast resistant gene of rice
Pi-1 and Pi-2 gene is authorized for 2016 by Hubei Province.
Fertility identification: the present invention judges pollen fertility by observation pollen staining and shape.The normal pollen grain of fertility
Containing more amylum body, I is met2The rounded atropurpureus of-KI, and sterile pollen grain is through I2- KI dyeing has allusion quotation to lose and justify to lose two kinds,
Pollen grain development is unsound, and shape shrinkage is irregular, or round, no content, to I2The reaction of-KI dye-free, noncapacitation power,
Commonly referred to as allusion quotation loses (typical abortive pollen), and pollen not shrinkage is still circle, and part is dyed, and also noncapacitation power, then be circle
It loses.
Embodiment 1:
One, First Year spring carries the sterile line E agriculture 1S of the endogenous blast resistant gene Pi-1 and Pi-2 of rice in Hainan
It is male parent for maternal, the rice homozygous lines mfb-MH86 with marker-free transform insect-resistant gene Cry1Ab, the two hybridizes
To F1Seed；
Two, F is planted in the same year positive Wuhan Ji Hai1Seed obtains F1Then group is returned with E agriculture 1S and obtains BC1F1Seed,
The next spring plants BC in Hainan1F1To obtain BC1F1Group, then to BC1F1Group takes blade to carry out Cry1Ab egg by strain
White test strips detection, the plant for selecting anti insect gene Cry1Ab to be expressed are returned as male parent and E agriculture 1S, obtain BC2F1
Seed, wherein Cry1Ab albumen test strips detection specifically: 1-2cm rice seedling or Mature Leaf is first taken to be placed in 2mL EP
Guan Zhong is added 400mL sterile water and is ground into homogenate on beveller, stand after centrifugation, then Bt Cry1Ab protein immunization is taken to chromatograph
Test strips are put into EP pipe, so that the liquid level of EP pipe is no more than the detection line of test strips, test strips are observed after 5min, if in test strips
There is control band and These positive bands then judge that anti insect gene Cry1Ab is expressed, if only occurring control band in test strips
Judge that anti insect gene Cry1Ab is not expressed；
Three, positive season in the same year plants BC in Wuhan2F1Seed obtains BC2F1Group, to BC2F1Group carries out Cry1Ab egg by strain
White test strips detection, the plant for selecting anti insect gene Cry1Ab to be expressed are returned as male parent and E agriculture 1S, obtain BC3F1
Seed, spring in third year plant BC in Hainan3F1Seed is to obtain BC3F1Group, then in BC3F1Molecular Detection is selected in group
As a result single plant sowing is carried out for 6 plants of positive and excellent economical character plant, obtains 6 BC3F2Strain seed, is denoted as respectively
14WT85,14WT86,14WT87,14WT88,14WT89 and 14WT90, wherein the Molecular Detection is blast resistant gene Pi-
1, the PCR of Pi-2 and anti-snout moth's larva gene C ry1Ab are detected, and the total volume of the PCR reaction system of PCR detection is 50 μ L, including slow
Fliud flushing, two final concentrations are the primer of 1pM/L, the dNTP of final concentration of 200 μM/L, the Taq enzyme that vigor is 2U, sterile pair
Water, Plasmid DNA template and sample template are steamed, the volume of the buffer is 5 μ L, and the volume of described two primers is respectively 1 μ L, institute
The volume for stating dNTP is 1 μ L, and the volume of the Taq enzyme is 1 μ L, and the volume of the distilled water is 41 μ L, the blast resisting base
Primer used by PCR detection because of Pi-1 is L:5'-ATTGCTGCAAAGTGGGAGAC-3', R:3'-
Primer used by the PCR of AAGTGGAGGCAGTTCACCAC-5', the blast resistant gene Pi-2 are detected is L:5'-
GTGCATGAGTCCAGCTCAAA-3', R:3'-GTGTACTCCCATGGCTGCTC-5', the anti-snout moth's larva gene C ry1Ab's
Primer used by PCR is detected is L:5'-AAGTTCCTCTCTTGTCCGTGTACG-3', R:3'-
GATGAATCCATGAGAACATAGGAGC-5'；
Four, positive season in the same year plants above-mentioned BC in Wuhan3F2Strain seed, each strain plant the group of 100 seedlings, by strain into
Then the detection of row Cry1Ab albumen test strips and Fertility identification select Fertility identification qualification, anti insect gene Cry1Ab to be expressed
And 25 plants of plant that economical character is excellent, retain the rice root and stem of certain plants, wherein the Fertility identification is 1%I2- KI solution stained pollen method,
Specifically: first use 1%I2- KI solution dyes pollen, then passes through 3 days observation pollen staining situations of continuous microscopy and shape
Select pollen sterile rate be not less than 99.5% single plant, wherein when microscopy, taken on every fringe each three grain husks in upper, middle and lower spend in flower
Medicine is placed on a glass slide and dyes, and every 2 ﹣ of each selection observes pollen stainings and shape to judge pollen fertility, no in 3 visuals field
Educate pollen rate=pollen sterile number/total pollen number * 100%；
Five, the 4th year Spring Rice root and stem of certain plants Hainan breeding, obtains 25 BC3F3Strain seed, positive season in the same year plant this 25
BC3F3Strain seed, each strain plant 50 seedling groups, carry out the detection of Cry1Ab gene molecule and economical character by strain in seedling stage
It investigates, and Fertility identification is carried out using the processing of artificial water cooling pond, Fertility al-teration origin temp is selected to be lower than 23.5 DEG C, Cry1Ab base
Because of homozygous and economical character and the smallest strain 15WT11 of E agriculture 1S difference, Cry1Ab albumen test strips are carried out by strain to the strain
Detection, the anti insect gene Cry1Ab of each single plant of the strain is expressed as the result is shown, retains the list of 10 fertility qualifications
The strain rice root and stem of certain plants, wherein the Cry1Ab gene molecule is detected as the PCR detection of anti-snout moth's larva gene C ry1Ab, the artificial water cooling pond
Processing refers to: plant is dug out potting from crop field in rice young panicle the 4th~7 phase of differentiation and is placed on nature down toward plant work by elder generation
The root and stem of certain plants, then the single plant of the potting root and stem of certain plants living is put into water cooling pond, it pours into cold water treatment 6~8 days and moves back to growing under natural conditions, then
It selects sword-like leave and two leaf pulvinus is away from single fringe in ± 2cm, the microscopy after earing, wherein water temperature is arranged during the processing
Are as follows: 23.1 DEG C of turn-on temperature, 22.8 DEG C of shutdown temperature；
Six, the 5th year spring bred 10 rice root and stem of certain plants in Hainan, obtained BC3F4Strain seed, summer in the same year plant in Wuhan
BC3F4Strain, retains its rice root and stem of certain plants, and band Hainan breeding obtains BC3F5Strain, the identified strain inheritance stability, is named as
KS11。
Claims (8)
1. the breeding method of a kind of blast resisting, anti-snout moth's larva transgenic paddy rice sterile line, it is characterised in that:
The method successively the following steps are included:
One, the first sterile line E agriculture 1S to carry the endogenous blast resistant gene Pi-1 and Pi-2 of rice is female parent, has without selection mark
Remember that the rice homozygous lines mfb-MH86 of transform insect-resistant gene Cry1Ab is male parent, the two hybridizes to obtain F1Seed, then plant F1Seed
It is returned afterwards with female parent and obtains BC1F1Seed；
Two, BC is first planted1F1Seed is to obtain BC1F1Group, then in BC1F1Anti insect gene Cry1Ab is selected to obtain table in group
The plant and maternal backcrossing reached obtains BC2F1Seed；
Three, BC is first planted2F1Seed is to obtain BC2F1Group, then in BC2F1Anti insect gene Cry1Ab is selected to obtain table in group
The plant and maternal backcrossing reached obtains BC3F1Seed；
Four, BC is first planted3F1Seed is to obtain BC3F1Group, then in BC3F1Select more strain molecule testing results for sun in group
Property and the excellent plant of economical character, plantation obtains BC after single plant sowing3F2Strain；
Five, BC is first planted3F2Seed is to obtain BC3F2Strain, then in BC3F2Fertility identification qualification, pest-resistant base are selected in strain
Because of Cry1Ab is expressed and economical character is excellent more plants of plant, single plant sowing obtains BC3F3Seed；
Six, BC is first planted3F3Seed obtains BC3F3Strain, then in seedling stage to BC3F3Strain carries out Cry1Ab gene molecule by strain
Detection, and by artificial water cooling pond processing selection Fertility al-teration origin temp lower than 23.5 DEG C, Cry1Ab gene molecule detection knot
Fruit is homozygous more plants of strain, filters out economical character and the smallest strain of maternal difference wherein, harvest the strain seed after
Continuous plantation obtains BC3F4Seed；
Seven, BC is first planted3F4Seed is to obtain BC3F4Then group harvests multiple single plants and obtains BC3F5Strain is chosen in seed, breeding
It is the Rice New Material that obtained selfed seed as cultivates obtained blast resisting, anti-snout moth's larva, at this point, completing breeding.
2. the breeding method of a kind of blast resisting according to claim 1, anti-snout moth's larva transgenic paddy rice sterile line, feature
It is:
It is described Step 2: in three, five, the method whether detection anti insect gene Cry1Ab is expressed is Cry1Ab albumen test strips
Detection.
3. the breeding method of a kind of blast resisting according to claim 2, anti-snout moth's larva transgenic paddy rice sterile line, feature
It is:
The Cry1Ab albumen test strips detection are as follows: first take 1 ﹣ 2cm rice seedling or Mature Leaf to be placed in EP pipe, nothing is added
Bacterium water is ground into homogenate, stands after centrifugation, then Bt Cry1Ab protein immunization chromatograph test strip is taken to be put into EP pipe, makes EP pipe
Liquid level is no more than the detection line of test strips, test strips is observed after 5min, if control band and These positive bands all occur in test strips
Judge that anti insect gene Cry1Ab is expressed, judges that anti insect gene Cry1Ab does not have if only occurring control band in test strips
It is expressed.
4. a kind of breeding of blast resisting, anti-snout moth's larva transgenic paddy rice sterile line described in any one of ﹣ 3 according to claim 1
Method, it is characterised in that:
In the step 4, the PCR that Molecular Detection is blast resistant gene Pi-1 and Pi-2 and anti-snout moth's larva gene C ry1Ab is examined
It surveys；
In the step 5, Fertility identification 1%I2- KI solution stained pollen method；
In the step 6, Cry1Ab gene molecule is detected as the PCR detection of anti-snout moth's larva gene C ry1Ab.
5. the breeding method of a kind of blast resisting according to claim 4, anti-snout moth's larva transgenic paddy rice sterile line, feature
It is:
The total volume of the PCR reaction system of the PCR detection is 50 μ L, is drawing for 1pM/L including buffer, two final concentrations
Object, the dNTP of final concentration of 200 μM/L, Taq enzyme, sterile distilled water, Plasmid DNA template and the sample template that vigor is 2U,
The volume of the buffer is 5 μ L, and the volume of described two primers is respectively that the volume of 1 μ L, the dNTP is 1 μ L, the Taq enzyme
Volume be 1 μ L, the volume of the distilled water is 41 μ L.
6. the breeding method of a kind of blast resisting according to claim 4, anti-snout moth's larva transgenic paddy rice sterile line, feature
It is:
Primer used by the PCR of the blast resistant gene Pi-1 is detected are as follows:
L:5'-ATTGCTGCAAAGTGGGAGAC-3'
R:3'-AAGTGGAGGCAGTTCACCAC-5'；
Primer used by the PCR of the blast resistant gene Pi-2 is detected are as follows:
L:5'-GTGCATGAGTCCAGCTCAAA-3'
R:3'-GTGTACTCCCATGGCTGCTC-5'；
Primer used by the PCR of the anti-snout moth's larva gene C ry1Ab is detected are as follows:
L:5'-AAGTTCCTCTCTTGTCCGTGTACG-3'
R:3'-GATGAATCCATGAGAACATAGGAGC-5'。
7. the breeding method of a kind of blast resisting according to claim 4, anti-snout moth's larva transgenic paddy rice sterile line, feature
It is:
In the step 5,1%I2- KI solution stained pollen method refers to: first using 1%I2- KI solution dyes pollen, then
The single plant for selecting pollen sterile rate to be not less than 99.5% by continuous microscopy 3 days observation pollen staining situations and shape, wherein mirror
When inspection, taken on every fringe each three grain husks in upper, middle and lower spend in anther be placed on a glass slide and dye, 2 ﹣ 3 views of every each selection
Pollen staining and shape are observed to judge pollen fertility, pollen sterile rate=pollen sterile number/total pollen number * 100% in open country.
8. the breeding method of a kind of blast resisting according to claim 4, anti-snout moth's larva transgenic paddy rice sterile line, feature
It is:
In the step 6, artificial water cooling pond processing refers to: first digging out plant from crop field in rice young panicle the 4th~7 phase of differentiation
Potting is placed on nature down toward the plant root and stem of certain plants living, then the single plant of the potting root and stem of certain plants living is put into water cooling pond, pour into cold water treatment 6~
It moves back within 8 days to growing under natural conditions, then selects sword-like leave and two leaf pulvinus are away from single fringe in ± 2cm, the mirror after earing
Inspection, wherein water temperature is arranged during the processing are as follows: 23.1 DEG C of turn-on temperature, 22.8 DEG C of shutdown temperature.
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