CN112438198A - Application of cross incompatibility gene in preparation of insect-resistant transgenic corn shelter - Google Patents
Application of cross incompatibility gene in preparation of insect-resistant transgenic corn shelter Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
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Abstract
The invention discloses application of a cross incompatibility gene in preparation of an insect-resistant transgenic maize shelter. The invention provides a method for arranging insect-resistant transgenic plant refuge, which comprises the following steps: 1) preparing shelter hybrid and transgenic insect-resistant plant hybrid; the refuge hybrid, which contains a unidirectional cross-incompatibility gene that makes the plant unidirectional cross-incompatible, and is a plant hybrid that is not insect-resistant; the transgenic insect-resistant plant hybrid comprising an insect-resistant gene that renders the plant insect-resistant; 2) and mixing the seeds of the refuge hybrid seeds with the seeds of the transgenic insect-resistant plant hybrid seeds, and sowing to realize the refuge setting of the insect-resistant transgenic plants. The method uses the non-transgenic corn hybrid which contains the one-way cross incompatible gene Ga1-S and is not insect-resistant as a refuge, can realize the refuge effect on field pests, and can play a role in generating resistant insects to insect-resistant transgenic corn.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a cross-incompatible gene in preparation of an insect-resistant transgenic maize shelter.
Background
In the industrialization process of insect-resistant transgenic corn, in order to avoid the generation of resistance of pests such as corn borer, armyworm and the like to insect-resistant genes, in the process of planting insect-resistant transgenic corn, non-transgenic corn needs to be planted around a transgenic corn field or in the field, so as to avoid the generation of resistance of corresponding pests to insecticidal proteins, and the strategy is a high-dose/refuge strategy in the industrialization of insect-resistant transgenic corn.
The high-dose/refuge strategy is an important method in the process of controlling the resistant insects, plays an important role in the popularization process of the American transgenic corns, and data shows that the population density of the resistant insects is not increased and is always stabilized under a controllable density for 20 years after the American transgenic corns are popularized, so that the high-dose/refuge strategy plays a key role in the process. At present, two methods for arranging refuge are mainly used in countries such as the United states where insect-resistant transgenic corn is planted, one method is to plant non-transgenic corn around a transgenic corn field, insert the non-transgenic corn in the field and in the vicinity of the transgenic corn field (fig. 1, the gray part is insect-resistant transgenic corn, and the grey area is refuge), and more than 5% of the non-transgenic corn is required to be planted in a non-transgenic corn planting field to provide refuge (fig. 1); more than 20% of non-transgenic corn needs to be planted in the transgenic cotton planting field to provide shelter for the insect-resistant transgenic corn (figure 1, lower panel); another method for refuge establishment in the United states is seed-mixed sowing, which is popular with seed industry and farmers due to its simplicity of operation, but because of the susceptibility of refuge plants to contamination of their ear and seed with transgenic pollen, refuge efficacy is also diminished. At present, countries such as the United states planting insect-resistant transgenic corn are large-scale planting, refuge is easy to set, and the current mainstream method is the first method, and plays an important role in preventing insects from generating resistance. In China, the planting mode of corn is fundamentally different from that of western countries such as the United states, and there is no way to set shelter according to the United states. The second method, mixing non-transgenic hybrid in transgenic seed, but because the filaments of non-transgenic corn plants must receive Pollen from the surrounding transgenic corn during the flowering and pollination process, the result is that the seeds on the ears of non-transgenic corn are mostly transgenic corn seeds and are in a heterozygous state, and are diluted twice with the dose of the insect-killing protein, thus not only failing to achieve the effect of refuge, but also accelerating the generation of resistant pests Manag Sci. (2017)). Nevertheless, the mixed seeding method still has a great market prospect from the perspective of seed enterprise selling seeds and farmer seeding, and is just lack of effective method. Therefore, there is an urgent need to provide a method or strategy that overcomes the disadvantages of the hybrid shelter method.
Disclosure of Invention
In order to break through and eliminate the defects of mixed-method shelters and better popularize and apply the strategy, the invention provides the following technical scheme:
one object of the present invention is to provide a method for providing refuge to insect-resistant transgenic plants.
The method provided by the invention comprises the following steps:
1) preparing shelter hybrid and transgenic insect-resistant plant hybrid;
the refuge hybrid, which contains a unidirectional cross-incompatibility gene that makes the plant unidirectional cross-incompatible, and is a plant hybrid that is not insect-resistant;
the transgenic insect-resistant plant hybrid comprising an insect-resistant gene that renders the plant insect-resistant;
the refuge hybrid and the transgenic insect-resistant plant hybrid have the same gene background except for the insect-resistant gene and the unidirectional cross-incompatible gene;
2) and mixing the seeds of the refuge hybrid seeds with the seeds of the transgenic insect-resistant plant hybrid seeds, and sowing to realize the refuge setting of the insect-resistant transgenic plants.
In the above method, the refuge hybrid is prepared by introducing the unidirectional cross-incompatible gene into a non-insect-resistant plant to obtain a plant hybrid which contains the unidirectional cross-incompatible gene and is not insect-resistant;
or, the transgenic insect-resistant plant hybrid is obtained by introducing the insect-resistant gene into the non-insect-resistant plant.
In the above method, the introduction is by backcross transformation or other biological methods.
In the above method, said introducing a unidirectional cross-incompatibility gene into said non-insect-resistant plant is achieved by backcrossing a plant containing said unidirectional cross-incompatibility gene with said non-insect-resistant plant;
or, the non-insect-resistant plant is a non-insect-resistant transgenic plant (e.g., herbicide-resistant transgenic corn), or a non-insect-resistant non-transgenic plant;
in the method, in the step 2), the mixing proportion is set according to the actual requirements of different corn ecological regions.
The actual need setting according to the ecological district of maize of difference is specifically: 10-20% of shelter needs to be arranged in transgenic cotton planting areas, such as Xinjiang; in non-cotton-planting areas, 5% of shelters can be set (the cotton areas in China are all in Xinjiang, so that the non-Xinjiang areas can be provided with 5% of shelters). This is because cotton is also used as an insect-resistant gene and can kill insects. Therefore, a higher proportion of shelter is required in cotton areas.
In the method, the mixing proportion is less than or equal to 25 percent of the seeds of the refuge hybrid seeds in the mass percent of the mixed seeds.
In the above method, the mixing ratio is 5%, 10%, 15%, 20% or 25% of the seeds of the refuge hybrid by mass.
In the above method, the one-way cross-incompatibility gene is any gene capable of serving as a one-way cross-incompatibility;
or the one-way cross-incompatible gene is from an animal, plant or microorganism;
in the embodiment of the invention, the one-way hybridization incompatibility gene is Ga1-S, and the nucleotide sequence of the one-way hybridization incompatibility gene is shown as a sequence 1.
Or the insect-resistant gene is derived from the trichomonad bacillus thuringiensis (Bt for short) or other insect-killing genes (can be artificially synthesized); the insect-resistant gene can be Cry1Ab or Vip3a or Cry1F or Cry2Ab and the like.
In the embodiment of the invention, the insect-resistant genes can be Cry1Ab and Cry2 Ab.
The application of the hybrid which contains the one-way cross-incompatible gene and is not resistant to insects in at least one of the plants such as 1) to 3) in the above-mentioned method is also within the protection scope of the present invention:
1) the pest resistance to insecticidal protein is avoided in the planting of the insect-resistant transgenic plant;
2) setting up an insect-resistant transgenic plant shelter;
3) refuge insect-resistant transgenic plants.
The invention also provides the following method:
the invention provides a method for preparing insect-resistant transgenic plant refuge, which comprises the steps of 1) preparing refuge hybrid seeds in the method for arranging insect-resistant transgenic plant refuge;
alternatively, the invention provides a method for refuge of insect-resistant transgenic plants, which is to set refuge of insect-resistant transgenic plants according to the method for refuge of insect-resistant transgenic plants.
Or the application of the one-way cross incompatible gene in refuge of insect-resistant transgenic plant is also the protection scope of the present invention.
In the above, the shelter is suitable for planting insect-resistant transgenic plants in insect-resistant transgenic corns in any country around the world; is especially suitable for China.
The present invention proposes pollen of corn gene containing homozygous Ga1-S gene and utilizing the phenomenon of single-way cross incompatibility in corn, which is controlled by a single-way cross incompatibility gene Ga 1-S. Therefore, Ga1-S gene is introduced into the same background as insect-resistant transgenic corn by a conventional breeding method or a genetic engineering method, hybrid seeds containing Ga1-S gene are mixed into a packaging bag of insect-resistant transgenic corn hybrid seeds in a commercialization process, non-transgenic corn can be randomly distributed in transgenic corn fields in the sowing process, and shelters with different proportions are arranged according to the actual needs of different corn ecoregions. The non-transgenic corn hybrid which contains the one-way cross incompatibility gene Ga1-S and is not insect-resistant is used as a refuge, so that the refuge effect on field pests can be realized, and insects which are resistant to insect-resistant transgenic corn can be generated.
Drawings
FIG. 1 shows the intent of the refuge adopted in the United states (the gray portion is the transgenic maize; the white portion is the non-transgenic maize refuge; the top is a 5% proportion of the non-transgenic maize refuge; the bottom is a 20% non-transgenic maize refuge).
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples, the detection of the insect resistance of the corn is carried out by using an American Agdia transgenic insect-resistant Bt-Cry1Ab/1Ac detection test strip, and if the test strip is positive, the test strip contains an insect-resistant gene Cry1Ab/1 Ac).
Some of the biomaterials in the following examples are described below:
the insect-resistant transformant 2A-5 was prepared as follows: infecting a receptor corn by using a pCAMBIA3301+ mCry1Ab + mCry2Ab vector through agrobacterium to obtain T0 generation transgenic corn, namely an insect-resistant transformant 2A-5; through detection, the transformant is insect-resistant and contains an insect-resistant gene Cry1 Ab).
The receptor corn is an inbred line X178 (provided by national corn improvement center of Chinese agricultural university; recorded in the following documents, miRNA expression difference analysis before and after pollination of the corn inbred line X178 and fluid 478; Yanlin Zhao Yong from Shanghai Longwang Wei of province Yang, Yangxi, 30-36 pages in 5 th stage of 2018 in corn science).
The vector pCAMBIA3301+ mCry1Ab + mCry2Ab is a vector obtained by introducing an expression cassette of an insect-resistant gene mCry1Ab and an expression cassette of an insect-resistant gene mCry2Ab into the space between LB and RB of pCAMBIA3301 (available from Hainanhui Hu, Biotech Co., Ltd.).
The expression cassette of the target gene mCry1Ab consists of a 35S promoter, a gene mCry1Ab shown in a sequence 2 and an NOS terminator;
the expression cassette of the target gene mCry2Ab comprises a 35S promoter, a gene mCry2Ab shown in sequence 3 and an NOS terminator group.
Zhengdan 958, approved serial No.: jade 20000009 was examined domestically.
Popcorn 401D Ga1-S (MGS-25776), source: the Maize stock center of US contains a one-way cross-incompatible gene Ga1-S, the nucleotide sequence of which is SEQ ID NO. 1.
Example 1 insect-resistant transgenic maize refuge and uses thereof
Obtaining insect-resistant transgenic corn hybrid
The insect-resistant transformant 2A-5 is utilized to carry out backcross transformation on the parent of the good hybrid Zhengdan 958 which is popularized in a large area in production, and the insect-resistant transgenic corn hybrid Zhengdan 958K is obtained.
The specific method comprises the following steps:
1. backcross transformation
Such as:
in 12 months in 2015, respectively hybridizing a maize inbred line Zheng 58 and a maize inbred line Chang 7-2 serving as female parents and an insect-resistant transformant 2A-5 serving as male parents to obtain hybrid progeny F1 (from Zheng 58) and hybrid progeny F1 (from Chang 7-2);
in 2 months in 2016, taking hybrid offspring BC1F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively, and backcrossing for 1 time to obtain BC1F1 (from Zheng 58) and BC1F1 (from Chang 7-2);
in 2016 for 6 months, taking hybrid offspring BC1F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively, and backcrossing for 2 times to obtain BC2F1 (from Zheng 58) and BC2F1 (from Chang 7-2);
in 10 months in 2016, using hybrid offspring BC2F1 as male parent, using Zheng 58 and Chang 7-2 as recurrent parent respectively, backcrossing for 3 times to obtain BC3F1 (from Zheng 58) and BC3F1 (from Chang 7-2);
in 12 months in 2016, using hybrid offspring BC3F1 as male parent, using Zheng 58 and Chang 7-2 as recurrent parent respectively, backcrossing for 4 times to obtain BC4F1 (from Zheng 58) and BC4F1 (from Chang 7-2);
in 2 months of 2017, taking hybrid offspring BC4F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively, and backcrossing for 5 times to obtain BC5F1 (from Zheng 58) and BC5F1 (from Chang 7-2);
in 6 months in 2017, the hybrid progeny BC5F1 (from Zheng 58) and BC5F1 (from Chang 7-2) are respectively selfed to obtain BC5F2 (from Zheng 58) and BC5F2 (from Chang 7-2);
and (3) detecting the insect resistance by adopting a transgenic insect-resistant Bt-Cry1Ab/1Ac detection test strip.
The plants having the insect resistance were selected as BC5F2 (from Zheng 58) and BC5F2 (from Chang 7-2) insect-resistant plants from BC5F2 (from Zheng 58) and BC5F2 (from Chang 7-2), respectively.
In 10 months of 2017, the BC5F2 (from Zheng 58) insect-resistant plant and the BC5F2 (from Chang 7-2) insect-resistant plant are respectively subjected to continuous selfing to obtain a BC5F3 (from Zheng 58) insect-resistant plant and a BC5F3 (from Chang 7-2) insect-resistant pure plant;
in 2 months of 2018, a BC5F3 (from Zheng 58) insect-resistant plant and a BC5F3 (from Chang 7-2) insect-resistant plant are crossed to obtain an insect-resistant hybrid Zhengdan 958K (identified by using a transgenic insect-resistant Bt-Cry1Ab/1Ac detection test strip).
Secondly, obtaining refuge hybrid which contains the one-way cross incompatible gene Ga1-S and is not insect-resistant
Utilizing a one-way hybridization incompatibility gene Ga1-S (the nucleotide sequence is shown as sequence 1), carrying out backcross transformation on parents of excellent hybrid seeds such as Zhengdan 958 and the like popularized in large area in production, and obtaining hybrid seeds Zhengdan 958G and the like containing the one-way hybridization incompatibility gene Ga1-S, namely refuge hybrid seeds containing the one-way hybridization incompatibility gene Ga1-S and being not resistant to insects.
The specific method comprises the following steps:
1. backcross transformation
Such as:
in 12 months in 2013, respectively hybridizing a maize inbred line Zheng 58 and a maize inbred line Chang 7-2 which serve as female parents and a cracked maize 401D Ga1-S (MGS-25776) which serves as male parents to obtain hybrid progeny F1 (from Zheng 58) and hybrid progeny F1 (from Chang 7-2);
in 6 months in 2014, taking hybrid progeny F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively, and backcrossing for 1 time to obtain BC1F1 (from Zheng 58) and BC1F1 (from Chang 7-2);
performing backcross for 2 times by taking hybrid offspring BC1F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively in 12 months in 2015 to obtain BC2F1 (from Zheng 58) and BC2F1 (from Chang 7-2);
in 2016 for 2 months, taking hybrid offspring BC2F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively, and backcrossing for 3 times to obtain BC3F1 (from Zheng 58) and BC3F1 (from Chang 7-2);
in 2016 for 6 months, taking hybrid offspring BC3F1 as male parents and Zheng 58 and Chang 7-2 as recurrent parents respectively, and backcrossing for 4 times to obtain BC4F1 (from Zheng 58) and BC4F1 (from Chang 7-2);
in 12 months in 2016, using hybrid offspring BC4F1 as male parent, using Zheng 58 and Chang 7-2 as recurrent parent respectively, backcrossing for 5 times to obtain BC5F1 (from Zheng 58) and BC5F1 (from Chang 7-2);
selfing filial generations BC5F1 (from Zheng 58) and BC5F1 (from Chang 7-2) for 1 time in 2 months in 2017 to obtain BC5F2 (from Zheng 58) and BC5F2 (from Chang 7-2);
ga1/Ga1 homozygous plants were identified by the molecular markers shown in Table 1, and plants consistent with the banding pattern of popcorn 401D Ga1-S (MGS-25776) were selected as BC5F2 (from Zheng 58) Ga1/Ga1 homozygous plants and BC5F2 (from Chang 7-2) Ga1/Ga1 homozygous plants.
TABLE 1
In 6 months of 2017, selfing BC5F2 (from Zheng 58) Ga1/Ga1 homozygous plant and BC5F2 (from Chang 7-2) Ga1/Ga1 homozygous plant for 2 times respectively to obtain BC5F3 (from Zheng 58) and BC5F3 (from Chang 7-2);
ga1/Ga1 homozygous plants were identified by the molecular markers shown in Table 1, and plants consistent with the banding pattern of popcorn 401D Ga1-S (MGS-25776) were selected as BC5F3 (from Zheng 58) Ga1/Ga1 homozygous plants and BC5F3 (from Chang 7-2) Ga1/Ga1 homozygous plants.
In 12 months of 2017, BC5F3 (from Zheng 58) Ga1/Ga1 homozygous plant and BC5F3 (from Chang 7-2) Ga1/Ga1 homozygous plant are crossed to obtain a non-transgenic corn hybrid Zheng 958G which contains the unilaterally-hybridized incompatible gene Ga1-S and is not insect-resistant as a refuge hybrid (identified by using a transgenic insect-resistant Bt-Cry1Ab/1Ac detection test strip).
Third, transgenic corn or non-transgenic corn hybrid containing unidirectional hybridization incompatibility gene Ga1-S and being not insect-resistant refuges insect-resistant transgenic corn hybrid
Mixing the two prepared non-transgenic corn hybrid Zhengdan 958G (refuge hybrid) containing the one-way cross-incompatible gene Ga1-S and being not insect-resistant with the seeds of the one prepared insect-resistant transgenic corn hybrid Zhengdan 958K (insect-resistant transgenic corn) at different mass ratios, and setting five gradients of the ratio of refuge seeds to total seeds: 5%, 10%, 15%, 20%, 25%. And sowing the mixed seeds in 2018 in 6 months, and culturing to obtain a shelter hybrid plant (a plant with a negative insect-resistant gene detection result) and an insect-resistant transgenic corn hybrid plant (a plant with a positive insect-resistant gene detection result).
Seeds on the refuge plant harvested in 2018 and 9 months are detected for insect-resistant genes, and the proportion of pollen of transgenic corn which can be accepted by the clusters on the refuge plant is determined, wherein the specific method comprises the following steps:
and (3) respectively carrying out fruit ear sampling 20 days after the refuge plants are pollinated by each gradient, respectively sampling 150 fruit ears for each hybridization, and detecting by adopting a transgenic insect-resistant Bt-Cry1Ab/1Ac detection test strip indoors.
As shown in Table 2, no target insect-resistant gene was detected in any of the refuge plants obtained by mixed sowing of five gradients of 5%, 10%, 15%, 20% and 25%.
Table 2 Zhengdan 958G and Zhengdan 958K mix the detection result of the insect-resistant gene of grain on the ear of non-transgenic plant containing the unidirectional hybridization incompatible gene Ga1-S and not resistant to insects
The results show that the non-transgenic corn hybrid which contains the one-way cross incompatibility gene Ga1-S and is not insect-resistant is used as a refuge, so that the refuge effect on field pests can be realized, and insects which are resistant to insect-resistant transgenic corn can be played.
Comparative example, non-transgenic corn hybrid containing no one-way cross incompatibility gene refuges insect-resistant transgenic corn hybrid
Non-transgenic corn hybrid no-Zhengdan 958 (non-transgenic corn hybrid) not containing the one-way cross-incompatibility gene and anti-insect transgenic corn hybrid Zhengdan 958K (anti-insect transgenic corn hybrid) prepared one of example 1 were mixed in various ratios according to the method of the third of example 1, and the non-transgenic corn hybrid seed to the total seed mass ratio was set to five seed mixing gradients: 5%, 10%, 15%, 20%, 25%. And sowing the mixed seeds in 2018 and 6 months, and culturing to obtain non-transgenic corn hybrid plants (plants with negative insect-resistant gene detection results) and insect-resistant transgenic corn hybrid plants (plants with positive insect-resistant gene detection).
And (4) harvesting seeds on the non-transgenic corn hybrid plants in 2018 and 9 months for detecting insect-resistant genes, and determining the proportion of the pollen of the transgenic corn which can be accepted by the ears on the non-transgenic plants. The method comprises the following specific steps:
and (3) carrying out fruit ear sampling 20 days after the non-transgenic corn hybrid plants are pollinated by each seed mixed gradient, respectively sampling 150 fruit ears for each hybrid, and carrying out rapid detection on insect-resistant genes indoors.
The results are shown in Table 3 below,
table 3 detection result of insect-resistant genes of seeds on fruit ears of Zhengdan 958 and Zhengdan 958K mixed non-transgenic plants
As can be seen from the table, the proportion of transgenic seeds on the ears of the non-transgenic plants gradually decreases with the increase of the proportion of non-transgenic seeds, but the proportion is all above 70%.
Thus, comparing example 1, it can be seen that refuge of field pests can be effectively achieved using a non-transgenic corn hybrid, which contains the one-way cross-incompatibility gene Ga1-S and is not insect-resistant, as a refuge.
SEQUENCE LISTING
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aactactgca tcaacaccta ccagagcgct ttcaagggcc tcaacacccg tctgcacgac 720
atgctggagt tcaggaccta catgttcctc aacgtgttcg agtacgtgtc catctggtcc 780
ctgttcaagt accagagcct gctcgtctcc tccggcgcca acctgtacgc cagcggctcc 840
ggcccccagc agacccagag cttcacctcc caggactggc ccttcctgta ctccctcttc 900
caggtcaact ccaactacgt cctgaacggc ttcagcggcg cccgcctgag caacaccttc 960
cccaacatcg tcggcctccc cggctccacc accacccacg ccctgctggc tgcccgcgtg 1020
aactactccg gcggcatctc ctccggcgac atcggcgcca gccccttcaa ccagaacttc 1080
aactgctcca ccttcctccc ccccctgctg acccccttcg tgcgctcctg gctcgactcc 1140
ggctccgacc gcgagggcgt cgccaccgtc accaactggc agaccgagag cttcgagacc 1200
accctgggcc tgaggtccgg cgccttcacc gctcgtggca acagcaacta cttccccgac 1260
tacttcatcc gcaacatctc cggcgtcccc ctcgtcgtgc gcaacgagga cctgcgcagg 1320
cccctgcact acaacgagat ccgcaacatc gcctccccca gcggcacccc cggcggcgcc 1380
cgtgcctaca tggtgtccgt ccacaaccgc aagaacaaca tccacgccgt ccacgagaac 1440
ggctccatga tccacctcgc tcccaacgac tacaccggct tcaccatcag ccccatccac 1500
gccacccagg tcaacaacca gacccgcacc ttcatctccg agaagttcgg caaccagggc 1560
gacagcctga ggttcgagca gaacaacacc accgcccgct acaccctgcg cggcaacggc 1620
aactcctaca acctctacct gcgtgtgtcc tccatcggca acagcaccat ccgcgtcacc 1680
atcaacggca gggtgtacac cgccaccaac gtcaacacca ccaccaacaa cgacggcgtc 1740
aacgacaacg gcgcccgctt cagcgacatc aacatcggca acgtggtcgc ttcctccaac 1800
tccgacgtcc ccctggacat caacgtgacc ctcaactccg gcacccagtt cgacctgatg 1860
aacatcatgc tggtccccac caacatcagc cccctctact aa 1902
Claims (10)
1. A method for arranging insect-resistant transgenic plant refuge comprises the following steps:
1) preparing shelter hybrid and transgenic insect-resistant plant hybrid;
the refuge hybrid, which contains a unidirectional cross-incompatibility gene that makes the plant unidirectional cross-incompatible, and is a plant hybrid that is not insect-resistant;
the transgenic insect-resistant plant hybrid comprising an insect-resistant gene that renders the plant insect-resistant;
the refuge hybrid and the transgenic insect-resistant plant hybrid have the same gene background except for the insect-resistant gene and the unidirectional cross-incompatible gene;
2) and mixing the seeds of the refuge hybrid seeds with the seeds of the transgenic insect-resistant plant hybrid seeds, and sowing to realize the refuge setting of the insect-resistant transgenic plants.
2. The method of claim 1, wherein:
the refuge hybrid is prepared by introducing the unidirectional hybridization incompatibility gene into a non-insect-resistant plant to obtain a plant hybrid which contains the unidirectional hybridization incompatibility gene and is not insect-resistant;
or, the transgenic insect-resistant plant hybrid is obtained by introducing the insect-resistant gene into the non-insect-resistant plant.
3. The method of claim 2, wherein: the introduction is by backcross transformation or other biological methods.
4. The method of claim 3, wherein:
the introduction of the unidirectional cross-incompatibility gene into the non-insect-resistant plant is achieved by backcrossing and transferring a plant containing the unidirectional cross-incompatibility gene and the non-insect-resistant plant;
or, the non-insect-resistant plant is a non-insect-resistant transgenic plant or a non-insect-resistant non-transgenic plant.
5. The method according to any one of claims 1-4, wherein:
in the step 2), the mixing proportion is set according to the actual requirements of different corn ecological regions.
6. The method according to any one of claims 1-5, wherein: the mixing proportion is less than or equal to 50 percent of the mass of the seeds of the shelter hybrid seeds in the mixed seeds.
7. The method according to any one of claims 1-5, wherein: the mixing proportion is 5-50% of the seeds of the shelter hybrid seeds in percentage by mass of the mixed seeds.
8. The method according to any one of claims 1-7, wherein: the one-way cross incompatibility gene is any gene capable of playing a role in one-way cross incompatibility;
or the one-way cross-incompatible gene is from an animal, plant or microorganism;
or the insect-resistant gene is an insecticidal gene derived from the brevibacterium thuringiensis or other insecticidal genes.
9. Use of a hybrid of a plant containing a unidirectional cross-incompatibility gene and which is not insect-resistant according to any of the methods of claims 2 to 8 in at least one of the following 1) to 3):
1) the pest resistance to insecticidal protein is avoided in the planting of the insect-resistant transgenic plant;
2) setting up an insect-resistant transgenic plant shelter;
3) refuge the insect-resistant transgenic plant;
or, a method of producing a refuge from a transgenic insect-resistant plant, comprising crossing a refuge produced in step 1) of any of the methods of claims 1-8;
or, a method of refuge of a transgenic insect-resistant plant by providing refuges of a transgenic insect-resistant plant according to the method of any of claims 1-8;
or the use of the one-way cross-incompatibility gene of any of the methods of claims 1-8 in the setting of refuge in insect-resistant transgenic plants.
10. The method of any one of claims 1 to 8 or the use of claim 9, wherein: the shelter is suitable for planting insect-resistant transgenic plants in any country around the world.
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| CN201910812771.3A CN112438198A (en) | 2019-08-30 | 2019-08-30 | Application of cross incompatibility gene in preparation of insect-resistant transgenic corn shelter |
| PCT/CN2020/107656 WO2021036741A1 (en) | 2019-08-30 | 2020-08-07 | Use of hybrid incompatibility gene in preparation of insect resistant transgenic corn refuge |
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| CN201910812771.3A CN112438198A (en) | 2019-08-30 | 2019-08-30 | Application of cross incompatibility gene in preparation of insect-resistant transgenic corn shelter |
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Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102317461A (en) * | 2009-02-19 | 2012-01-11 | 先锋国际良种公司 | Mixing No Attack Area through the manipulation of cenospecies production period is carried out is disposed |
| CN102458093A (en) * | 2009-05-03 | 2012-05-16 | 孟山都技术有限公司 | Systems and processes for combining different types of seeds |
| US20130036520A1 (en) * | 2011-08-05 | 2013-02-07 | Dow Agrosciences Llc | USE OF DIG3 INSECTICIDAL CRYSTAL PROTEIN IN COMBINATION WITH Cry1Ab FOR MANAGEMENT OF RESISTANCE IN EUROPEAN CORNBORER |
| CN104313036A (en) * | 2014-09-19 | 2015-01-28 | 中国农业大学 | Insect resistant gene mCry2Ab and application thereof |
| CN107249297A (en) * | 2015-02-18 | 2017-10-13 | 先锋国际良种公司 | The effect that enhancing blending is protected |
| CN107383177A (en) * | 2017-08-16 | 2017-11-24 | 中国农业大学 | The artificial synthesized Bt killing genes mcry1Ab for transgenic anti-insect plants |
| CN108329383A (en) * | 2018-02-08 | 2018-07-27 | 中国科学院遗传与发育生物学研究所 | A kind of and not affine relevant albumen of corn hybridization and its encoding gene and application |
| CN108794610A (en) * | 2018-06-21 | 2018-11-13 | 中国科学院遗传与发育生物学研究所 | Corn hybridization is not affine GAP-associated protein GAP ZmGa1S and its encoding gene and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| MX2009005285A (en) * | 2006-12-22 | 2009-05-28 | Pioneer Hi Bred Int | Resistance management strategies. |
| AU2011242578B2 (en) * | 2010-04-23 | 2016-04-21 | Dow Agrosciences Llc | Combinations including Cry34Ab/35Ab and Cry3Aa proteins to prevent development of resistance in corn rootworms (Diabrotica spp.) |
| CA2886104A1 (en) * | 2012-09-20 | 2014-03-27 | E.I. Du Pont De Nemours And Company | Management of corn rootworm and other insect pests |
-
2019
- 2019-08-30 CN CN201910812771.3A patent/CN112438198A/en active Pending
-
2020
- 2020-08-07 WO PCT/CN2020/107656 patent/WO2021036741A1/en active Application Filing
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102317461A (en) * | 2009-02-19 | 2012-01-11 | 先锋国际良种公司 | Mixing No Attack Area through the manipulation of cenospecies production period is carried out is disposed |
| CN102458093A (en) * | 2009-05-03 | 2012-05-16 | 孟山都技术有限公司 | Systems and processes for combining different types of seeds |
| US20130036520A1 (en) * | 2011-08-05 | 2013-02-07 | Dow Agrosciences Llc | USE OF DIG3 INSECTICIDAL CRYSTAL PROTEIN IN COMBINATION WITH Cry1Ab FOR MANAGEMENT OF RESISTANCE IN EUROPEAN CORNBORER |
| CN104313036A (en) * | 2014-09-19 | 2015-01-28 | 中国农业大学 | Insect resistant gene mCry2Ab and application thereof |
| CN107249297A (en) * | 2015-02-18 | 2017-10-13 | 先锋国际良种公司 | The effect that enhancing blending is protected |
| BR112017017703A2 (en) * | 2015-02-18 | 2018-04-10 | Pioneer Hi Bred Int | corn seed mix, method of reducing pollination incidence among plants grown from different seed types in a corn seed mix and insect resistance management method |
| CN107383177A (en) * | 2017-08-16 | 2017-11-24 | 中国农业大学 | The artificial synthesized Bt killing genes mcry1Ab for transgenic anti-insect plants |
| CN108329383A (en) * | 2018-02-08 | 2018-07-27 | 中国科学院遗传与发育生物学研究所 | A kind of and not affine relevant albumen of corn hybridization and its encoding gene and application |
| CN108794610A (en) * | 2018-06-21 | 2018-11-13 | 中国科学院遗传与发育生物学研究所 | Corn hybridization is not affine GAP-associated protein GAP ZmGa1S and its encoding gene and application |
Non-Patent Citations (4)
| Title |
|---|
| FEI YANG等: "Pollen contamination in seed mixture increases the dominance of resistance to Bt maize in Spodoptera frugiperda (Lepidoptera: Noctuidae)", 《PEST MANAGEMENT SCIENCE 》 * |
| SCHNABLE PS等: "Zea mays chromosome 4 clone CH201-396P14", 《EMBL-EBI DATABASE》 * |
| 刘旭: "玉米单向异交不亲和基因Ga1的遗传分析及精细定位", 《中国优秀博硕士学位论文全文数据库(博士) 农业科技辑》 * |
| 赵贤容等: "玉米单向异交不亲和基因研究进展及其在玉米育种上的应用", 《玉米科学》 * |
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