CN1226924C - Bt-transgene pest-resistant cotton modifier and application thereof - Google Patents
Bt-transgene pest-resistant cotton modifier and application thereof Download PDFInfo
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- CN1226924C CN1226924C CN 200410037505 CN200410037505A CN1226924C CN 1226924 C CN1226924 C CN 1226924C CN 200410037505 CN200410037505 CN 200410037505 CN 200410037505 A CN200410037505 A CN 200410037505A CN 1226924 C CN1226924 C CN 1226924C
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Abstract
The present invention belongs to the field of pesticides, which relates to a Bt-transgene pest-resistant cotton regulator and the application thereof; more specifically, the present invention provides a regulator containing amino acid, a plant growth regulator and a gene activator, and the regulator can be used for preparing a seed coating agent or be sprayed to the leaf surface of transgenic cotton after being diluted. The regulator can overcome the defect that the Bt-transgene pest-resistant cotton has unstable pest resistance; simultaneously, the regulator regulates and optimizes the morphological index and the yield component index of a cotton plant and finally achieves the goals of enhancing yield and quality at the time of enhancing the pest resistance.
Description
[technical field]
The invention belongs to pesticide field, relate to a kind of soil of transgenic Bt cotton conditioning agent and application thereof.
[background technology]
Soil of transgenic Bt cotton resistance to insects instability, show as, there is significant difference in the effect that different parts organ and different growing blade are resisted cotton bollworm harm, at first, at Bt cotton plant different parts blade different (Shu Chune, 1998, cotton journals with Different Organs poisoning protein content, 1998,10 (1): 301-306; Cui Jinjie, 1999 cotton journals, 1999,11 (3): 141-146; Dong Zhiqiang, cotton journal, 2000,12 (3): 113-117); Secondly, along with the propelling of cotton development stage, the expression intensity of Bt gene in blade weakens (Chen Song, cotton journal, 2000,12 (4): 189-193) gradually; And under environment stresses such as high temperature, arid, the cotton exogenous gene expression of Bt exists inactivation and gene " silence " phenomenon (Yang Jinshui, biotechnology progress, 1995,15 (3): 41-45; Kaiser, Science, 1996,273:423).The unstable situation of resistance to insects very easily causes the generation of bollworm resistance colony.Indoor eliminating selected evidence, and cotton bollworm is remarkable ascendant trend (Xia Jingyuan, cotton journal, 1999,11 (2): 57-64) to the resistance of desinsection toxalbumin along with eliminating the increase of selecting number of times; Feed continuously with the cotton blade of Bt and to raise cotton bollworm, through 12-18 generation eliminate choosing after, the resistance to insects of Bt cotton almost thoroughly disappear (Guo Sandui, Scientia Agricultura Sinica, 1999,32 (3): 1-7).Thereby, if fundamentally do not solve the unstable problem of expressing of the cotton resistance to insects of Bt, will be more serious to the harm that Cotton Production causes.The at present general ancillary method that sprays chemical insecticide or spray the Bt biological bactericide that adopts is is in time prevented and treated according to index for control.Like this, both run counter to the original intention of soil of transgenic Bt cotton invention, and caused the cotton grower simultaneously, increased planting cost simultaneously in operational trouble, also unfavorable to environmental protection.
[summary of the invention]
[technical problem that will solve]
The conditioning agent product that the purpose of this invention is to provide a kind of scientific formulation, increase soil of transgenic Bt cotton resistance to insects easy to prepare, in order to overcome the unsettled defective of soil of transgenic Bt cotton resistance to insects, regulate morphological index and the output composing indexes of optimizing cotton plant simultaneously, finally reach when increasing resistance to insects the purpose that improves the yield and quality.Simultaneously, this conditioning agent can be applicable to spray in transgene cotton blade face agricultural chemicals, coating agent for seed.
[technical scheme]
Conditioning agent of the present invention has been introduced chemicals that promotes Bt gene expression and the plant growth regulator that improves protein stability, and is aided with the plant growth regulator of regulating cotton morphological index and yield index.
In the described conditioning agent, amino acid 4.90～5.10%: amino acid can be alleviated the competition between conventional albumen and the insecticidal proteins, produces the Bt insecticidal proteins of q.s in the cotton difference is grown period and different parts blade;
Amino acid of the present invention is amino acid additive common on the market, generally contain several amino acids, join new chemical industry Co., Ltd's agricultural amino acid (pulvis) product as Zhengzhou, Henan, contain 17 seed amino acid principal components, total amino acid content 〉=50%, moisture≤2.0%, ash content, salinity, alkali branch, ammonia content≤2.0%;
Table 1: aminoacid ingredient content (%)
| The amino acid title | Content (%) |
| Threonine aspartic acid serine glutamic acid cystine proline glycine alanine lysine | 3.56％ 3.45％ 4.49％ 9.12％ 2.45％ 3.50％ 2.43％ 2.20％ 2.30％ |
| Histidine valine methionine isoleucine leucine tyrosine phenylalanine arginine | 0.90％ 3.10％ 0.23％ 1.70％ 2.80％ 1.02％ 1.78％ 5.20％ |
Plant growth regulator accounts for 4.90%～5.10%: wherein, promote that cell chromosome duplicates, improve cell sap concentration, the plant growth regulator of regulating cotton morphological index and yield component comprises two classes: the first kind: selected chlormequat (chlormequat, CCC), first piperazine (mepiquat-chloride, Pix), uniconazole P (uniconazole, PP
333), daminozide (daminozide, B9), preferred chlormequat and first piperazine, in conjunction with yield traits and practicality, the first piperazine be an optimum.Second class: the basic element of cell division (cytokinin), 2-chloro-4-pyridine radicals-3-phenylurea (forchlorfenuron, 4PU-30), brassinosteroid (brassinolide, BR) and 6-benzyl aminopurine (accel, 6-BA), the preferred oil rape lactone is optimum.
Wherein, first piperazine (Pix): molecular formula C
7H
8CLN, molecular weight 149.7, pure product are white crystals, and are soluble in water, are insoluble in acetone, ethyl acetate.The first piperazine suppresses the biosynthesis of red enzyme element, suppresses cell elongation, and dwarfed plant improves assimilational capacity, promotes maturation, increases output.
Brassinosteroid (BR): molecular formula C
28H
48O
6, molecular weight 480, fusing point 274-275 ℃, pure product are white crystalline powder, are soluble in methyl alcohol and ethanol.Promote the elongation and the division of cell, promote photosynthesis, improve resistance.
Gene activation accounts for 25%～50%: select 5-aminosalicylic acid (5-AminosalicylicAcid), salicylic acid (Salicylic Acid) and aspirin (Aspirin) for use, preferred salicylic acid.
Wherein, salicylic acid (SA): C
7H
6O
3, molecular weight: 138.12, fusing point: 159 ℃, 211 ℃ of boiling points (20mmHg), 76 ℃ of distillations, white needle-like crystals is slightly soluble in water, is soluble in organic solvent and hot water.Sharply phenol and carbonic acid gas are resolved in heating.Salicylic acid can promote the exogenous Bt expression of gene.Qin (The Plant Cell, 1994, in the promotor that research report trans Bt gene cotton 6:863-874) is used one section asl sequence that activated by salicylic acid is arranged, Gatz (Current opinion in Biotech., 1996,7:168-172) research points out that salicylic acid can be induced genetically modified expression.
Also can contain conventional activating agent and spreader-sticker in the conditioning agent of the present invention, concentration generally accounts for 1.95～2.05%: select TritonX (Triton) and tween (Tween) class for use, be preferably Tweens, comprise polysorbas20, polysorbate40, polysorbate60, Tween 80 and polysorbate85.
Surplus is a solvent, and the present invention is preferably water, methyl alcohol and ethanol, and three's volume ratio is 1: 11～17: 4～6.
In above-mentioned prescription, salicylic acid can activate Bt foreign gene promotor, promotes duplicating of exogenous Bt gene, exogenous Bt gene normal expression in the cotton difference is grown period and different parts blade, the Bt insecticidal proteins of generation q.s; Brassinosteroid can improve cytoactive, increases the stability of Bt insecticidal proteins in the cotton body, guarantees that the Bt insecticidal proteins keeps Stability Analysis of Structures in adverse circumstance; Increase the pest-resistant stability of soil of transgenic Bt cotton.The first piperazine can increase cytoplasmic concentration, promote duplicating of cell chromosome, simultaneously, can optimize the morphological index (comprising plant height, blade size, leaf area, leaf inclination angle etc.) and yield index (comprising into bell position, one-tenth bell number, bell weight and lint percentage etc.) of regulating cotton.In concrete enforcement, three kinds of components have significant synergy.Selecting first piperazine, salicylic acid and brassinosteroid is key component, 0,1,2 three concentration gradient is set, and (concentration gradient sees Table 2, the amino acid of adding 5%), carrying out permutation and combination, amount to 26 and handle 1 contrast, serves as the test kind with soil of transgenic Bt cotton GK-12 (following general designation Bt cotton), indoor seed soaking 12 hours, sand culture, after emerging, pouring Hogland complete nutrition liquid.Gather in the crops tri-leaf period, measures the content of toxalbumin in the true leaf.As shown in table 3, the result shows that three kinds of components have significant synergy, and toxalbumin content has increased by 98.98%.
Table 2: first piperazine (PIX), salicylic acid (g) and brassinosteroid (B) concentration gradient table (concentration: ppm)
| Code | B | PIX | S |
| 0 1 2 | 0 0.02 0.04 | 0 100 200 | 0 500 1000 |
Table 3, first piperazine (P), salicylic acid (g) and the experiment of brassinosteroid (B) synergy
| Handle | CK | S1 | S2 | P1 | P2 | B1 | B2 | B1P1S1 |
| Toxalbumin content (ng.g-1FW) | 52.83 | 60.15 | 64.66 | 45.76 | 33.08 | 28.46 | 60.85 | 105.12 |
| Comparison is according to increasing (%) | +13.86 | +22.39 | -13.38 | -37.38 | -46.12 | +15.19 | +98.98 |
The collocation method of described conditioning agent is: with the water-soluble retired piperazine of formula rate, then with ethanol difference dissolved oil rape lactone and salicylic acid, use dissolve with ethanol amino acid, press formula rate afterwards and mix, use methanol constant volume.
This conditioning agent can all can be used in first flower bud phase of soil of transgenic Bt cotton, full-bloom stage and Sheng bell phase, converted water when using and was diluted to 300～700 times of liquid, sprayed in the blade face then.
The present invention simultaneously also provides a kind of coating agent for seed, and the preparation method according to the seed coating medicine of routine prepares by the above-mentioned conditioning agent that makes.
The application of seed coating medicine matches with foliage-spray and carries out, and it is time bar about 40 days on the 30th 20-May of April that the effect of seed coating medicine is limited to after planting before budding.
If without seed coating medicine, also will do seed soaking and handle before the sowing with the seed-soaking liquid of conditioning agent configuration of the present invention.
[beneficial effect]
Conditioning agent of the present invention is a kind of product that strengthens resistance to insects and improve the output dual-use function that has, it have nontoxic, have no side effect, cost is low, characteristics such as easy to use.Again because the prescription of this product is novel unique, once use and to play multiple efficacies, the anti-agent that increases of comparing the plant growth regulator of existing adjusting cotton morphological index and yield index and strengthening resistance to insects, have small investment, sphere of action wide, can improve the soil of transgenic Bt cotton resistance to insects comprehensively and improve the advantage of output, thereby the present invention production has positive impetus to Cotton in China.Observe in the multiple spot demonstration test of Hebei province's Xingtai City and Hengshui City, Henan Province, Shanxi Province, Shandong Province according to the present patent application people, use this product after, insecticidal proteins content improves 96%～283% in the soil of transgenic Bt cotton blade, output improves more than 30%.
1. activate the Bt killing gene, promote Bt killing gene normal expression in adverse circumstance, strengthen cell viability, promote the synthetic of insecticidal proteins, thereby reduced the chemical medication number of times and the dosage in Bt cotton field.
2. promote cotton root system to grow, downgrade plant height, shorten length of internode, improve plant type, coordinate to nourish and grow and the ratio of reproductive growth, for the cotton high yield of Bt is put up the high yield shelf.
3. impel cotton knot bell to concentrate, the knot bell is many, and the knot bell is big, the next and in to enclose bell many, make the cotton boll blowing smoothness, quality better output height.
4. improve the cotton chlorophyll content in leaf blades of Bt, prolong leaf longevity, strengthen photosynthetic intensity, improve cotton quality, the flower ratio improves cotton quality before the increase frost.
Adopt not limited embodiment to be enumerated explanation hereinafter.
[embodiment]
Plant growth regulator of the present invention is selected amino acid (Zhengzhou, Henan connection letter chemical industry Co., Ltd for use, registration card number: LD2021), (the State Scientific and Technological Commission Beijing rural area technology development co. of doing jointly of first piperazine, registration card number: LS91328) and brassinosteroid (the big scientific and technological industry of Kunming cloud limited company, agriculture chemical registration card number: LS94573), gene activation is selected salicylic acid (flood Xinghua, Jiangsu Province factory for use, the pharmaceutical grade registration card number: BP93198), polysorbas20 is as activating agent and spreader-sticker.
Embodiment 1
At first, with 48ml water-soluble retired piperazine 50g, with 10ml dissolve with ethanol brassinosteroid 0.01g, 150ml dissolve with ethanol salicylic acid 250g, 60ml dissolve with ethanol amino acid 50g mixes above-mentioned solution afterwards, adds the 20ml polysorbas20, with methanol constant volume to 1000ml.
Embodiment 2
At first, with 48ml water-soluble retired piperazine 50g, with 10ml dissolve with ethanol brassinosteroid 0.008g, 150ml dissolve with ethanol salicylic acid 300g, 60ml dissolve with ethanol amino acid 50g mixes above-mentioned solution afterwards, adds the 20ml polysorbas20, with methanol constant volume to 1000ml.
Embodiment 3
At first, with 48ml water-soluble retired piperazine 50.5g, with 10ml dissolve with ethanol brassinosteroid 0.008g, 150ml dissolve with ethanol salicylic acid 350g, 60ml dissolve with ethanol amino acid 49g mixes above-mentioned solution afterwards, adds the 20ml polysorbas20, with methanol constant volume to 1000ml.
Embodiment 4
At first, with 48ml water-soluble retired piperazine 49.5g, with 10ml dissolve with ethanol brassinosteroid 0.012g, 150ml dissolve with ethanol salicylic acid 400g, 60ml dissolve with ethanol amino acid 51g mixes above-mentioned solution afterwards, adds the 20ml polysorbas20, with methanol constant volume to 1000ml.
Embodiment 5
At first, with 48ml water-soluble retired piperazine 50g, with 10ml dissolve with ethanol brassinosteroid 0.01g, 150ml dissolve with ethanol salicylic acid 450g, 60ml dissolve with ethanol amino acid 50g mixes above-mentioned solution afterwards, adds the 20ml polysorbas20, with methanol constant volume to 1000ml.
Embodiment 6
At first, with 48ml water-soluble retired piperazine 50g, with 10ml dissolve with ethanol brassinosteroid 0.01g, 150ml dissolve with ethanol salicylic acid 500g, 60ml dissolve with ethanol amino acid 50g mixes above-mentioned solution afterwards, adds the 20ml polysorbas20, with methanol constant volume to 1000ml.
Embodiment 7 foliage-spray conditioning agents
The foliage-spray conditioning agent can be used in first flower bud phase of soil of transgenic Bt cotton, full-bloom stage and Sheng bell phase, when using the conditioning agent of embodiment 2 was converted water and was diluted to 500 times of liquid, sprayed in the blade face then.
Embodiment 8 coating agent for seed
Embodiment 3 gained conditioning agents----------------------------2ml
Polyvinylpyrrolidone-------------------------------300 grams
Carmine---------------------------------------0.1 gram
Collocation method is: with water-soluble polyvinylpyrrolidone and the famille rose separated of 600ml, add conditioning agent afterwards, be settled to 1000ml with water.
Seed coating medicine can be to carrying out Cotton seeds before the soil of transgenic Bt cotton sowing, and the 1000ml conditioning agent can dressing 100kg seed.
Experimental example 1
1. this test and Selection first piperazine (Pix), salicylic acid (SA) and brassinosteroid (B) are key component, 0,1,2 three concentration gradient is set, and (concentration gradient sees Table 2, the amino acid of adding 5%), carrying out permutation and combination, amount to 26 and handle 1 contrast, serves as the test kind with soil of transgenic Bt cotton GK-12 (following general designation Bt cotton), indoor seed soaking 12 hours, sand culture, after emerging, pouring Hogland complete nutrition liquid.Gather in the crops tri-leaf period, measures the content of toxalbumin in the true leaf.The result shows: have 16 processing to have positive-effect in 27 processing, the content increasing degree of Bt albumen is 4.70%-190.63%, wherein B1P1S0, B1P2S0, B1P1S1, B2P1S0 and five of B2P1S2 are treated to the first echelon, and Bt protein content amplification is between 96.97%-190.63%.Second echelon's amplification is between 41.33%-70.37%.Third echelon amplification is between 4.70%-28.60%.In view of the above, choose B1P1S0, B1P2S0, B1P1S1, B2P1S0 and B2P1S2 processing and carry out field experiment.
2. being test material with soil of transgenic Bt cotton GK-12, arranging field trial, B1P1S0, B1P1S1, B1P2S0, B2P1S0 and B2P1S2 system chemical control are set handle, is contrast with the clear water.Broadcasting preceding seed soaking (200 μ g/g) and containing flower bud phase (07-20,150 μ g/g) foliage-spray respectively.Small plot experiment, randomized arrangement, three repetitions; Sub-district area 11m * 4.8m.Size row plantation (50cm, 100cm), spacing in the rows 22.22cm, density 52,500 strain/hectares.The result shows: 1. B1P1S0, B1P2S0, B1P1S1, B2P1S0 four kinds of processing seed cotton yield and lint yield are higher than contrast, yield component is that single-strain bell-forming number, Dan Lingchong, lint percentage all are higher than contrast, and 1-2 really saves with 1-10 fruit branch high-quality knot bell position bolling rate and is higher than contrast.B2P1S2 handles the difficulty of emerging, but yield component all is higher than contrast.2. B1P1S0, B1P2S0, B1P1S1, B2P1S2, five processing of B2P1S0 seedling flower bud phase, full-bloom stage and Sheng bell phase to spire, functional leaf and old and feeble blade in most of period of content of Bt albumen be synergistic effect, it is the most outstanding that wherein B1P1S1, B1P2S0 and B2P1S2 handle performance, is synergistic effect substantially in 3 breeding times, 3 class blades.
3. biology measurement result: respectively at June 20 (seedling flower bud phase), July 20 (full-bloom stage) and August 20 (containing the bell phase) getting different disposal spire, functional leaf, old and feeble blade and bract, flower bud, the young bell of flower (petal, calyx, stamen and gynoecium), the indoor feeding first-instar young, observing larval growth grows and death state, the result shows: 1. soil of transgenic Bt cotton significantly increases at seedling flower bud phase insecticidal effect under B1P1S0, B1P2S0, B1P1S1, B2P1S2, five kinds of processing of B2P1S0, and the lethality of cotton bollworm is 100%.2. full-bloom stage and Sheng bell phase, the pest-resistant effect of soil of transgenic Bt cotton blade resistance to insects and output organ is the most outstanding with B1P1S1 and B2P1S2 processing performance under B1P1S0, B1P2S0, B1P1S1, B2P1S2, five kinds of processing of B2P1S0.Yet B2P1S2 handles seed soaking and handles and cause the cotton difficulty of emerging, and foliage-spray burns seedling, and the therefore final B1P1S1 of determining is for increasing the Chemical Regulation agent prescription of transgenic cotton against pests resistance to insects.
Table 4, output and output constitute table
| Handle | Average single-strain bell-forming number (individual) | Average bell heavy (g) | Average seed cotton yield (Kg.hm 2) | Average lint yield (Kg.hm 2) | Lint percentage (%) |
| CK B1P1S1 | 16.64 31.00 | 5.37 6.68 | 2405.25 3114.45 | 915.15 1250.4 | 38.05 40.15 |
Claims (6)
1, soil of transgenic Bt cotton conditioning agent, this conditioning agent contains following components in weight percentage:
Amino acid 4.90～5.10%;
Plant growth regulator 4.90%～5.10%;
Gene activation 25%～50%;
Wherein, described plant growth regulator is first piperazine and brassinosteroid, and the gene activity agent is a salicylic acid.
2, conditioning agent as claimed in claim 1, wherein first piperazine and brassinosteroid, both weight ratios are 5000: 0.8～1.2.
3, the method for preparation claim 1 or 2 described conditioning agents, this method may further comprise the steps: water dissolving chlormequat chloride, with ethanol difference dissolved oil rape lactone and salicylic acid, use dissolve with ethanol amino acid, mix afterwards, use methanol constant volume.
4, claim 1 or 2 described conditioning agents are used to prepare the application that sprays in the agricultural chemicals on transgene cotton blade face.
5, claim 1 or 2 described conditioning agents are used to prepare the application of the soak that soaks the transgenic cotton flower seed.
6, claim 1 or the 2 described conditioning agents application that is used to prepare coating agent for seed.
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| CN 200410037505 CN1226924C (en) | 2004-04-23 | 2004-04-23 | Bt-transgene pest-resistant cotton modifier and application thereof |
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|---|---|---|---|
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| CN103688934B (en) * | 2013-12-18 | 2016-05-04 | 重庆市优胜科技发展有限公司 | Promote crop yield dry to improve antiviral multifunctional preparation of local flavor and preparation method thereof |
| CN105010346B (en) * | 2015-07-15 | 2018-01-12 | 四川国光农化股份有限公司 | A kind of plant growth regualting composition and its preparation and application |
| CN105409619B (en) * | 2015-11-19 | 2018-07-20 | 中国农业科学院棉花研究所 | A method of improving transgenic Bt cotton external source insecticidal proteins content |
| CN106359381A (en) * | 2016-08-25 | 2017-02-01 | 四川国光农化股份有限公司 | Plant growth regulation composition, preparation therefrom, and application thereof |
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