CN113789351A - Method for improving exogenous DNA (deoxyribonucleic acid) conversion efficiency of corn - Google Patents
Method for improving exogenous DNA (deoxyribonucleic acid) conversion efficiency of corn Download PDFInfo
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Abstract
The invention discloses a method for improving the efficiency of transforming exogenous DNA into corn. After the corn is artificially pollinated, removing bracts and all filaments in the female ear, covering a sealed container, fixing, turning over the whole corn plant, fixing or hanging, adding exogenous DNA solution into the sealed container, soaking the female ear without filaments in the DNA solution, and finally restoring the corn plant. The method has the advantages of long soaking time of the exogenous DNA solution, short introduction path, full utilization of pollen tube channels, easy operation and low cost, and can improve the efficiency of transforming exogenous DNA into corn.
Description
Technical Field
The invention belongs to the field of plant transgenic breeding, and particularly relates to a method for improving the efficiency of exogenous DNA (deoxyribonucleic acid) transformation of corn by reversely soaking female ears and utilizing a pollen tube channel.
Background
Chinese scientists put forward a breeding idea of transforming crops by using pollen tube channels for the first time in 1978, and succeeded in cotton. This technology has been rapidly developed and widely used in crop breeding, and has achieved remarkable results. The main principle of the pollen tube channel transformation method is that after pollination, exogenous DNA can permeate along the pollen tube and enter embryo sac through nucellar channel to transform ovum, zygote or early embryo cell without normal cell wall. The pollen tube channel method utilizes the unique advantage that the reproductive system of the pollen tube channel method is used as a carrier, overcomes some obstacles of distant hybridization, enters partial DNA fragments or target genes into a receptor genome, is easy to integrate, and the characters controlled by a transfer gene are easy to stabilize in a receptor, thereby being a simple, convenient and rapid breeding way. The channel of pollen tube is used to introduce exogenous DNA into plant, and currently, the injection method and the dropping method are mainly used. The injection method is that at a certain time after pollination, that is, during the time when the pollen tube passes through pistil tissue to reach embryo sac after pollen germinates, exogenous DNA is injected into the tissue with pollen tube channel or the inside of the tissue by using an injector, so that the DNA enters and is integrated into genome, and then transformed seeds are obtained through natural fructification. The dropping method is also that after pollination, in a certain time period, namely in the time period that the pollen tube passes through pistil tissue and reaches embryo sac after pollen germinates, the style is cut off, exogenous DNA is dropped on the section, or the style is not cut off, exogenous DNA is directly dropped on the stigma, so that DNA enters the embryo sac along the pollen tube channel and is integrated into the genome of zygote, and then transformed seeds are obtained through natural fructification. The pollen tube channel transformation technology is suitable for all sexual propagation angiosperms, does not depend on a tissue culture technology, is simple to operate, does not need expensive instruments and equipment, and can directly obtain transformed seeds, so that the pollen tube channel transformation technology is popular with people. Corn is one of the more crops transformed using the pollen tube pathway, and has been developed: pollen tube channel transformation methods such as stigma dropping method, pollen particle carrying method, ovary microinjection method, and bract opening method. However, these methods either have too long a path for the exogenous DNA to enter the embryo sac along the filament, or have too short a contact time with the exogenous DNA, and the transformation efficiency is very low, which severely restricts the application development. How to further increase the introduction rate of the foreign DNA will be the key point of long-term research in the future.
Disclosure of Invention
In view of the above-mentioned drawbacks and deficiencies of the prior art, it is an object of the present invention to provide a method for improving the efficiency of transforming exogenous DNA into corn by inverting soaked ears and using pollen tube channels.
The invention relates to a method for improving the efficiency of exogenous DNA (deoxyribonucleic acid) conversion of corn by reversely soaking female ears and utilizing a pollen tube channel, which is realized by the following technical scheme: after the corn is artificially pollinated, removing bracts and all filaments in the female ear, covering a sealed container, fixing, turning over the whole corn plant, fixing or hanging, adding exogenous DNA solution into the sealed container, soaking the female ear without filaments in the DNA solution, and finally restoring the corn plant.
Preferably, the corn is potted, and specifically comprises the following steps: the plant is located in the center of the pot, the soil surface is 2-3 cm away from the edge of the pot, 2 half-moon plates with the same size as the pot are placed on the soil surface, then a mesh bag is sleeved outside the pot, and the opening of the bag is fastened.
Preferably, the meniscus is made of one of plastic, foam, wood board and the like, a semicircular depression with the diameter of 1.5-2.5 cm is arranged on the midpoint of the edge with the semicircular diameter, and the mesh bag is made of one of plastic, hemp and the like.
Preferably, in the time period from the time that the corn is artificially pollinated for 16-22 hours, the pollen tube reaches the embryo sac and begins to divide the zygote for the first time, the bracts are longitudinally cut by 3 equal parts, the bracts in the inner layer are removed, the outermost 2-3 layers are remained, and all filaments are removed from the base.
Preferably, the container is a plastic bag, is made of polyethylene plastic, is narrow at the bottom and wide at the opening, is 1.5-2.0 cm wide at the bottom, is 2.0-3.0 cm wide at the opening and is 5.0-7.0 cm long, is properly folded according to the size of the female ear, and is fixed outside the female ear.
Preferably, said whole corn plant is inverted, in particular: and slightly inverting the planting pot and the plants, hooking meshes of the net bag at the bottom of the planting pot by using an S-shaped iron hook, hanging the other end of the hook on a support, and enabling the female ears to be 30-100 cm away from the ground, wherein the leaves cannot be broken.
Preferably, the exogenous DNA solution is added into the sealed container, and specifically: the exogenous DNA solution is added into the plastic bag small container along the gap between the plastic bag and the female ear by a dropper until the plastic bag small container is full.
Preferably, the concentration of the exogenous DNA solution is 300-500 mu g/ml.
Preferably, the soaking time is 20-30 min, and the plastic bag and the female ears are lightly vibrated every 4-6 min.
The invention has the following beneficial effects:
1. the exogenous DNA solution is less in dosage, can be added according to needs in the treatment process, and even if the container covering the female ears is filled, the volume of the added exogenous DNA is only 3-5 ml, and can be recovered after the test is finished;
2. the soaking time can be controlled according to the needs, and the whole female ear is evenly soaked;
3. as the filament is removed from the base part, the exogenous DNA can directly enter the embryo sac from the base part of the filament through the pollen tube channel, and the distance of the exogenous DNA entering the embryo sac is greatly shortened, so that the exogenous DNA introduction path is very short, and the efficiency of transforming the exogenous DNA into the corn is improved;
4. easy operation and low cost.
In summary, the method is long in soaking of the exogenous DNA solution, short in introduction path and capable of improving the efficiency of exogenous DNA transformation of the corn by fully utilizing the pollen tube channel, and is easy to operate and low in cost.
Drawings
FIG. 1: potted corn plantlets;
FIG. 2: bagging the male ear and the female ear when the corn blossoms;
FIG. 3: plastic bags with various specifications special for the female ears are sleeved;
FIG. 4: inverting the plastic mesh bag used for the flowerpot and the corn plant;
FIG. 5: a foam meniscus for blocking mud soil when the flowerpot is inverted;
FIG. 6: removing the corn ears of the bracts and the filaments on the inner layer;
FIG. 7: sleeving the female ears with a special plastic bag and fixing;
FIG. 8: before the plants are inverted, the flowerpot and the plants are wrapped by a foam meniscus and a plastic mesh bag;
FIG. 9: the corn plants are hung on a frame after being inverted;
FIG. 10: adding exogenous DNA solution into the plastic bag to soak the female ears;
FIG. 11: restoring the outer bract of the female ear after soaking;
FIG. 12: seedlings of corn seeds obtained after the dendrobium officinale genome is transformed germinate on a culture medium;
FIG. 13: flowering and seed setting conditions of first-generation corn seeds harvested after the dendrobium officinale genome is transformed in the field;
FIG. 14: detecting an electrophoresis strip of the corn transformed by the saccharum arundinaceum genome DNA by using an Alu-like marker, wherein 1 is a positive control, 2 is a Hainan 92-77 variety (saccharum arundinaceum), 3 is a cliff 96-40 variety (filial generation of saccharum arundinaceum), and 4-23 are T0Transgenic plants are generated, and M is marker.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof. The following examples are given without specifying the particular experimental conditions and methods, and the technical means employed are generally conventional means well known to those skilled in the art.
Example 1: corn transformed by exogenous DNA of dendrobium officinale
Sowing a maize inbred line variety HZ1 in a flowerpot for potting, transplanting the plantlets into a plastic flowerpot with the height of 22cm, the upper inner diameter of 23cm and the lower inner diameter of 17cm when the plantlets grow to the height of 10-15 cm, filling planting soil to a position 2-3 cm away from the upper edge of the flowerpot opening, placing the flowerpot in a place with sufficient sunlight, and carrying out normal water and fertilizer management. The tassels and the female ears are bagged before pollen shedding to prevent pollen bunching (as shown in figure 2). After pollen of the tassel is scattered, artificial pollination is carried out, pollination time is registered, and the tassel is continuously bagged to prevent pollen from being mixed. Then, the method for transforming maize with exogenous DNA of the present invention is performed, comprising the following steps:
(1) preparing 2 foam menisci for blocking mud soil when inverting flowerpots according to the sizes of the flowerpots, wherein a semicircular depression with the diameter of 1.5-2.5 cm is formed in the midpoint of the semicircular diameter edge of each foam meniscus (as shown in figure 5), a plastic mesh bag (as shown in figure 4) used for inverting the flowerpots and corn plants is prepared, small plastic bags (as shown in figure 3) with various specifications and width of 2-3 cm and length of 5-7 cm are specially used for covering female ears, the corn plants are located in the center of the flowerpots, the soil surface is 2-3 cm away from the edge of the flowerpots (as shown in figure 1), 2 foam menisci matched with the sizes of the flowerpots are placed on the soil surface, then a plastic mesh bag is sleeved on the outer side of the flowerpots, and bag openings are tightened;
the dendrobium officinale has the effects of promoting the production of body fluid, nourishing the stomach, nourishing yin, clearing heat, moistening the lung, tonifying the kidney, improving eyesight, strengthening waist and the like, and the dendrobium officinale DNA is introduced into the corn, so that the corn can obtain some excellent properties of the dendrobium officinale, and the nutritional ingredients and the medicinal value of the corn are improved. Extracting the genomic DNA of the dendrobium officinale by adopting a CTAB method, purifying and then storing in a refrigerator for later use. Then preparing a 300-500 mu g/ml exogenous DNA solution (containing 0.02% Silwet L-77), wherein the exogenous DNA is the dendrobium officinale genome DNA.
(2) During the time period from the artificial pollination of the corn for 16-22 hours until the pollen tube reaches the embryo sac and the zygote is split for the first time, the bracts of the female ear are longitudinally split into 3 equal parts by using an operating blade, the bracts of the inner layer are removed, the outermost 2-3 layers are remained, and all filaments are removed from the base (as shown in figure 6);
(3) selecting a plastic bag with proper size, wherein the plastic bag is made of polyethylene plastic, the bottom is narrow and wide, the bottom is 1.5-2.0 cm wide, the mouth is 2.0-3.0 cm wide, and the length is 5.0-7.0 cm, properly folding according to the size of the female ear, covering the female ear with the filaments removed, and stably and fixedly pricking with transparent adhesive, a clip and the like (as shown in figure 7);
(4) flattening the mud of the flowerpot, paving a foam meniscus around the plant base, and sheathing and binding the flowerpot by using a plastic mesh bag (as shown in figure 8);
(5) slightly inverting the flowerpot and the plant, hooking the plastic mesh bag at the bottom of the flowerpot by using an S-shaped iron hook, and then hooking the plastic mesh bag on a frame by using the iron hook, wherein the distance between the female ear and the ground is 30-100 cm, and the leaves cannot be broken (as shown in figure 9);
(6) dripping the exogenous DNA solution into the plastic bag along the gap between the plastic bag and the filament-free female ear by using a dropper until the plastic bag is filled with the exogenous DNA solution, wherein the volume of the added exogenous DNA solution is 3-5 ml (shown in figure 10);
(7) soaking the filament-free female ears in the exogenous DNA solution for 30 minutes, and slightly vibrating the plastic bag and the female ears every 5 minutes;
(8) after soaking, loosening the plastic bag from the female ear, recovering the exogenous DNA solution in the plastic bag, taking down the flowerpot and the plant from the shelf, and recovering (as shown in figure 11);
(9) after the soaked female ears are dried in the air, the bracts are recovered, and are bound by a rubber, and bagging is continued to prevent the powder from being mixed;
(10) timely harvesting and drying the seeds for later use after the seeds are mature, and then transforming the dendrobium officinale genome to obtain corn seeds which are subjected to MS (Mass Spectrometry) without sugar or growth regulator0Germination is carried out on the culture medium, the germinated plantlets are shown in figure 12, and the transformed plantlets are morphologically very different from a test variety HZ 1: when a leaf just grows from the transformed seedling, a node grows, the leaf is not naturally opened, the boundary between the leaf sheath and the leaf is not obvious, and the whole trumpet-shaped leaf is purple red; the test HZ1 seedlings are not subjected to node pulling, only pseudostems are provided, the pseudostems are formed by leaf sheaths, the leaf sheaths and the leaves are obviously demarcated, and the leaves are naturally opened and are green;
the first generation corn seeds harvested after the dendrobium officinale genome transformation successfully bloom and seed in the field, as shown in fig. 13, the transformed seedlings grow slowly, the plant height is 20cm after emergence of seedlings, only 3 pieces of corn seeds begin to sprout, the female ears are basically stuck to the ground, and only 10-15 seeds are formed; the tested variety HZ1 generally begins to sprout, and the ear shoots, the female ears, the male ears, the fruit sets and the like after 13-19 leaves grow 40-48 days after the seedling.
Example 2: corn transformed by exogenous DNA of stipa arundinacea
The method comprises the steps of sowing a maize inbred line variety WYS2 in a flowerpot for potting, transplanting seedlings into a plastic flowerpot with the height of 22cm, the upper inner diameter of 23cm and the lower inner diameter of 17cm when the seedlings grow to the height of 10-15 cm, filling planting soil to a position 2-3 cm away from the upper edge of a flowerpot opening, placing the flowerpot in a place with sufficient sunlight, and carrying out normal water and fertilizer management. The tassels and the female ears are bagged before pollen shedding to prevent pollen bunching (as shown in figure 2). After pollen of the tassel is scattered, artificial pollination is carried out, pollination time is registered, and the tassel is continuously bagged to prevent pollen from being mixed. Then, the method for transforming maize with exogenous DNA of the present invention is performed, comprising the following steps:
(1) preparing 2 foam menisci for blocking mud soil when inverting flowerpots according to the sizes of the flowerpots, wherein a semicircular depression with the diameter of 1.5-2.5 cm is formed in the midpoint of the semicircular diameter edge of each foam meniscus (as shown in figure 5), a plastic mesh bag (as shown in figure 4) used for inverting the flowerpots and corn plants is prepared, small plastic bags (as shown in figure 3) with various specifications are specially used for covering female ears and with the width of 2-3 cm and the length of 5-7 cm, the corn plants are located in the center of the flowerpots, the soil surface is 2-3 cm away from the edge of the flowerpots, 2 foam menisci matched with the sizes of the flowerpots are placed on the soil surface, then a plastic mesh bag is sleeved outside the flowerpots, and bag openings are tightened;
the saccharum arundinaceum has the characteristics of strong vigor, wide adaptability, drought resistance, disease resistance, lean tolerance and the like, and the saccharum arundinaceum DNA is introduced into the corn, so that the corn can obtain some excellent properties of the saccharum arundinaceum, and the stress resistance and the adaptability of the corn are improved. In the embodiment, a CTAB method is adopted to extract the genomic DNA of the festuca arundinacea, the purified festuca arundinacea is placed in a refrigerator for storage for later use, then a foreign DNA solution (containing 0.02 percent Silwet L-77) of 300 to 500 mu g/ml is prepared,
(2) during the time period from the artificial pollination of the corn for 16-22 hours until the pollen tube reaches the embryo sac and the zygote is split for the first time, the bracts of the female ear are longitudinally split into 3 equal parts by using an operating blade, the bracts of the inner layer are removed, the outermost 2-3 layers are remained, and all filaments are removed from the base (as shown in figure 6);
(3) selecting a plastic bag with proper size, wherein the plastic bag is made of polyethylene plastic, the bottom is narrow and wide, the bottom is 1.5-2.0 cm wide, the mouth is 2.0-3.0 cm wide, and the length is 5.0-7.0 cm, properly folding according to the size of the female ear, covering the female ear with the filaments removed, and stably and fixedly pricking with transparent adhesive, a clip and the like (as shown in figure 7);
(4) flattening the mud of the flowerpot, paving a foam meniscus around the plant base, and sheathing and binding the flowerpot by using a plastic mesh bag (as shown in figure 8);
(5) slightly inverting the flowerpot and the plant, hooking the plastic mesh bag at the bottom of the flowerpot by using an S-shaped iron hook, and then hooking the plastic mesh bag on a frame by using the iron hook, wherein the distance between the female ear and the ground is 30-100 cm, and the leaves cannot be broken (as shown in figure 9);
(6) dripping the exogenous DNA solution into the plastic bag along the gap between the plastic bag and the filament-free female ear by using a dropper until the plastic bag is filled with the exogenous DNA solution, wherein the volume of the added exogenous DNA solution is 2-5 ml (shown in figure 10);
(7) soaking the filament-free female ears in the exogenous DNA solution for 30 minutes, and slightly vibrating the plastic bag and the female ears every 5 minutes;
(8) after soaking, loosening the plastic bag from the female ear, recovering the exogenous DNA solution in the plastic bag, taking down the flowerpot and the plant from the shelf, and recovering (as shown in figure 11);
(9) after the soaked female ears are dried in the air, the bracts are recovered, and are bound by a rubber, and bagging is continued to prevent the powder from being mixed;
(10) and harvesting the mature seeds in time and drying the seeds in the sun for later use.
The above steps are the same as the operation steps of embodiment 1, and therefore the demonstration drawings corresponding to the above steps are also the same as embodiment 1.
Detection test for transforming corn by exogenous DNA of festuca arundinacea
Extracting the genome DNA of the festuca arundinacea, adopting the method for reversely soaking the female ears and improving the efficiency of transforming exogenous DNA into corn by using the pollen tube channel to transform the inbred line variety WYS2 of the corn, operating and transforming 18 plants, wherein 16 plants can harvest T0Corn seeds, 172 grains in total. 85T plants after sowing0The seedling survives.
T obtained after transformation of exogenous DNA0After sowing the generation of corn seeds, primarily screening the corn seeds according to the difference of observed leaves and common corn leaves in the form during the seedling stage, and then collecting the leaves of plants to extract genome DNA. Analysis of T Using Alu-like labeling technique0Whether the generation maize seedlings contain the blood margin of the festuca arundinacea or not is detected by adopting the following detection primers: AGRP 80: 5'GGG TTG TCY TTG CCA TCA TA3', AGRP 81: 5'GAG YAG CRC AGA GGT TAC GA3', PCR amplified for 35 cycles, and detected by 1% agarose gel electrophoresis.
The Alu-like marker is a unique analysis technology for detecting the genomic DNA of the festuca arundinacea, and about 270bp bands can be amplified when the genomic DNA of the festuca arundinacea is contained, otherwise, no bands exist (as shown in FIG. 14). The detection test shows that the electric lanes 1, 2 and 3 are clear and respectively represent a positive control, a Hainan 92-77 variety (the festuca arundinacea) and a cliff 96-40 variety (the F1 generation of the hybrid of the festuca arundinacea); lanes 4, 5, 6, 7, 13, 15 and 16 show bands in 7 lanes, which preliminarily prove that the introduction of the DNA of the festuca arundinacea into the corn by soaking is successful. Press 85T0The conversion rate is 8.24% by seedling calculation, the conversion rate is far higher than the data recorded by other conversion methods in the existing literature, and the conversion rate of other methods is generally 2-3%.
Claims (9)
1. A method for improving the efficiency of transforming corn with exogenous DNA includes such steps as artificial pollination, removing bracts and all filaments from female ear, enclosing it in a sealed container, fixing, turning over the whole corn plant, fixing or hanging, adding exogenous DNA solution to the sealed container, immersing the female ear without filaments in DNA solution, and restoring the corn plant.
2. The method according to claim 1, wherein the corn is potted, specifically: the plant is located in the center of the pot, the soil surface is 2-3 cm away from the edge of the pot, 2 half-moon plates with the same size as the pot are placed on the soil surface, then a mesh bag is sleeved outside the pot, and the opening of the bag is fastened.
3. The method of claim 2, wherein said meniscus is made of one of plastic, foam, and wood, and has a semicircular depression with a diameter of 1.5-2.5 cm at the midpoint of the semicircular diameter edge, and said mesh bag is made of one of plastic and hemp.
4. The method of claim 1, wherein within 16-22 hours of artificial pollination of corn, the time period from the pollen tube reaching the embryo sac to the first division of the zygote, the bracts are cut longitudinally by 3 equal parts, the inner bracts are removed, the outermost 2-3 layers are retained, and all filaments are removed from the base.
5. The method according to claim 1, wherein the container is a plastic bag made of polyethylene plastic, has a narrow bottom and a wide mouth, has a bottom width of 1.5 to 2.0cm, a mouth width of 2.0 to 3.0cm and a length of 5.0 to 7.0cm, is appropriately folded according to the size of the ear, and is fixed outside the ear.
6. The method of claim 1, wherein the whole corn plant is inverted, in particular: and slightly inverting the planting pot and the plants, hooking meshes of the net bag at the bottom of the planting pot by using an S-shaped iron hook, hanging the other end of the hook on a support, and enabling the female ears to be 30-100 cm away from the ground, wherein the leaves cannot be broken.
7. The method according to claim 1, characterized in that the exogenous DNA solution is added in a sealed container, in particular: the exogenous DNA solution is added into the plastic bag small container along the gap between the plastic bag and the female ear by a dropper until the plastic bag small container is full.
8. The method according to claim 1, wherein the concentration of the exogenous DNA solution is 300 to 500. mu.g/ml.
9. The method according to claim 1, wherein the soaking is performed for 20-30 min, and the plastic bag and the female ear are gently vibrated every 4-6 min.
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Application publication date: 20211214 |