CN107529551B - Efficient induction method of mixed tea trees with transgenic hairy roots - Google Patents
Efficient induction method of mixed tea trees with transgenic hairy roots Download PDFInfo
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Abstract
The invention discloses a high-efficiency induction method of a mixed tea tree with transgenic hairy roots, which belongs to the technical field of crop genetic breeding, and the method uses an agrobacterium rhizogenes A4 strain to infect the hypocotyl of a seedling by a needle punching method to obtain 90% of rooting rate, so that the infected tea tree has developed hairy roots and lateral roots, and a high-efficiency and stable tea tree transgenic hairy root system is established.
Description
Technical Field
The invention relates to the technical field of crop genetic breeding, in particular to a high-efficiency induction method of a mixed tea tree with transgenic hairy roots.
Background
Tea is second only to water, the first largest non-alcoholic beverage with remarkable health benefits and is widely welcomed. However, the tea trees planted in China are widely planted, are planted in hills and mountains with poor soil, and are frequently stressed by various biotic and abiotic adversities. The establishment of broad spectrum resistant tea germplasm has been the goal of the industry. A mixed tea plant with transgenic hairy roots should be a way to achieve this goal. The tea tree is difficult to realize genetic transformation through mediation of agrobacterium. Even though there are some successful reports of agrobacterium-mediated tea plant transgenes, the reports are difficult to repeat. This may be related to the oxidation of polyphenol oxides in tea plant tissue and its inhibitory effect on agrobacterium.
Tea is second only to water, the first largest non-alcoholic beverage with remarkable health benefits and is widely welcomed. Tea yields in china (1467467 tons) were the first in the world, followed by india (991180 tons), kenya, srilanca and turkish. The worldwide tea yield reaches 4520000 tons, and the daily average tea consumption in the uk is 644.1 tons. Tea tree roots are rich in unique L-theanine synthesized from the roots, but accumulate in the leaves of the tea tree. In recent years, L-theanine has been increasing in importance because it has beneficial effects on reduction of anxiety, suppression of hypertension, improvement of memory and promotion of relaxation. The tea is rich in polyphenol substances, which are substances with the functions of resisting cancers, allergy, virus, inflammation and bacteria and enhancing immunity. Natural polyphenols may have a beneficial antioxidant effect on the human body, since they can eliminate the activity of free radicals in the body via secondary metabolic pathways. The content of monomeric flavonol catechin in green tea with the highest content is 30-40% (DW), wherein epigallocatechin gallate (EGCG) has the highest content. Galloyl groups in ECG and EGCG have been reported to produce antibacterial effects by binding directly to the peptidoglycan layer of bacteria and interfering with its biosynthesis.
Tea trees are a crop of a rainwater irrigation and single crop system which grows in different countries and under different weather conditions and the optimal growth state is determined by the climate change. However, the impact of climate change on economics limits the yield of tea. In view of the drought environment and the increase in soil salinity, it is necessary to improve the agricultural traits of tea plants by analyzing the principles of molecular and physiological processes related to tea plant salinity and drought tolerance. In recent years, gene transfer using agrobacterium-mediated transformation has been used extensively in the production of transgenic plants to improve the agronomic and nutritional characteristics of the plants, but has been an obstacle to biotechnological development for genomic applications in tea-like agronomically important crops.
Although some examples of genetic transformation of tea have been reported in clones of the species assam, the recalcitrance of tea to Agrobacterium-mediated transformation has hampered studies in tea biology and the development of well-known varieties of tea. To overcome this bottleneck, it is necessary to establish a genetic transformation method for tea. Normally, polyphenols with high bacteriostatic concentrations are toxic to plant tissues and also have high reactivity with tannins formed by oxidation of tannins in adverse and traumatic conditions. Although less polyphenols are present in leaf callus, accumulation of polyphenolic substances is moderately increased under inoculation and stress conditions such as explant excision, wounding, and co-culture with Agrobacterium, antibiotic selection, exposure conditions, and disinfectant conditions applied to the surface of the explant. In the in vitro Agrobacterium-mediated transformation process, most crops typically require a co-cultivation time of two to three days. However, tea plants require five to six days to increase transformation efficiency. The increase in time may cause excessive browning of the explant. To optimize the agrobacterium-mediated transformation efficiency of tea tree and control phenolic oxidation of explant tissue, many scholars have used different culture conditions and media to overcome the challenge of inducing hair roots in vitro. Although different adsorbents and antioxidants are used in the media to slow tissue browning (necrosis), these substances may have important negative effects on agrobacterium-mediated transformation as well as preventing gene expression.
Research to expand genetic analysis and gene function requires efficient transformation techniques. Different explants of tea, such as shoots, hypocotyls, cotyledons, cotyledonary nodes, etc., have been used to produce transgenic plants using either particle gun injection or Agrobacterium-mediated transformation. However, most of these methods are limited in their effectiveness and labor intensive. The genes related to root biology are studied in aspects of nutrition absorption, pathogen interaction, symbiotic relationship and hormone transport. These problems can be solved by Agrobacterium rhizogenes-mediated transformation. The extrachromosomal replicon Ri plasmid from agrobacterium rhizogenes transfers its oncogene into plant genomic DNA and then initiates the massive formation of independent hairy roots and can be analyzed in a short time.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a high-efficiency induction method of a mixed tea tree with transgenic rooting so as to solve the technical problem of low agrobacterium-mediated tea tree transgenic efficiency.
The invention is realized by the following technical scheme:
the invention provides a high-efficiency induction method of a mixed tea tree with transgenic hairy roots, which comprises the following steps:
(1) screening full tea tree seeds, disinfecting, germinating in sterilized nutrient soil at 26 +/-2 ℃ for two months to obtain tea tree seedlings, and screening healthy tea tree seedlings for later use;
(2) transforming the exogenous gene into an agrobacterium rhizogenes A4 strain to obtain recombinant agrobacterium rhizogenes, inoculating the recombinant agrobacterium rhizogenes into an A4 improved solid culture medium for culture, and obtaining and collecting agrobacterium rhizogenes colonies; the method for obtaining the recombinant agrobacterium rhizogenes and the culture process can be realized by adopting a conventional genetic engineering method, and the specific references can be found in the following documents: methods in molecular Biology, vol.343: agrobacterium Protocols, 2/e, volume 1;
(3) picking the agrobacterium rhizogenes bacterial colony in the step (2) by using a needle head, and poking the bacterial colony into the hypocotyl of the tea tree seedling in the step (1), wherein the needle head penetrates through the middle part of the hypocotyl; dipping some agrobacterium rhizogenes bacterial colonies obtained in the step (2) by using a triangular glass rod, and smearing the bacterial colonies on a needle punching area of a hypocotyl; obtaining dip-dyed seedlings;
(4) putting the impregnated seedlings into a humidity box, watering the seedlings by using a suspension containing 10% (volume ratio) of A4 strain for the first two weeks, and irrigating the seedlings by using 1% (volume ratio) of MS culture medium and water for 3 months in the later period to obtain the mixed tea trees with transgenic hairy roots, wherein the mixed tea trees have developed hairy root lateral roots, the root systems are transgenic, and overground branch leaves are non-transgenic.
Further, in the step (1), the method for screening the full tea tree seeds comprises the following steps: washing fresh tea tree seeds with water, soaking in water overnight, screening to remove seed shells and floating seeds, and selecting the seeds with the bottom sinking, namely the full tea tree seeds.
Further, in the step (1), the formula of the nutrient soil is as follows: mixing the Ping's matrix peat soil and the vermiculite according to the mass ratio of 3: 1.
Further, in the step (2), the preparation method of the modified solid culture medium A4 comprises the following steps: mixing 10g of sucrose and 0.2g of MgSO4·7H2O、0.5g K2HPO4、0.2g CaSO4、0.1gNaCl、1％(wt/vol)1.0ml NaMoO4、1％(wt/vol)1ml C6H5FeO41% (wt/vol) of boric acid 1ml, 1.0g of yeast extract were dissolved in 900ml of sterile distilled water, and thenMaking volume to 1000ml, adjusting pH to 6.8-7.0, adding 1% agar, sterilizing at 121 deg.C, and storing at 4 deg.C.
Further, in the step (4), a suspension containing 10% (by volume) of the a4 strain is prepared by: activating the recombinant agrobacterium rhizogenes by using an A4 modified liquid culture medium, carrying out expanded culture to obtain a stock solution, taking 100ml of the stock solution, and fixing the volume to 1000ml by using sterile distilled water, wherein the preparation method of the A4 modified liquid culture medium comprises the following steps: mixing 10g of sucrose and 0.2g of MgSO4·7H2O、0.5g K2HPO4、0.2g CaSO4、0.1gNaCl、1％(wt/vol)1.0ml NaMoO4、1％(wt/vol)1ml C6H5FeO41% (wt/vol) of boric acid 1ml and yeast extract 1.0g were dissolved in sterile distilled water 900ml, and then the volume was adjusted to 1000ml, pH was adjusted to 6.8-7.0, and after sterilization at 121 ℃, the mixture was stored at 4 ℃ for further use.
Further, in the step (4), the preparation method of 1% (by volume) MS medium comprises: respectively adding 25ml of 10 XMS major salt, 5ml of 100 XMS minor salt and 5ml of 100 XFe EDTA into 500ml of sterile distilled water, then fixing the volume to 1000ml, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 minutes to obtain an MS culture medium, taking 10ml of MS culture medium into 990ml of sterile distilled water when the temperature of the culture medium is reduced to 45 ℃, and storing at 25 ℃ in the absence of light.
Compared with the prior art, the invention has the following advantages: the invention provides a high-efficiency induction method of a mixed tea tree with transgenic rooting, which is characterized in that agrobacterium rhizogenes A4 strain is used for infecting hypocotyls of seedlings to obtain 90% rooting rate and developed lateral roots of hair roots, and a high-efficiency and stable tea tree transgenic hair root system is established.
Drawings
FIG. 1 is a comparison graph of the rooting of tea shoots, wherein A (1, 2) is the control of uninfected tea shoots, A (3, 4) is the control of uninfected tea shoots, and B is the control of needle-infected tea shoots;
FIG. 2 shows the result of agarose gel electrophoresis for molecular characterization.
Detailed Description
Example 1
1. Material
1.1 reagents
1) Agrobacterium rhizogenes strain A4[ Agricultural Culture Collection of China (ACCC) ]
2)10 × MS major salts: purchased from chemical reagents of national drug group, Ltd, prepared according to the product specification, and stored at 4 ℃.
3)100 × MS majorsalts: purchased from chemical reagents of national drug group, Ltd, prepared according to the product specification, and stored at 4 ℃.
4)100 × Fe EDTA: purchased from chemical reagents of national drug group, Ltd, prepared according to the product specification, and stored at 4 ℃.
5)100pmol rolC primer:
an upstream primer: 5'-CCAAGCTTGTCAGAAAACTTCAGGG-3'
A downstream primer: 5'-CCGGATCCAATACCCAGCGCTTT-3'
6)100pmol of aux1 primer:
an upstream primer: 5'-CCAAGCTTGTCAGAAAACTTCAGGG-3'
A downstream primer: 5'-CCGGATCCAATACCCAGCGCTTT-3' are provided.
7) 4% carbendazim: 4g of carbendazim was dissolved in 900ml of sterile distilled water, and then the volume was fixed to 1000ml and stored at room temperature.
8)0.1％HgCl2: 1g of HgCl2Dissolving in 900ml of sterile distilled water, metering to 1000ml, and storing at room temperature.
9) A4 modified liquid medium (Agrobacterium rhizogenes growth): mixing 10g sucrose, 0.2g MgSO4·7H2O、0.5gK2HPO4、0.2g CaSO4、0.1g NaCl、1％(wt/vol)1.0ml NaMoO4、1％(wt/vol)1ml C6H5FeO41% (wt/vol) of boric acid 1ml, 1.0g of yeast extract were dissolved in 900ml of sterile distilled water, and then the volume was fixedTo 1000 ml. Adjusting pH to 6.8-7.0, and storing at 4 deg.C.
10) A4 modified solid medium: agar was added to the modified liquid medium A4 in an amount of 1% by mass.
11) 1% (wt/vol) agar plate: 2g of agar were added to 200ml of distilled water. The mixture was sterilized at 121 ℃ for 15 minutes, and 10ml of the mixture was poured into a sterile petri dish (90X 15mm) after the temperature was lowered to 40 ℃.
12) A4 strain suspension containing 10% (by volume): activating recombinant Agrobacterium rhizogenes with A4 improved liquid culture medium, performing amplification culture to obtain stock solution, collecting 100ml stock solution, and diluting with sterile distilled water to 1000ml
13) 1% (by volume) of MS medium (water-infested plants): respectively adding 25ml of 10 XMS major salt, 5ml of 100 XMS minor salt and 5ml of 100 XFe EDTA into 500ml of sterile distilled water, then fixing the volume to 1000ml, adjusting the pH value to 5.8, and sterilizing at 121 ℃ for 15 minutes to obtain the MS culture medium. When the temperature is reduced to 45 ℃, 10ml to 990ml of sterile distilled water is taken, mixed evenly and poured into 100ml of bottle, and the bottle is stored at 25 ℃ under the dark condition.
14) Filling a cultivation pot: adding the sterilized Ping's matrix peat soil and vermiculite into the pot at a ratio of 3:1, and cooling to 20 deg.C.
1.2 apparatus
1) Sterilizable glass culture dish (90 x 15mm)
2) Plastic dish (700 x 500 x 50mm)
3) Needle 21G with 1.25Prime (0.8 with 32mm)
4) Sealing film (4IN 125FT)
5) Triangular glass rod with handle
6) Humidity box
7) Electrophoresis apparatus
8) Artificial growth box
9) Shaking table (Zhicheng 100H)
1.3 device configuration
1) A humidity chamber: in order to maintain humidity, the mini plastic film cover should be covered after the cultivation pot is put into the plastic tray. The trays should be sterilized beforehand with 70% ethanol.
2) An artificial growth chamber: the photoperiod of the growth chamber should be adjusted to 16h light period and 8h dark period at 26 + -2 deg.C. The light source used luminescence (40-50mmol m-2 s-1).
3) A bacteria incubator: the temperature for culturing Agrobacterium rhizogenes was kept at 28 ℃ for hard drying.
4) Shaking table: agrobacterium rhizogenes was mixed with 10% A4 liquid medium and kept at 150rpm to obtain a homogeneous bacterial suspension.
5) PCR cycling program and electrophoresis: a reaction system containing 1Unit Easy-Taq polymerase (Beijing Omegano gold Biotechnology Co., Ltd.), 2.5. mu.L of 10 xbuffer, 2. mu.L of dNTP (2.5 mmol. multidot.L-1), 1. mu.L (10 mmol. multidot.L-1) of each of the primers, 10ng of gDNA and 17.5. mu.L of water was prepared for PCR in a total of 25. mu.L. The procedure of the PCR machine (Bio-Rad S1000) was initial denaturation temperature 95 ℃ for 3min, 30 cycles of amplification (95 ℃ for 30S, 58 ℃ for 30S and 72 ℃ for 1min), and then 72 ℃ for 10 min. The PCR product was electrophoresed in 1.5% agar gel and then stained with ethidium bromide for observation.
2 method
2.1 Collection of seeds (autumn)
Mature seeds collected from a seven-year-old agricultural resistance early variety in a tea experimental base of the university of Anhui agriculture;
2.2 screening of seeds
In order to make the tea tree seedlings grow hairy roots, fresh seeds are washed and selected by tap water; the seed coat was removed by overnight soaking. Then filtering, separating floating and sinking seeds, and selecting the sinking seeds for sowing to remove the floating seeds.
2.3 seed surface Disinfection
(1) 100 seeds were placed in a 25ml conical flask and washed twice with distilled water.
(2) The mixture was soaked in 4% (by volume) of sterile water overnight and then washed with distilled water to remove the fungus.
(3) With 0.1% (by volume) of HgCl2The solution was used to disinfect the seed surface for 4 minutes, then washed three times with distilled water.
(4) 100ml of 70% (vol/vol) ethanol was added and gently agitated for two minutes and then filtered.
(5) The residual ethanol was removed with a large amount of water and the seeds were washed.
2.4 Germination of seeds
The seeds were planted in pots filled with sterilized Ping's matrix peat soil and vermiculite and germinated in growth chamber (26 + -2 deg.C) for two months to obtain tea tree seedlings.
2.5 preparation of inoculum (Agrobacterium rhizogenes)
Agrobacterium rhizogenes, which had been removed from a-80 ℃ freezer and stored with 50% glycerol, was streaked onto A4 modified solid medium to grow a single colony. The plates with Agrobacterium rhizogenes strain A4 were cultured for two days in an incubator at 28 ℃ and then a single colony was picked up and cultured for 4 days in a new A4 medium plate.
2.6 hairy root Induction
The selected healthy two month old tea seedlings grown in the growth chamber were used for the transgenic experiments and the colonies grown in step 2.5 were collected in a petri dish. Picking a small group of agrobacterium with a needle, and then poking the seedling into the hypocotyl of the seedling, wherein the needle penetrates through the middle part of the hypocotyl. Some agrobacteria were then dipped with a triangular glass rod and applied to the needled area of the hypocotyl. The infested seedlings were placed in a humidity chamber and were watered with a suspension containing 10% of the A4 strain for the first two weeks and later with 1% (by volume) MS medium and water for 3 months.
2.7 collecting root tissue
Collecting infected and non-infected roots, washing the roots with tap water for 1 hour, then washing with sterile distilled water, and finally sterilizing the surfaces of the roots with 70% alcohol to be used as a material for molecular identification.
2.8 molecular characterization
Root genomic DNA is extracted, and the rolC and auxI genes of the T-DNA region of Agrobacterium rhizogenes are cloned by taking the root genomic DNA as a template so as to confirm whether the T-DNA on the Agrobacterium rhizogenes plasmid is integrated into the genome sequence of the plant. The PCR results for rolC and auxI genes are shown in FIG. 2.
2.9 statistics of results
TABLE 1 statistical table of rooting efficiency of tea seedlings
As can be seen in Table 1, hairy roots are obtained with great probability (90%) by this method. This technique can be used to control the growth of roots to increase the production of L-theanine. Furthermore, the transgenic A4 strain carrying the target gene can be used to promote the root system to adapt plants to biotic and abiotic stresses. The method can obtain similar transformation effect in all tea plant varieties or other woody and medicinal plants.
3. Conclusion
The invention successfully infects recalcitrant Chinese tea tree varieties with the agrobacterium-mediated living transformation method to grow hairy roots, discovers that T-DNA on agrobacterium plasmids is successfully integrated into the genome of the newly grown hairy roots by molecular identification of the grown hairy roots, proves that any exogenous gene can be introduced into tea trees by the agrobacterium-mediated method to obtain transgenic hairy root lateral roots, overcomes the limitation of the in vitro method in the application of the tea trees, establishes a high-efficiency and stable tea tree transgenic hairy root system, and can be used as an important tool for research in the aspects of root system biology and secondary metabolism.
The above is a detailed embodiment and a specific operation process of the present invention, which are implemented on the premise of the technical solution of the present invention, but the protection scope of the present invention is not limited to the above-mentioned examples.
Claims (3)
1. A high-efficiency induction method of mixed tea trees with transgenic hairy roots is characterized by comprising the following steps:
(1) screening full tea tree seeds, disinfecting, germinating in sterilized nutrient soil at 26 +/-2 ℃ for two months to obtain tea tree seedlings, and screening healthy tea tree seedlings for later use;
(2) transforming the exogenous gene into an agrobacterium rhizogenes A4 strain to obtain recombinant agrobacterium rhizogenes, inoculating the recombinant agrobacterium rhizogenes into an A4 improved solid culture medium for culture, and obtaining and collecting agrobacterium rhizogenes colonies;
(3) picking the agrobacterium rhizogenes bacterial colony in the step (2) by using a needle head, and poking the bacterial colony into the hypocotyl of the tea tree seedling in the step (1), wherein the needle head penetrates through the middle part of the hypocotyl; dipping some agrobacterium rhizogenes bacterial colonies obtained in the step (2) by using a triangular glass rod, and smearing the bacterial colonies on a hypocotyl needling area; obtaining dip-dyed seedlings;
(4) putting the impregnated seedlings into a humidity box, watering the seedlings by using an A4 strain suspension containing 10% by volume for two weeks, and irrigating the seedlings by using an MS culture medium containing 1% by volume and water for 3 months at the later stage to obtain mixed tea trees with transgenic hairy roots;
wherein: in the step (2), the preparation method of the A4 modified solid medium comprises the following steps: mixing 10g sucrose, 0.2g MgSO4·7H2O、0.5g K2HPO4、0.2g CaSO40.1g NaCl, 1.0ml 1% wt/vol NaMoO41ml of 1% wt/volC6H5FeO41ml of 1% wt/vol boric acid and 1.0g of yeast extract are dissolved in 900ml of sterile distilled water, then the volume is determined to 1000ml, the pH value is adjusted to 6.8-7.0, 1% mass ratio of agar is added, the mixture is sterilized at 121 ℃, and then the mixture is stored at 4 ℃ for standby;
in the step (4), the preparation method of the suspension containing 10% by volume of the A4 strain comprises the following steps: activating the recombinant agrobacterium rhizogenes A4 strain by using an A4 improved liquid culture medium, carrying out amplification culture to obtain a stock solution, taking 100ml of the stock solution, and fixing the volume to 1000ml by using sterile distilled water, wherein the preparation method of the A4 improved liquid culture medium comprises the following steps: mixing 10g sucrose, 0.2g MgSO4·7H2O、0.5g K2HPO4、0.2g CaSO40.1g NaCl, 1.0ml 1% wt/vol NaMoO41ml of 1% wt/volC6H5FeO41ml of 1% wt/vol boric acid, 1.0g yeast extract in 900ml sterile distilled water, then constant volume to 1000ml, adjusting pH to 6.8-7.0, 12Sterilizing at 1 deg.C, and storing at 4 deg.C;
in the step (4), the preparation method of the MS culture medium with the volume ratio of 1% comprises the following steps: adding 25ml of 10 XMS majorsalt, 5ml of 100 XMS minor salt and 5ml of 100 XFe EDTA into 500ml of sterile distilled water, then fixing the volume to 1000ml, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15 minutes to obtain an MS culture medium, taking 100ml of MS culture medium into 900ml of sterile distilled water when the temperature of the culture medium is reduced to 45 ℃, and storing at 25 ℃ in the absence of light.
2. The method for inducing hybrid tea plant with transgenic hairy roots according to claim 1, wherein the method for screening the plump tea plant seeds in the step (1) comprises the following steps: washing fresh tea tree seeds with water, soaking in water overnight, screening to remove seed shells and floating seeds, and selecting the seeds with the bottom sinking, namely the full tea tree seeds.
3. The method for inducing hybrid tea plant with transgenic hairy roots according to claim 1, wherein in the step (1), the nutrient soil is prepared from the following formula: mixing the Ping's matrix peat soil and the vermiculite according to the mass ratio of 3: 1.
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