Method for establishing transgenic system of dendrocalamus malabaricus
阅读说明：本技术 马来甜龙竹转基因体系的建立方法 (Method for establishing transgenic system of dendrocalamus malabaricus ) 是由 林新春 鲁海雯 周小红 侯丹 洪彬 张慧聪 刘容秀 于 2021-08-20 设计创作，主要内容包括：本发明公开了一种马来甜龙竹转基因体系的建立方法,采用以下步骤：1)愈伤组织诱导；2)农杆菌培养；3)侵染及共培养；4)脱菌及筛选培养；5)抗性愈伤组织分化及抗性苗生根；6)抗性植株分子鉴定。本发明以马来甜龙竹愈伤组织为受体,通过农杆菌介导法从472块愈伤组织中成功获得34棵抗性植株,转化率为7.20%,抗性植株均有花青素积累表型,提取其中6棵抗性植株进行RNA提取,并反转录得到其cDNA进行PCR检测,结果证实玉米花青素基因已转入竹子基因组中。本发明转化效率高,步骤简单,周期短,效果显著,对其他竹种的转基因体系建立具有一定的参考作用。(The invention discloses a method for establishing a dendrocalamus margaritae transgenic system, which comprises the following steps: 1) inducing callus; 2) culturing agrobacterium; 3) infection and co-culture; 4) removing bacteria and screening culture; 5) differentiation of the resistant callus and rooting of the resistant seedlings; 6) and (5) identifying the resistant plant molecules. The method takes the dendrocalamus malabaricus callus as an acceptor, 34 resistant plants are successfully obtained from 472 callus by an agrobacterium-mediated method, the transformation rate is 7.20%, the resistant plants all have anthocyanin accumulation phenotype, 6 resistant plants are extracted for RNA extraction, and reverse transcription is carried out to obtain cDNA for PCR detection, and the result proves that the zeaxanthin gene is transferred into a bamboo genome. The method has the advantages of high transformation efficiency, simple steps, short period and obvious effect, and has a certain reference function for establishing transgenic systems of other bamboo species.)
1. The method for establishing the transgenic system of the dendrocalamus malabaricus is characterized by comprising the following steps of:
1) callus induction: selecting aseptic seedling axillary buds of the dendrocalamus malabaricus as explants, performing induction culture in a callus induction culture medium for 30 +/-2 days to obtain callus, selecting milky yellow callus with hard texture, transferring the milky yellow callus to a proliferation culture medium, and culturing to obtain embryonic callus of the dendrocalamus malabaricus for subsequent transgenosis of the dendrocalamus malabaricus;
2) and (3) agrobacterium culture: selecting Agrobacterium containing the desired plasmid to 50 mg.L-1Kanamycin (Kanamycin, Kan) and 50 mg.L-1The Rifampicin (Rif) YEP (Yeast extract Peptone) solid plate of (Rifampicin, Rif) is streaked, dark culture is carried out at 28 ℃ for 1-3 d, thus obtaining single colony, and the single colony is selected and inoculated to 1mL containing 50 mg.L-1Kanamycin and 50 mg. L-1YEP liquid medium of Rifampicin (II) in (III) was cultured at 28 ℃ and 200rpm with shaking to OD600The value is between 0.5 and 0.6, and then the whole 1mL of bacterial liquid is inoculated into 50mL of bacterial liquid containing 50 mg.L-1Kanamycin and 50 mg. L-1YEP liquid medium of Rifampicin (II) in (III) was cultured at 28 ℃ and 200rpm with shaking to OD600The value is 0.5-0.6, OD600Bacteria capable of reaching use standardAdding Acetosyringone (AS) into the solution until the concentration reaches 200 μ M, and performing shake culture at 28 deg.C and 200rpm for 30min for infection;
3) infection and co-culture: mixing the embryonic callus of the dendrocalamus margaritae with a bacterial solution, shaking for 15min, pouring out the bacterial solution, sucking off the surface redundant bacterial solution on sterile filter paper, transferring the surface redundant bacterial solution to a co-culture medium, and performing dark culture for 4d at 25 +/-2 ℃;
4) bacteria removal and screening culture: taking out the callus after co-culture for 4 days, sterilizing, washing the callus with sterile water for 3-4 times until the waste liquid is no longer turbid, and adding 200 mg.L-1Cleaning Timentin (Timentin, Tim) with sterile water, soaking for 3-5 min, removing bacteria, drying surface water with sterile filter paper, blowing for 1h on an ultra-clean workbench with sterile wind until the surface of the callus is dry, transferring the dried callus into a screening culture medium, carrying out screening culture under the condition of dark culture at 25 +/-2 ℃, carrying out subculture once every 4 weeks, and obtaining resistant callus after 3 subcultures;
5) differentiation of resistant callus and rooting of resistant seedlings: transferring the resistant callus obtained by screening culture to a differentiation medium I, and culturing at a light intensity of 45 [ mu ] mol-m-2 s-1Culturing for 16/8h in a photoperiod at 25 + -2 deg.C for differentiation; transferring the regenerated bud after differentiation culture into a differentiation culture medium II, transferring the regenerated bud into a rooting culture medium after the regenerated bud grows to 3-4 cm, and performing light intensity of 45 mu mol-m-2 s-1The light cycle is 16/8h, the rooting induction is carried out under the culture condition of the temperature of 25 +/-2 ℃, and the rooting is carried out for about 30 days;
6) and (3) identifying the resistant plant molecules: and detecting the obtained regeneration plant by using a PCR method, determining that the exogenous gene is integrated into the genome DNA of the dendrocalamus margaritaceae and expressing at the RNA level.
2. The method for establishing the dendrocalamus margaritifera transgenic system according to claim 1, wherein the callus induction culture medium in the step 1) has a formula of MS +3 mg-L-1 2，4-D+0.5mg·L-1 NAA+500mg·L-1 CH+500mg·L-1 Pro+500mg·L-1Gln+sucrose 30g·L-1+gelrite 3.3g·L-1pH 5.7; the formula of the proliferation culture medium is as follows: MS +2 mg. L-1 2，4-D+0.5mg·L-1 6-BA+500mg·L-1 CH+20g·L-1sucrose+3.3g·L- 1gelrite，pH 5.7。
3. The method for establishing the phyllostachys margarita transgenic system according to claim 1, wherein the bacterial liquid in step 2) contains acetosyringone AS 200. mu.M.
4. The method for establishing the dendrocalamus margaritifera transgenic system according to claim 1, wherein the formula of the co-culture medium in the step 3) is MS +2 mg-L-1 2，4-D+0.5mg·L-1 6-BA+200μM AS+30g·L-1 sucrose+3.3g·L-1 gelrite，pH 5.7。
5. The method for establishing the dendrocalamus margaritifera transgenic system according to claim 1, wherein the formula of the screening medium in the step 4) is MS +2 mg-L-1 2，4-D+0.5mg·L-1 6-BA+150mg·L-1Hyg+200mg·L-1 Tim+30g·L-1 sucrose+3.3g·L-1 gelrite，pH 5.7。
6. The method for establishing the dendrocalamus margaritifera transgenic system according to claim 1, wherein the formula of the differentiation medium I in the step 5) is MS +0.02 mg-L-1 TDZ+20g·L-1sucrose+150mg·L-1Hyg+200mg·L-1 Tim+3.3g L-1gelrite, pH 6.0; the formula of the differentiation medium II is as follows: MS +1 mg. L-1 6-BA+1mg·L-1KT+0.25mg·L-1NAA+500mg·L-1CH+150mg·L-1Hyg+200mg·L-1Tim+20g·L-1+sucrose 3.3g L-1gelrite, pH 5.7; the rooting medium comprises the following components: 1/2MS + IBA 3 mg.L-1+3.3g·L-1gelrite，pH5.7。
Technical Field
The invention belongs to the technical field of plant transgenosis, and particularly relates to a method for establishing a dendrocalamus malabaricus transgenic system.
Background
Malaysia odorata (A)Dendrocalamus asper) Is prepared from the genus of Musa (Dendrocalamus) Large sympodial bamboos, an important economic bamboo species, are widely cultivated in south-east asia countries, particularly in philippines, malaysia, thailand, and the like. The method is also suitable for introducing and cultivating Hongkong, Taiwan and Yunnan provinces, Guangzhou and Fujian provinces in China. The problems of long flowering period, difficult prediction of flowering period, low fruiting rate and the like of the dendrocalamus malabaricus always prevent the development of breeding research of the dendrocalamus malabaricus. The problem of difficult breeding can be effectively solved by using modern biotechnology and plant transgenic technology to improve the dendrocalamus margaritaceae. At present, the successful establishment of bamboo transgenic research is mainly reported by dendrocalamus latiflorus, and a transgenic system of the dendrocalamus malabaricus has not been reported yet.
Disclosure of Invention
According to the invention, callus obtained by inducing aseptic seedling axillary buds of dendrocalamus malabaricus is used as an acceptor material, and resistant callus is obtained by agrobacterium infection, co-culture and screening culture. And differentiating the resistant callus on a differentiation culture medium to obtain a resistant plant, determining the resistant plant to be transgenic dendrocalamus malabaricus through molecular detection, inducing the transgenic dendrocalamus malabaricus to root on a rooting culture medium, and acclimatizing and transplanting. The invention provides a method for establishing a dendrocalamus margaritae transgenic system.
The invention is realized by the following technical scheme:
the method for establishing the dendrocalamus margaritae transgenic system comprises the following steps:
1) callus induction: selecting aseptic seedling axillary buds of the dendrocalamus malabaricus as explants, performing induction culture in a callus induction culture medium for 30 +/-2 days to obtain callus, selecting milky yellow callus with hard texture, transferring the milky yellow callus to a proliferation culture medium, and culturing to obtain embryonic callus of the dendrocalamus malabaricus for subsequent transgenosis of the dendrocalamus malabaricus;
2) and (3) agrobacterium culture: selecting Agrobacterium containing the desired plasmid to 50 mg.L-1Kanamycin (Kan) and 50 mg. L-1Drawing a YEP solid plate of rifampicin (Rif), performing dark culture at 28 ℃ for 1-3 d to obtain a single colony, selecting the single colony, and inoculating the single colony to 1mL of a liquid containing 50 mg.L-1Kanamycin and 50 mg. L-1In YEP liquid Medium for Rifampicin, cultured at 28 ℃ and 200rpm with shaking to OD600The value is between 0.5 and 0.6, and then the whole 1mL of bacterial liquid is inoculated into 50mL of bacterial liquid containing 50 mg.L-1Kanamycin and 50 mg. L-1YEP liquid medium of Rifampicin (II) in (III) was cultured at 28 ℃ and 200rpm with shaking to OD600The value is 0.5-0.6, OD600Adding AS into the bacterial liquid until the concentration reaches 200 mu M after reaching the use standard, and continuously carrying out shake culture at 28 ℃ under the culture condition of 200rpm for 30min for infection;
3) infection and co-culture: mixing the embryonic callus of the dendrocalamus margaritae with a bacterial solution, shaking for 15min, pouring out the bacterial solution, sucking off the surface redundant bacterial solution on sterile filter paper, transferring the surface redundant bacterial solution to a co-culture medium, and performing dark culture for 4d at 25 +/-2 ℃;
4) bacteria removal and screening culture: taking out the callus after co-culture for 4d for degerming, washing the callus with sterile water for 3-4 times until the waste liquid after washing is no longer turbid, and then using the sterile liquid containing 200mgL-1Washing and soaking timentin (Tim) in sterile water for 3-5 min, removing bacteria, then sucking surface water by using sterile filter paper, blowing for 1h in an ultra-clean workbench by using sterile wind until the surface of the callus is dry, transferring the blow-dried callus into a screening culture medium, carrying out screening culture under the condition of dark culture at 25 +/-2 ℃, carrying out subculture once every 4 weeks, and obtaining resistant callus after 3 times of subculture;
5) differentiation of resistant callus and rooting of resistant seedlings: transferring the resistant callus obtained by screening culture to a differentiation medium I, and culturing at a light intensity of 45 μmol · m-2·s-1The photoperiod is 16/8h, the differential culture is carried out under the culture condition of the temperature of 25 +/-2 ℃, the regeneration bud obtained by the differential culture is transferred into a differential culture medium II, the regeneration bud is transferred into a rooting culture medium after growing to 3-4 cm, and the light intensity is 45 mu mol.m-2·s-1The light cycle is 16/8h, the rooting induction is carried out under the culture condition of the temperature of 25 +/-2 ℃, and the rooting is carried out for about 30 days;
6) and (3) identifying the resistant plant molecules: and detecting the obtained regeneration plant by using a PCR method, determining that the exogenous gene is integrated into the genome DNA of the dendrocalamus margaritaceae and expressing at the RNA level.
Further, the formula of the callus induction culture medium in the step 1) is MS +3 mg.L-1 2，4-D + 0.5 mg·L-1 NAA +500 mg·L-1 CH + 500 mg·L-1 Pro + 500 mg·L-1 Gln + sucrose 30 g·L-1 + gelrite 3.3 g·L-1pH 5.7; the formula of the proliferation culture medium is as follows: MS +2 mg. L-1 2，4-D + 0.5 mg·L-1 6-BA + 500 mg·L-1 CH + 20 g·L-1 sucrose + 3.3 g·L-1 gelrite，pH 5.7。
Further, the bacterial liquid in the step 2) contains 200 mu M of Acetosyringone (AS).
Further, the formulation of the co-culture medium in the step 3) is MS +2 mg.L-1 2，4-D + 0.5 mg·L-1 6-BA + 200 μM AS+ 30 g·L-1 sucrose + 3.3 g·L-1 gelrite，pH 5.7。
Further, step 4)Medium screening culture medium formula MS +2 mg.L-1 2，4-D + 0.5 mg·L-1 6-BA + 150 mg·L-1Hyg + 200 mg·L-1 Tim + 30 g·L-1 sucrose + 3.3 g·L-1 gelrite，pH 5.7。
Further, the formula of the differentiation medium I in the step 5) is MS +0.02 mg.L-1 TDZ + 20 g·L-1sucrose + 150 mg·L-1Hyg + 200 mg·L-1 Tim +3.3 gelrite g L-1pH is 6.0; the formula of the differentiation medium II is as follows: MS +1 mg. L-1 6-BA + 1 mg·L-1 KT + 0.25 mg·L-1 NAA + 500 mg·L-1 CH + 150 mg·L-1Hyg + 200 mg·L-1 Tim +20 g·L-1 + sucrose 3.3 g L-1gelrite, pH 5.7; the rooting medium comprises the following components: 1/2MS + IBA 3 mg.L-1+3.3 g·L-1 gelrite，pH5.7。
The method takes the dendrocalamus malabaricus callus as an acceptor, 34 resistant plants are successfully obtained from 472 callus by an agrobacterium-mediated method, the transformation rate is 7.20%, the resistant plants all have anthocyanin accumulation phenotype, 6 resistant plants are extracted for RNA extraction, and reverse transcription is carried out to obtain cDNA for PCR detection, and the result proves that the zeaxanthin gene is transferred into a bamboo genome. The method has the advantages of high transformation efficiency, simple steps, short period and obvious effect, and has a certain reference function for establishing transgenic systems of other bamboo species.
Drawings
FIG. 1 is a diagram showing the state of a callus before infection with Agrobacterium of the present invention;
FIG. 2 is a diagram showing the state of co-culture of Agrobacterium and embryogenic callus according to the present invention;
FIG. 3 is a diagram showing the status of callus of the present invention in a screening medium;
FIG. 4 is a diagram showing the state of the callus of the present invention in differentiation medium I;
FIG. 5 is a diagram showing the state of the callus of the present invention in differentiation medium II;
FIG. 6 is a transgenic plant of the invention;
FIG. 7 shows the PCR detection result of the transgenic plant of the present invention;
in the figure, DL 2000: DL 2000 Marker; WT: (ii) untransformed plants; h2O: water replaces the DNA template for amplification; L1-L6: transgenic lines nos. 1 to 6; +: to containLcThe plasmid of (a) is amplified without a template as a positive control;
FIG. 8 shows the transgenic plants of the present invention.
Detailed Description
The invention will be described in further detail below with reference to the accompanying drawings to better understand the technical solution.
Example 1: callus acquisition
Selecting aseptic dendrocalamus malabaricus seedling, inoculating young axillary bud thereof into callus induction culture medium, wherein the callus induction culture medium basic culture medium is MS (Murashige)&Skoog), adding exogenous hormone 2, 4-dichlorophenoxyacetic acid (2, 4-D) 3 mg.L-10.5 mg.L of 1-naphthylacetic acid (NAA)-1Then, 500 mg.L of hydrolyzed casein (Casein Hydrolysate, CH) was added-1Proline (L-Proline, Pro) 500 mg.L-1Glutamine (Gln) 500 mg.L-1Then adding 30 g.L of sucrose (sucrose)-1Gelrite (gelrite) 3.3 g.L-1The pH was adjusted to 5.7. Culturing at 25 + -2 deg.C, inducing for about 30d to generate callus, selecting cream yellow compact embryogenic callus, transferring to callus proliferation culture medium containing MS as basic culture medium and 2 mg.L-12，4-D，0.5 mg·L-16-benzyladenine (N- (Phenylmethyl) -9H-purin-6-amine, 6-BA), 500 mg.L-1CH，20 g·L-1sucrose and 3.3 g.L-1gitril, pH 5.7. Proliferation medium was changed every 4 weeks as shown in FIG. 1.
Example 2: agrobacterium culture
The Agrobacterium strains used in the experiments wereEHA105The plasmid ispCAMBIA1301Hygromycin phosphotransferase gene with CaMV35S promoter: (HPT) And zeaxanthin gene using UBI promoter: (Lc) (ii) a The agrobacterium carrying the target plasmidThe bacteria content is 50 mg.L-1Kanamycin (Kan) and 50 mg. L-1The solid YEP medium of Rifampicin (Rif) was plated on a plate and activated, after dark cultivation at 28 ℃ for 2-3 days, a single colony was picked and inoculated to 1mL of a medium containing 50 mg.L-1Kanamycin (Kan) and 50 mg. L-1In the liquid YEP medium of Rifampicin (Rif), cultured at 28 ℃ with shaking at 200rpm, as OD of the bacterial liquid600After reaching 0.5-0.6, inoculating all 1mL of bacterial liquid into 50mL of bacterial liquid containing 50 mg.L-1Kanamycin (Kan) and 50 mg. L-1In the liquid YEP medium of Rifampicin (Rif) of (Rif), the culture was carried out at 28 ℃ with shaking at 200rpm until the OD of the bacterial liquid was reached600Reaching 0.5-0.6, then adding AS until the concentration reaches 200 mu M, and continuing shaking culture for 30 min.
Example 3: infection and Co-cultivation
Soaking the embryogenic callus in prepared infection solution, shaking continuously, infecting for about 15min, and taking out the callus. Sucking dry the bacteria solution on the surface of the callus with sterile filter paper, and inoculating into co-culture medium containing MS as basic medium and 2 mg/L-1 2，4-D，0.5 mg·L-1 6-BA，200 μM AS，30 g·L-1sucrose and 3.3 g.L-1gentril. The callus and the agrobacterium are cultured in a co-culture medium for 4 days under the culture condition of 25 +/-2 ℃ and in dark, and the figure 2 shows.
Example 4: screening culture
Taking out the callus after the co-culture for 4 days, washing the callus after the co-culture for 3-4 times by using sterile water until the washed waste liquid is not turbid any more, and then using 200 mg.L-1Washing timentin (Tim) with sterile water, and soaking for 5 min. After cleaning, transferring the callus to sterile filter paper, absorbing surface moisture, and blowing the callus for 1-2 h in an ultra-clean workbench by using sterile wind until the surface of the callus is dry. Then transferring the culture medium into a screening culture medium for screening culture, wherein the formula of the screening culture medium is as follows: MS is used as a basic culture medium and is added with 2 mg.L-1 2，4-D，0.5 mg·L-1 6-BA，150 mg·L-1 Hygromycin (Hygromycin B, Hyg), 200 mg.L-1 Tim，30 g·L-1sucrose and 3.3 g.L-1Gentril, pH 5.7, 25. + -. 2 ℃ in dark. Subculture was performed every 4 weeks, and after subculture twice, resistant callus was obtained, as shown in FIG. 3.
Example 5: differentiation of resistant callus and rooting of resistant seedling
Using the callus obtained in the screening culture, transferring the callus into a differentiation culture medium I, wherein the formula of the differentiation culture medium is as follows: MS is used as a basic culture medium, and 0.02 mg.L is added-1Thidiazuron (TDZ), 200 mg.L-1 Gln，150 mg·L-1 Hyg，200 mg·L-1 Tim，20 g·L-1sucrose and 3.3 g.L-1gelrite, pH 6.0, at a light intensity of 45. mu. mol. m-2·s-1And performing differentiation culture for 16/8h in a photoperiod at 25 +/-2 ℃, obtaining resistant seedlings within about 30d, and then transferring the resistant seedlings into a differentiation culture medium II (shown in figure 4), wherein the formula of the differentiation culture medium II is shown in figure 5: MS is used as a basic culture medium, and 1 mg.L is added-1 6-BA，1 mg·L-1 KT，0.25 mg·L-1 NAA，500 mg·L-1 CH，150 mg·L-1 Hyg，200 mg·L-1 Tim，20 g·L-1sucrose and 3.3 g.L-1gelrite, pH 5.7, light intensity 45. mu. mol. m-2·s-1Light period 16/8h, temperature 25 + -2 deg.C. Transferring the resistant plants to a rooting culture medium after the resistant plants grow to 3-4 cm in a differentiation culture medium II, wherein the formula of the rooting culture medium is as follows: 1/2MS is used as a minimal medium, and 3 mg.L is added-1Indole butyric acid (Indole-3-Butytric acid, IBA), 30 g.L-1sucrose and 3.3 g.L-1gelrite, pH 5.7, light intensity 45. mu. mol. m-2·s-1Light period 16/8h, temperature 25 + -2 deg.C. Rooting can be seen after about 1 week, and after 30 days, the root becomes thick and long, as shown in FIG. 6.
Example 6: molecular identification of resistant plants
Extracting transgenic dendrocalamus margaritae leaf RNA, performing reverse transcription to obtain cDNA, performing PCR detection, and collecting zeaxanthin geneLcThe primer sequence of (a) is: 5'-CGACGCTTTGTTCACCCTGT-3', and 5'-ACGGGAGCAGCACAGGAAAT-3'. The PCR procedure was: pre-denaturation at 95 ℃ for 5 min; then denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and circulating for 30 times; 72 deg.CExtending for 5 min; the results are shown in FIG. 7, and the electrophoresis results of 6 transgenic plants are positive.
Example 7: plant domestication and transplantation
And taking the identified transgenic plant out of the culture bottle, carefully washing under running water to remove the residual culture medium on the root, and planting the transgenic plant in a matrix after the culture medium is washed clean. The substrate supplies sufficient water, the plants are covered by a plastic packaging bag to prevent water loss, and the light intensity is 45 mu mol.m-2·s-1And performing acclimation for 16/8h at 25 +/-2 ℃, cutting an opening of about 2 cm at the top of the plastic package bag after one week, expanding the opening once every 1-2 d until the top of the plastic package bag is completely opened, removing the plastic package bag after 2-3 d is maintained until plants have no obvious water loss phenomenon, and obtaining a diagram of fig. 8 for the acclimated transgenic Malaysia bambusicola.
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