Method for establishing populus euphratica transgenic system based on hygromycin screening

文档序号：900148 发布日期：2021-02-26 浏览：3次中文

阅读说明：本技术 一种基于潮霉素筛选的大青杨转基因体系建立的方法 (Method for establishing populus euphratica transgenic system based on hygromycin screening ) 是由杨静莉李成浩杨佳李文龙于 2020-11-27 设计创作，主要内容包括：本发明提供了一种基于潮霉素筛选的大青杨转基因体系建立的方法,属于植物育种技术领域。基于潮霉素筛选的大青杨转基因体系建立的方法,将含报告基因的重组载体导入农杆菌中培养,用农杆菌菌液侵染大青杨外植体后共培养,脱菌后,将大青杨外植体接种至含潮霉素的叶片不定芽分化培养基上进行筛选培养得到抗性芽,将抗性芽进行生根培养,获得大青杨转基因再生植株。本发明采用潮霉素作为筛选标记能有效降低转基因再生植株的假阳性率,同时本发明还具体限定了潮霉素筛选工作浓度、根癌农杆菌菌株、农杆菌菌液OD值、侵染时间、共培养时间以及温度等能大大提高抗性芽诱导率的因素,为大青杨育种提供稳定、可靠的材料。(The invention provides a method for establishing a transgenic system of populus davidiana based on hygromycin screening, and belongs to the technical field of plant breeding. A method for establishing a transgenic populus davidiana system based on hygromycin screening comprises the steps of introducing a recombinant vector containing a reporter gene into agrobacterium for culture, infecting a populus davidiana explant with agrobacterium liquid for co-culture, after bacteria removal, inoculating the populus davidiana explant to a leaf adventitious bud differentiation culture medium containing hygromycin for screening culture to obtain resistant buds, and performing rooting culture on the resistant buds to obtain a transgenic regeneration plant of the populus davidiana. The method adopts hygromycin as a screening marker, can effectively reduce the false positive rate of transgenic regeneration plants, and also specifically limits the factors of hygromycin screening working concentration, agrobacterium tumefaciens strains, agrobacterium tumefaciens liquid OD value, infection time, co-culture time, temperature and the like, which can greatly improve the induction rate of resistant buds, thereby providing stable and reliable materials for breeding populus davidiana.)

1. A method for establishing a transgenic system of populus megalobii based on hygromycin screening is characterized by comprising the following steps:

1) introducing the recombinant vector containing the target gene into agrobacterium and culturing to obtain agrobacterium liquid;

2) infecting the populus euphratica explants with the agrobacterium liquid, co-culturing, and removing bacteria to obtain the infected populus euphratica explants;

3) inoculating the infected populus euphratica explant to a hygromycin-containing leaf adventitious bud differentiation culture medium for screening culture to obtain an adventitious bud;

4) and carrying out rooting culture on the adventitious bud to obtain a transgenic regeneration plant of the populus euphratica.

2. The method for establishing the transgenic system of populus megalophylla based on hygromycin screening according to claim 1, wherein the concentration of hygromycin in the adventitious bud differentiation culture medium for leaves in the step 3) is 2.5-3.2 mg/L.

3. The method for establishing a transgenic system of populus tremuloides based on hygromycin screening according to claim 1 or 2, wherein the basic backbone vector of the recombinant vector containing the target gene in step 1) is pCAMBIA1302 vector.

4. The method for establishing the transgenic system of populus tremuloides based on hygromycin screening according to claim 1, wherein the agrobacterium is agrobacterium tumefaciens in the step 1);

the strain of agrobacterium tumefaciens includes EHA101, EHA105, or LBA 4404.

5. The method for establishing the transgenic system of populus davidiana based on hygromycin screening according to claim 1 or 4, wherein the OD value of the Agrobacterium liquid in the step 1) is 0.5-0.8;

the infection time of the agrobacterium liquid is 25-50 min.

6. The method for establishing the transgenic populus tremuloides system based on hygromycin screening according to claim 1, wherein the co-culture time of the agrobacterium tumefaciens liquid and the populus tremuloides explant in the step 2) is 2-3.5 days.

7. The method for establishing the transgenic system of populus tremuloides based on hygromycin screening according to claim 1, wherein the co-culture temperature of the agrobacterium tumefaciens liquid and the populus tremuloides explants in the step 2) is 20-27 ℃.

8. The method for establishing the transgenic system of populus tremuloides based on hygromycin screening according to claim 1, wherein the explants of populus tremuloides in step 2) comprise stem segments and leaves.

9. The method for establishing a transgenic populus megalophylla system based on hygromycin screening according to any one of claims 1, 2, 4 and 6 to 8, wherein the adventitious bud differentiation culture medium for leaves in step 2) is an MS culture medium containing 0.5 to 2mg/L of 6-BA, 0.2mg/L of NAA and 0 to 0.1mg/L of TDZ.

10. The method for establishing the transgenic system of populus tremuloides based on hygromycin screening according to claim 9, wherein the rooting medium in step 4) is 1/2MS medium.

Technical Field

The invention belongs to the technical field of plant breeding, and particularly relates to a method for establishing a populus euphratica transgenic system based on hygromycin screening.

Background

The Populus (Populus ussuriensis Kom.) is a main local tree species in northeast forest regions, is cold-resistant and fast-growing, is one of main tree species for building fast-growing timber forests in mountainous regions, and is also the tree species which is most widely distributed and has the highest utilization value in Populus pie tree species in northeast China. The dry shape is straight, the wood is white, and the wood is an excellent raw material for paper making and plywood. With the continuous felling of natural forests, people began to build artificial forests of populus euphratica. The forest land condition often needs a new variety of populus euphratica with strong stress resistance and adaptability. However, the conventional breeding cycle is long, the process is complex, and the influence of the external environment is large, so that the requirement of purposefully and directionally breeding a new poplar variety in a short period cannot be met. The new poplar variety can be purposefully and directionally cultivated by genetic engineering breeding, and the breeding period is greatly shortened. Therefore, the establishment of the transgenic system of the populus euphratica is particularly important.

At present, a poplar transgenic system is researched, kanamycin is often adopted for screening in the process of constructing the poplar transgenic system, but the screening effect of the screening method is poor, about half of the probability is false positive, and the breeding of transgenic plants of the populus is hindered.

Disclosure of Invention

In view of the above, the invention aims to provide a method for establishing a transgenic populus davidiana system based on hygromycin screening, so that false positive screening results are reduced, and a foundation is laid for establishing transgenic plants of populus davidiana.

The invention provides a method for establishing a transgenic system of populus davidiana based on hygromycin screening, which comprises the following steps:

1) introducing the recombinant vector containing the target gene into agrobacterium and culturing to obtain agrobacterium liquid;

2) infecting the populus euphratica explants with the agrobacterium liquid, co-culturing, and removing bacteria to obtain the infected populus euphratica explants;

3) inoculating the infected populus euphratica explant to a hygromycin-containing leaf adventitious bud differentiation culture medium for screening culture to obtain an adventitious bud;

4) and carrying out rooting culture on the adventitious bud to obtain a transgenic regeneration plant of the populus euphratica.

Preferably, the concentration of hygromycin in the adventitious bud differentiation medium for leaves in the step 3) is 2.5-3.2 mg/L.

Preferably, the basic backbone vector of the recombinant vector containing the target gene in step 1) is pCAMBIA 1302.

Preferably, the agrobacterium in step 1) is agrobacterium tumefaciens;

the strain of agrobacterium tumefaciens includes EHA101, EHA105, or LBA 4404.

Preferably, the OD value of the agrobacterium liquid in the step 1) is 0.5-0.8;

the infection time of the agrobacterium liquid is 25-50 min.

Preferably, the co-culture time of the agrobacterium liquid and the populus euphratica explant in the step 2) is 2-3.5 days.

Preferably, the co-culture temperature of the agrobacterium liquid and the populus euphratica explant in the step 2) is 20-27 ℃.

Preferably, the populus euphratica explant in step 2) comprises a stem section and a leaf.

Preferably, the adventitious bud differentiation culture medium for leaves in the step 2) is an MS culture medium containing 0.5-2 mg/L6-BA, 0.2mg/LNAA and 0-0.1 mg/LTDZ.

Preferably, the rooting culture medium in the step 4) is 1/2MS medium.

The method for establishing the poplar transgenic system based on hygromycin screening provided by the invention is characterized in that an agrobacterium-mediated method is utilized to transfer a target gene into a genome of populus Davidii so as to establish a transgenic receptor system of the populus Davidii, and meanwhile, hygromycin is limited as a screening marker after agrobacterium infection, so that the false positive of a transformant obtained by screening hygromycin is relatively low, the positive rate of the transformant generating resistance reaches more than 95% after molecular detection, and the problems of poor screening effect and high false positive rate caused by the fact that kanamycin is conventionally adopted as the screening marker in the prior art are solved, so that the aim of optimizing the poplar transformation system is fulfilled. Experiments prove that the stable genetic transformation efficiency in a transgenic system exceeds 10 percent.

Furthermore, the invention specifically defines various parameters which can influence the transformation rate in the agrobacterium-mediated method, including hygromycin screening working concentration, agrobacterium tumefaciens strains, agrobacterium liquid OD value, infection time, co-culture time, temperature and other factors, and can greatly improve the induction rate of resistant buds. Experiments prove that the induction rate of the resistant buds reaches 30-37% by strictly limiting the value range of parameters influencing the conversion rate, and meanwhile, the pollution rate can be reduced.

Drawings

FIG. 1 is a flow chart of transgenic system establishment of populus davidiana based on hygromycin screening;

FIG. 2 shows GUS activity assay of transgenic plants; left panel non-transgenic plants (WT); the right panel shows the transgenic plant (CaMV 35S:: GUS);

FIG. 3 is the process of transgenic system establishment of populus deltoids; a is to screen resistant buds in a culture medium added with 2mg/L hygromycin; B-C is a resistant bud growing from the wound; d is proliferation of the resistant bud, and E-F is rooting and plant regeneration of the resistant bud; g is the propagation result of the screened hygromycin-resistant transgenic regeneration plant; h is PCR detection at the DNA level; + represents PCR with plasmid DNA with GUS gene as template as positive control; -represents a negative control with non-transgenic populus megalobii DNA as template; i is QRT-PCR detection result.

Detailed Description

The invention provides a method for establishing a transgenic system of populus davidiana based on hygromycin screening, which comprises the following steps:

1) introducing the recombinant vector containing the target gene into agrobacterium and culturing to obtain agrobacterium liquid;

2) infecting the populus euphratica explants with the agrobacterium liquid, co-culturing, and removing bacteria to obtain the infected populus euphratica explants;

3) inoculating the infected populus euphratica explant to a hygromycin-containing leaf adventitious bud differentiation culture medium for screening culture to obtain an adventitious bud;

4) and carrying out rooting culture on the adventitious bud to obtain a transgenic regeneration plant of the populus euphratica.

The invention introduces a recombinant vector containing a target gene into agrobacterium for culture to obtain agrobacterium liquid.

The method of constructing the recombinant vector containing the target gene of the present invention is not particularly limited, and a method of constructing a recombinant vector known in the art may be used. The present invention is not particularly limited in kind of the target gene, and a target gene of interest in the art may be used. In order to illustrate the implementation process of the genetic transformation system constructed by the invention, the GU reporter gene is used as a target gene, GUS-transformed plants are detected by using X-Gluc staining solution, and the transgenic efficiency is screened by a chromogenic reaction. Plants that are positive will appear blue in color. The reporter gene is convenient to use, and a positive transformant of the reporter gene can be easily detected.

In the present invention, the basic backbone vector of the recombinant vector containing the target gene is preferably a pCAMBIA1302 vector. The pCAMBIA1302 vector is available from the addge official website (https:// www.fenghbio.cn/.

The method for introducing the recombinant vector into Agrobacterium in the present invention is not particularly limited, and a transformation method well known in the art may be used. The agrobacterium is preferably agrobacterium tumefaciens. The strain of agrobacterium tumefaciens includes EHA101, EHA105, or LBA 4404. Experiments show that different strains of agrobacterium tumefaciens have different transfection effects, and compared with the GV3101 strain, the EHA101, EHA105 or LBA4404 strain has significant advantage in resistant bud induction rate, which reaches more than 30%.

In the invention, the OD value of the agrobacterium liquid is preferably 0.5-0.8, more preferably 0.6-0.8, and most preferably 0.8. Experiments show that the higher the OD value of the agrobacterium liquid is, the highest transgene efficiency is obtained, and the OD is₆₀₀After reaching 1.0, the induction rate of resistant shoots was reduced, when OD was reached₆₀₀The transgenic efficiency is highest when the concentration is 0.8, and the bacterial liquid concentration is too highHigh growth speed of bacteria, difficult bacteria removal and high pollution rate, so the OD is selected in the invention₆₀₀0.8 is most suitable.

After the agrobacterium liquid is obtained, the populus Davidii explant is infected by the agrobacterium liquid and then is co-cultured, and after bacteria are removed, the infected populus Davidii explant is obtained.

In the invention, the infection time of the agrobacterium liquid is preferably 25-50 min, more preferably 30-45 min, and most preferably 30 min. Experiments show that the dip-dyeing time directly influences the transgenic efficiency, the too short infection time is not beneficial to agrobacterium to transfer target genes into plant cells, and the too long time easily causes the reduction of the activity of explants. The induction rate of resistant buds is higher for 30min and 45min than for 15min, and there is no difference between 30min and 45min because 30min is selected to be suitable.

In the invention, the co-culture time of the agrobacterium tumefaciens liquid and the populus euphratica explant is preferably 2-3.5 days, and more preferably 2.5-3 days. Experiments show that the bacteria grow vigorously and are not easy to remove due to too long co-culture time, and the induction rate of the resistant buds is obviously reduced after the co-culture time exceeds 4 days, so that the selection of the co-culture time for 3 days is optimal. The co-culture temperature of the agrobacterium tumefaciens liquid and the populus euphratica explant is preferably 20-27 ℃, more preferably 22-25 ℃, and most preferably 23 ℃.

In the invention, during the co-culture, the agrobacterium liquid and the populus euphratica are placed in the adventitious bud differentiation culture medium of the leaf. The leaf adventitious bud differentiation culture medium is preferably an MS culture medium containing 0.5-2 mg/L6-BA, 0.2mg/L NAA and 0-0.1 mg/L LTDZ, and more preferably an MS culture medium containing 1-2 mg/L6-BA and 0.2mg/L NAA or an MS culture medium containing 1-2 mg/L6-BA, 0.2mg/L NAA and 0.1mg/L TDZ. The preparation and sterilization method of the adventitious bud differentiation medium for leaves in the invention is not particularly limited, and the preparation and sterilization method of the medium known in the field can be adopted.

In the present invention, the populus megalophylla explant preferably comprises a stem section and a leaf, more preferably a stem section. The source of the populus euphratica explant is preferably a sterile tissue culture seedling of populus euphratica. The preparation method of the tissue culture seedling of the populus euphratica comprises the steps of sterilizing and washing tissue materials of the populus euphratica to prepare explants, inoculating the explants to the adventitious bud differentiation culture medium of the leaves, and inoculating the obtained adventitious buds to a rooting culture medium for rooting culture to obtain the tissue culture seedling. The rooting medium is preferably 1/2MS medium without any auxin added. The rooting culture time is preferably four weeks.

In the invention, the method for removing the bacteria adopts sterilized water to wash the cultured populus euphratica explants for 3-5 times.

After the infected Chinese white poplar explant is obtained, the infected Chinese white poplar explant is inoculated to a leaf adventitious bud differentiation culture medium containing hygromycin for screening culture to obtain an adventitious bud.

In the invention, the adventitious bud differentiation medium of the leaf is preferably an MS medium containing 0.5-2 mg/L6-BA, 0.2mg/L LNAA and 0-0.1 mg/L LTDZ, more preferably an MS medium containing 2-1 mg/L6-BA and 0.2mg/L LNAA or an MS medium containing 2-1 mg/L6-BA, 0.2mg/L LNAA and 0.1mg/L TDZ, and most preferably an MS medium containing 2.0 mg/L6-BA and 0.2mg/L LNAA. The concentration of hygromycin in the adventitious bud differentiation culture medium for the leaves is preferably 2.5-3.2 mg/L, and more preferably 3.0 mg/L.

In the invention, the temperature of the screening culture is preferably 22-25 ℃, and most preferably 23 ℃. The time of the screening culture is preferably 12-17 d, and more preferably 15 d. The critical concentration of hygromycin for leaf differentiation is selected to be effective in inhibiting the growth of non-transformed cells to allow them to die slowly without affecting the normal growth of transformed cells. Experiments show that the number of differentiated adventitious buds is reduced along with the increase of hygromycin concentration, and only a small amount of adventitious buds can be generated when the hygromycin concentration is 1mg/L and 2mg/L, but buds cannot be differentiated to form when the hygromycin concentration is 3 mg/L. Therefore, when genetic transformation of populus Davidii is carried out, hygromycin concentration of 3mg/L is suitable.

In the present invention, the rooting medium is preferably 1/2MS medium. The populus euphratica is easy to root, the root system is developed, the adventitious bud can naturally extend and root when growing on a culture medium without hormone, and the transgenic plant can be obtained.

In the present invention, the obtained transgenic plant is tested. The detection method comprises physiological and biochemical detection and molecular detection. The physiological and biochemical tests were examined for the effect of a reporter gene. In the embodiment of the invention, GUS gene can be subjected to chemical staining, and leaves of plant tissues are discolored, so that the transgenic plant is judged to be a positive transgenic plant. The molecular detection preferably adopts PCR and qRT-PCR detection methods to detect target gene fragments, and whether positive transgenic plants exist is judged by analyzing the fragments and expression quantity of the target genes. According to the method, the genetic transformation efficiency of the populus euphratica transgenic system is detected through GUS histochemical staining, PCR and qRT-PCR, and the stable genetic transformation efficiency of the transgenic system is over 10%.

The method for establishing a transgenic populus tremuloides system based on hygromycin screening provided by the present invention is described in detail below with reference to the following examples, but they should not be construed as limiting the scope of the present invention.

Example 1

Screening of adventitious bud Induction Medium

When the annual populus deltoids seedlings grow for 2 months, the height of the seedlings is about 50cm, and top leaves are selected from the seedlings. Placing the mixture in a 100ml triangular flask, wrapping the bottle mouth with gauze, tightly binding, placing the mixture under a tap for washing for two to four hours, taking out the material, placing the material in 75% alcohol for disinfection for 30s to 1min, washing with autoclaved distilled water for 3 times, placing the material in 1% sodium hypochlorite solution for disinfection for 20min, and washing with sterilized water for 3 times. Selecting three growth regulators of 6-BA, NAA and TDZ by taking MS as a basic culture medium, setting different concentration gradients to obtain a plurality of culture media with different concentration gradients, respectively inoculating stem sections and leaves of the tissue culture seedlings of the populus euphratica to different screening culture media, culturing 10 explants in each culture dish, repeating each treatment for three times, subculturing once every four weeks, and recording the induction rate of the adventitious buds treated differently.

The results of the screening of the different types of screening media are shown in Table 1.

TABLE 1 Induction of adventitious buds by different growth-regulating substances

The results in Table 1 show that the differentiation of adventitious buds after the combination of 6-BA, NAA and TDZ with different concentrations has the highest differentiation efficiency of adventitious buds under the conditions of 1.0mg/L of 6-BA, 0.2mg/LNAA and 0.1mg/LTDZ (see Table 1), and the differentiation efficiency of adventitious buds reaches 96.3 percent.

Example 2

Influence of different parameters of Agrobacterium tumefaciens mediated method on transformation rate

Inserting a reporter gene GUS into a plant expression vector pCAMBIA1302, transforming the reporter gene GUS into agrobacterium tumefaciens, culturing the reporter gene GUS in a culture medium of 1.0 mg/L6-BA +0.2mg/LNAA +0.1mg/LTDZ, and culturing the positive agrobacterium tumefaciens transformed with GUS to a certain concentration through identification to obtain agrobacterium tumefaciens bacterial liquid.

Respectively infecting the populus Davidii explants by using agrobacterium tumefaciens bacterial liquid, adding the adventitious bud induction culture medium obtained by screening in the embodiment 1 after infection is finished, co-culturing, and detoxifying to obtain the infected populus Davidii explants. The infected populus Davidii explants are subjected to induction culture in the hygromycin-containing adventitious bud induction culture medium obtained by screening in example 1 to obtain adventitious buds. Transferring the adventitious bud to 1/2MS culture medium to obtain transgenic populus euphratica regeneration plant.

1) Effect of Agrobacterium tumefaciens strains on the Induction Rate of resistant shoots

Effect of using different agrobacterium strains EHA101, EHA105, LBA4404, GV13014 on populus euphratica transgenic efficiency. Transferring the constructed vector into the 4 strains, detecting to be qualified, and culturing to OD₆₀₀And when the value is 0.8, collecting the agrobacterium liquid. Infecting tender leaves of populus euphratica for 30min at normal temperature with agrobacterium liquid, infecting 50 explants each time, repeating the three times of biology, and statistically calculating the induction rate (%) of the resistant bud according to formula I after culturing for one month.

The induction rate (%) of the resistant shoot is the number of leaves from which the resistant shoot grew/total number of leaves of the transgenic explant (number) formula I.

TABLE 2 results of the different Agrobacterium strains on the rate of induction of resistant shoots

The results show (table 2) that different agrobacterium strains also have certain influence on the induction rate of resistant buds, and compared with GV3101, EHA101, EHA105 and LBA4404 have obvious advantages on the induction of resistant buds, wherein the induction rate of EHA105 on the resistant buds of populus euphratica is the highest and is as high as 35.3%, so that the EHA105 strain is used for subsequent transgenosis.

2) Influence of Agrobacterium tumefaciens liquid OD value on induction rate of resistant bud

The infection concentration of Agrobacterium is also a key factor affecting the transformation efficiency of transgenes, respectively using OD₆₀₀Mixing EHA105 strain liquid of 0.2, 0.5, 0.8, 1.0 and 1.2 with explants, and dip-dyeing at normal temperature for 30min, wherein 50 explants are dip-dyed each time, and biological repetition is carried out for three times. The infected explants are inoculated to the adventitious bud induction culture medium selected in the example 1 for co-culture, washed by sterilized water and sterilized, and then inoculated to the adventitious bud induction culture medium containing hygromycin for screening culture, and the induction rate (%) of the resistant buds is counted after one month of culture.

TABLE 3 influence of Agrobacterium concentration on the rate of induction of resistant shoots

The results show (Table 3) that the induction rate of resistant shoots increases with increasing concentration of Agrobacterium solution, but OD₆₀₀After reaching 1.0, the induction rate of resistant shoots began to decrease, when OD was reached₆₀₀The transgenic efficiency is highest when 0.8 is taken, and the bacterial liquid concentrationToo high, the growth rate of the bacteria is relatively fast, the bacteria are difficult to remove, the pollution rate is relatively high, and therefore, the OD is selected₆₀₀And most preferably 0.8.

3) Effect of Dip time on Induction Rate of resistant shoots

To investigate the effect of the time of the transfection on the efficiency of the transgenesis, OD was used₆₀₀Respectively infecting the populus Davidiana explants for 15min, 30min and 45min by using 0.8 EHA105 strain liquid, carrying out screening culture on an adventitious bud induction culture medium containing 3mg/L hygromycin after 3 days of culture, and counting the induction rate (%) of resistant buds after culturing for one month.

TABLE 4 Effect of Dip time on Induction Rate of resistant shoots

The results show (Table 4) that the induction rate of resistant buds is higher for 30min and 45min than for 15min, and there is no difference between 30min and 45min, because 30min is selected appropriately.

4) Effect of Co-cultivation time on Induction Rate of resistant shoots

In order to study the influence of the co-culture time on the transgenic efficiency, four time periods of 1d, 2d, 3d and 4d are selected on the basis of the culture method to judge the influence of the time periods on the induction rate of the resistant buds.

TABLE 5 Effect of Co-cultivation time on the Induction Rate of resistant shoots

The results show (Table 5) that the efficiency was highest in the co-culture for 3 days, followed by 2 days. Too long co-culture time can cause the bacteria to grow vigorously and not to be easy to be killed, so that the induction rate of the resistant buds is obviously reduced after 4 days. Therefore, the selection of 3 days co-culture resulted in the best results.

5) Temperature of co-cultivation

Co-culture temperature sometimes also has an influence on the transgenic efficiency, and co-culture is carried out for 3 days under the culture conditions of 21 ℃, 23 ℃ and 25 ℃, respectively, 50 explants are infected each time, and biological repetition is carried out for three times. After 3 days of co-culture, the induction rate (%) of resistant shoots was counted after one month of selection culture in adventitious shoot induction medium containing 3mg/L of hygromycin.

TABLE 6 Effect of Co-cultivation temperature on the Induction Rate of resistant shoots

The results showed (Table 6) that the induction rate of resistant shoots was the highest at the co-cultivation temperature of 23 ℃ but the induction rates of resistant shoots were not greatly different at the three temperatures.

6) Determination of hygromycin screening concentration

On the basis of the screening results, the wild type populus Davidiana explants are taken as materials, and 6 hygromycins with the concentration of 0, 1, 2, 3, 4, 5 and 6mg/L are respectively added into a solid differentiation medium for screening for half a month. And (3) culturing on the basis of the screening result because the hygromycin is high in toxicity, counting the survival rate for half a month, and finally taking the critical concentration which is just dead as a sensitivity test of the hygromycin with proper concentration for screening the transformants.

TABLE 7 Effect of different concentrations of hygromycin on adventitious bud Induction in populus Davidii

The young leaf is inoculated on a control of a leaf adventitious bud differentiation culture medium which does not contain hygromycin, the leaf can almost generate a large number of adventitious buds after inoculation, and the leaf has different reactions on culture media added with hygromycin with different concentrations. As can be seen from Table 2, the number of adventitious buds differentiated decreases with an increase in hygromycin concentration, and only a small number of adventitious buds can be produced at hygromycin concentrations of 1mg/L and 2mg/L, but buds cannot be differentiated to form at a concentration of 3 mg/L. The critical concentration of hygromycin for leaf differentiation is chosen to be both effective in inhibiting the growth of non-transformed cells, allowing them to die slowly, and not to affect the normal growth of transformed cells. Therefore, when genetic transformation of populus Davidii is carried out, hygromycin concentration of 3mg/L is suitable.

Hygromycin is selected as a screening marker, so that the transformant obtained by screening is relatively low in false positive, more than 95% of transformants generating resistance are positive after molecular detection, and compared with the false positive result with half the probability about the screening effect of the kanamycin as the screening marker gene selected previously, the accuracy of the populus euphratica transformation system can be improved by using hygromycin as the screening marker.

7) The explant type: selecting stem and leaf of well-grown tissue culture seedling as explant, and culturing with Agrobacterium liquid (OD)₆₀₀0.8) for 30min, 50 explants per time, three biological replicates, and the induction rate (%) of resistant shoots was counted after one month of culture.

TABLE 8 Effect of different explants on the Induction Rate of resistant shoots

The results show (Table 8) that the efficiency of leaf stainings was somewhat higher, that resistant shoots were obtained from the leaves as explants in higher numbers than from the stem sections, and that the stem sections were less susceptible to bacterial shedding. However, the differentiation rate of the resistant buds exceeds 20 percent, and the screening time can be controlled within one month to achieve the expected technical goal.

Example 3

A method for establishing a transgenic system of populus megalophylla based on hygromycin screening comprises the following steps:

1) inserting GUS gene into plant expression vector PCAMBIA1302, transforming the obtained recombinant vector into Agrobacterium tumefaciens EHA105 strain, culturing, and performing amplification reaction by using specific primer F of GUS gene: 5'-AATCCATCGCAGCGTAATGCTCT-3' (SEQ ID NO: 1); r: 5'-GCTGGCCTGCCCAACCTTTCGGTAT-3' (SEQ ID NO:2) to obtain a positive agrobacterium liquid containing GUS gene;

2) by OD₆₀₀Infecting sterilized young leaf of David poplar with 0.8 of Agrobacterium liquid for 30min, adding adventitious bud differentiation culture medium (containing 2 mg/L6-BA and 0.2 m)g/LNAA MS culture medium) is co-cultured for 3d at the temperature of 23 ℃, and is sterilized after being washed for 3-4 times by using sterilized water, so as to obtain the infected populus euphratica explant;

3) inoculating the infected populus Davidii explants to a leaf adventitious bud differentiation culture medium containing 3mg/L hygromycin, and screening and culturing for 1 month at 23 ℃ to obtain a large number of adventitious buds; counting the number of adventitious buds, and calculating the induction rate of resistant buds, wherein the induction rate of resistant buds is up to more than 35%;

4) and (3) inoculating the adventitious bud into an 1/2MS culture medium without any auxin for continuous culture for four weeks to obtain a transgenic regeneration plant of the populus euphratica.

5) Detecting the obtained transgenic regeneration plant material of the populus euphratica by using X-Gluc staining solution, and simultaneously taking wild type populus euphratica as a control. The transgenic efficiency is screened by a color reaction, and most of screened resistant buds are positive (more than 98%). The results are shown in FIG. 2.

Compared with a control group, the poplar transgenic regeneration system constructed by the invention is blue, which indicates that the constructed poplar transgenic regeneration system successfully constructs a regeneration system containing a GUS gene.

And simultaneously extracting DNA and RNA of transgenic plant materials with positive dyeing results, detecting GUS genes and expression conditions thereof by respectively adopting a common PCR amplification method and a qRT-PCR method, and simultaneously taking wild type populus megalophylla (WT) as a control.

Common PCR amplification method

Designing a specific primer F of the GUS gene: 5'-AATCCATCGCAGCGTAATGCTCT-3' (SEQ ID NO: 1); r: 5'-GCTGGCCTGCCCAACCTTTCGGTAT-3' (SEQ ID NO: 2). Randomly selecting 14 GUS positive transgenic plant strains, extracting genome DNA, and carrying out PCR amplification detection by using the genome DNA as a template and using a specific primer F and a specific primer R as a primer pair, wherein the PCR program is pre-denaturation at 94 ℃ for 3 min; denaturation at 94 deg.C for 30s, annealing at 58 deg.C for 30s, extension at 72 deg.C for 1min, and circulation for 30 times; total extension at 72 ℃ for 7 min. The results are shown in FIG. 3 as H, and 7 strains are positive after PCR detection.

② real-time fluorescent quantitative PCR (qRT-PCR) analysis method

The RNA of the 7 strains is extracted by a kit method, a PuActin gene (primer sequence: 5'-TGTTGCCCTTGACTATGAGCAGGA-3', SEQ ID NO: 5; 5 'acggaatctctcagctccaatggt-3', SEQ ID NO: 6) is used as an internal reference gene, and the RNA of the 7 strains is extracted by a kit method, wherein the RNA of the 7 strains is expressed by the following genes: 5'-TACGGCAAAGTGTGGGTCAA-3' (SEQ ID NO:3) and 5'-TCTTGCCGTTTTCGTCGGTA-3' (SEQ ID NO:4) as primers for qRT-PCR amplification, wherein the total reaction system of qRT-PCR is 20. mu.L: 1 uL cDNA template, 1 uL forward and reverse primers respectively, 10 uL 2 × Easy Taq PCR Supermix enzyme, and 7 uL deionized water. Reaction procedure: pre-denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, 60 ℃ for 30s, for 45 cycles; plates were read every 0.5 ℃ from 55 ℃ to 99 ℃ for 1 s.

As a result, as shown by I in FIG. 3, the expression level of GUS gene was varied in 7 lines, but the expression level was many times higher in 7 lines than in WT.

Example 4

A method for establishing a transgenic system of populus megalophylla based on hygromycin screening comprises the following steps:

1) inserting GUS-containing gene into plant expression vector pCAMBIA1302 to obtain recombinant vector, transforming the recombinant vector into Agrobacterium tumefaciens EHA101 strain, culturing, and performing PCR amplification and identification to obtain positive Agrobacterium tumefaciens bacterial liquid containing GUS gene;

2) by OD₆₀₀After infecting the disinfected young stem section of the populus Davidii for 30min by using 0.7 agrobacterium liquid, adding a leaf adventitious bud differentiation culture medium (an MS culture medium containing 1mg/L6-BA, 0.2mg/LNAA and 0.1 mg/LTDZ) to perform co-culture for 3d at the temperature of 23 ℃, washing for 3-4 times by using sterilized water, and then removing bacteria to obtain an infected populus Davidii explant;

4) and (3) inoculating the adventitious bud into an 1/2MS culture medium without any auxin for continuous culture for four weeks to obtain a transgenic regeneration plant of the populus euphratica, wherein the false positive rate is lower than 2%.

5) And (3) carrying out dyeing detection on the obtained transgenic regeneration plant material of the populus euphratica by using an X-Gluc dyeing solution, and simultaneously using wild type populus euphratica as a control.

The genetic transformation efficiency of the populus deltoids transgenic system is detected through GUS histochemical staining, PCR and qRT-PCR, and the stable genetic transformation efficiency of the transgenic system is over 10 percent.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> northeast university of forestry

<120> method for establishing populus euphratica transgenic system based on hygromycin screening

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