Agrobacterium rhizogenes-mediated pear transgenic method

Abstract

The invention discloses an efficient pear transgenosis method which is mediated by agrobacterium rhizogenes and belongs to the technical field of genetic transformation. According to the method, agrobacterium rhizogenes K599 containing a selection marker gene mCherry is used for infecting leaves of tissue culture seedlings of sorbose No.1 and Coanvens, and transgenic positive roots can be obtained by accurately controlling pre-culture time, bacterial liquid OD600 value, infection time, co-culture time, hairy root induction culture medium components and dark culture time. The method is high in transformation efficiency and short in period, the generated hairy roots can autonomously grow on a culture medium without adding exogenous hormones, and the transferred target genes can be stably expressed in the hairy roots for a long time. In addition, the expression vector contains red fluorescent protein mCherry as a reporter gene, so that transgenic positive roots can be directly screened through fluorescent signals.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic transformation, and more specifically relates to a pear transgenic method mediated by Agrobacterium rhizogenes. Background Art

[0002] Pear is a plant of the genus Pyrus (Pyrus.L) in the subfamily Maloideae of the family Rosaceae. It is one of the important deciduous fruit trees in the world. It can be divided into Oriental pear and Western pear according to its origin. Oriental pear mainly includes white pear, autumn pear, sand pear and Xinjiang pear. 'Yamali No. 1' is a wild species of autumn pear, which has strong cold resistance and stress resistance, strong adaptability, and is often used as a rootstock; 'Confrance' is a cultivated variety of Western pear, with moderate cold resistance and poor resistance to rot, but the fruit has high commercial value.

[0003] As one of the important fruit trees in my country, pear trees have a long breeding cycle and low efficiency due to their self-incompatibility and long juvenile period. Therefore, it has become an urgent task for the pear industry to accelerate the cultivation of new pear varieties with excellent traits using genetic engineering technology. Establishing an efficient pear genetic transformation system is not only conducive to in-depth research on the biological functions of key genes, but also provides technical support for the precise improvement of quality traits. However, it usually takes 5 to 6 months to obtain positive buds using the transformation method mediated by Agrobacterium tumefaciens, and the transformation efficiency is only about 2%. In addition, chimeras may appear during the transformation process, which increases the complexity of the transformation work and the time required for functional gene verification.

[0004] Agrobacterium rhizogenes, like Agrobacterium tumefaciens, belongs to the genus Agrobacterium under the family Rhizoliaceae. It has a circular genomic DNA, called the Ri plasmid, which has the ability to replicate genetically independently of the cell nucleus. The T-DNA on the Ri plasmid carries genes related to the formation of hairy roots and auxin synthesis, allowing the recipient plant to produce hairy roots that can grow spontaneously on a culture medium without the addition of plant hormones. The hairy roots produced by Agrobacterium rhizogenes transformation have a single-cell origin, fast growth rate, high degree of differentiation, physiological and biochemical and genetic stability, and can obtain stable transformed roots in a relatively short period of time without the presence of chimeras. Therefore, the establishment of a simple and efficient Agrobacterium rhizogenes transformation system is of great significance for the genetic improvement of pears. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a pear transgenic method mediated by Agrobacterium rhizogenes.

[0006] The invention discloses a pear transgenic method mediated by Agrobacterium rhizogenes, comprising constructing a recombinant expression vector containing a screening marker gene mCherry and a target gene, transferring the correctly identified recombinant expression vector into Agrobacterium rhizogenes, preparing an infection solution by using the Agrobacterium rhizogenes, and using the Agrobacterium rhizogenes to infect leaves of pre-cultured subcultured tissue culture seedlings, followed by co-cultivation and induction to obtain regenerated transgenic positive roots.

[0007] As a preferred embodiment of the present invention, the expression vector construction method is: the screening marker mCherry (FPbase accession number: ZERB6) and the target gene are constructed into the vector pGG8-17 (SEQ ID NO.1) by the Golden gate assembly method, mCherry is driven by the cauliflower mosaic virus (CaMV) 35S promoter, and the target gene is driven by the promoter of the Arabidopsis thaliana UBQ10 gene (AT4G05320).

[0008] As a preferred embodiment of the present invention, the correctly identified recombinant vector is transformed into Agrobacterium rhizogenes K599 competent cells using freeze-thaw transformation method.

[0009] As a preferred embodiment of the present invention, the preparation method of the Agrobacterium rhizogenes infection solution is as follows: Agrobacterium rhizogenes is streaked and activated on a TY solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, cultured at 28°C for 2 days, and then a single colony is picked and inoculated on a medium containing 50 mg / L kanamycin, 50 mg / L streptomycin, 1M CaCl 2 TY liquid medium, shake at 28℃ and 180rpm, take the overnight culture liquid and add it to a new medium containing 50mg / L kanamycin, 50mg / L streptomycin, 1MCaCl 2 The cells were cultured in TY liquid medium at 28°C with shaking at 180 rpm until the logarithmic growth phase. The cells were collected by centrifugation at 5000 rpm for 5 min, the supernatant was discarded, and the infection solution was diluted to the required OD using MS liquid medium (100 μM acetosyringone, pH 5.8) as the resuspension medium. 600 value for subsequent experiments.

[0010] As a preferred embodiment of the present invention, the old leaves of the pear tissue culture seedlings are removed and placed on MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar medium for growth for 1 month, then the tender leaves at the top of the tissue culture seedlings are selected, the petioles are cut off, and the leaves are placed on MS solid medium for pre-culture for 0-1d to obtain the leaves of the pre-cultured subculture tissue culture seedlings.

[0011] As a preferred embodiment of the present invention, the pear tissue culture seedling variety is selected from 'Yamanashi No. 1' or 'Confrens'.

[0012] As a preferred embodiment of the present invention, 'Shanli No. 1' is placed on MS solid culture medium for pre-culture for 1 day, while 'Confrens' does not require pre-culture.

[0013] As a preferred embodiment of the present invention, the process of Agrobacterium rhizogenes infection is as follows: the leaves of the pre-cultured subcultured tissue culture seedlings are immersed in the Agrobacterium rhizogenes infection solution; the inoculated leaves are dried with the bacterial solution and placed in a co-culture medium for culture; the leaves are transferred to a hairy root induction medium for dark culture, and then light culture is performed for 26 to 28 days to obtain transgenic pear roots.

[0014] As a further preferred embodiment of the present invention, the infection time of Agrobacterium rhizogenes is preferably 10 min for 'Shanpi No. 1' and 20 min for 'Confrens'.

[0015] As a further preferred embodiment of the present invention, the co-culture medium is MS+30 g / L sucrose+100 μM acetosyringone+7 g / L agar, with a pH of 5.80-5.85; the co-culture time for 'Shanpi No. 1' is 5 days, and that for 'Confrens' is 3 days.

[0016] As a further preferred embodiment of the present invention, the hairy root induction medium: 'Shanxi No. 1' is 1 / 4MS + 30g / L sucrose + 500mg / L Timentin + 7g / L agar, pH 5.80-5.85, and 'Confrance' is 1 / 8MS + 30g / L sucrose + 125mg / L Timentin + 7g / L agar, pH 5.80-5.85; the dark culture time for 'Shanxi No. 1' is 1d, and that for 'Confrance' is 5d.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The present invention uses Agrobacterium rhizogenes K599 to infect the leaves of the tissue culture seedlings of 'Yamanashi No. 1' and 'Confrens'. The pre-culture time, OD value of the bacterial solution, and the 600 The transgenic positive roots were obtained by controlling the value, infection time, co-culture time, hairy root induction medium composition, and dark culture time. The induction ability of transgenic hairy roots of different varieties was significantly different. The induction efficiency of hairy roots of 'Yamanashi No. 1' was much higher than that of 'Confrens', which was about 75%. In addition, the transgenic method of the present invention is simple to operate, the culture conditions are easy to control, the cycle is short, and the hairy roots can grow autonomously on a culture medium without adding exogenous hormones, and the transferred target gene can be stably expressed in the hairy roots for a long time.

[0019] (2) The present invention uses the red fluorescent protein mCherry as a screening marker, so the transgenic roots have obvious red fluorescent signals, while the untransformed roots do not have this signal. A dual-wavelength fluorescent protein excitation light source LUYOR-3415RG (LUYOR, Shanghai, China) can be used in conjunction for rapid screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Flow chart of pear transgenic mediated by Agrobacterium rhizogenes in the embodiment of the present invention

[0021] Figure 2 This is the effect of pre-culture time on the transformation efficiency of hairy roots of "Yamanashi No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0022] Figure 3 3. The effect of different concentrations of Agrobacterium rhizogenes on the transformation efficiency of hairy roots of "Yamanashi No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0023] Figure 4 The figure shows the effect of different concentrations of acetosyringone on the transformation efficiency of hairy roots of "Shanli No. 1" and "Conflens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0024] Figure 5 1 and Confrens, respectively. The figure shows the effect of infection time on the transformation efficiency of hairy roots of "Yamanashi No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown above the figure.

[0025] Figure 6 This is the effect of co-cultivation time on the transformation efficiency of hairy roots of "Yamanashi No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0026] Figure 7 This is the effect of different types of basic culture media on the transformation efficiency of hairy roots of "Yamanashi No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0027] Figure 8 This is the effect of sucrose concentration in the screening culture medium on the transformation efficiency of hairy roots of "Shanli No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0028] Fig. 9 This is the effect of the antibiotic timentin concentration on the transformation efficiency of hairy roots of "Shanli No. 1" and "Confrens" in the examples of the present invention. The root number and GUS staining rate are shown above the figure.

[0029] Fig.10 This is the effect of dark culture time on the transformation efficiency of hairy roots of "Yamanashi No. 1" and "Conflens" in the examples of the present invention. The root number and GUS staining rate are shown on the top of the figure.

[0030] Fig.11 Graph showing the comparison of root transformation efficiency of six marker genes in the present invention.

[0031] Fig.12 The transgenic root obtained by infection using the embodiment of the present invention is shown in FIG.

[0032] Fig.13 The pGG8-17 plasmid map DETAILED DESCRIPTION

[0033] Example 1

[0034] The specific operation process of constructing the GUS reporter gene expression vector is as follows: First, the commercial plant binary expression vector pRI 101-AN (TaKaRa, catalog number: 3262) was linearized by double restriction digestion using KpnI and BamHI endonucleases. Then, the GUS (β-Glucuronidase) reporter gene was cloned. The cloning system and PCR procedure were referred to GXL DNA Polymerase (TaKaRa, Catalog No.: R050A) product manual. The size of the PCR product fragment was detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision using FastPure Gel DNA Extraction Mini Kit (Vazyme, Catalog No.: DC301-01). Finally, the GUS gene was connected to the linearized expression vector using the In-Fusion cloning method to form a recombinant vector. The cloning system was referred to the In- HD Cloning Kit (TaKaRa, Cat. No. 639649). The transcriptional expression of the GUS gene is driven by the cauliflower mosaic virus (CaMV) 35S promoter.

[0035] Example 2

[0036] We conducted a preliminary experiment using the hairy root transformation system reported in other species, using the leaves of subcultured tissue culture seedlings of 'Yamanashi No. 1' as the material. No pre-culture was performed in the experiment, and OD 600 The root-inducing Agrobacterium liquid was 0.3, and 100 μmol / L acetosyringone was added, and the infection time was set to 10 minutes. Subsequently, the leaves were co-cultured in the dark on MS solid medium containing 100 μmol / L acetosyringone for 3 days, and then transferred to the root-inducing medium (1 / 2MS, 30 g / L sucrose, 250 mg / L timentin) for light culture for 28 days. Although transgenic positive roots can be successfully obtained, the transformation efficiency is low. Based on this, we used a single-factor experimental method, using the leaves of subcultured tissue culture seedlings of 'Yamali No. 1' and 'Confrens' as materials, to explore the effects of the following 9 factors on the root transformation efficiency, and screened the optimal marker gene. At the same time, we updated the transformation conditions in real time, aiming to establish an efficient genetic transformation system for pear hairy roots:

[0037] 1) Effect of pre-culture time on root transformation efficiency

[0038] Pre-culture can promote cell division at the wound of the explant, make it easier for the recipient cells to integrate foreign genes, and improve the transformation efficiency. In this experiment, the top tender leaves of the tissue culture seedlings were selected, the petioles were removed, and they were placed on MS solid medium for pre-culture for 0, 1, 3, and 5 days in order to screen the most suitable pre-culture time. The hairy root induction rate and GUS staining rate of 'Yamanashi No. 1' first increased and then decreased with the increase of pre-culture time. When the pre-culture time was 1 day, the hairy root induction rate was the highest, which was 97.78%, and the GUS staining rate was 50.54%, which was significantly higher than that of other pre-culture time treatment groups (Table 1 and Figure 2 ). The hairy root induction rate of 'Confrens' gradually decreased with the extension of pre-culture time. When the pre-culture time was 0 days, the hairy root induction rate was the highest, which was 42.22%. There was no significant difference in the hairy root induction rate between 0 days and 1 day of pre-culture. The GUS staining rate of all pre-culture time treatment groups reached 100.00% (Table 2 and Figure 2 ). According to the above results, the optimal pre-culture time of 'Yamanashi No. 1' is 1 day, and the optimal pre-culture time of 'Confrens' is 0 day.

[0039] Table 1 Effect of pre-culture time on root transformation efficiency of 'Shanli No. 1'

[0040]

[0041] Table 2 Effect of pre-culture time on root transformation efficiency of 'Confrens' pear

[0042]

[0043] 2) Effect of Agrobacterium rhizogenes concentration on root transformation efficiency

[0044] The concentration of Agrobacterium tumefaciens liquid can affect the infection efficiency of Agrobacterium rhizogenes to a certain extent. In this experiment, OD 600 The four bacterial concentrations of 0.1, 0.3, 0.5, and 0.7 were used to screen the optimal bacterial concentration. The process of Agrobacterium rhizogenes infection was as follows: the leaves of the subcultured tissue culture seedlings after pre-culture were immersed in the Agrobacterium rhizogenes infection solution; the inoculated leaves were dried with bacterial solution and placed in the co-culture medium for co-culture; the leaves were transferred to the hairy root induction medium for dark culture, and then cultured under light for 28 days to obtain the transgenic pear roots. The results are shown in Tables 3, 4 and Figure 3 As shown in the figure, with the increase of bacterial solution concentration, the hairy root induction rate of 'Shanli No. 1' showed an upward trend. 600 The peak value was 64.44% when the bacterial solution OD was 0.7. 600When the concentration of OD was 0.5, the GUS staining rate was the highest, which was 53.73%. The differences in the hairy root induction rate and GUS staining rate among the treatment groups were not significant. Therefore, the bacterial solution concentration had little effect on the transformation of hairy roots of 'Yamanashi No. 1'. The hairy root induction rate of 'Confrens' showed a downward trend with the increase of bacterial solution concentration. 600 The highest concentration was 15.55% when the concentration was 0.1, which was significantly higher than that of other treatment groups, and the GUS staining rate of each treatment group reached 100.00%. Based on the above results, the optimal bacterial solution concentration for Agrobacterium rhizogenes to infect 'Yamanorashi No. 1' was 0.7, and that for 'Confrens' was 0.1.

[0045] Table 3 Bacterial solution OD 600 Effects on the root transformation efficiency of 'Shanli No. 1'

[0046]

[0047] Table 4 Bacterial solution OD 600 Effects on the transformation efficiency of 'Confrens' roots

[0048]

[0049] 3) Effect of acetosyringone concentration on root transformation efficiency

[0050] During the infection of Agrobacterium rhizogenes, the transformation efficiency of plants can be improved by adding acetosyringone to the infection solution. In this experiment, three acetosyringone concentrations of 0, 100, and 200 μmol / L were added to the MS liquid culture medium in order to screen the optimal acetosyringone concentration.

[0051] The results are shown in Tables 5, 6 and Figure 4 As shown. With the increase of acetosyringone concentration, the rooting rates of 'Yaman Pear No. 1' and 'Conforense' showed a trend of first increasing and then decreasing, reaching a peak at 100μmol / L, which were 71.11% and 44.44%, respectively. However, the GUS staining rate of 'Yaman Pear No. 1' reached a maximum of 100.00% at 0μmol / L acetosyringone, while the GUS staining rate of 'Conforense' reached 100.00% in all treatment groups. Based on the above results, the optimal acetosyringone concentration for 'Yaman Pear No. 1' and 'Conforense' was 100μmol / L.

[0052] Table 5 Effect of acetosyringone concentration on root transformation efficiency of 'Shanli No. 1'

[0053]

[0054] Table 6 Effect of acetosyringone concentration on transformation efficiency of 'Confrens' roots

[0055]

[0056]

[0057] 4) Effect of infection time on root transformation efficiency

[0058] This experiment set four different infection times, namely 5, 10, 15, 20, and 25 minutes, in order to screen the optimal infection time for Agrobacterium rhizogenes. Figure 5 It can be seen that with the increase of infection time, the root induction rate and root quantity of 'Yaman Pear No. 1' and 'Confolence' generally showed a trend of first rising and then falling. The root induction rate of 'Yaman Pear No. 1' reached a peak of 64.45% at 10 minutes of infection, and the root induction rate of 'Confolence' reached a maximum of 40.00% at 20 minutes of infection; there was no significant difference in the GUS staining rate of the root hairs of the two pears in each treatment group. The staining rate of 'Yaman Pear No. 1' was the highest at 10 minutes of infection, which was 57.58%, while the GUS staining rate of each treatment group of 'Confolence' was 100.00%. Based on the above results, the optimal infection time of Agrobacterium rhizogenes infection of 'Yaman Pear No. 1' is 10 minutes, and the optimal infection time of 'Confolence' is 20 minutes.

[0059] Table 7 Effect of infection time on root transformation efficiency of 'Shanli No. 1'

[0060]

[0061] Table 8 Effect of infection time on root transformation efficiency of 'Confrens'

[0062]

[0063] 5) Effect of co-cultivation time on root transformation efficiency

[0064] During the co-cultivation, Agrobacterium rhizogenes transfers its own T-DNA to the plant body and integrates it into the plant cell genome. The duration has an important influence on the induction of hairy roots. This experiment set up four co-cultivation time treatments of 1, 3, 5, and 7 days in order to screen the optimal co-cultivation time. The results are shown in Tables 9, 10 and Figure 6As shown. All four co-culture times can induce hairy roots in ‘Yaman Pear No. 1’ and ‘Confolence’. With the extension of co-culture time, the rooting rate showed a trend of first increasing and then decreasing. The hairy root induction rate of ‘Yaman Pear No. 1’ was the highest at 5 days of co-culture, which was 51.11%, and there was no significant difference in the induction rate at 3 days, 5 days, and 7 days of co-culture; the hairy root induction rate of ‘Confolence’ was the highest at 3 days of co-culture, which was 77.78%, significantly higher than other treatment groups; there was no significant difference in the GUS rate of hairy roots in each treatment of the two pears, and the GUS staining rate of each treatment of ‘Confolence’ reached 100.00%. Based on the above results, the optimal co-culture time for Agrobacterium rhizogenes to infect ‘Yaman Pear No. 1’ was 5 days, and the optimal co-culture time for ‘Confolence’ was 3 days.

[0065] Table 9 Effect of co-cultivation time on root transformation efficiency of 'Shanli No. 1'

[0066]

[0067] Table 10 Effect of co-cultivation time on root transformation efficiency of 'Confrens'

[0068]

[0069] 6) Effect of root induction medium on root transformation efficiency

[0070] To study the effect of culture medium type on hairy root induction, leaves after co-culture were placed on MS, 1 / 2MS, 1 / 4MS, 1 / 6MS, and 1 / 8MS culture media for hairy root induction in order to screen the most suitable culture medium for rooting. The results are shown in Tables 11, 12 and Figure 7 As shown. With the decrease of MS content in the culture medium, the root induction rates of 'Yaman Pear No. 1' and 'Confolence' showed a trend of first increasing and then decreasing. The root induction rate of 'Yaman Pear No. 1' reached a maximum of 40.00% at 1 / 4MS; 'Confolence' had better induction effects at 1 / 6MS and 1 / 8MS, which were 42.22% and 40%, respectively, but the average root number of leaves on 1 / 8MS medium was higher than that of 1 / 6MS, which was 0.82 roots / piece. The GUS staining rates of 'Yaman Pear No. 1' at 1 / 2MS, 1 / 4MS, 1 / 6MS, and 1 / 8MS were significantly higher than those of the MS treatment, and there was no significant difference in the GUS staining rates of these four treatments; the GUS staining rates of hairy roots of 'Confolence' in each treatment reached 100.00%. Based on the above results, the optimal culture medium for hairy root induction of 'Yaman Pear No. 1' is 1 / 4MS, and the optimal culture medium for 'Confolence' is 1 / 8MS.

[0071] Table 11 Effects of root induction medium on root transformation efficiency of 'Yamanashi No. 1'

[0072]

[0073]

[0074] Table 12 Effect of root induction medium on root transformation efficiency of 'Confrens'

[0075]

[0076] 7) Effect of sucrose concentration on root transformation efficiency

[0077] In order to study the effect of different sucrose concentrations on the genetic transformation efficiency of hairy roots of 'Shanli No. 1' and 'Confrens', four different concentrations of sucrose, 3.75, 7.5, 15, and 30 g / L, were added to the rooting medium in order to screen the optimal sucrose concentration. With the increase of sucrose concentration, the hairy root induction rate of 'Shanli No. 1' and 'Confrens' generally showed an upward trend. 'Shanli No. 1' had a better effect at sucrose concentrations of 15 g / L and 30 g / L, and there was no significant difference between the two. However, at 30 g / L sucrose, the number of leaves with roots was more, with an average of 2.29 roots / piece (Table 13 and Table 14). Figure 8 ); the rooting induction rate of 'Kong Fulunsi' was the highest at 30 g / L sucrose concentration, which was 22.22%, significantly higher than that of other treatment groups (Table 14 and Figure 8 ); There was no significant difference in the GUS rate of hairy roots in the two pear varieties, and the GUS staining rate of each treatment of 'Converence' reached 100.00%. Based on the above results, it was found that the optimal sucrose concentration for hairy root induction in 'Yaman Pear No. 1' and 'Converence' was 30g / L.

[0078] Table 13 Effect of sucrose concentration on root transformation efficiency of 'Shanli No. 1'

[0079]

[0080] Table 14 Effect of sucrose concentration on root transformation efficiency of 'Confrens'

[0081]

[0082] 8) Effect of Timentin Concentration on Root Transformation Efficiency

[0083] Appropriate antibiotic concentration is an important factor affecting the induction of hairy roots. In order to explore the effect of antibiotic concentration on the induction of hairy roots, 125, 250, 375, and 500 mg / L of timentin were added to the induction medium in order to screen the most suitable antibacterial concentration. With the increase of timentin concentration, the hairy root induction rate of 'Yaman Pear No. 1' showed an overall upward trend, reaching a peak of 66.67% at 500 mg / L. The average number of hairy roots at this time was also the highest, at 1.62 / piece. At the same time, the GUS staining rate of hairy roots in each treatment of 'Yaman Pear No. 1' reached more than 50.00%, and there was no significant difference among the treatments; while the rooting rate of 'Confrens' gradually decreased with the increase of antibiotic concentration, and the induction effect was the best at 125 mg / L, which was 57.78%. At the same time, when the timentin concentration reached more than 375 mg / L, the induction of hairy roots was completely inhibited. Based on the above results, it was found that the optimal concentration of timentin for adventitious root induction in 'Yamanashi No. 1' was 500 mg / L, and that in 'Confrens' was 125 mg / L.

[0084] Table 15 Effect of Timentin concentration on root transformation efficiency of 'Shanli No. 1'

[0085]

[0086] Table 16 Effect of Timentin concentration on root transformation efficiency of 'Confrens'

[0087]

[0088] 9) Effect of dark culture time on root transformation efficiency

[0089] In order to study the effect of different dark culture time on the genetic transformation efficiency of 'Yaman 1' and 'Confrens' mediated by Agrobacterium rhizogenes, this experiment set five dark treatment times of 0, 1, 3, 5, and 7 days on the hairy root induction medium after co-culture, in order to screen the optimal dark culture time. With the extension of dark culture time, the hairy root induction rate of 'Yaman 1' gradually decreased. There was no significant difference in the hairy root induction rate between 0d and 1d of dark culture, but the number of hairy roots in 1d of dark culture was significantly higher than that of 0d, which was 3.38 / piece. The GUS rate of hairy roots in each treatment was about 50.00% and there was no significant difference (Table 17 and Fig.10 ); The hairy root induction rate of 'Confrens' showed a trend of first increasing and then decreasing with the extension of dark culture time, reaching a peak of 57.78% at 5 days of dark culture, at which the average number of hairy roots was 0.64 / piece, and the GUS staining rate of hairy roots in each treatment reached 100.00% (Table 18 and Fig.10 ).

[0090] Table 17 Effect of dark culture time on root transformation efficiency of 'Shanli No. 1'

[0091]

[0092]

[0093] Table 18 Effect of dark culture time on root transformation efficiency of 'Confrens'

[0094]

[0095] 10) Screening of optimal marker genes for Agrobacterium rhizogenes-mediated pear genetic transformation system

[0096] In genetic transformation experiments, the GUS gene is often used as a marker gene to screen positive materials. However, after the plant material is stained, its cells have died and subsequent in vitro regeneration experiments cannot be carried out. Therefore, it is particularly important to screen a suitable marker gene to screen out transgenic materials without damaging the plant material. By comparing the hairy root induction rates of 'Yamanashi No. 1' and 'Confrance', it was found that the hairy root induction rate of 'Yamanashi No. 1' was significantly higher than that of 'Confrance'. Therefore, the hairy root induction system of 'Yamanashi No. 1' was used to screen marker genes. In this experiment, six marker gene vectors driven by p35s, DsRed, EGFP, mCherry, TdTomato, dTomato, and RUBY, were constructed. Through Agrobacterium rhizogenes transformation, it was found that the hairy root induction efficiency of mCherry, DsRed, and EGFP was better, and there was no significant difference among the three, but the transformation efficiency of mCherry was significantly higher than that of other marker genes, at 57.93% ( Fig.11 ab); It can be seen that mCherry has the best fluorescence expression effect, so the red fluorescent protein mCherry gene was selected as the marker gene for the screening of transgenic roots ( Fig.11 ci).

[0097] Example 3

[0098] (1) Grow the tissue culture seedlings of 'Confrens' on a subculture medium of MS + 0.5 mg / L 6-BA + 0.1 NAA + 30 g / L sucrose + 7.5 g / L agar for one month, select the top tender leaves, cut off the petioles, and place them on MS solid culture medium for later use. 'Confrens' does not need to be pre-cultured.

[0099] (2) Streak the Agrobacterium rhizogenes containing the recombinant plasmid on TY solid medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, and incubate at 28°C for 2-3 days. Pick a single colony and inoculate it on a plate containing 50 mg / L kanamycin, 50 mg / L streptomycin, and 1 M CaCl 2Add the overnight cultured bacterial solution to the new TY liquid medium and shake at 180 rpm at 28°C until the logarithmic growth phase. Centrifuge at 5000 r / min for 5 min to collect the bacterial cells and dilute and resuspend the bacterial solution with MS liquid medium to OD 600 The concentration was 0.1, and 100 μM acetosyringone was added to the resuspension to prepare the Agrobacterium rhizogenes infection solution.

[0100] (3) The explants were immersed in the Agrobacterium infection solution for 20 min; the leaves were removed and placed on sterile filter paper to absorb the bacterial solution, and the explants were placed on a co-culture medium (MS + 30 g / L sucrose + 100 μM acetosyringone + 7 g / L agar, pH 5.80-5.85) covered with sterile filter paper for co-culture for 3 days; the explants were transferred to a hairy root culture medium (1 / 8 MS + 30 g / L sucrose + 125 mg / L timentin + 7 g / L agar, pH 5.80-5.85) and cultured in the dark for 5 days to induce hairy roots, and then cultured in the light for 28 days to obtain transgenic pear roots.

[0101] Example 4

[0102] We used the Golden gate assembly method to construct the selection marker mCherry and PbrMYB169 gene (NCBI accession number: MG594365) into the vector pGG8-17 (SEQ ID NO. 1), in which mCherry was driven by the cauliflower mosaic virus (CaMV) 35S promoter and PbrMYB169 was driven by the promoter of the Arabidopsis thaliana UBQ10 gene (AT4G05320) (see Fig.12 a). The expression vector construction process is as follows:

[0103] (1) Design cloning primers for each fragment according to the Golden Gate assembly method and add appropriate adapter sequences at the 5' end (Table 19);

[0104] Table 19 List of cloning primers designed by Golden gate assembly strategy

[0105]

[0106] (2) Amplify CaMV35S promoter, mCherry, 35S terminator, Arabidopsis UBQ10 promoter, PbrMYB169 and UBQ10 terminator respectively. The cloning system and PCR procedure refer to GXL DNA Polymerase (TaKaRa, Catalog No.: R050A) product manual. The size of the PCR product fragment was detected by agarose gel electrophoresis, and the target fragment was recovered by gel excision using FastPure Gel DNA Extraction Mini Kit (Vazyme, Catalog No.: DC301-01);

[0107] (3) The recovered fragments were connected to the expression vector pGG8-17 using the Golden Gate assembly method. The cloning system and PCR procedure were described in Golden Gate Assembly Kit(BsaI- v2) (NEB, catalog number: E1601) product instructions. The ligation product was transformed into Escherichia coli DH5α competent cells by heat shock method, the correct colonies were identified, and the recombinant plasmid was extracted and transformed into Agrobacterium rhizogenes K599 competent cells.

[0108] Subsequently, the following methods were used to verify whether the hairy root transformation system could be used for gene function verification:

[0109] (1) Grow the tissue culture seedlings of 'Yamanashi No. 1' on a subculture medium of MS + 0.5 mg / L 6-BA + 0.1 NAA + 30 g / L sucrose + 7.5 g / L agar for one month, select the top tender leaves, cut off the petioles, and place them on MS solid culture medium for pre-culture for 1 day.

[0110] (2) Same as Example 2 (2), but OD 600 The concentration is 0.7.

[0111] (3) The explants were placed in the Agrobacterium infection solution for infection for 10 min, the leaves were removed and placed on sterile filter paper to absorb the bacterial solution, and the explants were placed on a co-culture medium (MS + 30 g / L sucrose + 100 μM acetosyringone + 7 g / L agar, pH 5.80-5.85) covered with sterile filter paper for co-culture for 5 days. The explants were then transferred to a hairy root medium (1 / 4 MS + 30 g / L sucrose + 500 mg / L timentin + 7 g / L agar, pH 5.80-5.85) and cultured in the dark for 1 day to induce hairy roots. After 28 days of light culture, transgenic pear roots were obtained.

[0112] Screening of transgenic hairy roots by observing the red fluorescent protein mCherry, such as Fig.12 b. qPCR analysis showed that the expression level of PbrMYB169 in the PbrMYB169 overexpression (MYB169-OE) line was significantly increased compared with the wild-type (WT) and empty vector (EV) control lines (see Fig.12 c). The lignin content of these MYB169-OE hairy roots was significantly higher than that of the WT and EV controls (see Fig.12 d). We also examined the expression levels of genes related to secondary wall biosynthesis in empty vector control and PbrMYB169 transgenic roots by qPCR. Consistent with the increase in lignin content, overexpression of PbrMYB169 in transgenic roots also led to a significant increase in the expression of genes related to a specific branch of monomeric lignin biosynthesis, while having no significant effect on cellulose and xylan biosynthesis ( Fig.12 e) These results indicate that PbrMYB169 regulates secondary wall lignin biosynthesis as a transcriptional activator, which is consistent with previous research results and shows that the Agrobacterium rhizogenes transformation system of the present invention can be used for gene function verification.

Claims

1. A method for transgenic pear mediated by Agrobacterium rhizogenes, characterized in that: A recombinant expression vector containing a screening marker gene mCherry and a target gene is constructed, and the correctly identified recombinant expression vector is transferred into Agrobacterium rhizogenes. The Agrobacterium rhizogenes is used to prepare an infection solution, which is used to infect the leaves of pre-cultured subcultured tissue culture seedlings. Subsequently, co-cultivation is performed to induce regenerated transgenic positive roots.

2. The pear genetic modification method according to claim 1, characterized in that: The method for constructing the recombinant expression vector is as follows: the screening marker mCherry with FPbase accession number ZERB6 and the target gene are constructed into the vector pGG8-17 by using the Golden gate assembly method to obtain the recombinant expression vector, wherein mCherry is driven by the cauliflower mosaic virus 35S promoter, and the target gene is driven by the promoter of the Arabidopsis thaliana UBQ10 gene.

3. The pear genetic modification method according to claim 1, characterized in that: The correctly identified recombinant vector was transformed into Agrobacterium rhizogenes K599 competent cells using the freeze-thaw transformation method.

4. The pear genetic modification method according to claim 1, characterized in that: The preparation method of the infection solution of Agrobacterium rhizogenes is as follows: the Agrobacterium rhizogenes is activated by streaking on a TY solid culture medium containing 50 mg / L kanamycin and 50 mg / L streptomycin, and after culturing at 28°C for 2 days, a single colony is picked and inoculated into a TY liquid culture medium containing 50 mg / L kanamycin, 50 mg / L streptomycin, and 1M CaCl2, and the culture is propagated by shaking at 180 rpm on a shaker at 28°C, and the overnight cultured bacterial solution is added to a new TY liquid culture medium containing 50 mg / L kanamycin, 50 mg / L streptomycin, and 1M CaCl2, and the culture is shaken at 180 rpm on a shaker at 28°C until the logarithmic growth phase, and the bacteria are collected by centrifugation at 5000 r / min for 5 min, the supernatant is discarded, and the infection solution is diluted to the required OD with MS liquid culture medium at pH 5.8 containing 100 μM acetosyringone as the resuspension solution. 600 value for subsequent experiments.

5. The pear genetic modification method according to claim 1, characterized in that: The old leaves of the pear tissue culture seedlings were removed and placed on MS + 0.5 mg / L 6-BA + 0.1 mg / L NAA + 30 g / L sucrose + 7.5 g / L agar medium for 1 month. Then, the tender leaves at the top of the tissue culture seedlings were selected, the petioles were cut off, and the leaves were placed on MS solid medium for pre-culture for 0-1 days to obtain the pre-cultured subculture tissue culture seedling leaves.

6. The pear genetic modification method according to claim 5, characterized in that: The pear tissue culture seedling variety is selected from 'Yamanashi No. 1' or 'Confrens'.

7. The pear genetic modification method according to claim 6, characterized in that: 'Yamanashi No. 1' was pre-cultured on MS solid medium for 1 day, while 'Confrens' did not require pre-culture.

8. The pear genetic modification method according to claim 7, characterized in that: The process of infecting 'Yamanashi No. 1' with Agrobacterium rhizogenes is as follows: the leaves of the pre-cultured subculture seedlings are immersed in OD 600 The method comprises the following steps: placing the leaves in an infection solution with a concentration of 0.7 for 10 minutes; then drying the bacterial liquid on the surface of the leaves and placing the leaves in a co-culture medium for culturing for 5 days; transferring the leaves to a hairy root induction medium for culturing in the dark for 1 day for hairy root induction, and then culturing under light for 26 to 28 days to obtain transgenic pear roots; wherein the formula of the co-culture medium is: MS + 30 g / L sucrose + 100 μM acetosyringone + 7 g / L agar, with a pH of 5.80 to 5.85; the formula of the hairy root induction medium is: 1 / 4 MS + 30 g / L sucrose + 500 mg / L timentin + 7 g / L agar, with a pH of 5.80 to 5.

85.

9. The pear genetic modification method according to claim 7, characterized in that: The process of infecting 'Confrens' with Agrobacterium rhizogenes is as follows: the cut leaves of the subcultured seedlings are directly immersed in OD 600 The method comprises the following steps: placing the leaves in a 0.1 infection solution for 20 minutes; then drying the bacterial liquid on the leaf surface and placing the leaves in a co-culture medium for 3 days; transferring the leaves to a hairy root induction medium for dark culture for 5 days for hairy root induction, and then culturing the hairy roots under light for 26 to 28 days to obtain transgenic pear roots; wherein the co-culture medium formula is: MS + 30 g / L sucrose + 100 μM acetosyringone + 7 g / L agar, pH 5.80 to 5.85; the hairy root induction medium formula is: 1 / 8 MS + 30 g / L sucrose + 125 mg / L timentin + 7 g / L agar, pH 5.80 to 5.85.

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