Toona sinensis BLH3 gene and application thereof in efficient genetic transformation and regeneration of Toona sinensis

Abstract

The invention relates to the technical field of plant tissue culture, in particular to a cedrela sinensis BLH3 gene and application thereof in efficient genetic transformation and regeneration of cedrela sinensis. The nucleotide sequence of the cedrela sinensis BLH3 gene is shown as SEQ ID NO.3, and the cedrela sinensis BLH3 gene has the effect of promoting root growth. When the genetic transformation and regeneration method is used for cedrela sinensis tissue culture, the cedrela sinensis tissue culture has the characteristics of high plant proliferation multiple, short rooting time, more and robust root systems and high genetic transformation efficiency.

Description

Technical Field

[0001] The invention relates to the technical field of plant tissue culture, in particular to a toon BLH3 gene and application thereof in efficient genetic transformation and regeneration of toon. Background Art

[0002] Toona sinensis (A. Juss.) Roem. is a perennial woody plant of the Meliaceae family and the genus Toona. Toona sinensis leaves are a seasonal specialty, rich in vitamins and secondary metabolites such as flavonoids, and contain volatile aromatic organic compounds such as toona sinensis, which not only help to enhance the body's immune function, but also strengthen the spleen and stimulate appetite; Toona sinensis wood is yellow-brown with red rings, beautiful texture, durability, corrosion resistance, high density, antibacterial and other advantages, and has a wide range of uses, and is known as the "Chinese mahogany".

[0003] At present, along with the in-depth study of the medicinal value of Chinese toon buds, as the only woody vegetable, a Chinese toon craze has arisen on the market, and the open-field cultivation mode that was limited to spring harvest in the past can no longer meet people's needs. Because the amount of Chinese toon seeds is seldom, resources are limited, and dioecious, the seed propagation offspring traits are easily separated and degraded, and the reproduction coefficient by methods such as cutting seedling, root burying, tillering is very low, and there is dormancy phenomenon and slow growth rate in the terminal bud, which is difficult to meet production needs. Although tissue culture has become a kind of important propagation pathway of Chinese toon, most of them have adventitious bud proliferation multiples lower (3~5 times), rooting efficiency is extremely low (about 25%) and the cycle is long, and the genetic transformation system lacks, which has seriously hindered the development of Chinese toon transgenic breeding.

[0004] Therefore, providing a method for efficient genetic transformation and regeneration of Toona sinensis is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0005] The object of the present invention is to provide a BLH3 gene of Toona sinensis and its application in efficient genetic transformation and regeneration of Toona sinensis. The Toona sinensis BLH3 gene and method of the present invention are used to carry out Toona sinensis tissue culture, which has the characteristics of high plant regeneration efficiency, short rooting time and strong root system, and high genetic transformation efficiency.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a Toona sinensis BLH3 gene, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0008] The present invention also provides an application of the toon BLH3 gene in the efficient genetic transformation and regeneration of toon.

[0009] The present invention also provides a method for efficient genetic transformation and regeneration of Toona sinensis, comprising the following steps:

[0010] (1) After disinfecting and sterilizing the stem segments of Toona sinensis with axillary buds, inoculate them in MS culture medium and culture them for one month to obtain sterile lateral shoots; transfer the sterile lateral shoots to differentiation medium and culture them until callus tissue is formed at the base;

[0011] (2) Connecting the Toona sinensis BLH3 gene to the expression vector pBI121-GFP to construct a recombinant expression vector pBI121-BLH3-GFP;

[0012] (3) transforming the recombinant expression vector pBI121-BLH3-GFP into Agrobacterium, suspending the Agrobacterium containing the recombinant expression vector pBI121-BLH3-GFP in a sterilized MS liquid medium, adding acetosyringone to obtain an infection solution; infecting callus tissue with the infection solution, and after infection, transferring the callus tissue into a co-culture medium for dark culture;

[0013] (4) transferring the co-cultivated callus tissue to a screening medium and culturing it in the dark until adventitious buds are formed, and then transferring it to the light and continuing to culture it until its height is ≥3 cm to obtain transformed plantlets;

[0014] (5) The transformed plantlets were transferred to a rooting medium and cultured until 4 to 6 young roots were formed, the root system was 3 to 4 cm long, and the seedling height was ≥ 4 cm, and then complete regenerated transgenic plants were obtained.

[0015] Preferably, the components of the differentiation medium in step (1) are 0.8-1.2 mg / L 6-BA, 0.8-1.2 mg / L KT, 0.04-0.06 mg / L NAA, 4-8 mg / L sodium nitroprusside, 25-35 g / L sucrose, 5-8 g / L agar, supplemented to 1 L with MS medium, pH 5-6.

[0016] Preferably, the nucleotide sequences of the primers used in the construction of the recombinant expression vector in step (2) are shown in SEQ ID NOs. 4 to 5.

[0017] Preferably, the components of the culture medium for co-cultivation in step (3) are 6-BA 0.8-1.2 mg / L, KT 0.8-1.2 mg / L, NAA 0.04-0.06 mg / L, sodium nitroprusside 4-8 mg / L, sucrose 25-35 g / L, agar 5-8 g / L, acetosyringone 80-120 μmol / L, supplemented to 1 L with MS culture medium, pH 5-6.

[0018] Preferably, the components of the screening culture medium in step (4) are 6-BA 0.8-1.2 mg / L, KT 0.8-1.2 mg / L, NAA 0.04-0.06 mg / L, sodium nitroprusside 4-8 mg / L, sucrose 25-40 g / L, agar 5-7 g / L, kanamycin 20-40 mg / L, timentin 150-300 mg / L, supplemented to 1 L with MS culture medium, pH 5-6.

[0019] Preferably, the rooting medium in step (5) comprises 0.8-1.0 mg / L IBA, 0.4-0.6 mg / L paclobutrazol, 15-25 g / L sucrose, 5-7 g / L agar, 150-300 mg / L timentin, supplemented to 1 L with 1 / 2 MS medium, pH 5-6.

[0020] Preferably, except for dark culture, the culture conditions in steps (1) to (5) are: temperature 23-25° C., humidity 70-80%, light intensity 1500-2000 lx, and duration 16 h / day.

[0021] Preferably, the regenerated transgenic plants of step (5) need to be transplanted into a plant culture room after being obtained. The conditions of the plant culture room are as follows: temperature 23-27°C, humidity 60-80%, light intensity 200-400 μmol / m 2 / s, duration 14h / day.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] By using the regeneration method of the present invention to carry out tissue culture of Chinese toon, the proliferation multiple of the plant can reach 10 times, the root system is well developed and grows to 3-4 cm, the plant proliferation multiple is high and the root system is strong; in addition, the BLH3 gene of the present invention also has the function of promoting root growth, and the Chinese toon BLH3 gene of the present invention is transformed into Chinese toon for Chinese toon tissue culture, the root system of the genetically modified plant grows better, the root system is well developed and grows to 3-4 cm after rooting for 25 days, the number of roots can reach 6-8, and the conversion rate is as high as 54.2%. It has multiple advantages such as short rooting time, a large number of strong roots, high genetic transformation efficiency and high transplant survival rate. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram of the germination of the stem segment of Toona sinensis with axillary buds in Example 1;

[0025] Figure 2 This is a diagram of the differentiation of adventitious buds of Toona sinensis in Example 1;

[0026] Figure 3 This is a diagram of callus differentiation and proliferation of adventitious buds of Toona sinensis in Example 1;

[0027] Figure 4 This is the competent transformation identification diagram of Agrobacterium EHA105 in Example 2; wherein M: DL2000Marker; 2-11 is pBI121-BLH3-GFP, 1 is a negative control; 12 is a positive control;

[0028] Figure 5 This is a diagram of callus tissue infected by Agrobacterium in Example 2;

[0029] Figure 6 This is a diagram of adventitious bud differentiation of callus tissue after dark culture in Example 2;

[0030] Figure 7 This is a diagram of rooting of transformed plantlets in Example 2;

[0031] Figure 8 This is a diagram of transplanting regenerated transgenic plants in Example 2;

[0032] Fig. 9 This is a diagram for identifying transgenic Toona sinensis lines, where M: DL2000 Marker; + is the positive control with the plasmid, - is the negative control with non-transgenic Toona sinensis, and Ts1~Ts8 are regenerated transgenic plants. DETAILED DESCRIPTION

[0033] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0034] Example 1

[0035] A method for efficiently regenerating toona sinensis, comprising the following steps:

[0036] 1. Collect the stem segments with axillary buds from the wild and bring them back to the laboratory. Rinse them with running water overnight, disinfect them with 70% alcohol for 1 min and sodium hypochlorite for 50 min in a clean bench, and inoculate them in MS medium. After one month, sterile side shoots were obtained (see Figure 1 ).

[0037] 2. Transfer the sterile lateral branches to the differentiation medium and culture for 7 days. Callus and callus clusters begin to differentiate at the base of the stem (see Figure 2 ), and continue to culture for 18 days until the clustered buds elongate to 2 cm, obtaining adventitious buds.

[0038] The composition of differentiation medium is MS medium + 6-BA 1.0mg / L + KT 1.0mg / L + NAA 0.05mg / L + sodium nitroprusside 6mg / L + sucrose 30g / L + agar 6.5g / L, pH 5.8;

[0039] 3. Transfer the adventitious buds to the rooting medium and culture for 10 days. 4 to 6 young roots are formed and the rooted tissue culture seedlings are obtained;

[0040] The rooting medium composition is 1 / 2MS medium + IBA 1.0 mg / L + paclobutrazol 0.5 mg / L + sucrose 20 g / L + agar 6.5 g / L, pH 5.8;

[0041] 4. Harden the seedlings and transplant them.

[0042] The rooted tissue culture seedlings were cultured for 10 days until the root system was well developed and 3 to 4 cm long and the seedling height was ≥ 4 cm. They were then transplanted into a cultivation medium with a volume ratio of peat soil: vermiculite: perlite of 3:3:1 and placed in a plant culture room for cultivation.

[0043] Tissue culture room conditions: temperature 24 °C, humidity 75%, light intensity 1800 lx, duration 16 h / day.

[0044] The conditions of the plant culture room were as follows: temperature 25°C, humidity 70%, and light intensity 300 μmol / m 2 / s, duration 14h / day.

[0045] In this Example 1, the present invention also took another adventitious bud from step 2 and transferred it to a fresh differentiation medium for subculture proliferation culture for 25 days. As a result, a large number of callus tissues and callus clustered buds were differentiated from the stem base, and 10 adventitious buds could be separated after cutting. The average proliferation multiple was 10 (see Figure 3 ).

[0046] Embodiment 2 A method for efficient genetic transformation and regeneration of Toona sinensis comprises the following steps:

[0047] 1. Preparation of binary plant recombinant expression vector pBI121-BLH3-GFP

[0048] 1. Cloning of the Toona sinensis BLH3 gene

[0049] Using the Chinese toon genome (CNSA: https: / / db.cngb.org / search / proj ect / CNP0000958 / ) as a reference, primers for amplifying BLH3 were designed. The primer sequences are shown in SEQ ID NOs. 1 to 2. The cDNA sequence was amplified from Chinese toon and sequenced by Zhejiang Shangya Company. After sequencing, the sequence of the Chinese toon BLH3 gene was shown in SEQ ID NO. 3 below.

[0050] F1 (shown in SEQ ID NO. 1): 5'-TCTCGATTTCAAGGAGAGCA-3';

[0051] R1 (shown in SEQ ID NO. 2): 5'-ACCCCATATTTGCAGCTACC-3'.

[0052] The sequencing sequence (shown in SEQ ID NO.3) is:

[0053]

[0054] The PCR reaction system is shown in Table 1 below:

[0055] Table 1 PCR reaction system (25 μL)

[0056]

[0057]

[0058] The PCR reaction program is:

[0059]

[0060] 2. Enzyme digestion of vector plasmid

[0061] The linearized vector plasmid pBI121-GFP was obtained by double restriction digestion, and its reaction system is shown in Table 2 below.

[0062] Table 2 Double enzyme digestion reaction system

[0063] Components Volume (μL) Vector plasmid pBI121-GFP 2μg XbaⅠ 2μL KpnⅠ 2μL Cutsmart 5μL <![CDATA[ddH2O]]> Add to 50 μL

[0064] Place the prepared enzyme digestion system in a PCR tube, pipette and mix well, let stand at 37°C for 3 h, then take out and purify and recover it using a PCR product purification kit for later use.

[0065] 3. PCR amplification

[0066] To ensure the consistency of the target sequence, the template is the nucleotide sequence described in SEQ ID NO.3. According to the characteristics of seamless assembly, a primer sequence with a vector linker is designed. The primer sequences are shown in the following SEQ ID Nos. 4 to 5.

[0067] F2 (as shown in SEQ ID No.4):

[0068] 5'-agaacacgggggactctagaATGGGTGCTTATAATCCAAG-3',

[0069] R2 (as shown in SEQ ID NO.5):

[0070] 5'-ctcaccatggtacccccgggCACTACAAAATCATGCATCA-3'.

[0071] The PCR reaction system and procedure were the same as the gene cloning step in step 1, and the amplified product was purified and recovered using a PCR product purification kit.

[0072] 4. Seamless assembly and construction of recombinant plasmid

[0073] The seamless assembly reaction system (10 μL) was prepared as shown in Table 3 below.

[0074] Table 3 Seamless assembly reaction system

[0075]

[0076] The prepared reaction system was placed in a 37°C water bath and allowed to stand for 30 minutes before being taken out.

[0077] 5. The reaction mixture was transformed into competent E. coli DH5α (Vidi Biotechnology, DL1001S) using the heat shock method.

[0078] 6. Identification of positive clones

[0079] Pick a single colony from the overnight culture and inoculate it into liquid LB medium containing 50 mg / L kanamycin resistance, shake the bacteria, and then perform bacterial liquid PCR verification (see Figure 4 ), primers F2 and R2, PCR reaction system (25 μL), as shown in Table 4 below.

[0080] Table 4 PCR reaction system

[0081] Components Volume (μL) F2 1 R2 1 2 × PhantaMaxMasterMix 12.5 Bacterial liquid 1 <![CDATA[ddH2O]]> 9.5

[0082] The PCR reaction program was

[0083]

[0084] The PCR was verified to be correct and then sequenced, and the sequencing result was 2079 bp, which was the recombinant expression vector pBI121-BLH3-GFP.

[0085] 2. Transformation of Agrobacterium

[0086] Transform the competent cells of Agrobacterium tumefaciens EHA105 by electroporation. Add 4 μl of the recombinant expression vector pBI121-BLH3-GFP to the competent cells EHA105 dissolved on ice, mix well, and then transfer to the pre-cooled electroporation transformation cup. Transform according to the instrument instructions, and set the voltage of the electroporator to 2.0 kV. Immediately add 1 ml of LB culture medium to the transformation cup, transfer to a 1.5 ml centrifuge tube, and culture at 28°C for 4 hours. After slight centrifugation to remove the appropriate amount of culture medium, spread it on a plate containing an appropriate amount of 50 mg / L of kanamycin and culture it at 28°C for 3 days. Pick a single colony and use PCR to identify whether the target plasmid has been transferred (the method is the same as the identification of Escherichia coli positive clones).

[0087] 3. Infection, transformation and co-cultivation

[0088] Agrobacterium EHA105 containing the recombinant expression vector pBI121-BLH3-GFP was cloned in LB liquid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and the culture was shaken at 28°C for 3 days. The Agrobacterium cells were collected and suspended in sterilized MS liquid medium. The cell OD was adjusted. 600 is 0.4-0.6, and then acetosyringone is added at a final concentration of 100 μmol / L to obtain the infection solution;

[0089] During infection, callus tissue (0.5 cm × 0.5 cm) from the base of the aseptic lateral branch stem in Example 1 was selected and infected with 200 ml of infection solution. After 30 minutes of infection, the callus tissue was taken out and the residual infection solution was dried and transferred to the co-culture medium for dark culture for 3 days (see Figure 5 );

[0090] The co-cultivation medium components are:

[0091] MS medium + 6-BA 1.0 mg / L + KT 1.0 mg / L + NAA 0.05 mg / L + sodium nitroprusside 6 mg / L + sucrose 30 g / L + agar 6.5 g / L + acetosyringone 100 μmol / L, pH 5.8.

[0092] 4. Screening and differentiation of callus tissue

[0093] The callus tissue after co-cultivation was transferred to the screening medium and cultured in the dark for 10 days to differentiate adventitious buds, and then moved to the light to grow for 10 days until the height was ≥3 cm, and the transformed plantlets were obtained (see Figure 6 );

[0094] The screening medium components are:

[0095] MS medium + 6-BA 1.0 mg / L + KT 1.0 mg / L + NAA 0.05 mg / L + sodium nitroprusside 6 mg / L + sucrose 30 g / L + agar 6.5 g / L + kanamycin 30 mg / L + timentin 200 mg / L, pH 5.8.

[0096] 5. Rooting of Transformed Plantlets

[0097] The transformed plantlets were transferred to the rooting medium and cultured for 25 days until 6 to 8 young roots were formed (see Figure 7 ), when the root length was 3-4 cm and the seedling height was ≥4 cm, the complete regenerated transgenic plants were obtained and transplanted into the plant culture room (see Figure 8 ).

[0098] The rooting medium composition is:

[0099] 1 / 2MS medium + IBA 1.0 mg / L + paclobutrazol 0.5 mg / L + sucrose 20 g / L + agar 6.5 g / L + kanamycin 30 mg / L + timentin 200 mg / L, pH 5.8.

[0100] Tissue culture room conditions: temperature 24 °C, humidity 75%, light intensity 1800 lx, duration 16 h / day.

[0101] Dark culture conditions were: temperature 24°C, humidity 75%.

[0102] The conditions of the plant culture room were as follows: temperature 25°C, humidity 70%, and light intensity 300 μmol / m 2 / s, duration 14h / day.

[0103] In Example 2, the transgenic plants proliferated 4 times due to the presence of the antibiotic kanamycin. The present invention extracted genomic DNA from leaves of 24 complete transgenic plants, and used the primers described in SEQ ID NO.1 and SEQ ID NO.2 for PCR identification. The PCR verification was correct and sequencing was performed. The sequencing result was 2079 bp (see Fig. 9 ), and finally 13 transgenic Toona sinensis positive plants were obtained, with a statistical transformation rate of 54.2%.

[0104] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A Toona sinensis BLH3 gene, characterized in that: Its nucleotide sequence is shown in SEQ ID NO.

3.

2. Use of the toon BLH3 gene according to claim 1 in efficient genetic transformation and regeneration of toon.

3. A method for efficient genetic transformation and regeneration of Toona sinensis, characterized in that: The following steps are involved: (1) After disinfecting and sterilizing the stem segments of Toona sinensis with axillary buds, inoculate them in MS culture medium and culture them for one month to obtain sterile lateral shoots; transfer the sterile lateral shoots to differentiation medium and culture them until callus tissue is formed at the base; (2) connecting the Toona sinensis BLH3 gene described in claim 1 to the expression vector pBI121-GFP to construct a recombinant expression vector pBI121-BLH3-GFP; (3) Transforming the recombinant expression vector pBI121-BLH3-GFP into Agrobacterium, suspending the Agrobacterium containing the recombinant expression vector pBI121-BLH3-GFP in a sterilized MS liquid medium, adding acetosyringone to obtain an infection solution; infecting callus tissue with the infection solution, and after infection, transferring the callus tissue to a co-culture medium for dark culture; (4) transferring the co-cultivated callus tissue to a screening medium and culturing it in the dark until adventitious buds are generated, and then transferring it to light and continuing to culture it until its height is ≥3 cm to obtain transformed plantlets; (5) The transformed plantlets were transferred to a rooting medium and cultured until 6 to 8 young roots were formed, the root system was 3 to 4 cm long, and the seedling height was ≥ 4 cm, and then complete regenerated transgenic plants were obtained.

4. The method according to claim 3, characterized in that The components of the differentiation medium in step (1) are 6-BA 0.8-1.2 mg / L, KT 0.8-1.2 mg / L, NAA 0.04-0.06 mg / L, sodium nitroprusside 4-8 mg / L, sucrose 25-35 g / L, agar 5-8 g / L, and the volume is supplemented to 1 L with MS medium, with a pH of 5-6.

5. The method according to claim 3, characterized in that: The nucleotide sequences of the primers used in the construction of the recombinant expression vector in step (2) are shown in SEQ ID NOs. 4 to 5.

6. The method according to claim 3, characterized in that The components of the culture medium for co-cultivation in step (3) are 0.8-1.2 mg / L 6-BA, 0.8-1.2 mg / L KT, 0.04-0.06 mg / L NAA, 4-8 mg / L sodium nitroprusside, 25-35 g / L sucrose, 5-8 g / L agar, 80-120 μmol / L acetosyringone, and the volume is supplemented to 1 L with MS culture medium, with a pH of 5-6.

7. The method according to claim 3, characterized in that The components of the screening culture medium in step (4) are 6-BA 0.8-1.2 mg / L, KT 0.8-1.2 mg / L, NAA 0.04-0.06 mg / L, sodium nitroprusside 4-8 mg / L, sucrose 25-40 g / L, agar 5-7 g / L, kanamycin 20-40 mg / L, timentin 150-300 mg / L, supplemented to 1 L with MS culture medium, pH 5-6.

8. The method according to claim 3, characterized in that The rooting medium in step (5) comprises 0.8-1.0 mg / L IBA, 0.4-0.6 mg / L paclobutrazol, 15-25 g / L sucrose, 5-7 g / L agar, 150-300 mg / L timentin, and 1 / 2 MS medium is added to make up to 1 L, with a pH of 5-6.

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