A gene for regulating poplar plant type development and its application

Abstract

The present invention provides a gene for regulating the development of poplar plant type and its application, belonging to the technical field of plant breeding. The present invention obtains the 84K poplar PagSUPa gene by gene cloning, constructs a PagSUPa gene overexpression vector, and successfully obtains PagSUPa gene overexpression poplar plants OE-2 and OE-7 by Agrobacterium-mediated genetic transformation of 84K poplar. Compared with non-transgenic control poplar (CK) plants, the PagSUPa gene overexpression poplars are significantly shorter, the internodes are shortened, and the leaves are smaller, curled, and wrinkled. It shows that the PagSUPa gene can regulate the poplar plant type, and has application significance in the field of forest genetic engineering and the cultivation of new poplar greening varieties.

Description

Technical Field

[0001] The invention belongs to the technical field of plant breeding, and in particular relates to a gene for regulating the development of poplar plant type and an application thereof. Background Art

[0002] Poplars belong to the Salicaceae family. As a fast-growing variety, poplar wood is widely used. Many countries and regions choose poplar wood as raw material in many production and life applications. In recent years, with the completion of the sequencing of the poplar genome, a lot of research has been done on improving the quality of poplar wood, increasing the growth rate of wood, and improving the quality of wood using molecular breeding methods such as genetic engineering. Poplars are also often used as street trees. Changes in tree crown shape, leaf shape, and leaf color are important indicators for selecting urban greening tree species. The posture and number of leaves are also important factors in determining differences in material production. According to breeding requirements, requirements for poplar plant type are also applied to poplar breeding.

[0003] SUPERMAN is a member of the C2H2 and C2HC zinc finger superfamily protein (Zink Finger Protein), and SUP is a Q-type C2H2 zinc finger protein. It is characterized by containing two typical C2H2 zinc finger structures, one is the conserved QALGGH motif that is essential for its DNA binding activity, and the other is the inhibitory factor EAR associated with the ethylene response element binding factor. Among them, transcription factors containing the inhibitory motif EAR play a vital role in regulating plant growth, development and response to stress. The EAR motif reduces the potential transcription level of the reporter gene and the transcriptional activation activity of other transcription factors. Studies have found that SUP is an active repressor in Arabidopsis, and it also has a class II EAR motif at the carboxyl terminus, which can bind to DNA as an inhibitory domain and has obvious transcriptional repression activity. Therefore, SUP may have similar effects and mechanisms to Q-type C2H2 zinc finger proteins and participate in the regulation of cell division and differentiation.

[0004] As a regulator of cell proliferation and cell growth, SUPERMAN's role may be mediated by hormone changes, which may involve the regulation of auxin and cytokinin. Overexpression of the SUP gene in tobacco, moderate expression levels of SUP can promote cell growth and proliferation, while excessive expression levels are inhibitory. In addition, SUP can also affect the cell cycle through hormone signals. Studies have reported that the acceleration of cell proliferation caused by overexpression of SUP in Arabidopsis and tobacco is consistent with the upregulation of cell cycle gene expression in SUP overexpressing curly seedlings, indicating that SUP is likely to affect cell proliferation by regulating the cell cycle. Moreover, the growth retardation and proliferation inhibition shown in SUP-poor plants and in vitro tissues indicate that SUP activity has a threshold, and above this threshold, SUP will inhibit plant growth. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a gene for regulating the development of poplar plant type and its application, by cloning the PagSUPa gene in 84K (P.alba×P.glandulosa) poplar, constructing an overexpression vector, and transforming to obtain PagSUPa transgenic overexpression poplar. Combining genetic engineering and molecular biology, analyzing the regulation of leaf development and plant height growth of poplar PagSUPa gene is of great significance for cultivating new poplar varieties and changing plant type.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] A gene for regulating the development of poplar plant type is named PagSUPa gene, the nucleotide sequence of which is shown in SEQ ID NO.1, and the encoded amino acid sequence is shown in SEQ ID NO.2.

[0008] The present invention also provides a primer pair capable of amplifying the PagSUPa gene, wherein the nucleotide sequence of the forward primer of the primer pair is shown as SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown as SEQ ID NO.4.

[0009] The present invention also provides a recombinant expression vector for regulating the development of poplar plant type, comprising the PagSUPa gene.

[0010] The present invention also provides a method for constructing the recombinant expression vector, comprising the following steps: the expression vector pMDC43 is selected, the nucleotide sequence is shown as SEQ ID NO.6, the vector contains a 35S promoter, and the vector construction method uses the Gateway construction method.

[0011] The present invention also provides a recombinant strain for regulating the development of poplar plant type, comprising the PagSUPa gene or the recombinant expression vector.

[0012] The present invention also provides the application of the PagSUPa gene, recombinant expression vector or recombinant strain in changing the plant type of poplar plants, and in breeding new poplar varieties.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] The present invention obtains the 84K poplar PagSUPa gene by gene cloning, constructs a PagSUPa gene overexpression vector, and successfully obtains PagSUPa gene overexpression poplar plants OE-2 and OE-7 by Agrobacterium-mediated genetic transformation of 84K poplar.

[0015] Compared with the non-transgenic control poplar (CK), the PagSUPa gene-overexpressing poplars were significantly shorter, with shorter internodes and smaller, curled, and wrinkled leaves. This indicates that the PagSUPa gene can regulate the plant type of poplars and has application significance in the field of forest genetic engineering and the cultivation of new poplar greening varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Example 5 Expression level of transgenic plants;

[0017] Figure 2 : Example 5 Transgenic plant phenotype;

[0018] Figure 3 : Example 5 Transgenic plant leaf phenotype;

[0019] Figure 4 : Example 5 Transgenic plant stem phenotype. DETAILED DESCRIPTION

[0020] 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.

[0021] Example 1

[0022] According to sequence comparison, the gene in the genome of 84K (P. alba × P. glandulosa) with the closest homology to the Arabidopsis thaliana AtSUP (AT3G23130.1) was obtained and named PagSUPa gene. Its nucleotide sequence is shown in SEQ ID NO.1 and its amino acid sequence is shown in SEQ ID NO.2.

[0023] Example 2

[0024] The cloning method of the gene PagSUPa obtained in Example 1 comprises the following steps:

[0025] Snapgene software was used to design full-length primers for the gene sequence, and PagSUPa forward and reverse primers with Gateway linkers were designed, as shown in SEQ ID NO.3 and SEQ ID NO.4.

[0026] PagSUPa with Gateway vector adapter primers was amplified from Populus argentea 84K cDNA using 2× phanta max master mix. The reaction system was (20 μL), as shown in Table 1:

[0027] Table 1 2× phanta max master mix PCR amplification system

[0028]

[0029] The reaction procedure is:

[0030] Pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 56°C for 15 s, extension at 72°C for 60 s, denaturation to extension cycles for 35 times, and complete extension at 72°C for 5 min.

[0031] The final obtained full-length cDNA sequence of the gene was 603bp and was named PagSUPa gene.

[0032] Example 3

[0033] Construction of expression vector of gene PagSUPa

[0034] The expression vector selected was pMDC43, which contains a 35S promoter. The vector was constructed using the Gateway construction method. The specific steps are as follows:

[0035] The forward and reverse primers of PagSUPa were added with Gateway adapters, and the PCR products were obtained by high-fidelity enzyme PCR. The products were recovered after agarose gel electrophoresis, and BP reaction was performed. The reaction system is shown in Table 2. The product was connected to the entry vector pDNOR207 using Gateway BP Clonase II Enzyme Mix. The sequence of the entry vector pDNOR207 is shown in SEQ ID NO.5. Then, Escherichia coli was transformed using DH5α competent cells. The culture dish was placed in a 37°C incubator for 8-12 hours, and then the monoclonal colonies were picked for PCR detection and sequencing verification. After the sequencing results were correct, the PagSUPa-pDNOR207 plasmid was obtained. LR reaction was performed. The reaction system is shown in Table 3. PagSUPa was recombined from the pDNOR207 plasmid to the pMDC43 vector using Gateway LR Clonase II Enzyme Mix. The sequence of the plant expression vector pMDC43 is shown in SEQ ID NO.6. The ligation product was directly used for the next step of E. coli transformation. After PCR detection and sequencing verification, the PagSUP-pMDC43 plasmid was obtained.

[0036] Table 2 Gateway BP reaction system

[0037]

[0038] The reaction procedure was: 25°C for 4 h.

[0039] Table 3 Gateway LR reaction system

[0040]

[0041] The reaction procedure was: 25°C for 4 h.

[0042] Example 4

[0043] Genetic transformation of PagSUP gene

[0044] The PagSUP-pMDC43 overexpression vector constructed in Example 3 was transferred into Agrobacterium GV3101 by freeze-thaw method, and the PagSUP gene was transferred into Populus argentatus 84K by Agrobacterium-mediated transfection. The steps are as follows:

[0045] (1) Preparation of callus tissue

[0046] Take the leaves of wild-type 84K tissue culture seedlings with good growth status, scratch the leaves perpendicular to the veins in the clean bench using sterile and disinfected instruments, and spread them on callus induction medium. Add 2 mg / L 2,4-D, 0.1 mg / L KT and 0.2 mg / L NAA to the callus induction medium, and culture them in the dark at 24-26℃ for 15-25 days.

[0047] (2) Preparation of bacterial solution and re-suspending of cells

[0048] Single clones were picked from the plate containing the PagSUPa-pMDC43 expression vector for colony detection. Positive single colonies were inoculated into 1 mL of liquid LB medium with resistance and cultured at 28°C, 200 rpm for 14-18 h. 100 μL of the cultured bacterial solution was transferred to 100 mL of liquid LB medium with resistance and cultured at 28°C, 200 rpm for 15 h until OD 600 =0.4-0.6, at which point the bacterial solution has good activity. The bacterial solution was centrifuged at 3500rpm for 15min, and the suspension was used (100μmol / L AS was added before the trial to fully suspend the bacteria until OD 600 =0.4-0.6.

[0049] (3) Infection of callus tissue

[0050] In the clean bench, use tweezers to cut the cultured callus into small pieces, transfer them to the resuspended bacterial solution and infect for 10-15 minutes, gently shake the bacterial solution by hand to make the callus fully immersed in the bacterial solution. Take out the infected callus and place it on sterile filter paper to dry (the surface of the callus turns obviously white).

[0051] (4) Co-culture

[0052] The callus was transferred to a co-culture medium containing 100 μM AS and cultured at 24-26°C in the dark for 3 days.

[0053] (5) Differentiation induction culture

[0054] The co-cultured callus was transferred to a differentiation induction medium containing 3 mg / L hygromycin B and 200 mg / L timentin. 0.5 mg / L 6-BA and 0.05 mg / L NAA were added to the differentiation induction medium. The callus tissue was induced to produce adventitious buds under the conditions of a culture temperature of 23-25°C and a light intensity of 16h / 8h (day / night). Resistant adventitious buds could be screened after about 30 days of culture.

[0055] (6) Rooting culture

[0056] The resistant adventitious buds with sufficient stem length were transferred to 1 / 2MS rooting medium containing 3mg / L hygromycin B and 200mg / L timentin, and 1 / 2MS rooting medium was supplemented with 0.05mg / L IBA and 0.02mg / L NAA, and the plants were induced to root under the conditions of culture temperature of 23-25℃ and light of 16h / 8h (day / night). DNA was extracted after the rooted plants reached a height of about 2cm, and positive plants were identified by PCR.

[0057] Example 5

[0058] Analysis of traits of PagSUPa transgenic positive plants

[0059] The RNA of the positive plants obtained in Example 4 was extracted and reverse transcribed into cDNA, and the transcription level of the transgenic poplars overexpressing PagSUPa was detected using the SYBR Premix Ex TaqTM Kit (TaKaRa) kit and the ACTIN gene as an internal reference using the Therme QuantStudio3 real-time quantitative instrument.

[0060] It can be seen that compared with the non-transgenic poplar (CK), the expression levels of the transgenic poplars (OE-2 and OE-7) were significantly higher, 203 and 109 times that of the control, respectively ( Figure 1 ). After culturing the transgenic plants for 3 months, it was observed that the two lines of PagSUPa transgenic poplar (OE-2 and OE-7) were significantly shorter than the control plants (55.6 cm in height), 8.9 cm and 27.2 cm respectively. The ground diameter of the transgenic plants was larger than that of the control plants, and the difference in the number of internodes was not significant ( Figure 2 The leaves of PagSUPa transgenic poplars were significantly smaller than those of the control, and the leaf area, leaf length and leaf width were significantly smaller than those of the control ( Figure 3 The stems of PagSUPa transgenic poplars were thinner at the top and thicker at the bottom compared with the control ( Figure 4 ).

[0061] 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. The application of PagSUPa gene in changing the plant type of poplar plants is characterized by: The nucleotide sequence of the PagSUPa gene is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The amino acid sequence encoded by the PagSUPa gene is shown as SEQ ID NO.

2.

3. The use according to claim 1, characterized in that: The nucleotide sequence of the forward primer of the primer pair for amplifying the PagSUPa gene is shown in SEQ ID NO.3, and the nucleotide sequence of the reverse primer is shown in SEQ ID NO.

4.

4. A recombinant expression vector for regulating the development of poplar plant type, characterized in that: Comprising the PagSUPa gene according to claim 1.

5. The method for constructing the recombinant expression vector according to claim 4, characterized in that: The following steps are involved: The expression vector pMDC43 was selected, and the nucleotide sequence was shown in SEQ ID NO.

6. The vector contained a 35S promoter, and the vector was constructed using the Gateway construction method.

6. A recombinant strain for regulating the development of poplar plant type, characterized in that: Comprising the PagSUPa gene according to claim 1 or the recombinant expression vector according to claim 4.

7. Use of the recombinant expression vector according to claim 4 or the recombinant strain according to claim 6 in changing the plant type of poplar plants.

8. Use of the PagSUPa gene according to claim 1, the recombinant expression vector according to claim 4 or the recombinant strain according to claim 6 in breeding new poplar varieties, characterized in that: The direction of the cultivation is that the plants become shorter, the internodes become shorter, and the leaves become smaller, curled, and wrinkled.

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