Liriodendron tulipifera LcPORA1 gene VIGS silencing system as well as construction method and application thereof

Abstract

The invention discloses a liriodendron tulipifera LcPORA1 gene VIGS silencing system as well as a construction method and application thereof, and relates to the technical field of plant genetic engineering. The invention discloses a specific nucleotide fragment for silencing a liriodendron LcPORA1 gene. The sequence of the specific nucleotide fragment is as shown in SEQ ID NO. 1; the LcPORA1 is connected to a pTRV2 vector, and a vector TRV2-LcPORA1 transformed agrobacterium competent cell is obtained through construction; mixing TRV2-LcPORA1 and TRV1 positive bacterium solutions according to a ratio, and then dip-dyeing liriodendron in-vitro petals; the liriodendron petals of which the LcPORA1 gene expression quantity is obviously reduced are obtained. The result shows that when the OD600 of the positive bacterium solution is 0.8, the expression quantity of the gene is obviously reduced, and the petals are degreased. Compared with a control group, the chlorophyll content in the tulip tree petals is obviously reduced after the tulip tree petals are infected with the TRV2-LcPORA1.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a VIGS silencing system of Liriodendron chinense LcPORA1 gene and a construction method and application thereof. Background Art

[0002] Virus induced gene silencing (VIGS) is an efficient and simple method that does not rely on transgenic technology and directly uses recombinant viruses to silence target genes in specific tissues of plants. Among them, the VIGS system mediated by Tobacco rattle virus (TRV) has many advantages such as high infection efficiency, long silencing time, and mild symptoms. It is currently the most widely used viral vector plasmid in VIGS technology. It uses viral vectors carrying effective cDNA fragments of target genes to induce the degradation of specific homologous gene mRNA in plants, thereby achieving post-transcriptional gene silencing, thereby triggering the appearance of relevant phenotypes in plants.

[0003] There are currently only two species of Liriodendron in nature, namely, Liriodendron chinense, which is distributed in the area south of the Yangtze River in my country and some mountainous areas in northern Vietnam, and Liriodendron tiliaceus, which is distributed in the eastern part of North America. Both are tall trees with similar phenotypes. However, there are certain differences in some tissues and organs, such as petal size, petal color, leaf shape and other aspects. Among them, the variation in flower color is one of the most significant phenotypes to distinguish the two. The petals of Liriodendron chinense are almost dark green as a whole, while the petals of Liriodendron tiliaceus are light green and there is a yellow stripe in the middle of the petals. Previous researchers conducted an analysis based on the differences between the petals of the two, but due to the lack of effective direct verification methods, the molecular mechanism of the petal coloring differences of the analyzed Liriodendron genus plants only exists in the guesswork stage. Therefore, the development of an efficient and direct gene function verification method can provide effective technical support for revealing the molecular genetic mechanism of flower color variation in this species.

[0004] The TRV-based plant VIGS system has been successful in many plants, but there is no report on the study of gene function in Liriodendron plants. Therefore, it is urgent to establish a method for TRV-mediated gene silencing in Liriodendron petals to achieve functional research on genes related to the coloring of Liriodendron petals. However, the existing technology usually selects the PDS (phytoene dehydrogenase) gene as a reporter gene, lacks other reporter genes that can be used for gene function verification, and has not successfully used VIGS technology in Liriodendron to achieve efficient gene silencing, resulting in many difficulties in verifying the function of related genes. Therefore, the establishment of a VIGS system for Liriodendron petals is very important for the subsequent functional research of related genes. 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 specific nucleotide fragment for silencing the LcPORA1 gene of Liriodendron chinense. Another technical problem to be solved by the present invention is to provide a VIGS silencing system for the LcPORA1 gene of Liriodendron chinense. Another technical problem to be solved by the present invention is to provide a method for constructing the VIGS silencing system for the LcPORA1 gene of Liriodendron chinense. Another technical problem to be solved by the present invention is to provide an application of the VIGS silencing system for the LcPORA1 gene of Liriodendron chinense, which is used for rapid, simple and low-cost verification of the gene function of Liriodendron chinense.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A specific nucleotide fragment for silencing the LcPORA1 gene of Liriodendron chinense, the sequence of which is shown in SEQ ID NO.1.

[0008] The method for constructing the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense comprises: connecting the specific nucleotide fragment for silencing the LcPORA1 gene of Liriodendron chinense as shown in SEQ ID NO.1 to the pTRV2 vector to construct the vector TRV2-LcPORA1, i.e. the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense.

[0009] The application of the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense in inhibiting the expression of the LcPORA1 gene of Liriodendron chinense comprises the following steps:

[0010] 1) Construction of the VIGS silencing system of the LcPORA1 gene in Liriodendron chinense, namely the vector TRV2-LcPORA1;

[0011] 2) Transform TRV1, TRV2 and the recombinant plasmid containing the TRV2-LcPORA1 vector fragment into Agrobacterium competent cells respectively, and collect the positive bacterial liquid after cultivation;

[0012] 3) TRV2 and TRV2-LcPORA1 positive bacterial solutions were mixed with TRV1 positive bacterial solution in proportion and then used to infect the petals of Liriodendron chinense in vitro;

[0013] 4) Cultivate, screen and obtain petals of Liriodendron chinense with significantly reduced expression of LcPORA1 gene.

[0014] The mixing ratio is 1:1.

[0015] The OD of the positive bacterial solution 600 is 0.8.

[0016] Application of the VIGS silencing system of the LcPORA1 gene in Liriodendron chinense to identify the function of the LcPORA1 gene in Liriodendron chinense.

[0017] Application of the VIGS silencing system of the LcPORA1 gene in regulating the chlorophyll content in the petals of Liriodendron chinense.

[0018] Application of the VIGS silencing system of the LcPORA1 gene in regulating the stability of chloroplast structure in the petals of Liriodendron chinense.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1) The specific nucleotide fragment for silencing the LcPORA1 gene of Liriodendron chinense disclosed for the first time in the present invention is shown in SEQ ID NO. 1. The specific nucleotide fragment for silencing the LcPORA1 gene of Liriodendron chinense was connected to the pTRV2 vector to construct the vector TRV2-LcPORA1; after transformation of Agrobacterium competent cells; the resuspended bacterial liquid containing TRV2 and TRV2-LcPORA1 was mixed with the resuspended bacterial liquid containing TRV1 at a volume ratio of 1:1, and then the petals of Liriodendron chinense in vitro were infected. The results showed that the petals of Liriodendron chinense at different developmental stages could not be infected by conventional injection method, and the petal immersion method in vitro was the best VIGS infection method for Liriodendron chinense petals.

[0021] 2) The present invention mixed TRV2-LcPORA1 and TRV1 positive bacterial solutions of different bacterial solution concentrations in proportion and then infected the petals of Liriodendron chinense in vitro; cultivated and screened to obtain Liriodendron chinense petals with significantly reduced LcPORA1 gene expression. The results showed that compared with the empty control, when the positive bacterial solution OD 600 When the value was 0.8, the expression level of the gene decreased significantly, and there was an obvious phenotypic change, that is, the petals became chlorotic.

[0022] 3) In the LcPORA1 gene-silenced plant of Liriodendron chinense constructed by the present invention, the chlorophyll content in the petals of Liriodendron chinense significantly decreased after being infected with TRV2-LcPORA1 compared with the control group; the thylakoid membrane structure in the chloroplasts in the control group was clearly visible, with relatively tight stacking and regular arrangement, while the thylakoid stacking of the chloroplasts in the TRV2-LcPORA1 experimental group became loose or even partially missing, with disordered arrangement, and the stacking of the membrane was no longer clear. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is the phenotype of Liriodendron tulipifera flowers subjected to gene silencing;

[0024] Figure 2 This is a comparison of the infection effects of different concentrations of infection solutions on the isolated petals of Liriodendron chinense.

[0025] Figure 3 This is a graph showing the relative expression level of the LcPORA1 gene after infection with different concentrations of Agrobacterium;

[0026] Figure 4 This is a graph showing the changes in chlorophyll content in the petals of Liriodendron chinense after infection with TRV2-LcPORA1;

[0027] Figure 5 This is a transmission electron micrograph of chlorophyll in the petals of Liriodendron chinense infected with TRV2-LcPORA1. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out by referring to the methods listed in the book "Molecular Cloning Laboratory Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0029] The materials used in this application are petals of Liriodendron chinense, collected from the Baima Campus of Nanjing Forestry University.

[0030] Example 1

[0031] 1. Cloning of the silencing fragment of LcPORA1 from Liriodendron chinense

[0032] Total RNA was extracted from petals of Liriodendron chinense (SteadyPure Plant RNA Extraction Kit), and the RNA quality was determined by agarose gel electrophoresis, followed by reverse transcription into cDNA (HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper)). A 300 bp specific nucleotide fragment (3' region fragment of LcPORA1 gene) for silencing LcPORA1 gene was then amplified from cDNA, and the sequence is shown in SEQ ID NO. 1. The primer sequences are as follows:

[0033] LcPORA1-F: 5'-gagtaaggttaccgaattctAGCTAGCCCAACAAGCTTCTC-3',

[0034] LcPORA1-R: 5'-cgagacgcgtgagctTTCGCTTCCCTTTACCCTGGC-3'.

[0035] PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s; annealing at 95°C for 15 s; extension at 60°C for 15 s; full extension at 72°C for 30 s; and the number of cycles was 35.

[0036] 2. Vector Construction

[0037] TRV2 was double-digested with XbaⅠ and SacⅠ, and the large fragment was recovered as a ligation vector. The PCR amplification product was recovered and ligated to the pTRV2 vector, thus completing the construction of the TRV2-LcPORA1 vector (the nucleotide sequence of the TRV2-LcPORA1 vector is shown in SEQ ID NO.2). The ligation product was transformed into Escherichia coli DH5α competent cells, and then a monoclonal strain was selected. The correct recombinant plasmid was screened by bacterial liquid PCR identification and sequencing, and finally the recombinant plasmid containing the TRV2-LcPORA1 vector fragment was transferred into the Agrobacterium strain EHA105.

[0038] 3. Preparation of bacterial solution

[0039] The recombinant plasmids of TRV1, TRV2 and TRV2-LcPORA1 vector fragment were transferred into Agrobacterium strain EHA105; 1 mL of each plasmid was inoculated into 50 mL of LB liquid medium (containing 50 mg·L -1 of kanamycin and 50 mg·L -1 acetosyringone) at 28°C and 200 r·min- 1 Activation culture for 6-8h at 4℃ and 5000r·min -1 Centrifuge for 10 min to collect the cells, discard the supernatant and resuspend with 10 mmol / L -1 MgCl2, 10 mmol·L -1 MES and 150 mmol·L -1 AS, pH = 5.7) to resuspend the bacteria, and obtain resuspended bacterial solutions containing TRV1, TRV2 and TRV2-LcPORA1, respectively. Then, the OD of the resuspended bacterial solution containing TRV2-LcPORA1 was measured. 600 , and are adjusted to OD 600 =0.8.

[0040] Finally, the resuspended bacterial solutions containing TRV2 and TRV2-LcPORA1 were mixed with the resuspended bacterial solution containing TRV1 at a volume ratio of 1:1, and the mixture was allowed to stand for 2 h at room temperature in the dark before infecting the detached petals of Liriodendron chinense.

[0041] Example 2

[0042] 1. Different methods of infecting petals of Liriodendron chinense

[0043] Select Liriodendron tulipifera buds that have no obvious pests and diseases and are about to reach the pollination period ( Figure 1 ), transport the petals alive at low temperature to the laboratory, remove the outer sepals and rinse with running water for 1 hour, then sterilize in the clean bench: rinse with 75% alcohol for 30-60 seconds, rinse with sterile water 2-3 times, rinse with 10% hydrogen peroxide for 10 minutes, add appropriate amount of Tween 20, rinse with running water 5-7 times and place in a sterile bottle. ① Immerse and infect the petals in vitro: Then use tweezers and a scalpel to peel off the petals in vitro and immerse them in the target bacterial solution at 28℃ and 200r·min -1 Infect for 10 minutes, rinse with sterile water 2-3 times to sterilize, dry the surface water, place in 1 / 2MS solid culture medium and culture at room temperature for 24 hours in the dark; finally, place the plant material in a growth room at 20-25℃ for normal culture. ② Conventional injection method: The petals of Liriodendron chinense were infected by conventional injection method.

[0044] The results showed that the conventional injection method could not infect the petals of Liriodendron chinense at different developmental stages. The infection solution was extremely difficult to inject into the petals, and the few petals that could be injected would have local necrosis or no phenotype under field conditions due to environmental influences. The in vitro petal immersion infection method can effectively avoid the influence of field environmental changes, and the immersion infection method does not cause obvious damage to the surface of the petals. Therefore, the petals can retain high biological activity and avoid death for a certain period of time. Moreover, the corresponding phenotypic changes after gene silencing are significant. Therefore, the in vitro petal immersion method is the best VIGS infection method for Liriodendron chinense petals.

[0045] 2. Different bacterial concentrations infected the petals of Liriodendron chinense

[0046] OD 600 The detached petals of Liriodendron chinense were infected with bacterial solution at concentrations of 0.2, 0.4, 0.6, and 0.8, and the phenotypic changes of the plants were observed and the changes in the relative expression level of the LcPORA1 gene were detected.

[0047] The results are as follows Figure 2 As shown, only when OD 600 When the OD value is 0.8, the phenotypic changes after gene silencing can be clearly observed. 600 When the value is 0.2, 0.4, or 0.6, the phenotypic changes are not obvious.

[0048] The results are as follows Figure 3 As shown, compared with the empty load control, OD 600 When OD was 0.2 and 0.4, the expression level of LcPORA1 gene did not decrease significantly (P>0.05); 600 When OD was 0.6 and 0.8, the expression level of LcPORA1 gene was significantly lower than that of the empty control (P<0.01), but 600When the value was 0.8, the expression level of the gene decreased significantly, and there was an obvious phenotypic change, that is, the petals became chlorotic.

[0049] 3. Changes in chlorophyll content of transgenic lines

[0050] The results are as follows Figure 4 As shown in the figure, compared with the control group, the chlorophyll content in the petals of Liriodendron chinense decreased significantly after 7 days of infection with TRV2-LcPORA1.

[0051] 4. Changes in thylakoid structure of transgenic lines

[0052] The results are as follows Figure 5 As shown, the thylakoid membrane structure in the chloroplasts in the control group was clearly visible, with relatively tight stacking and regular arrangement, while the thylakoid stacking of the chloroplasts in the TRV2-LcPORA1 experimental group became loose or even partially missing, with disordered arrangement, and the stacking of the membrane was no longer clear.

[0053] In summary, LcPORA1 is a key gene in the chlorophyll synthesis pathway. The silencing of this gene will lead to the restriction of chlorophyll synthesis, which further affects the development and function of the thylakoid membrane. Combined with the determination of chlorophyll content and the observation of thylakoid electron microscopy, it can be concluded that the LcPORA1 gene in the petals of Liriodendron chinense infected with TRV2-LcPORA1 was effectively silenced, and its gene function was verified, indicating that this system can effectively verify the function of related genes in the petals of Liriodendron chinense directly.

[0054] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A specific nucleotide fragment for silencing the LcPORA1 gene of Liriodendron chinense, the sequence of which is shown in SEQ ID NO.

1.

2. A method for constructing a VIGS silencing system for the LcPORA1 gene of Liriodendron chinense, characterized in that: include: The specific nucleotide fragment used for silencing the LcPORA1 gene of Liriodendron chinense as shown in SEQ ID NO.1 was connected to the pTRV2 vector to construct the vector TRV2-LcPORA1, namely the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense.

3. The VIGS silencing system of the LcPORA1 gene of Liriodendron chinense constructed by the construction method of claim 2.

4. Use of the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense according to claim 3 in inhibiting the expression of the LcPORA1 gene of Liriodendron chinense.

5. The use according to claim 4, characterized in that: The steps include: 1) Construction of the VIGS silencing system of the LcPORA1 gene in Liriodendron chinense, namely the vector TRV2-LcPORA1; 2) Transform the TRV1 and recombinant plasmids containing the TRV2-LcPORA1 vector fragment into Agrobacterium competent cells respectively, and collect the positive bacterial liquid after cultivation; 3) Mix the TRV2-LcPORA1 positive bacterial solution and the TRV1 positive bacterial solution in proportion and then infect the petals of Liriodendron chinense in vitro; 4) Cultivate, screen and obtain petals of Liriodendron chinense with significantly reduced expression of LcPORA1 gene.

6. The use according to claim 5, characterized in that: The mixing ratio is 1:

1.

7. The use according to claim 5, characterized in that: The OD of the positive bacterial solution 600 is 0.

8.

8. Use of the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense according to claim 2 in identifying the function of the LcPORA1 gene of Liriodendron chinense.

9. Use of the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense according to claim 2 in regulating the chlorophyll content in petals of Liriodendron chinense.

10. Use of the VIGS silencing system of the LcPORA1 gene of Liriodendron chinense according to claim 2 in regulating the stability of chloroplast structure in petals of Liriodendron chinense.

Images