Application of CsFIP37 gene in regulating somatic embryogenesis in citrus

Abstract

The present invention relates to the application of CsFIP37 gene in regulating citrus somatic embryogenesis, by constructing m 6 A modified A-modified overexpression vector for CsFIP37, a key component of the methyltransferase complex, was transformed into calli of the cultivar 'Guoqing No. 1,' which has lost its ability to produce somatic embryos. The study revealed that CsFIP37 promotes somatic embryogenesis in citrus. This study provides insights into the regulatory mechanisms of somatic embryogenesis, providing new insights into fundamental aspects of somatic embryogenesis and potentially significant for improving the yield and quality of agronomically important crops.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the CsFIP37 gene in regulating citrus somatic embryogenesis. Background Art

[0002] Citrus is an economically important perennial evergreen fruit tree, with a production rate that ranks among the highest among all fruits. However, as one of the world's most important fruit crops, its characteristics, such as polyembryonic, long-young, and male-female sterility, hinder conventional citrus breeding. Furthermore, the embryonic potential of citrus embryonic callus decreases or even disappears over long periods of subculture. Therefore, genetic improvement of citrus using biotechnology is an effective breeding approach.

[0003] Somatic embryogenesis (SE) refers to the process by which somatic cells, under induced conditions, undergo a series of biological processes to produce somatic embryos. Plant somatic embryogenesis boasts advantages such as ubiquity, genetic stability, low mutation rates, and high reproductive coefficients. Since its first description in the 1950s, somatic embryogenesis has become a powerful tool in plant biotechnology for propagating endangered species, with important applications in large-scale asexual reproduction and germplasm conservation. Somatic embryogenesis is also a key regenerative step in genetic improvement through biotechnological approaches such as screening for somatic clone variation, somatic hybridization, and genetic transformation.

[0004] N 6 -Adenylate methylation (N 6 -methyladenosine,m 6 A) modification is one of the most critical internal modifications of RNA, which can enrich and regulate the genetic information of eukaryotic organisms and play an important role in the zygotic embryonic development of plants. 6 A modification is through m 6 FIP37 is an important m-methyltransferase in plants. 6 AtFIP37, one of the A methyltransferases, is expressed during embryogenesis and throughout plant development, such as in undifferentiated cells (such as meristems or embryonic cells) and highly differentiated cells (such as epidermal hairs). It is highly expressed in actively proliferating tissues. Disruption of AtFIP37 leads to premature cessation of seed development and embryonic development at the globular stage. Arabidopsis fip37 mutants exhibit embryonic lethality phenotypes. The fip37 mutant restricts the expression of two key shoot apical meristem regulatory factors, STM and WUS, affecting the decay rate of STM and WUS mRNA, leading to excessive proliferation of the shoot meristem. At the same time, m 6A loss of RNA modification throughout the transcriptome. In rice, loss-of-function mutants of OsFIP37 exhibit significant meiotic defects during microspore development and are accompanied by a corresponding decrease in endogenous auxin content. OsFIP37 interacts with the RNA-binding protein OsFAP1 in rice anthers, mediating mRNA expression on the auxin biosynthesis gene OsYUCCA3 during microspore development. 6 A RNA modification thereby increases the abundance of OsYUCCA3 transcripts and promotes local auxin biosynthesis in anthers during male meiosis, which is essential for meiosis and subsequent rice pollen development.

[0005] At the same time, although somatic embryos and seed embryos are both forms of embryonic development, they differ significantly in their origins, developmental processes, and applications. That is, the development of seed embryos and somatic embryos is controlled by complex gene regulatory networks that may contain the same genes, but the effects of these genes may be affected by other factors (such as hormones, transcription factors, epigenetic modifications, etc.), resulting in different activities of these factors in different embryo types, or genes may be expressed at specific stages of seed embryos, while exhibiting different spatiotemporal expression patterns in the development of somatic embryos. In addition, the development of somatic embryos usually requires specific in vitro culture conditions that may affect gene expression and function. Therefore, even key developmental genes in seed embryos may exhibit different functions under in vitro conditions. Summary of the Invention

[0006] The present invention constructs m 6 The overexpression vector of CsFIP37, a key component of the methyltransferase complex modified by A, was then transformed into calli of 'National Day No. 1' that had lost the ability to produce somatic embryos, in order to explore the role of CsFIP37 in citrus somatic embryogenesis and preliminarily explore the role of m 6 The mechanism of action of A modification in citrus somatic embryogenesis. Further investigation of the regulatory mechanisms of somatic embryogenesis will lead to the development of various biotechnological applications and provide new opportunities for understanding fundamental aspects of somatic embryogenesis, which is of great significance for improving the yield and quality of agronomically important crops.

[0007] The present invention provides the use of a CsFIP37 gene, an expression cassette and / or a recombinant vector containing the same in improving plant somatic embryogenesis. The protein sequence encoded by the CsFIP37 gene is shown in SEQ ID NO: 1.

[0008] Furthermore, the plant somatic embryo is a citrus somatic embryo.

[0009] The present invention also provides a method for improving plant somatic embryogenesis, wherein an expression cassette, a recombinant vector and / or a recombinant microorganism that overexpresses the CsFIP37 gene is introduced into plant callus tissue to construct a CsFIP37 overexpressing callus line. The plant somatic embryo is a citrus somatic embryo.

[0010] Furthermore, the constructed CsFIP37 gene overexpression vector was transformed into Agrobacterium, and transgenic positive calli were screened to obtain the transgenic positive calli. The embryogenesis rate of the transgenic positive calli was significantly improved.

[0011] Further, the following steps are included:

[0012] S1. Grow the induced callus on MT medium for about 15-20 days, pick the calli with good growth and transfer them to liquid suspension medium, and culture them on a shaker at 110-120 rpm for 4 days before infection with Agrobacterium.

[0013] S2. Discard the suspension culture medium in the callus tissue and add the EHA105 Agrobacterium culture medium containing the CsFIP37 gene overexpression plasmid to the callus tissue. Shake for 5 minutes and then let it stand for 20 minutes. Blot the culture medium on the callus surface with filter paper and spread it onto the co-culture medium containing acetosyringone with filter paper. Co-culture at 23°C for 3 days.

[0014] S3. The co-cultured callus tissue was transferred to a screening medium supplemented with antibiotics, and screened and cultured in an incubator at 28° C. until positive resistant calli grew out.

[0015] The present invention also provides a method for citrus somatic embryogenesis and plant regeneration, comprising the following steps:

[0016] S1. Grow the induced callus on MT medium for about 15-20 days, pick the calli with good growth and transfer them to liquid suspension medium, and culture them on a shaker at 110-120 rpm for 4 days before infection with Agrobacterium.

[0017] S2. Discard the suspension culture medium in the callus tissue and add the Agrobacterium culture medium containing the CsFIP37 gene overexpression plasmid to the callus tissue. Shake for 5 minutes and then let it stand for 20 minutes. Use filter paper to absorb the culture medium on the callus surface and spread it onto the co-culture medium containing acetosyringone with filter paper. Co-culture at 23°C for 3 days.

[0018] S3. Transfer the co-cultured callus to a screening medium supplemented with antibiotics and culture in a 28°C incubator until resistant calli grow out;

[0019] S4. Pick a small piece of freshly grown resistant callus from each group and place it in the screening medium for further culture and purification until the callus tissue grows stably;

[0020] S5. Further induce the well-growing callus to differentiate into complete plants.

[0021] Beneficial effects:

[0022] By studying the expression pattern of CsFIP37, the present invention speculates that it may be involved in the embryonic development of citrus. The expression level of CsFIP37 in the seeds of 'Fulingxia' orange is higher than that in other organ tissues, and the expression level of CsFIP37 in the embryonic callus of 'Fulingxia' is higher than that in the callus of 'National Day No. 1', which has lost the ability to produce somatic embryos.

[0023] This study investigated the role of CsFIP37 in citrus somatic embryogenesis by constructing CRISPR knockout and overexpression vectors for CsFIP37. The CsFIP37 knockout vector was transformed into embryonic callus from the 'Fulingxia' variety, while the CsFIP37 overexpression vector was transformed into callus from the 'National Day One' variety, which has lost the ability to produce somatic embryos. DNA testing revealed that the embryonic callus from the 'Fulingxia' variety transformed with the CRISPR vector died. This could be due to operational errors during the transformation process, or perhaps the transformation was successful, but the loss of CsFIP37 caused callus cell death. DNA testing of calli from the overexpressing transgenic lines confirmed successful transformation. Expression levels of the positive overexpression lines were then measured, resulting in three CsFIP37 overexpression lines. After induction with glycerol induction medium, all CsFIP37-overexpressing callus lines generated green somatic embryos, while the empty control callus lines did not generate somatic embryos. These results indicate that CsFIP37 can promote somatic embryogenesis in citrus.

[0024] Embryogenic callus of citrus is typically generated from nucellar tissue as an explant through hormone induction. Non-embryogenic callus of citrus is typically obtained from stem segments as explants after dark culture on MT medium supplemented with hormones such as 2,4-D. Embryogenic callus of 'Fulingxia' orange has small cells with dense and regular cell arrangement, while non-embryonic callus has large cells with loose and irregular cell arrangement. Cytological observation of embryogenic callus from CsFIP37-overexpressing callus lines using paraffin sectioning revealed that cells in CsFIP37-overexpressing callus lines were densely and orderly arranged, with dense cytoplasm. In contrast, callus from the empty control line showed no discernible regular arrangement, with loosely arranged cells and pale cytoplasm. Logo staining of yellow-green calli generated eight weeks after induction from CsFIP37-overexpressing calli and calli from a control line revealed that the cells in the control line were large and had unclear nuclei, while the cells in the CsFIP37-overexpressing calli were small and had clearly visible nuclei. Cell morphology was consistent with paraffin sections: cells in the control line were loosely and irregularly arranged, while cells in the CsFIP37-overexpressing calli were densely and regularly arranged. These results indicate that overexpression of CsFIP37 altered the cytological morphology of the 'National Day No. 1' callus, which had lost the ability to generate somatic embryos, toward an embryogenic callus capable of somatic embryogenesis.

[0025] The present invention deeply explores the relationship between the CsFIP37 gene and the regulatory mechanism of somatic embryogenesis, provides new ideas for understanding citrus somatic embryogenesis, and is of great significance for improving crop yield and quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other relevant drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 This is the expression analysis of CsFIP37 gene.

[0028] Figure 2 Construction of a CsFIP37 gene subcellular localization vector. a): Amplification of the CsFIP37 gene target fragment. M: DL5000 molecular weight marker; 1-4: Amplification of the CsFIP37 gene target fragment; b): Transformation of the CsFIP37 gene expression vector into E. coli. M: DL5000 molecular weight marker; 1-6: Transformation results of the CsFIP37 gene; c): Schematic diagram of the pCAMBIA1300-35S-GFP vector.

[0029] Figure 3 The subcellular localization of CsFIP37 protein is shown in Figure 5. The scale bar is 10 μm.

[0030] Figure 4 The CsFIP37 gene CRISPR vector was obtained. a) Construction of the CsFIP37 gene expression cassette. M: DL2000 molecular weight marker; 1-4: Construction of the CsFIP37 gene T1 expression cassette; 5-8: Construction of the CsFIP37 gene T2 expression cassette; b) Transformation of E. coli with the CsMTA and CsFIP37 gene CRISPR vectors. M: DL2000 molecular weight marker; 1-4: Transformation results of the CsFIP37 gene; c) Schematic diagram of the pYLCRISPRCAS9-P35S-N vector.

[0031] Figure 5 Obtaining the CsFIP37 gene entry vector. a): Amplification of the CsMTA and CsFIP37 gene target fragments. M: DL5000 molecular weight marker; 1-4: Amplification of the CsFIP37 gene target fragment; b): Transformation of the CsFIP37 gene entry vector into E. coli. M: DL5000 molecular weight marker; 1-6: CsFIP37 gene transformation results; c): Schematic diagram of the PDONR207 vector.

[0032] Figure 6 The CsFIP37 gene overexpression vector was obtained. a) Transformation of the CsFIP37 gene overexpression vector into E. coli. M: DL5000 molecular weight marker; 1-6: CsFIP37 gene transformation results; b) Schematic diagram of the pK7WG2D vector.

[0033] Figure 7 Positive detection of CsFIP37 CRISPR transgenic callus. a) Kanamycin resistance gene primers were used to identify CRISPR transgenic callus; b) Vector-derived primers were used to identify CRISPR transgenic callus; c) CsACT7 internal reference gene primers were used to identify CRISPR transgenic callus. M: DL2000 molecular weight standard; 1 and 15: Water (negative control); 2 and 16: Wild type; 3 and 4, 17 and 18: Empty vector lines; 19-28: CsFIP37 transgenic callus.

[0034] Figure 8Identification of CsFIP37-overexpressing transgenic callus lines using kanamycin-resistance primers and 35S-F / Adapter_attB2 primers. The upper panel shows identification of CsFIP37-overexpressing transgenic callus lines using kanamycin-resistance primers. M: DL2000 molecular weight standard; 1-2: water (negative control); 3-4: wild type; 5-8: empty vector line; 9-20: CsFIP37 transgenic callus lines. The lower panel shows identification of CsFIP37-overexpressing transgenic callus lines using 35S-F / Adapter_attB2 primers. M: DL2000 molecular weight standard; 1-2: water (negative control); 3-4: wild type; 5-8: empty vector line; 9-20: CsFIP37 transgenic callus lines.

[0035] Figure 9 Figure 3. Phenotypic analysis and embryogenesis rate of CsFIP37-overexpressing transgenic callus lines. E0 represents culture on MT selection medium, while E8, E10, E12, and E14 represent 8, 10, 12, and 14 weeks of induction on glycerol induction medium, respectively. Scale bar: 2 mm.

[0036] Figure 10 Cytological observation of CsFIP37-overexpressing transgenic callus lines. a) Paraffin sections of CsFIP37 transgenic callus lines and empty vector control callus lines (OE-EV). Scale bar: 100 μm. b) Logo staining of CsFIP37 transgenic callus lines and empty vector control callus lines. Scale bar: 50 μm. DETAILED DESCRIPTION

[0037] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention with this. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0038] Roots and stems of the 'Fulingxia' sweet orange were obtained from seedlings cultured in culture medium. Leaves, flowers, and fruits were collected from mature 'Fulingxia' sweet orange trees in the resource garden of the National Citrus Breeding Center at Huazhong Agricultural University. Embryogenic callus from the 'Fulingxia' tree and callus from the 'Guoqing No. 1' variety, which has lost its somatic embryogenic capacity, were obtained from the Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University and propagated and preserved in this laboratory.

[0039] Strains and vectors:

[0040] The strains and plasmid vectors used in this invention are as follows: Escherichia coli strain DH5α was obtained from Beijing Quanshijin Biotechnology Co., Ltd., and Agrobacterium strain EHA105 was purchased from Angyu Biotechnology Co., Ltd. The pDNOR207 and pK7WG2D vectors used for overexpression of the target gene, as well as the pCAMBIA1300 vector for subcellular localization analysis, were obtained from Professor Xu Qiang (Key Laboratory of Horticultural Plant Biology, Ministry of Education, Huazhong Agricultural University).

[0041] Example 1 Sequence Analysis of CsFIP37 Protein

[0042] The protein sequence was downloaded and homology searched using the NCBI website (https: / / www.ncbi.nlm.nih.gov / ). CsFIP37 is a single-copy gene in citrus, and its protein sequence is:

[0043] MASRTHLDDDDDDFGGDFSANHNSRRSGSKRSFGDIEDDEDDIFGSRKANSKVEETAPGVATGMILSLRESLQNCKDTLATCQLELEAAKLEIQKWHSSFQNELFIPPGTSPEPRLVINYLQTLKSSEEMLKEQLEKAKKKEAAFIVTFAKREQEIAELKSAVRDLKAQLKPPL MQARRLLLDPAIHEEFRRLKNLVEEKDKKVKELEENIAAVSFTANSKMGKALMAKCKTLQEENDEIGRQNEEGETHQLSVKLALQKSLNAELKSQFEALYKHMDGLTDDVERSNEMVLMLREKLEEKDHELEKLKHELRQKSVLEEDKNDSVSDKNIGNDVTVSGEAVS(SEQ ID NO:1).

[0044] Example 2 Analysis of tissue-specific expression of the CsFIP37 gene

[0045] 'Fulingxia' sweet orange fruits were collected from the resource garden of the National Citrus Breeding Center of Huazhong Agricultural University and stored at 4°C until seed removal. Seeds were sterilized with 1 mol / L NaOH and 3% sodium hypochlorite solution and then washed 3-4 times with sterile water for 10 minutes each time. After peeling the seed coat, seeds were sown on MT medium and vernalized in a refrigerator at 4°C for at least one week. They were then incubated in a culture room at 26°C with 16 hours of light and 8 hours of darkness. After one month of incubation, roots and stems were harvested and stored frozen in liquid nitrogen. 'Fulingxia' leaves, flowers, and fruits were collected from mature 'Fulingxia' sweet orange trees in the resource garden of the National Citrus Breeding Center of Huazhong Agricultural University and stored frozen in liquid nitrogen.

[0046] After complete collection of materials, total RNA was extracted and reverse transcribed into cDNA. The reverse-transcribed cDNA was diluted 10-fold, and the expression of the target gene in different tissues was analyzed by real-time quantitative PCR (qRT-PCR) using CsACT7 as the reference gene. The qRT-PCR reaction was performed for 45 cycles, and the gene expression level of each sample was repeated four times as technical replicates. qRT-PCR primer sequences (5'-3'): CsActin7-F: ACCTGCTGGAAGGTGCTGAG, CsActin7-F: CCAAGCAGCATG AAGATCAA. CsFIP37-qF: CTGCAAACAGCAAGATGGGG, CsFIP37-qR: GATGTGTCTCGCCTTCCTCA.

[0047] The expression pattern of CsFIP37 gene was analyzed by using the above-mentioned technique in wild-type callus tissue (G1) that lost the ability of somatic embryogenesis, wild-type embryogenic callus tissue (FLX) of 'Guoqing No. 1', and roots, stems, leaves, flowers, fruits and seeds of 'Fulingxia' sweet orange. Figure 1 As shown, the CsFIP37 gene is highly expressed in seeds. There is no significant difference in CsFIP37 expression in embryonic callus from the 'Fulingxia' variety compared to other organs and tissues. However, CsFIP37 expression in embryonic callus from the 'Fulingxia' variety is higher than in callus from the 'Guoqing No. 1' variety, which has lost its ability to produce somatic embryos. This suggests that CsFIP37 may play a role in citrus embryogenesis.

[0048] Example 3 Subcellular localization of CsFIP37 gene

[0049] 1. Construction of the subcellular localization vector of CsFIP37 gene

[0050] pCAMBIA1300 was selected as the vector for subcellular localization. First, the pCAMBIA1300 empty vector plasmid was extracted, and then the pCAMBIA1300 vector plasmid was digested with BamHI enzyme. The fragment was verified by agarose gel electrophoresis and the target fragment was recovered from the gel. The enzyme digestion reaction system was as follows:

[0051] Table 1 Single enzyme digestion reaction system

[0052]

[0053] Place the PCR tube containing the enzyme digestion system at 37°C for 30 minutes. After the reaction is complete, detect the digestion products using 1.0% agarose gel electrophoresis. Recover the correct PCR product from the gel to obtain the target fragment.

[0054] Primers were designed to amplify the CsFIP37 gene by PCR, verified by agarose gel electrophoresis, and the target fragment was recovered by gel recovery. The pCAMBIA1300 vector recovered by enzyme digestion was ligated with the recovered CsFIP37 gene fragment. Figure 2 The ligation reaction system is:

[0055] Table 2 Ligation reaction system

[0056]

[0057] Place the PCR tube containing the ligation reaction system at 50°C for 45 minutes to react. After the bacterial solution becomes obviously turbid, use the PCR method to identify its positive. After the PCR reaction program is completed, take 10μL of the amplified product for 1% agarose gel electrophoresis detection. For the bacterial solution that is identified as positive, take 500μL and send it to the sequencing company for sequencing. The sequencing primers (SP-L2: GTCGTGCTCCACATGTTGACCG, SP-R: CGCACCCGACATAGATGCAATAACTTC) are amplification primers, and take another 500μL of the bacterial solution and add an equal volume of 25% glycerol. Store it in a -80°C refrigerator and store the remaining bacterial solution in a 4°C refrigerator. After the sequencing results come out, pick the bacterial solution with correct sequencing to extract the plasmid.

[0058] 2. Subcellular localization of CsFIP37 protein

[0059] The pCAMBIA1300 vector containing the target gene was transformed into EHA105 Agrobacterium. The overnight cultured bacterial solution was tested by bacterial solution PCR. After the correct bacterial solution was added to 500 μL of 25% glycerol in an equal volume, it was placed in a -80°C refrigerator. Separately, 100 μL of bacterial solution was added to 3 ml of LB liquid culture medium containing kanamycin (50 mg / L). Each milliliter of liquid culture medium contained 1 μL of 50 mg / ml of acetosyringone. Nuclear localization marker-NCR Agrobacterium and P19 Agrobacterium were activated using the same method. After the expansion, the cells were collected by centrifugation, and 1 mL of 10 mmol / L MgCl2 solution was added to resuspend the cells. Each milliliter of MgCl2 contained 1 μL of 50 mg / mL of acetosyringone. The cells were then kept in the dark at room temperature for more than 3 hours. EHA105 Agrobacterium containing the target gene vector was mixed with nuclear localization marker-NCR Agrobacterium and P19 Agrobacterium in a ratio of 1:1:1, and then the OD value of the bacterial solution was adjusted with 10 mmol / L MgCl2. 600 To 0.5. Select tobacco plants that have been grown for about one month and inject the mixed bacterial solution into the back of the tobacco plants using a 1 mL syringe (without the needle). After 48 hours of incubation, observe the expression of the GFP tag under a confocal microscope.

[0060] Subcellular localization analysis of CsFIP37 protein was performed, and empty vector was used as blank control. Figure 3 As shown, in tobacco cells transformed with the CsFIP37 gene, obvious fluorescence signals could only be observed in the nucleus, while in empty-transformed cells, fluorescence signals were present throughout the cells. These results indicate that CsFIP37 is localized in the nucleus.

[0061] Example 4 Construction of Citrus Callus CsFIP37 Gene Vector

[0062] 1. Construction of CsFIP37 gene CRISPR vector

[0063] Primers were designed based on the target gene sequence, using the online website CRISPR-P2.0 (http: / / crispr.hzau.edu.cn / CRISPR2 / news.php). sgRNA expression cassettes were then constructed using the pYLgRNA-AtU6-1 and pYLgRNA-AtU6-26 plasmids as templates, respectively.

[0064] The expression cassette construction method is as follows: 2-5 ng of pYLgRNA-AtU6-1 and pYLgRNA-AtU6-26 plasmids were taken respectively, two expression cassettes were constructed for each gene, and four primers were used in the first round of PCR: UF and Target sequence-R (0.2 μM), Target sequence-F and gR-R (0.1 μM).

[0065] The first round PCR reaction system is as follows:

[0066] Table 3 First round PCR reaction system

[0067]

[0068] After mixing the reagents, centrifuge the PCR tube to fully mix the reagents and perform PCR reaction for 28 cycles. The first round of PCR reaction procedure is as follows:

[0069] Table 4 First round PCR amplification program

[0070]

[0071] PCR amplification was performed for 28 cycles. After the amplification was completed, 1 μL of the first-round PCR product was diluted 10 times as a template, and then the second-round PCR reaction was performed. The second-round PCR reaction was as follows:

[0072] Table 5 Second round PCR reaction system

[0073]

[0074] After mixing the reagents, centrifuge the PCR tube to fully mix the reagents and perform the PCR reaction. The PCR reaction procedure is the same as the first round PCR reaction procedure.

[0075] After the construction is successful, the enzyme digestion-ligation reaction of the binary vector pYLCRISPR / Cas9-DN and the sgRNA expression cassette is performed, such as Figure 4 The reaction system is as follows:

[0076] Table 6 Enzyme digestion-ligation reaction system

[0077]

[0078] After mixing the reagents, perform enzyme digestion and ligation using a variable temperature cycle: 37°C for 5 minutes, 10°C for 5 minutes, 20°C for 5 minutes, 15 cycles, and finally 37°C for 5 minutes. After completion, transform the ligation product into competent cells, select a single colony, and test the culture for positive PCR. Those that test positive are sent for sequencing. After sequencing is confirmed, the plasmid is extracted and introduced into Agrobacterium tumefaciens, stored at -80°C, and subsequently used to infect plant tissue.

[0079] 2. Construction of CsFIP37 gene plant transformation overexpression vector

[0080] Design primers for target genes:

[0081] CsFIP37-OX-F:GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGGCTTCACGGACTCATCT,

[0082] CsFIP37-OX-R: GGGGACCACTTTGTACAAGAAAGCTGGGTTTCAGCTT ACAGCTTCCCC;

[0083] Adapter-attB1:GGGGACAAGTTTGTACAAAAAAGCAGGCT,

[0084] Adapter-attB2: GGGGACCACTTTGTACAAGAAAGCTGGGT.

[0085] PCR amplification was performed using the cDNA of the leaves of the orange tree as a template. After the PCR was completed, agarose gel electrophoresis was performed to verify the fragments. The target gene fragments were recovered by gel recovery. After the target gene fragments were obtained, the recovered products were recombined into the entry vector pDNOR207 using BP enzyme. Figure 5 As shown. The BP reaction system is:

[0086] Table 7 BP reaction

[0087]

[0088] The reaction solution was incubated in a 25°C water bath for 4 hours and then used directly for E. coli transformation without stopping the reaction. After PCR detection, positive bacteria were selected for sequencing using Adapter-attB1 / B2 primers. The plasmids were extracted from the bacteria solution that had been sequenced correctly. The recombinant plasmids that had been sequenced correctly were then recombined into the pK7WG2D vector using LR enzyme, as shown in the following example: Figure 6 As shown. The LR reaction system is:

[0089] Table 8 LR reaction

[0090]

[0091] The reaction solution was incubated in a 25°C water bath for 4 hours. The reaction could be directly used for E. coli transformation without stopping the reaction. The positive bacteria were selected for sequencing. The bacteria solution with correct sequencing results was selected for bacterial preservation and plasmid extraction.

[0092] 2. Agrobacterium-mediated genetic transformation of callus tissue

[0093] The constructed target gene overexpression vector plasmid was transformed into EHA105 Agrobacterium tumefaciens. The Agrobacterium-mediated callus genetic transformation operation process was referred to Long Jianmei's doctoral dissertation (2017).

[0094] (1) Callus material preparation: Calli were grown on MT medium for about 15-20 days. Calli with good growth were selected and placed in liquid suspension medium. They were suspended on a shaker at 110-120 rpm for 4 days before infection with Agrobacterium.

[0095] (2) Preparation of Agrobacterium infection medium: Activate Agrobacterium carrying the target gene vector stored at -80°C by streaking onto LB medium (add the corresponding antibiotic). Once a single colony has grown, pick a single colony and streak it. Incubate in a 28°C incubator for approximately 2 days. Scrape the cells with a blade and place them into a liquid suspension medium. Incubate in a shaker at 28°C at 180-200 rpm for approximately 1 hour until the OD600 reaches between 0.6 and 0.8. This is then used for the next infection step.

[0096] (III) Infection and co-cultivation: Pour off the suspension culture medium in the callus tissue, add the Agrobacterium culture solution to the callus tissue, shake manually for about 5 minutes, and then let it stand for 20 minutes. Use filter paper to absorb the culture solution on the surface of the callus, spread it onto the co-culture medium (containing acetosyringone) with filter paper, and co-cultivate at 23°C for 3 days.

[0097] (IV) Screening culture: The co-cultured callus tissue was transferred to screening medium (with antibiotics added) and cultured in a 28°C incubator until resistant calli grew;

[0098] (5) Obtaining resistant callus: Pick a small piece visible to the naked eye from each cluster of freshly grown resistant callus and continue to culture and purify it in the screening medium until the callus tissue grows stably.

[0099] 3. Positive identification of transgenic callus and induction of somatic embryos

[0100] Extract DNA from transgenic callus. After successful DNA extraction, measure DNA concentration on a UV spectrophotometer (NanoDrop2000, Thermo Scientific). Then, for CRISPR transgenic callus DNA, use CsActin7 gene primers, vector self primers, and kanamycin resistance gene primers to PCR amplify the target gene fragment to detect transgenic material positive. For overexpression transgenic callus DNA, use 35S / Adapter-attB2 primers and kanamycin resistance gene primers to PCR amplify the target gene fragment to detect transgenic material positive. Figure 7 and 8 shown.

[0101] The CsFIP37 target gene CRISPR knockout vector was transformed into embryogenic callus of the 'Volinga' variety, which is capable of somatic embryogenesis. Empty vector was used as a control. DNA was then extracted from both transgenic and empty vector calli, and DNA concentration and quality were determined. Results showed low DNA concentration and poor quality. The extracted DNA was then tested for positive PCR using primers for the CsACT7 internal reference gene, kanamycin resistance gene primers, and vector-derived primers. The CsACT7 internal reference gene primers failed to amplify the target band, while the kanamycin resistance gene primers and vector-derived primers did. This indicates that the transgenic calli were dead. The target bands amplified by the kanamycin resistance gene primers and vector-derived primers were likely amplified by Agrobacterium containing the CsFIP37 target gene CRISPR knockout vector remaining on the callus surface. Therefore, the CsFIP37 target gene CRISPR knockout vector failed to transform embryogenic callus of the 'Volinga' variety, which is capable of somatic embryogenesis. like Figure 7 shown.

[0102] Select transgenic callus materials with positive DNA detection, extract RNA, reverse transcribe into cDNA and perform qRT-PCR to detect the expression level of the target gene in the transgenic material. The primer sequence and method are the same as Example 2.

[0103] The CsFIP37 overexpressing callus and the empty vector control callus were cultured on somatic embryo induction medium. Figure 9 As shown, when cultured on selection medium (E0), there was no significant difference between the CsFIP37-overexpressing callus and the empty vector control callus. After 8 weeks of induction on glycerol medium (E8), the CsFIP37-overexpressing callus exhibited a yellow-green color, while the empty vector control callus showed no significant color change. After 12 weeks of induction on glycerol medium (E12), the CsFIP37-overexpressing callus produced green somatic embryos, whereas the empty vector control callus did not.

[0104] These results indicate that overexpression of CsFIP37 can promote somatic embryogenesis in calli of the previously incapable 'Guoqing No. 1' strain. Furthermore, during embryogenesis induction, the embryogenesis rate of the CsFIP37-overexpressing callus reached 63.3%.

[0105]

[0106] Table 9

[0107] 4. Cytological Observation of Transgenic Callus

[0108] Paraffin sections: After embryogenesis, calli from CsFIP37-overexpressing transgenic plants and calli from the control strain were fixed in FAA fixative for 24 hours. The sections were then rinsed with distilled water to remove the fixative. The sections were then washed three times with 70% ethanol for 30 minutes each. Hematoxylin or safranin-fast green stain was added and the sections were allowed to stand at room temperature for 3 days. The sections were then dehydrated (gradually replacing all water in the material with a dehydrating agent) and cleared (replacing the dehydrating agent in the tissue with a clearing agent to allow paraffin to penetrate the plant tissue). The sections were then embedded in paraffin (replacing the clearing agent with paraffin by penetrating the tissue). Finally, the embedded paraffin blocks were sectioned and trimmed, and then sectioned using a microtome. After sectioning, the wax strips were stretched and dried using egg white glycerin on a heated slide. The sections were then dried in an incubator (38-40°C). The sections were then observed and photographed under a microscope.

[0109] Logul staining: Take the yellow-green CsFIP37 overexpressing transgenic callus material and the empty control callus material after induction for eight weeks, take a small piece of callus and flatten it on a slide, add appropriate amount of Logol staining solution to stain for 30 seconds, then wash with ddH2O for 15 minutes, add appropriate amount of transparent agent, cover with a coverslip, and place in a 4℃ refrigerator for 12 hours before observing under a differential interference microscope.

[0110] The cytological morphology of the embryogenic callus induced by the CsFIP37 overexpressing callus was observed using paraffin section technology. Figure 10 The results showed that callus cells in the CsFIP37-overexpressing callus were tightly packed and orderly, with dense cytoplasm, while callus cells in the empty control line showed loose, irregular arrangement and lighter cytoplasm. Differential interference microscopy revealed that cells in the empty control callus were larger, while cells in the yellow-green callus of the CsFIP37-overexpressing callus were smaller. Cell morphology was consistent with paraffin section observations. Cells in the empty control callus were loosely and irregularly arranged, with no clearly visible nuclei, while cells in the yellow-green callus of the CsFIP37-overexpressing callus were tightly packed and orderly, with clearly visible nuclei. However, logo staining revealed no significant difference in starch content between the two callus types. These results indicate that overexpression of CsFIP37 can promote somatic embryogenesis in callus of the "National Day No. 1" variety, which has lost its ability to produce somatic embryos.

[0111] That is, the present invention found that the CsFIP37 gene has the function of promoting citrus somatic embryogenesis, which is of great significance for improving the yield and quality of crops.

[0112] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details of the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the scope of protection of the present invention.

Claims

1. CsFIP37 Gene, containing the CsFIP37 Gene expression cassette and / or containing the CsFIP37 Application of a recombinant vector of a gene in improving plant somatic embryogenesis, the CsFIP37 The protein sequence encoded by the gene is shown in SEQ ID NO: 1, and the plant somatic embryo is a citrus somatic embryo.

2. A method for improving plant somatic embryogenesis, characterized in that: Overexpression CsFIP37 Gene expression cassette, recombinant vector and / or recombinant microorganism are introduced into plant callus to construct CsFIP37 Overexpression callus line; The constructed CsFIP37 The gene overexpression vector is transformed into Agrobacterium, and transgenic positive callus is obtained by screening, wherein the plant somatic embryo is citrus somatic embryo, and the CsFIP37 The protein sequence encoded by the gene is shown in SEQ ID NO:

1.

3. The method according to claim 2, characterized in that The steps include: S1. Grow the induced callus on MT medium for 15-20 days, pick the calli with good growth and transfer them to liquid suspension medium, and culture them on a shaker at 110-120 rpm for 4 days before infecting with Agrobacterium. S2. Pour off the suspension culture medium in the callus tissue and transfer the overexpressed CsFIP37 The EHA105 Agrobacterium tumefaciens solution containing the gene plasmid was added to the callus tissue, shaken for 5 minutes, and then allowed to stand for 20 minutes. The bacterial solution on the surface of the callus was blotted dry with filter paper and spread onto a co-culture medium containing acetosyringone with filter paper. The culture was then incubated at 23°C for 3 days. S3. The co-cultured callus tissue was transferred to a screening medium supplemented with antibiotics, and screened and cultured in an incubator at 28° C. until positive resistant calli grew out.

4. A method for citrus somatic embryogenesis and citrus plant regeneration, comprising the following steps: S1. Grow the induced callus on MT medium for 15-20 days, pick the calli with good growth and transfer them to liquid suspension medium, and culture them on a shaker at 110-120 rpm for 4 days before infecting with Agrobacterium. S2. Pour off the suspension culture medium in the callus tissue and transfer the overexpressed CsFIP37 The Agrobacterium tumefaciens liquid containing the gene plasmid was added to the callus tissue, shaken for 5 minutes, and then allowed to stand for 20 minutes. The liquid on the surface of the callus was blotted dry with filter paper, and the callus was spread onto a co-culture medium containing acetosyringone with filter paper and co-cultured at 23°C for 3 days. S3. Transfer the co-cultured callus to a screening medium supplemented with antibiotics and culture in a 28°C incubator until resistant calli grow out; S4. Pick a small piece of freshly grown resistant callus from each group and place it in the screening medium for further culture and purification until the callus tissue grows stably; S5, further inducing the well-grown callus to differentiate into complete citrus plants; in, described CsFIP37 The protein sequence encoded by the gene is shown in SEQ ID NO: 1.

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