Rapid, simple and convenient transgenic operation method for model diploid sweet potato sibling species

Abstract

The invention provides a model diploid sweet potato sibling species transgenic operation method, and belongs to the field of crop molecular breeding. The method comprises the following steps: when a model diploid sweet potato sibling species seedling grows to 4-6 true leaves, cutting off a stem section above a root, soaking the cut stem section in a bacteria solution of agrobacterium rhizogenes containing exogenous plasmids in a dark place for 1-3 hours, then smearing the agrobacterium rhizogenes containing the exogenous plasmids on the tangent plane of the bottom of the stem section, and then planting. The transgenic method provided by the invention is simple to operate, only watering is needed on time after transformation, transgenic seedlings can be obtained within 120 days, transgenic seeds can be obtained within 180 days, complicated and long-period tissue culture experiments are not needed, transgenic plants can be obtained more quickly after transformation, and the transgenic efficiency is greatly improved.

Description

Technical Field

[0001] The invention belongs to the field of molecular breeding of crops, and in particular relates to a rapid and convenient transgenic operation method for a model diploid sweet potato relative. Background Art

[0002] The genus Ipomoea has 800-900 species, including diploids, tetraploids and hexaploids, and is the largest genus in the Convolvulaceae family. Due to the high ploidy, complex genetic background and genotype dependence of the regeneration system, sweet potatoes face many difficulties in gene function research and new germplasm creation. The genome of closely related diploid wild species is relatively simple and has a low copy number, making them excellent materials for studying the genetic basis and variety improvement of sweet potatoes. At present, the regeneration systems of sweet potato diploids Ipomoea trifida (Zhang Cong), Ipomoeatriloba (Zhai Hong) and Ipomoea littoralis (Liu Aihua) have been published, and the regeneration tissue culture seedlings are obtained by utilizing the omnipotence of callus tissue. However, this method is difficult, has a long cycle, and has high technical requirements for operators. Therefore, it is urgent to provide a transgenic operation method for closely related diploid sweet potatoes to provide a good foundation for genetic improvement and gene function verification of the genus Ipomoea. Ipomoea cordatortriloba belongs to the same sweet potato group as sweet potato and is one of the most closely related diploid wild species of sweet potato. This species generally has good self-compatibility, high fruit set rate, low genome heterozygosity, and a shorter growing period than cultivated species, making it the best material as a model species in the sweet potato group. Summary of the invention

[0003] In view of this, the object of the present invention is to provide a method for transgenic operation of a model diploid sweet potato relative.

[0004] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0005] The present invention provides a method for genetically modifying a model diploid sweet potato relative, comprising the following steps:

[0006] When the model diploid sweet potato related species seedlings grow to 4-6 true leaves, the stem segments above the roots are cut, and the cut stem segments are immersed in a bacterial solution of Agrobacterium rhizogenes containing exogenous plasmids for 1-3 hours in the dark, and then the root-generating Agrobacterium rhizogenes containing exogenous plasmids are applied to the cut surface at the bottom of the stem segments before planting.

[0007] Preferably, the Agrobacterium rhizogenes containing the exogenous plasmid is obtained by transferring the exogenous plasmid into Agrobacterium rhizogenes.

[0008] Preferably, the exogenous plasmid is pCAMBIA1300 plasmid.

[0009] Preferably, the Agrobacterium rhizogenes is K599.

[0010] Preferably, the OD of the bacterial solution of Agrobacterium rhizogenes containing the exogenous plasmid is 600 It is 0.6~1.

[0011] Preferably, the Agrobacterium rhizogenes containing the exogenous plasmid is cultured in the dark at 20 to 30° C. for 18 to 30 hours on a TY solid culture medium before being applied.

[0012] Preferably, the planting matrix consists of vermiculite, coconut bran and perlite, and the mass ratio of the vermiculite, coconut bran and perlite is 4-8:2-5:0.5-1.5.

[0013] Preferably, the sweet potato diploid transgenic plants are obtained 110 to 130 days after planting, and the sweet potato diploid transgenic seeds are obtained 170 to 190 days after planting.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The transgenic method of the present invention is simple to operate. After the transformation, only watering is required on time. Transgenic seedlings can be obtained within 120 days, and transgenic seeds can be obtained within 180 days. There is no need for complex tissue culture experiments with long cycles. Transgenic plants can be obtained more quickly after transformation, and the transgenic efficiency is greatly improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a schematic diagram of the transgenic operation method;

[0017] Figure 2 The figures are the conditions of transgenic plants at different developmental stages (A is the fluorescent root after transformation, B and C are the fluorescent enlarged roots, and D and E are the germination of enlarged roots);

[0018] Figure 3 It is the result diagram of phenotypic analysis and molecular detection of wild-type seeds and transgenic seeds (A is the phenotype of wild-type seeds and transgenic seeds, B is the luminescence of wild-type seeds and transgenic seeds under green light excitation, and C is the gel image of molecular detection dsred fragment);

[0019] Figure 4 This is the result of molecular detection of the target gene MYB1 in the validation of the transgenic method;

[0020] Figure 5 This is a schematic diagram of the purple trait roots of the sweet potato diploid in the verification of the transgenic method;

[0021] Figure 6 These are the albino seedlings obtained by knocking out the PDS gene during the verification of the gene editing method. DETAILED DESCRIPTION

[0022] The present invention provides a method for genetically modifying a model diploid sweet potato relative, comprising the following steps:

[0023] When the model diploid sweet potato related species seedlings grow to 4-6 true leaves, the stem segments above the roots are cut, and the cut stem segments are immersed in a bacterial solution of Agrobacterium rhizogenes containing exogenous plasmids for 1-3 hours in the dark, and then the root-generating Agrobacterium rhizogenes containing exogenous plasmids are applied to the cut surface at the bottom of the stem segments before planting.

[0024] In the present invention, the model diploid sweet potato relative seedlings are obtained by cutting the seeds of the sweet potato relative diploid I. cordatotriloba and sowing them in a plug tray. The cutting is preferably done by cutting 0.5 to 2 mm with a corner of a nail clipper. 2 seed coat, the purpose is to allow water to pass through the seed coat, so that the seeds absorb water and swell; 8 to 12 days after sowing, the seeds germinate to obtain seedlings; when the I. cordatotriloba seedlings grow to 4 to 6 true leaves, the stem segments above the roots are cut off, and the cut stem segments are soaked in the bacterial solution of Agrobacterium rhizogenes containing exogenous plasmids for 1 to 3 hours in the dark, and the Agrobacterium rhizogenes containing exogenous plasmids is preferably obtained by transferring the exogenous plasmids into Agrobacterium rhizogenes; the exogenous plasmid is pCAMBIA1300 plasmid, and the pCAMBIA1300 plasmid is obtained by transferring the pCAMBIA1300 expression vector into Trans1-T1 Escherichia coli for cultivation and extraction; the Agrobacterium rhizogenes is K599; the OD of the bacterial solution of the Agrobacterium rhizogenes containing the exogenous plasmid is 600 Preferably, it is 0.6-1, and more preferably 0.7-0.9; the light-proof soaking time is more preferably 1.5-2.5h; after soaking, the root-generating Agrobacterium containing the exogenous plasmid is smeared on the bottom cut surface of the stem segment and then planted, and the root-generating Agrobacterium containing the exogenous plasmid is smeared after being dark-cultured at 20-30°C on a TY solid culture medium for 18-30h; the culture temperature is preferably 22-28°C, and the culture time is preferably 20-28h; the planting matrix consists of vermiculite, coconut bran and perlite, and the mass ratio of the vermiculite, coconut bran and perlite is preferably 4-8:2-5:0.5-1.5, and more preferably 5-7:3-4:0.7-1.4; the sweet potato closely related diploid I. cordatotriloba transgenic plants are obtained 110-130 days after planting, and the I. cordatotriloba transgenic seeds are obtained 170-190 days after planting.

[0025] 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.

[0026] Materials used in the present invention:

[0027] Diploid I. cordatotriloba germplasm material: National Sweet Potato Germplasm Bank (Xuzhou);

[0028] pCAMBIA1300 expression vector (containing dsred reporter gene): provided by the Plant Variety Testing Technology Laboratory of Xuzhou Agricultural Science Research Institute, Xuhuai District, Jiangsu Province.

[0029] pHSE401 gene editing vector and pCBC-DT1T2 plasmid: donated by Professor Chen Qijun's team from China Agricultural University.

[0030] Example 1

[0031] 1. Vector Construction

[0032] pCAMBIA1300 containing the dsred reporter gene was used as an expression vector and transferred into Trans1-T1 Escherichia coli (Shanghai Weidi Biotechnology Co., Ltd.) for cultivation to obtain positive single clones. After extracting the plasmid, it was transferred into K599 Agrobacterium rhizogenes (Shanghai Weidi Biotechnology Co., Ltd.).

[0033] 2. Establishment of transformation system

[0034] 1. Take the seeds of diploid I. cordatotriloba germplasm material, cut the seeds (use the corner of nail clippers to cut 1mm 2 After 10 days, when the seeds germinate and the seedlings grow to 4-6 true leaves, cut the stem segments above the roots (such as Figure 1 The wound cut surface area is as large as possible (as shown in A in Figure 1 (as shown in B in the figure).

[0035] 2. Activate the Agrobacterium rhizogenes containing the exogenous plasmid and shake until the bacterial solution OD 600 =0.8 for later use. Take 100 μL of bacterial solution and apply it on TY solid medium (Tryptone 5g / L, Yeastextract 3g / L, agar 15g / L, add 10mL of sterile 1M CaCl per liter of sterilized TY medium) 2 , 1 mL 50 mg / μL Kana and 0.5 mL 50 mg / μL STR) and cultured in the dark at 28°C for 1 day.

[0036] 3. Soak the cut stem segments in the bacterial solution in the dark for 2 hours (such as Figure 1 as shown in C in the figure).

[0037] 4. Take out the soaked stem segment and apply the root-inducing Agrobacterium on TY solid medium to the bottom cut surface of the stem segment (such as Figure 1 ).

[0038] 5. Mix vermiculite, coconut coir and perlite in a ratio of 6:3:1, place it in a large flowerpot of 80cm×30cm, plant the stem segments treated with bacterial solution, and water as needed.

[0039] After 100 days, when the tubers have expanded, dig out the tubers (such as Figure 2 ), and detected the dsred fluorescent marker, and found that the tuberous roots glowed red (as shown in B). Figure 2 ), place the tuberous roots that emit red light under green light in a flowerpot and wait for them to grow (as shown in C in the figure). Figure 2 It was observed that the seedlings showed red fluorescence under green light excitation (as shown in D Figure 2 ).

[0040] The transgenic seeds were obtained by planting the seedlings that emit red light under green light excitation. The transgenic seeds (dsred) and wild-type seeds (WT) were compared. Under green light excitation, the transgenic seeds emitted red fluorescence (e.g. Figure 3 As shown in Figure 2). After sowing wild-type seeds and transgenic seeds, DNA from young leaves was extracted, dsred fragments were amplified by PCR, and gel experiments were performed to verify. Figure 3 As shown in C, the wild type has no target band, and the transgenic has a target band in the range of 500-750bp.

[0041] Example 2

[0042] Determination of transgenic efficiency

[0043] Four groups of transformation experiments were carried out using the above method, and the positive rate was the number of positive enlarged roots divided by the number of stem segments used for transformation.

[0044] Experimental results: As shown in Table 1, the transgenic efficiency of the four transformation experiments was greater than 80%, and the highest transformation efficiency could reach 95.7%.

[0045] Table 1 Transgenic efficiency

[0046]

[0047] Example 3

[0048] Validation of transgenic methods

[0049] The MYB1 gene is an anthocyanin pathway gene. Overexpression of this gene can produce purple roots. To verify the effectiveness of this transformation system, an overexpression vector containing the target gene MYB1 was constructed. The pCAMBIA1300 vector was used as the backbone, and BamHI and KpnI were used as restriction sites for restriction digestion. The MYB1 gene with BamHI and KpnI connectors and the restriction digestion product were connected using a ligase to obtain the IbMYB1 vector. The vector was transferred into Trans1-T1 Escherichia coli for culture, and positive single clones were obtained. Single clones were selected for bacterial liquid PCR and gel experiments were performed to verify the vector. The PCR system is shown in Table 2, and the PCR program is shown in Table 3.

[0050] Table 2 PCR system

[0051]

[0052] Table 3 PCR program

[0053]

[0054]

[0055] Experimental results: PCR identification results are as follows Figure 4 The gel image showed the presence of vector and MYB1 bands, indicating that the vector was successfully constructed.

[0056] After the vector was successfully constructed, it was transferred into K599 rhizogenes Agrobacterium, and the transformation experiment was carried out according to the operation of Example 1, and roots that glowed red under green light excitation and had purple skin were obtained (such as Figure 5 shown).

[0057] Example 4

[0058] Validation of gene editing

[0059] Loss of plant olefin desaturase (PDS) function can lead to albino leaf phenotypes in plants. In order to determine whether this transformation system can edit the genes of I. cordatotriloba, we selected PDS as the target and constructed a CRISPR-Cas9 dual-target gene editing vector. Targets were designed for the two sites of the PDS (Icor_Chr13.00828) gene in I. cordatotriloba (as shown in Table 4), and the targets were integrated into the gRNA framework of plasmid pCBC-DT1T2. The gRNA-Sc of plasmid pCBC-DT1T2 was connected to the U6 promoter so that the U6 promoter drives the target site. The gRNA expression frame was connected to the pHSE401 plasmid by enzyme-linked sequencing to form the final CRISPR / Cas9 expression vector. After the vector was successfully constructed, it was transferred into K599 rhizogenes Agrobacterium, and the transformation experiment was carried out according to the operation of Example 1. Six transgenic positive roots were obtained from 10 infected explants, of which 5 produced transgenic positive buds and 2 produced albino buds (such as Figure 6 shown).

[0060] Table 4 Target sequences

[0061]

[0062] It can be seen from the above examples that the transgenic method of the present invention is simple to operate, and transgenic seedlings can be obtained within 120 days after transformation, and transgenic seeds can be obtained within 180 days. After transformation, transgenic plants can be obtained more quickly, and the transgenic efficiency is greatly improved.

[0063] 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 method for genetically modifying a model diploid sweet potato relative, characterized in that: The following steps are involved: When the model diploid sweet potato related species seedlings grow to 4-6 true leaves, the stem segments above the roots are cut, and the cut stem segments are immersed in a bacterial solution of Agrobacterium rhizogenes containing exogenous plasmids for 1-3 hours in the dark, and then the root-generating Agrobacterium rhizogenes containing exogenous plasmids are applied to the cut surface at the bottom of the stem segments before planting.

2. The operating method according to claim 1, characterized in that: The Agrobacterium rhizogenes containing the exogenous plasmid is obtained by transferring the exogenous plasmid into the Agrobacterium rhizogenes.

3. The operating method according to claim 2, characterized in that: The exogenous plasmid is pCAMBIA1300 plasmid.

4. The operating method according to claim 2, characterized in that: The Agrobacterium rhizogenes is K599.

5. The operating method according to claim 1, characterized in that: The OD of the bacterial solution of Agrobacterium rhizogenes containing the exogenous plasmid 600 It is 0.6~1.

6. The operating method according to claim 1, characterized in that: The Agrobacterium rhizogenes containing the exogenous plasmid is cultured on a TY solid culture medium in the dark at 20-30° C. for 18-30 hours before being applied.

7. The operating method according to claim 1, characterized in that: The planting matrix consists of vermiculite, coconut bran and perlite, and the mass ratio of the vermiculite, coconut bran and perlite is 4-8:2-5:0.5-1.

5.

8. The operating method according to claim 1, characterized in that: The sweet potato inbred diploid transgenic plants are obtained 110 to 130 days after planting, and the sweet potato inbred diploid transgenic seeds are obtained 170 to 190 days after planting.

Images