Method for creating cytoplasmic male sterile line by alfalfa transgenic technology

Abstract

The invention discloses a method for creating a cytoplasmic male sterile line by an alfalfa transgenic technology, and belongs to the technical field of bioengineering. The method comprises the following steps: S1, constructing pBI121-ATP8 of a recombinant expression vector; s2, constructing transgenic medicago sativa plants and obtaining medicago sativa male sterility germplasm resources; s3, breeding the medicago sativa transgenic cytoplasmic male sterile line. According to the invention, a brand-new gene, namely the alfalfa mitochondrial gene ATP8, is successfully cloned, and directional variation of male reproductive development of alfalfa is realized by regulating and controlling the gene and an encoding protein thereof, so that the pollen fertility of the alfalfa is accurately controlled; meanwhile, the invention further provides a maintainer line, the breeding efficiency is improved due to the existence of the maintainer line, and the sustainability of the hybrid seed production process is guaranteed. In perennial crops such as medicago sativa, long-term storage and utilization of the sterile line are facilitated.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, in particular to a method for creating a cytoplasmic male sterile line by using transgenic technology of alfalfa. Background Art

[0002] Alfalfa (Medicago sativa L.) is an important forage resource cultivated worldwide. With excellent feed value and adaptability, it is the forage of choice for the development of the livestock industry. As a typical monoecious, cross-pollinated plant, alfalfa exhibits self-incompatibility and is typically produced through hybridization. This plant exhibits significant hybrid vigor in hybrid seed production, primarily through artificial emasculation and hybridization using nuclear-cytoplasmic male sterile lines. The latter has become a research hotspot due to its ability to significantly reduce emasculation labor, lower seed production costs, and efficiently cultivate high-quality hybrid varieties.

[0003] With the continued growth in global demand for high-quality forage, revealing the molecular mechanism of alfalfa male sterility and utilizing its hybrid vigor to cultivate high-quality hybrids is of great significance for expanding the alfalfa industry and improving the efficiency of the forage-livestock industry. Although male sterility is detrimental to the natural reproduction of plants, as one of the survival strategies evolved in nature, it has become an important tool for genetic improvement. In alfalfa breeding, the use of male sterility traits for genetic improvement has received widespread attention. Pollen development is an extremely complex stage in the reproductive process of higher plants, involving the expression and regulation of a large number of genes. As the male gametophyte, pollen plays a key role in completing the fertilization process, and abnormalities in any stage of its development may lead to pollen abortion, exhibiting the characteristics of male sterility.

[0004] The occurrence of male sterility in alfalfa is spatiotemporal, and research into its associated genes and regulatory networks is crucial for uncovering its complex molecular mechanisms and for the application of male sterile lines in breeding practices. These findings will provide a theoretical basis for utilizing male sterile lines and heterosis in alfalfa to cultivate high-quality hybrids, thereby promoting its industrial development. However, as a tetraploid plant, alfalfa has a complex genome and a long growth cycle. Furthermore, the selection of high-quality male sterile lines and supporting maintainer lines is difficult, leading to numerous challenges in hybrid breeding and the utilization of heterosis. Future research should focus on core issues such as the genetic basis and inheritance patterns of fertility to further overcome these technical bottlenecks and accelerate the development of high-quality alfalfa hybrids. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for creating a cytoplasmic male sterile line by using transgenic technology of alfalfa, utilizing the characteristics of mitochondrial gene ATP8 and its protein in regulating pollen abortion of alfalfa, and controlling the male reproductive development of alfalfa by using transgenic technology, producing new alfalfa male sterile strains by inhibiting the protein, and restoring the male sterility of alfalfa by overexpressing the protein.

[0006] To achieve the above object, the present invention provides a method for creating a cytoplasmic male sterile line of alfalfa using transgenic technology, characterized in that it comprises the following steps:

[0007] S1. Construction of recombinant expression vector pBI121-ATP8

[0008] XbaI and SacI restriction sites were added to both ends of the full-length sequence of the mitochondrial gene ATP8 cloned from alfalfa, and the recombinant expression vector pBI121-ATP8 was constructed.

[0009] The nucleotide sequence of the mitochondrial gene ATP8 is shown in SEQ ID NO.1;

[0010] S2. Construction of transgenic alfalfa plants and acquisition of male sterile alfalfa germplasm resources

[0011] The recombinant expression vector pBI121-ATP8 was introduced into Gongnong No. 5 alfalfa using Agrobacterium-mediated genetic transformation to obtain transgenic plants. The transgenic plants were screened and identified using molecular biology techniques, and their fertility was examined during the flowering period. The male-sterile lines obtained from the screening were used as cytoplasmic male-sterile alfalfa germplasm resources.

[0012] S3. Breeding of transgenic cytoplasmic male sterile lines in alfalfa

[0013] (1) Breeding method of transgenic cytoplasmic homologous male sterile lines:

[0014] The transgenic male sterile germplasm resource in S2 is used as the female parent and backcrossed with the homologous maintainer line. When mature, the T1 generation seeds are harvested and sown, and the fertility performance of the T2 generation plants is observed. If the T2 generation plants still show male sterility characteristics, it indicates that the male sterility trait is determined by cytoplasmic inheritance. Subsequently, nuclear replacement saturation backcrossing is continued with the original backcross parent, and the male sterile line finally obtained is the cytoplasmic homologous cytoplasmic male sterile line;

[0015] (2) Breeding of transgenic cytoplasmic heterologous cytoplasmic male sterile lines: using the transgenic male sterile germplasm described in S3 or the male sterile plants of any generation in the breeding process of the above-mentioned transgenic cytoplasmic homologous cytoplasmic male sterile lines as the female parent, hybridizing with the heterologous maintainer line or conventional variety or line, and performing nuclear replacement backcrossing with the recurrent parent, the male sterile line thus bred is the cytoplasmic heterologous cytoplasmic male sterile line, and the recurrent parent is the maintainer line of the sterile line;

[0016] Preferably, the step S2 further comprises the following steps:

[0017] S2.1. Select the recipient material: Select vigorous, pest-free, and disease-free Gongnong No. 5 alfalfa plants;

[0018] S2.2 Plasmid Transformation: Agrobacterium-mediated genetic transformation was used to introduce Agrobacterium carrying the pBI121-ATP8 overexpression vector into pre-cultured alfalfa leaf tissue. After co-cultivation, screening, and regeneration, transgenic positive plants were obtained.

[0019] S2.3. Screening of male sterile lines: Transplant the positive transgenic alfalfa plants obtained from the above screening into the experimental field. After the plants enter the flowering period, systematically observe their anther development to screen out male sterile alfalfa lines.

[0020] Preferably, the maintainer line in step S3 is the alfalfa male sterile maintainer line MSJN-1B.

[0021] Beneficial effects of the present invention

[0022] The present invention successfully cloned a new gene - the alfalfa mitochondrial gene ATP8, and by regulating this gene and its encoded protein, achieved directed variation in the male reproductive development of alfalfa, thereby precisely controlling its pollen fertility. The male sterile alfalfa strain created had no significant differences in yield and quality traits from its source parent during the vegetative period, but after entering the reproductive growth stage, abnormal male reproductive development occurred, manifested as pollen abortion. In addition, the present invention also provides a maintainer line, which is mainly used to maintain the genetic stability of the sterile line in three-line hybridization and is the key to the continued use of the sterile line. It has the same genetic background as the sterile line, but has fertile cytoplasm, ensuring that sterility can be stably inherited. The existence of the maintainer line improves breeding efficiency and ensures the sustainability of the hybrid seed production process. In perennial crops such as alfalfa, it is more conducive to the long-term preservation and utilization of the sterile line. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the construction of the mitochondrial gene pBI121-ATP8 overexpression vector in Example 2 provided by the present invention;

[0024] Figure 2 Paraffin sections of Gongnong 5 and MSJN-1A provided by the present invention;

[0025] Figure 3 Schematic diagram of the inflorescence and anther phenotypes of Gongnong 5 and MSJN-1A provided by the present invention;

[0026] Figure 4 Schematic diagram of TTC staining of Gongnong 5, MSJN-1A and MSJN-2A pollen provided by the present invention. DETAILED DESCRIPTION

[0027] The present invention is further described in detail below by specific examples, which are only used to explain the technical solutions of the present invention, rather than to limit the scope of the present invention. In the following examples, if the experimental conditions are not particularly noted, they are all carried out according to conventional experimental conditions.

[0028] Example 1: Method for Creating Male Sterile Alfalfa Lines

[0029] 1.1 Cloning of the mitochondrial gene ATP8 controlling fertility in alfalfa

[0030] Using the wild-type alfalfa Gongnong 5 material, specific primers mitochondrial gene ATP8-F / R were designed according to the full-length sequence of mitochondrial gene ATP8:

[0031] Mitochondrial gene ATP8-F: 5′-AATAAGTGGCTCACGAGGAATGG-3′ and mitochondrial gene ATP8-R: 5′-CACCCATCCAGAATTTGAAGAAGAAG-3′

[0032] The steps were to extract total RNA from anthers, synthesize cDNA and amplify the full-length cDNA of mitochondrial gene ATP8 by PCR.

[0033] Sequencing identified the cDNA sequence as 486 bp in length, as shown in the nucleotide sequence of SEQ ID NO. 1. BLAST comparison showed that the gene belongs to the P-type ATPase family, a family of proteins that rely on ATP hydrolysis to drive transmembrane transport of ions or molecules.

[0034] 1.2 Overexpression of the mitochondrial gene ATP8 in alfalfa renders wild-type plants male sterile

[0035] The pBI121-ATP8 overexpression vector was constructed and transformed into the wild type Gongnong 5 alfalfa to obtain a sterile line. The recombinant vector construction map is shown in Figure 1 As shown, the vector comprises the nucleotide sequence shown in SEQ ID NO.1.

[0036] The specific method for constructing the pBI121-ATP8 overexpression vector is as follows:

[0037] XbaI and SacI restriction sites were added to both ends of the full-length mitochondrial gene ATP8, and specific primers mitochondrial gene ATP8-F-pBI / mitochondrial gene ATP8-R-pBI were designed and PCR amplified using high-fidelity enzymes.

[0038] Mitochondrial gene ATP8-F-pBI: 5'-GGAAGGTGGAACATGGATTG-3' and

[0039] Mitochondrial gene ATP8-R-pBI: 5'-TACGCCACGCCACAACTCTA-3'

[0040] The pBI-121 expression vector was digested with the same double enzyme at 37°C for 3 hours, and the target fragment was ligated with the linear vector after GUS removal. The cells were transformed into competent E. coli cells and cultured on LB / Kan solid medium. Single clones were picked for PCR detection and sequencing using vector primers M13-F / R.

[0041] M13-F: 5'-TGGGCTGTTGCTCCGGATTT-3' and

[0042] M13-R: 5'-AATCACCAGGTTGGAAGAGC-3'

[0043] The bacterial solution was propagated in LB / Kan liquid culture medium. After sequencing was correct, the pRNAi-ATP8 recombinant vector plasmid was transformed into Agrobacterium competent cells LBA4404. The resuspended bacteria were evenly spread on YEP / AMP solid medium (containing 20 mg / Lrif) and inverted and cultured at 28°C for 48 hours. Pick a single colony to the corresponding liquid culture medium to propagate the bacterial solution. The next day, 100 μL of the bacterial solution was aspirated and transferred to YEP liquid culture medium (without any antibiotics) at a ratio of 1 / 50 for propagation and activation. The bacteria were collected by centrifugation at 4000xg for 10 minutes, and the bacteria were resuspended in MS liquid culture medium (4.43 g / L MS + 30 g / L sucrose) to adjust the bacterial solution OD 600 When the value reaches 0.6-0.8, it can be used for genetic transformation of alfalfa.

[0044] Select the sterile seedlings of Gongnong No. 5 alfalfa that are growing vigorously and free of pests and diseases, and cut the leaves into 1cm thick wounds on all four sides. 2 Small pieces were used as explants and infected in Agrobacterium resuspension by shaking for 15 minutes. After no residual bacterial liquid was left, 9 pieces / dish were arranged on MS solid medium (MS519) with the upper surface of the leaves facing down.

[0045] 4.43 g / L + sucrose 30 g / L + agar powder 6.8 g / L) and co-cultured at 28°C in the dark for 3 days.

[0046] After co-cultivation, the explants were rinsed with sterile water (containing 500 mg / L Cef) and transferred to MS1 medium (MS + 2,4-D 2 mg / L + KT 0.25 mg / L + Kan 25 mg / L + TCC 500 mg / L) to induce callus growth. When they grew to 1 cm or more, they were cut and inoculated into MS2 medium (MS + KT 1.5 mg / L + Kan 25 mg / L + TCC 500 mg / L) for further differentiation culture. After about 30 days, they were transferred to MS3 medium (1 / 2MS + Kan 25 mg / L + TCC 500 mg / L) was added to induce rooting. After a large number of adventitious roots grew, molecular testing was performed on the regenerated seedlings. Genomic DNA from the leaves was extracted for PCR positive identification. The expression level of the mitochondrial gene ATP8 in the positive seedlings was analyzed by RT-PCR. Plants with effective target gene overexpression were comprehensively selected as male sterile plants. The obtained male sterile plants were hardened and transplanted to the experimental field for cultivation.

[0047] The specific method of the molecular identification is as follows:

[0048] Gene-specific primers were used for transgenic detection, and the primer sequences were as follows:

[0049] RNAi-F: 5'-ATGCCTCAACTGGATAAATT-3'

[0050] RNAi-R: 5'-TTTATTTTTTATTCTTCCTT-3'

[0051] The reaction system (10 μl) consisted of 1 μl (200 ng) of gDNA, 1 μl of 10× PCR Buffer, 0.2 μl of dNTPs (10 mmol / L), 0.3 μl of primers (10 μmol / L), 0.1 μl of Taq enzyme (5 U / μl), and 7.4 μl of ddH2O. The reaction conditions were: initial denaturation at 94°C for 3 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing for 30 s, and extension at 72°C for 60 s, followed by extension at 72°C for 10 min. PCR products were checked by 1% agarose gel electrophoresis.

[0052] 1.3 Deletion of the mitochondrial gene ATP8 leads to abnormal pollen development in alfalfa

[0053] After the transplanted male sterile plants entered the flowering period, their anther development was systematically observed. Figure 2 、 3As shown in the figure, compared with the non-transgenic Gongnong 5 phenotype, the pollen grains of the male sterile plants after overexpression of the mitochondrial gene ATP8 are shriveled, small in number, small in size, and missing; Figure 4 The results of microscopic examination after TTC staining showed that the pollen of the male sterile line had abnormal tapetal cells in the late tetrad stage, which led to microspore development defects, and then the release of pollen grains was small or could not be normal, resulting in abortion of alfalfa pollen, and creating a new male sterile alfalfa line.

[0054] 1.4 Application of the above-described male sterile line in alfalfa seed production

[0055] The mitochondrial ATP8 sterile line was backcrossed with fertile plants of the same variety (Gongnong 5) that same season, and T1 seeds were harvested at maturity. Plants were then sown and the fertility of the T2 generation plants observed, revealing continued male sterility. This was therefore inferred to be cytoplasmic male sterile (CMS). This line was then backcrossed with the non-transgenic Gongnong 5 that same season, and this process was repeated for three generations. Because its cytoplasmic nuclei were all derived from Gongnong 5, the transgenic cytoplasmic homologous male sterile line was designated MSJN-1A. Its recurrent parent, Gongnong 5, served as the maintainer line, MSJN-1B (Gongnong 5 was purchased commercially).

[0056] In addition, while selecting the cytoplasmic nuclear homologous male sterile line, it was hybridized with multiple heterologous varieties at the same time. Among them, the offspring of WL168HQ were found to have the trait of maintaining sterility, and nuclear replacement backcrossing was performed to select the cytoplasmic nuclear heterologous male sterile MSJN-2A (WL168HQ). Its recurrent parent WL168HQ is its maintenance line MSJN-2A (WL168HQ was purchased from the market).

[0057] The target gene can be detected in the T1 generation of the male sterile line. However, due to the fact that the nuclei of the transgenic plants are basically replaced after three generations of backcrossing with the maintainer line, the target gene cannot be detected in the sterile lines of higher generations.

Claims

1. A method for creating a cytoplasmic male sterile line using transgenic alfalfa technology, characterized in that: The following steps are involved: S1. Construction of recombinant expression vector pBI121-ATP8 XbaI and SacI restriction sites were added to both ends of the full-length sequence of the mitochondrial gene ATP8 cloned from alfalfa, and the recombinant expression vector pBI121-ATP8 was constructed. The nucleotide sequence of the mitochondrial gene ATP8 is shown in SEQ ID NO.1; S2. Construction of transgenic alfalfa plants and acquisition of male sterile alfalfa germplasm resources The recombinant expression vector pBI121-ATP8 was introduced into Gongnong No. 5 alfalfa using Agrobacterium-mediated genetic transformation to obtain transgenic plants. The transgenic plants were screened and identified using molecular biology techniques, and their fertility was examined during the flowering period. The male sterile lines obtained by screening were used as cytoplasmic male sterile germplasm resources of alfalfa; S3. Breeding of transgenic cytoplasmic male sterile lines in alfalfa (1) Breeding method of transgenic cytoplasmic homologous male sterile lines: The transgenic male sterile germplasm resource in S2 is used as the female parent and backcrossed with the homologous maintainer line. When mature, the T1 generation seeds are harvested and sown, and the fertility performance of the T2 generation plants is observed. If the T2 generation plants still show male sterility characteristics, it indicates that the male sterility trait is determined by cytoplasmic inheritance. Subsequently, nuclear replacement saturation backcrossing is continued with the original recurrent parent, and the male sterile line finally obtained is the cytoplasmic homologous cytoplasmic male sterile line; its recurrent parent is the maintainer line of the sterile line. (2) Breeding of transgenic cytoplasmic heterologous cytoplasmic male sterile lines: Using the transgenic male sterile germplasm described in S2 or the male sterile plants of any generation in the breeding process of the above-mentioned transgenic cytoplasmic homologous cytoplasmic male sterile lines as the female parent, hybridizing with its heterologous maintainer line or conventional variety or line, and performing nuclear replacement backcrossing with the recurrent parent, the male sterile line thus bred is the cytoplasmic heterologous cytoplasmic male sterile line, and its recurrent parent is the maintainer line of the sterile line.

2. The method for creating a cytoplasmic male sterile line using transgenic alfalfa technology according to claim 1, characterized in that: The S2 further comprises the following steps: S2.

1. Select the recipient material: Select vigorous, pest-free, and disease-free Gongnong No. 5 alfalfa plants; S2.2 Plasmid Transformation: Agrobacterium-mediated genetic transformation was used to introduce Agrobacterium carrying the pBI121-ATP8 overexpression vector into pre-cultured alfalfa leaf tissue. After co-cultivation, screening, and regeneration, transgenic positive plants were obtained. S2.

3. Screening of male sterile lines: Transplant the positive transgenic alfalfa plants obtained through the positive detection screening into the experimental field. After the plants enter the flowering period, systematically observe their anther development to screen out male sterile alfalfa lines.

3. The method for creating a cytoplasmic male sterile line using transgenic alfalfa technology according to claim 1, characterized in that: The homologous maintainer line in step S3 is the alfalfa male sterile maintainer line MSJN-1B.

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