CN116694660A - ShN/AINV3.1 gene for regulating sugarcane germination rate and application thereof - Google Patents

Abstract

The invention discloses a ShN/AINV3.1 gene regulating the germination rate of sugarcane and its application. The nucleotide sequence of the sugarcane ShN/AINV3.1 gene is shown in SEQ ID NO.1, and the amino acid sequence of its encoded protein is shown in SEQ ID NO.2. The research of the present invention finds that the overexpression of the ShN/AINV3.1 gene can significantly increase the germination rate of sugarcane, increase the germination length of sugarcane at the same time, and accelerate the germination rate of sugarcane. The invention provides technical support for improving sugarcane varieties and promoting the development of sugarcane industry.

Description

A ShN/AINV3.1 gene regulating the germination rate of sugarcane and its application

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a ShN/AINV3.1 gene regulating sugarcane germination rate and application thereof.

Background technique

Sugarcane (Saccharum spp.) is the most important sugar crop in my country and the world, and is the main raw material for sugar production in my country. About 90% of the country's total sugar production comes from sugarcane.

In plants, sucrose invertase (EC 3.2.1.26, INV), which catalyzes the irreversible hydrolysis of sucrose to glucose and fructose, is a key enzyme in regulating sucrose metabolism. In addition, sucrose invertases have also been shown to contribute to many aspects of plant growth and development, organ formation, sugar transport, stress response, carbon allocation, phloem unloading and source/sink regulation, and regulation of sugar composition and levels in sink tissues.

Sucrose invertase is divided into acid invertase sub-family (acid INV sub-family) and neutral/alkaline invertase sub-family (neutral/alkaline INV sub-family, namely N/AINV) according to different pH values. Among them, acid invertase can be subdivided into cell wall invertase (CWINV) and vacuole invertase (vacuole invertase, VINV; also known as soluble acid invertases, SAINV), and neutral/alkaline Sexual invertases are classified as cytoplasmic invertases.

The expression of AtINV gene in Arabidopsis is related to the growth and development of flower organs and seeds; the increase of Arabidopsis cell wall invertase activity will accelerate flowering and increase seed yield by nearly 30%. Suppression of N/AINV gene expression in tobacco leads to a block in pollen development and leads to male sterility. Inhibition of INVINH1 in tomato increased INV activity, delayed leaf senescence and improved seed and fruit yield.

Studies have shown that GhVIN1-mediated hexose signaling may act as an early event to control the expression of regulatory genes, thereby regulating the differentiation of ovule epidermal cells; GhVIN1 silencing also inhibits a group of regulatory genes through sugar signaling to prevent the germination of cotton fibers from the ovule epidermis. Using RNAi technology to inhibit the target gene GhVIN1 of cotton, the plants have impaired male and female fertilization ability, and the reduced expression of GhVIN1 in the seed coat is the main reason for the reduced fertility of female flowers. Silencing tomato SiN/AINV7 gene will increase pollen ROS content, reduce pollen activity and form parthenogenetic fruit. Mutation of rice OsVIN2 leads to smaller grains of the mutant, and OsVIN2 affects sucrose metabolism, thereby regulating grain size. The sorghum SbVIN1 gene was expressed at an early stage of seed development and reached its highest level 29 days after pollination.

Contents of the invention

The purpose of the present invention is to deeply explore the gene function of ShN/AINV3.1 gene in sugarcane seed germination, in order to have important guiding significance for cultivating sugarcane varieties with high seed germination rate by modern biotechnology.

The present invention provides a ShN/AINV3.1 gene that regulates the germination rate of sugarcane, its nucleotide sequence is shown in SEQID NO.1, specifically:

ATGAAGCGGGTGTCGTCGCACGTCTCGCTGGCCTCGGAGGCGGAGATCAATCTCGATCTGTCGCGC

CTCATCATCGACAGGCCGCAGCGGTTCACGCTGGAGCGGAAGCGCTCCTTCGACGAGCAGTCGTG

GAGCGAGCTCTCGCACTCCCACTCCCACCGCAACAACGACGGCTTCGACAGCGTGCTGCAGTCGC

CCGCATTCCCGTCCGGCGGATTCGACTCGCCTTTCTCCATCGGCACGCATTTCGGCGGGGGCGGCCC

GCACCCGCTGGTCAACGAGGCGTGGGAGGCGCTCAGGAAATCCGTCGTCTACTTCCGGGAACAGC

CCGTCGGTACCGTCGCTGCCGTGGATCATGCGTCCGAGGAAGTGTCCAACTATGATCAGGTCTTTGTGT

GAGGGATTTTGTTCCGAGTGCATTGGCTTTTCTGATGAACAATGAGACTGACATAGTGAAGAATTTT

CTCTTGAAAACTCTTCACCTTCAGAGCTCTGAGAAAATGGTAGACCGGTTCAAGCTTGGAGCAGGA

GCGATGCCTGCCAGTTTCAAGGTGGACCGTAACAAAAACAGAAACACTGAAACCTTAGTTGCTGAT

TTTGGTGAGAGTGCAATCGGCAGGGTGGCACCGGTTGACTCTGGATTTTGGTGGATCATTCTCCTTC

GGGCGTATACAAAGTACACCGGAGATGTTAGTTTGTCGGAATCACCTGATTGCCAGAAGTGCATGA

GGTTGATACTGAATCTCTGCTTATCTGAAGGATTTGATACTTTTCCAACTCTGCTTTGCACAGATGGC

TGCTCAATGATTGATCGTCGAATGGGTATATATGGTTATCCCATTGAGATCCAAGCCCTATTCTATATG

GCATTAAGATGTGCTCTCCAAATGCTCAAAGCCAGAGGGCGAAGGGAAGGATTTCATAGAGAAGATA

GGGCAACGGCTACATGCACTAACCTACCACATGAGGAACTACTTCTGGCTAGATTTTCACCAGCTGA

ATAACATATACAGATACAAAACAGAAGAGTATTCCCACACAGCTGTGAACAAGTTTAACGTCATTCC

GGATTCCATTCCTGATTGGGTGTTTGATTTCATGCCATGCCGAGGAGGCTACTTCCTTGGCAATGTCA

GCCCTGCTATGATGGATTTCCGGTGGTTTGCCCTTGGCAATTGCATTGCCATTGTATCATTCTCTAGCTA

CCCCAGAACAGTCAGTTGCTATAATGGATCTGATTGAGGAAAAGTGGGATGAGCTCGTTGGTGAGA

TGCCTCTGAAGATATGCTATCCTGCTCTCGAGAATCATGAGTGGAGAATTATCACTGGCTGTGACCC

CAAGAACACCCGGTGGAGTTACCACAATGGAGGATCGTGGCCAGTTCTTCTGTGGCTGCTGACAGC

AGCCTGCATCAAGACTGGTAGGCCACAGATGGCAAAACGTGCCATTGAGCTCGCTGAGTCGAGGCT

GCTCAAGGACGGCTGGCCGGAGTACTACGATGGCAAGCTAGGAAGATTCGTTGGTAAGCAGGCCA

GGAAGTTCCAAACCTGGTCCATTGCAGGTTACCTCGTCGCCCGCATGATGCTGGAGGACCCATCAA

CACTGATGATGATCTCCATGGAGGAGGACCGGCCTGTGAAGCCGACTATGCGGCGGTCAGCATCATGGAATGCCTGA.

The present invention also provides a protein encoded by the sugarcane ShN/AINV3.1 gene, the amino acid sequence of the protein is shown in SEQ ID NO.2, specifically:

MKRVSSHVSLASEAEINLDLSRLIIDRPQRFTLERKRSFDEQSWSELHSHSHSHRNNDGFDSVLQSPAFPS

GGFDSPFSIGTHFGGGGPHPLVNEAWEALRKSVVYFREQPVGTVAAVDHASEEVLNYDQVFVRDFVP

SALAFLMNNETDIVKNFLLKTLHLQSSEKMVDRFKLGAGAMPASFKVDRNKNRNTETTLVADFGESAI

GRVAPVDSGFWWIILLRAYTKYTGDVSLSESPDCQKCMRLILNLCLSEGFDTFPTLLCTDGCSMIDRRM

GIYGYPIEIQALFYMALRCALQMLKPEGEGKDFIEKIGQRLHALTYHMRNYFWLDFHQLNNIYRYKTE

EYSHTAVNKFNVIPDSIPDWVFDFMPCRGGYFLGNVSPAMMDFRWFALGNCIAIVSSLATPEQSVAIM

DLIEEKWDELVGEMPLKICYPALENHEWRIITGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQ

MAKRAIELAESRLLKDGWPEYYDGKLGRFVGKQARKFQTWSIAGYLVARMMLEDPSTLMMISMEEDRPVKPTMRRSASWNA*.

The protein encoded by the ShN/AINV3.1 gene is located in the Golgi body of the cell, and the Golgi body is a protein processing site, and is also related to the formation of plant cell walls, and can enhance the germination rate of sugarcane stems. The ShN/AINV3.1 gene and its biological material can be used to improve sugarcane germplasm resources and increase the germination rate of sugarcane seeds.

The invention also provides a biological material containing the sugarcane ShN/AINV3.1 gene. The biological material is a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

The recombinant expression vector can be constructed with existing plant expression vectors, such as binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. When using the ShN/AINV3.1 gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other In addition, when using the ShN/AINV3.1 gene to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG initiation codons or adjacent to The region start codon, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

The recombinant expression vector carrying the ShN/AINV3.1 gene can be transformed into plant cells or organization.

Said expression cassette especially refers to the expression cassette containing said sugarcane ShN/AINV3.1 gene.

The transgenic cell line especially refers to an artificially constructed transgenic cell line stably expressing the sugarcane ShN/AINV3.1 gene, or the protein encoded by the sugarcane ShN/AINV3.1 gene.

The recombinant bacterium especially refers to Escherichia coli or Agrobacterium constructed with ShN/AINV3.1 gene.

The present invention also provides a primer for amplifying the sugarcane ShN/AINV3.1 gene, the primer sequence is shown in SEQ ID NO.3-4, specifically:

F: 5'-CGA GGATCC ATGAAGCGGGTGTCGTCGCA-3'; (the underline is the restriction site of BamHI)

R: 5'-CGA AGGCCT GGCATTCCATGATGCTGACCG-3' (the underline is the Stul restriction site).

Further, the present invention also protects the application of the sugarcane ShN/AINV3.1 gene, the protein encoded by the sugarcane ShN/AINV3.1 gene or the biological material containing the gene in regulating the germination of sugarcane seeds and cane stems. The biological material refers to a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the sugarcane ShN/AINV3.1 gene.

Further, the present invention also protects the application of the sugarcane ShN/AINV3.1 gene, the protein encoded by the sugarcane ShN/AINV3.1 gene or the biological material containing the gene in the improvement of sugarcane germplasm resources. The biological material refers to a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the sugarcane ShN/AINV3.1 gene.

Further, the present invention also protects the application of the sugarcane ShN/AINV3.1 gene, the protein encoded by the sugarcane ShN/AINV3.1 gene or the biological material containing the gene in sugarcane breeding, the purpose of the breeding To breed sugarcane varieties with high germination rate. The biological material refers to a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium containing the sugarcane ShN/AINV3.1 gene.

The invention carries out the functional analysis of the sugarcane ShN/AINV3.1 gene through genetic transformation. Using Agrobacterium EHA105, the unique gene ShN/AINV3.1 in sugarcane was transferred into sugarcane for gene function verification. Germinate long, accelerate the germination speed of sugarcane. The research of the invention has good significance and effect on improving sugarcane varieties and promoting the development of sugarcane industry.

Description of drawings

Figure 1 is the sugarcane ShN/AINV3.1 gene clone.

Fig. 2 is PCR identification of transgenic sugarcane ShN/AINV3.1 gene.

Figure 3 is the PCR identification of Bar gene in transgenic sugarcane.

Fig. 4 is the protein identification of transgenic sugarcane ShN/AINV3.1.

Figure 5 is a long picture of sugarcane germination.

Figure 6 is the data analysis of sugarcane germination and sprout length.

Detailed ways

The following examples are to further illustrate the present invention, rather than limit the present invention.

Unless otherwise specified, the culture media in the examples are common commercially available products.

Example 1

1. CDS sequence of sugarcane ShN/AINV3.1 gene

The CDS sequence of the sugarcane ShN/AINV3.1 gene was cloned by homologous methods, and the forward primer (5'-CGAGGATCCATGAAGCGGGTGTCGTCGCA-3') and the reverse primer (5'-CGAAGGCCTGGCATTCCATGATGCTGACCG-3') were designed according to the rice OsN/AINV3 sequence ), using sugarcane cDNA as a template to amplify the target gene ShN/AINV3.1, the reagents used are Max DNA Polymerase (TaKaRa Code No. R045A). The PCR amplification reaction system and reaction conditions are shown in Table 1.

Table 1

Take 5 μL of the PCR product for 1% agarose gel electrophoresis. The result is shown in Figure 1. The result shows that the amplified product has a single band and a length of about 1674bp, which is consistent with the length of the target product, indicating that the PCR amplification was successful and the target length was obtained. of PCR products. The PCR product is sequenced to obtain the cDNA sequence of the sugarcane ShN/AINV3.1 gene, that is, the nucleotide sequence of the ShN/AINV3.1 gene of the present invention is shown in SEQ ID NO.1.

2. Amino acid sequence encoded by sugarcane ShN/AINV3.1 gene

According to the full-length cDNA sequence of the sugarcane ShN/AINV3.1 gene, the Snapgene software is used to convert it into an amino acid sequence. After determination, a total of 557 amino acids are encoded, which is the encoded protein of the ShN/AINV3.1 gene described in the present invention. The amino acid sequence of is shown in SEQ ID NO.2.

3. Construction of expression vector

The PCR product was subjected to electrophoresis using 1% agarose gel electrophoresis, and the gel recovery kit was used to cut and recover the gel, and the expression vector pCB302 (preserved in this laboratory, cited in the literature "Nannan Zhang, et al., Engineering Artificial MicroRNAs for Multiplex Gene Silencing and Simplified TransgenicScreen, Plant Physiology, Volume 178, Issue 3, November 2018, Pages 989-1001, https://doi.org/10.1104/pp.18.00828") carried out double enzyme digestion at the same time with no load, and then carried out vector ligation.

Enzyme digestion ligation reaction system (20 μL): Add 2 μL of 10x T4 DNA LigaseBuffer, 100 ng of the digested vector fragment, 100 ng of the target fragment, and 1 μL of T4 DNA Ligase in a centrifuge tube.

After ligation at 16°C for 30 min, 10 μL of the ligation product was taken to transform Escherichia coli DH5α competent cells, after transformation, spread on kanamycin-resistant LB solid medium, and culture at 37°C for 12 hours, and carry out bacterial liquid PCR identification. Pick 10 single colonies for PCR identification. The identification primers are as follows:

Forward primer 35S PPDK-F (5'-GTCACGTAGTAAGCAGCTCTCGG-3') and target gene reverse primer (5'-ATAACCATATATACCCATTCGACGATC-3').

The PCR amplification reaction system and reaction conditions are shown in Table 2.

Table 2

Take 5 μL of the above PCR product and detect the target band with a fragment of about 810 bp in 1% agarose gel electrophoresis. Take 100 μL of the bacterial liquid corresponding to 3-5 positive bands for sequencing, save the sequenced bacterial liquid for plasmid extraction, and transfect the plasmid into Agrobacterium EHA105 for genetic transformation.

4. Genetic transformation of sugarcane

(1) Select vigorously growing ROC22 plants from the field, put the young heart leaves of 5-7cm above the growth point in MY medium (MS+2mg/L 2,4-D+30g/L sucrose+7.0g/L agar powder), the induced callus was subcultured on the MY medium every 20 days, and after 2-3 subcultures, the vigorously growing callus was selected and prepared for the Agrobacterium transformation test.

(2) The single clone of the Agrobacterium strain EHA105 carrying the pCB302 plasmid was shaken in 25 mL liquid YEP medium containing 50 mg/L kanamycin and 50 mg/L rifampicin at 28 ° C until OD600 = 0.5, 5000 r/ The cells were collected by centrifugation for 5 min, and the cells were resuspended with an equal volume of liquid MS medium containing 150 μmol/L acetosyringone (AS), and shaken at 200 r/min for 2 h. The pretreated callus cells were placed in the bacterial solution for co-cultivation for 20 minutes, and then the bacterial solution was aspirated, and the infected cells were blotted dry on sterile filter paper and placed in a solution containing 2mg/L 2,4-D and 150μmol/ L AS was co-cultured on solid MS medium for 3 days, and the culture conditions were 28°C and dark.

The co-cultured embryogenic cell mass was washed with liquid MS medium containing 500 mg/L carbenicillin (Carb) and 2 mg/L 2,4-D, and then selected on MSC medium supplemented with 500 mg/L Carb nourish. Every 2 weeks, the resistant callus was selected for subculture, and after 4 weeks of selection and culture, the obtained resistant callus was transferred to the MD medium containing 30 mg/L glufosinate-ammonium and 500 mg/L Carb for differentiation. Induces the formation and germination of somatic embryos. When the resistant seedlings to be differentiated grow to about 2cm, they are transferred to root-promoting medium MR (1/2MS+50g/L sucrose+2.0mg/L NAA) containing 30mg/L glufosinate-ammonium and 500mg/L Carb. Promote root cultivation. When the roots of the seedlings grow to 5m long, move them outside for 2-3 days to harden the seedlings, then take out the seedlings, wash them and place them in the nutrient matrix.

5. Molecular identification of transgenic sugarcane

(1) PCR identification of transgenic sugarcane

Mark the surviving transgenic sugarcane with the serial number on the tip of the pipette, cut the sugarcane leaves to extract the transgenic sugarcane DNA, and use specific primers for PCR identification. Among them, the identification primer for the target gene ShN/AINV3.1 is: forward primer 35S PPDK -F (5'-GTCACGTAGTAAGCAGCTCTCGG-3') and target gene reverse primer (5'-ATAACCATATATACCCATTCGACGATC-3'). The identification primers of the Bar gene were: Bar-F: 5'-ACAAGCACGGTCAACTTCC-3', Bar-R: 5'-CTTCAGCAGGTGGGTGTAG-3'. The PCR products were detected using 1% agarose gel, and the results are shown in Fig. 2 and Fig. 3 .

It can be seen from Figures 2-3 that the transgenic sugarcane lines line1, line2, and line3 can all amplify specific target fragments and resistance genes (Bar genes), which proves that the target vector has been successfully integrated into the sugarcane genome.

In addition, among the 22 plants detected, 20 plants were PCR-positive plants, and the positive plants accounted for 90.9% of all the detected plants, and 4 of the positive plants could only detect the Basta glufosinate-ammonium resistance exogenous gene, This shows that some gene breaks occurred during the Agrobacterium transformation process, and the remaining 16 positive plants all contained the target gene ShN/AINV3.1 and the Basta glufosinate-ammonium resistance gene.

(2) Detection of protein expression of target gene in transgenic sugarcane

Transgenic sugarcane and wild-type WT leaf tissues were used to detect the protein translation of the target gene by WB experiment. Since the target gene was fused with the FLAG tag, we used Anti-FLAG antibody to detect the target gene. The results are shown in Figure 4. Near the 70kDa marker protein, the target protein was detected in the transgenic sugarcane lines line1, line2, and line3, while the wild-type WT could not detect the target protein. Therefore, the genes we selected are normally transcribed and translated in sugarcane, and are not in a state of gene silencing .

6. Germination test analysis of transgenic sugarcane

The sugarcane germination test was carried out in quartz sand, as shown in Figure 5 and Figure 6, when the sugarcane germinated for 3 days, the average bud length of the control group WT was 1.16 cm, and the buds of the transgenic sugarcane lines line1, line2, and line3 The lengths were 2.50, 2.58, and 2.21 cm, respectively. Using the data analysis in EXCEL, the difference between the control group WT and line1, line2, and line3 were analyzed for significant differences. P<0.01, that is, the data difference between the two groups is extremely significant.

When sugarcane germinated for 6 days, the average bud length of WT in the control group was 2.27 cm, and the bud lengths of transgenic sugarcane lines line1, line2, and line3 were 6.11, 6.84, and 6.32 cm, respectively. The results of variance analysis showed that the P values were all less than 0.01, and the differences were extremely significant .

On day 9 of sugarcane germination, the average bud length of WT in the control group was 5.42 cm, and the bud lengths of transgenic sugarcane lines line1, line2, and line3 were 10.29, 10.63, and 10.18 cm, respectively. The results of variance analysis showed that the P values were all less than 0.01, and the differences were extremely significant .

It can be seen that by overexpressing the ShN/AINV3.1 gene, the germination rate of sugarcane can be significantly increased, the germination length of sugarcane can be increased at the same time, and the germination rate of sugarcane can be accelerated.

The above are only preferred implementations of the present invention, and it should be noted that the above preferred implementations should not be regarded as limiting the present invention, and the scope of protection of the present invention should be based on the scope defined in the claims. For those skilled in the art, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, and these improvements and modifications should also be regarded as the protection scope of the present invention.

Claims (10)

1. A ShN/AINV3.1 gene regulating the germination rate of sugarcane, characterized in that its nucleotide sequence is as shown in SEQ ID NO.1. 2. The protein encoded by the ShN/AINV3.1 gene of claim 1, characterized in that its amino acid sequence is as shown in SEQ ID NO.2. 3. A biological material containing the ShN/AINV3.1 gene of claim 1. 4. The biological material according to claim 3, wherein the biological material is a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium. 5. The primer for amplifying the ShN/AINV3.1 gene according to claim 1 is characterized in that, the primer sequence is: F: 5'-CGAGGATCCATGAAGCGGGTGTCGTCGCA-3'; R: 5'-CGAAGGCCTGGCATTCCATGATGCTGACCG-3'. 6. The application of the ShN/AINV3.1 gene of claim 1, the protein of claim 2 or the biological material of claim 3 in the regulation of sugarcane seed and sugarcane stem germination. 7. The application of the ShN/AINV3.1 gene of claim 1, the protein of claim 2 or the biological material of claim 3 in the improvement of sugarcane germplasm resources. 8. The application of the ShN/AINV3.1 gene of claim 1, the protein of claim 2 or the biological material of claim 3 in sugarcane breeding. 9. The application according to claim 8, characterized in that the purpose of the breeding is to cultivate sugarcane varieties with high germination rate. 10. A method for promoting germination of sugarcane seeds and cane stems, characterized in that the expression of the ShN/AINV3.1 gene according to claim 1 in sugarcane is increased, and then the germination of sugarcane seeds and cane stems is promoted.