CN110144357A - Application of gene overexpression in improving plant nitrogen content and/or protein content or breeding high protein plant varieties - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to the application of gene overexpression in increasing the nitrogen content and/or protein content of plants or breeding high-protein plant varieties. The BnGS1 gene obtained by cloning of the present invention shows that the nitrogen content and crude protein content are 4.88% and 30.52% respectively through overexpression in tobacco, and the nitrogen content and crude protein content of wild-type tobacco leaves are 2.29% and 14.32% respectively, and the transgenic plants and wild There were significant differences in leaf nitrogen content and crude protein content of different types. This is also the first GS gene in ramie to achieve significant differences in nitrogen and nitrogen content through overexpression in tobacco, and this gene has a good application prospect. Cloning protein-related genes through molecular breeding can increase the protein content of ramie through overexpression, and the cycle of breeding new high-protein varieties is short and the protein content is higher.

Description

Overexpression of genes in increasing plant nitrogen and/or protein content or in breeding for high Application in protein plant varieties

technical field

The invention relates to the field of biotechnology, in particular to the application of gene overexpression in increasing the nitrogen content and/or protein content of plants or breeding high-protein plant varieties.

Background technique

The young stems and leaves of ramie are rich in nutrients and high in crude protein, which is a good protein raw material for plant feed. High-protein and high-quality forage grasses are extremely scarce in southern my country. There is a very broad prospect for vigorously developing ramie forage crops in southern China. Ramie is used as forage Utilization We must first detect the crude protein content, which is an important indicator to measure the quality of a forage ramie. The traditional selection of ramie varieties with high nitrogen content is carried out by hybridization and other means. This breeding method takes several years. It is difficult to have materials with particularly high nitrogen content in the ramie variety materials. Improving the nitrogen content of ramie is currently the The major problem to be solved in ramie breeding is that the material cycle of conventional breeding of high nitrogen content ramie varieties is very long, and it is difficult to obtain high nitrogen content varieties.

In plants, glutamine synthetase (Glutamine synthetase, GS) assimilates NH4 ⁺ into glutamine, and GOGAT (glutamate synthetase) is collectively referred to as the GS/GOGAT cycle. GS includes two major families, GS1 and GS2, in which GS1 is mainly located in the cytoplasm, and GS2 is located in the plastid. Plants generally have one GS2 gene and multiple GS1 genes. GS genes have effects on plant nitrogen use efficiency. In rice varieties with high nitrogen use efficiency and low nitrogen use efficiency, the activity of GS was significantly higher in the former than in the latter. Zheng JS et al. overexpressed BnGS1-2 in tobacco. The results showed that the NO ₃ ^- and NH ₄ ⁺ absorption capacity of transgenic tobacco was enhanced, NR activity was significantly increased, and the soluble protein content and nitrogen content were higher than those of the control group, but the difference was not significant. At the same time, the plant height, fresh weight and leaf soluble protein of BnGS2-1 and BnGS2-2 transgenic tobacco were all significantly increased; the nitrogen content was also increased, but there was no significant difference.

Therefore, it is of great practical significance to provide ramie nitrogen metabolism-related genes to increase ramie protein content and shorten the period of breeding high-protein varieties.

Contents of the invention

In view of this, the present invention provides the application of gene overexpression in increasing the nitrogen content and/or protein content of plants or in breeding high protein plant varieties. The invention adopts the method of molecular breeding to clone genes related to the nitrogen content of ramie to select and breed new ramie varieties with high nitrogen content, the time is relatively short, the nitrogen content increases greatly, and the varieties with high nitrogen content of ramie can be obtained quickly.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides the application of gene overexpression in increasing the nitrogen content and/or protein content of plants or in breeding high-protein plant varieties;

The gene has any one of the following nucleotide sequences:

I. have the nucleotide sequence of BnGS1; or

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in I; or

III. A sequence having at least 80% homology with the nucleotide sequence shown in I or a nucleotide sequence with the same or similar function as the protein expressed by the gene BnGS1 after translation; or

IV. The complementary sequence of the sequence shown in I, II or III.

In some embodiments of the invention, the plant is ramie or tobacco.

On the basis of the above research, the present invention also provides expression vectors, including overexpressed genes and

basic carrier;

The gene has any one of the following nucleotide sequences:

I. have the nucleotide sequence of BnGS1; or

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in I; or

III. A sequence having at least 80% homology with the nucleotide sequence shown in I or a nucleotide sequence with the same or similar function as the protein expressed by the gene BnGS1 after translation; or

IV. The complementary sequence of the sequence shown in I, II or III.

The present invention also provides a method for constructing the expression vector, including connecting the gene with the basic vector, transforming, picking bacteria, extracting the plasmid, and sequencing;

The gene has any one of the following nucleotide sequences:

I. have the nucleotide sequence of BnGS1; or

II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in I; or

III. A sequence having at least 80% homology with the nucleotide sequence shown in I or a nucleotide sequence with the same or similar function as the protein expressed by the gene BnGS1 after translation; or

IV. The complementary sequence of the sequence shown in I, II or III.

The present invention also provides host cells transformed or transfected with the expression vector.

The present invention also provides the application of the expression vector or the host cell in increasing the nitrogen content and/or protein content of plants or breeding high-protein plant varieties

In some embodiments of the invention, the plant is ramie or tobacco.

The invention also provides a method for increasing the nitrogen content in plants. The expression vector is transferred into the competent Agrobacterium, cultured, picked, and positive colonies are taken to infect the plants.

The invention also provides a method for increasing protein content in plants. The expression carrier is transferred into competent Agrobacterium, cultured, picked, and positive colonies are taken to infect plants.

The invention also provides a breeding method for high-protein plants. The expression vector is transferred into the competent Agrobacterium, cultivated, picked, and positive colonies are taken to infect the plants.

The BnGS1 gene obtained by cloning of the present invention shows that the nitrogen content and crude protein content are 4.88% and 30.52% respectively through overexpression in tobacco, and the nitrogen content and crude protein content of wild-type tobacco leaves are 2.29% and 14.32% respectively, and the transgenic plants and wild There were significant differences in leaf nitrogen content and crude protein content of different types. This is also the first GS gene in ramie to achieve significant differences in nitrogen and nitrogen content through overexpression in tobacco, and this gene has a good application prospect. Cloning protein-related genes through molecular breeding can increase the protein content of ramie through overexpression, and the cycle of breeding new high-protein varieties is short and the protein content is higher.

Description of drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings that are required in the description of the embodiments or the prior art.

Figure 1 shows the gel electrophoresis of the BnGS1 gene; wherein, Figure 1 (A) shows the PCR amplification product of the BnGS1 gene, and the lanes from left to right are 1: DNA marker, 2-3: BnGS1 gene; Figure 1 (B) It shows the PCR verification of TA monoclonal, the lanes from left to right are 1, 4: DNA marker, 2-3: BnGS1 gene;

Figure 2 shows the CDS sequence and amino acid sequence of BnGS1 (gray scale indicates the conserved domain);

Figure 3 shows BnGS1 protein structure analysis;

Figure 4 shows the expression of PEZR-(K)-LN-GFP: empty load in tobacco leaves; where, A, D: bright field; B, E: green fluorescence; C, F: synthesis;

Figure 5 shows single colony PCR identification; wherein Figure 5 (A) shows the BnDof14 gene, and the swimming lanes are 1: DNAmarker, 2-6: BnDof14 gene from left to right; Figure 5 (B) shows the BnGS1 gene, and the swimming lanes are from left to right 1, 6: DNA marker, 2-3: BnGS1 gene;

Figure 6 shows the PCR identification of Agrobacterium-positive transformed colonies; wherein, Figure 6 (A) shows the BnDof14 gene, and the lanes from left to right are 1: DNA marker, 2-5, 8: false positive colonies; 6-7: BnDof14 Gene-positive colonies; Figure 6 (B) shows the BnGS1 gene, and the lanes are 1-4 from left to right: positive colonies of the BnGS1 gene, 6: DNA marker;

Fig. 7 shows the regeneration of transgenic tobacco; Wherein, Fig. 7 (A) shows the callus of tobacco; Fig. 7 (B) shows young shoot rooting; Fig. 7 (C) shows that transgenic tobacco seedling is transplanted in the soil;

Figure 8 shows the DNA of transgenic tobacco; lanes from left to right are 1, 7: DNA marker, 2-6: DNA of transgenic tobacco;

Figure 9 shows the PCR product of transgenic tobacco; lanes from left to right are 1-3: BnGS1 gene, 4: DNA marker;

Figure 10 shows the relative expression of BnGS1 gene in BnGS1 transgenic and wild-type tobacco;

Figure 11 shows the GUS staining of transgenic tobacco leaves;

Figure 12 shows the phenotypes of BnGS1 transgenic tobacco and wild-type tobacco; wherein, Figure 12 (A) shows the contrast of plant height between transgenic tobacco and wild-type tobacco; Figure 12 (B) shows the color contrast of transgenic tobacco and wild-type tobacco leaves;

Figure 13 shows the nitrogen content of BnGS1 transgenic tobacco and wild-type tobacco leaves;

Fig. 14 shows the crude protein content of leaves of BnGS1 transgenic tobacco and wild type tobacco.

Detailed ways

The invention discloses the application of gene overexpression in increasing the nitrogen content and/or protein content of plants or in breeding high-protein plant varieties. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters to realize it. In particular, it should be pointed out that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and the relevant personnel can obviously make changes or appropriate changes and combinations to the method and application described herein without departing from the content, spirit and scope of the present invention to realize and Apply the technology of the present invention.

The raw materials and reagents used in the application of the overexpression of the gene provided by the invention in increasing the nitrogen content and/or protein content of plants or breeding high-protein plant varieties can be purchased from the market.

Below in conjunction with embodiment, further set forth the present invention:

Example 1 Ramie BnGS1 gene cloning and screening of TA monoclonal

The agarose gel electrophoresis image of the PCR product of ramie BnGS1 gene is shown in Figure 1(A). The PCR conditions are: (1) pre-denaturation at 94°C for 4 minutes, (2) denaturation at 94°C for 30 seconds, (3) renaturation at 54°C for 30 seconds, ( 4) Extend at 72°C for 1min 30s, cycle (2)-(4) 32 times, (5) extend at 72°C for 5min, store at 4°C. The BnGS1 gene is 1518bp long, and the gel map shows that the PCR product is consistent with the desired target fragment. After recovering the target fragment gel, connect it with pMD18-T, transform it into DH5α, smear the plate and culture it at 38°C, and select Vitiligo spp. on the plate. The results of bacterial liquid PCR are shown in Figure 1(B).

Example 2 Ramie BnGS1 gene bioinformatics analysis

The CDS sequence (as shown in SEQ ID No.1) and amino acid sequence (as shown in SEQ ID No.2) of the BnGS1 gene are shown in Figure 2, and the protein structure is shown in Figure 3. The CDS sequence of BnGS1 gene is 1071bp long, and the amino acid sequence is 356aa long. The molecular weight of the protein is 39.19Da, and the isoelectric point pI is 5.60. There is a conserved domain from amino acid 13 to 354, which is a unique domain of GS protein.

The subcellular localization of embodiment 3BnGS1 gene

PEZR-(K)-LN-BnGS1-GFP vector was constructed by double enzyme digestion experiment. The specific method is as follows:

(1) Use EcoRI and KpnI to perform double enzyme digestion on PEZR-(K)-LN, the system is as follows:

(2) Enzyme digestion at 37°C for 1 hour, and then gel recovery.

(3) Use EcoRI and KpnI restriction enzyme BnGS1 gene for double enzyme digestion, the system is as follows:

(4) Enzyme digestion at 37°C for 1 hour, and then gel recovery.

(5) Ligate the gel recovery products in (2) and (4) with T4DNA ligase, reaction system (10μL): plasmid DNA 100ng, target fragment 10ng, 10*Buffer 1μL, T4DNA ligase 0.5μL, add ddH ₂ O to 10 μL. Under the condition of 25°C, connect for 3h.

The constructed PEZR-(K)-LN-BnGS1-GFP vector was transformed into Agrobacterium GV3101, and the GV3101 containing the vector was injected into tobacco leaves with a syringe, cultured in the dark for 2 hours and then cultured for 3 days, and then observed under a laser confocal microscope The results of fluorescence expression in leaf slices are shown in Figure 4. The pictures of subcellular localization showed that the empty load produced green fluorescent signals in the cytoplasm and nucleus, indicating that the empty load was localized in the nucleus and cytoplasm. PEZR-(K)-LN-BnGS1-GFP only produces green fluorescence in the cytoplasm, so the BnGS1 protein is mainly distributed in the cytoplasm.

The acquisition of embodiment 4 transgenic tobacco

Construct the overexpression vector by single enzyme digestion method, the specific method is as follows:

(1) Use the TSAP Thermosensitive Alkaline Phosphatase Kit (Promega, USA) to perform single enzyme digestion on pBI121, and the reaction system is as follows (10 μL):

(2) Enzyme digestion at 37°C for 2 hours.

(3) Inactivation at 74°C for 15 minutes.

(4) Single-enzyme digestion of BnGS1 gene with SpeI, the reaction system is as follows (20 μL):

(5) Enzyme digestion at 37°C for 1 hour, and then gel recovery.

(6) Ligate the products in (3) and (5) with T4 DNA ligase, reaction system (10 μL): plasmid DNA 100 ng, target fragment 10 ng, 10*Buffer 1 μL, T4 DNA ligase 2 μL, add ddH ₂ O to 10 μL . Under the condition of 25°C, connect for 3h.

Transform the ligated overexpression vector into Escherichia coli DH5α competent, the transformation method is as follows:

(1) Thaw 100 μL of competent cells on ice, add 10 μL of pBI121 carrier with the target fragment under aseptic conditions, mix gently, and place on ice for 25 minutes.

(2) Heat shock at 42°C for 45s, then quickly transfer the centrifuge tube to ice and let it stand for 2min.

(3) Under sterile conditions, add 600 μL of LB liquid medium without antibiotics to the centrifuge tube, shake and culture for 1 hour at 37° C. and 200 rpm to recover the bacteria.

The revived cells were plated on a plate containing Kan, cultured at 37°C for 12 hours, and a single colony was picked and shaken. The bacterial liquid was verified by PCR, and the results are shown in Figure 5. Figure 5 shows that the selected single colony contains the fragment of interest. The plasmid was extracted and sequenced, and the result proved that the gene was successfully connected to the pBI121 vector.

Agrobacterium transformation and positive colony identification:

The constructed plant overexpression vector was transformed into Agrobacterium GV3101 competent by the heat shock method, as follows:

(1) Thaw Agrobacterium competent cells GV3101 stored at -70°C in an ice-water bath.

(2) Under sterile conditions, add 1 μg of plasmid DNA to the competent cells, mix gently, and let stand in an ice-water bath for 5 minutes.

(3) Place the centrifuge tube in liquid nitrogen for quick freezing for 5 minutes, and then quickly place the centrifuge tube in a 37°C water bath for 5 minutes. At this time, be careful not to shake the water surface; then put the centrifuge tube back into the ice water bath for 5 minutes.

(4) Add 800 μL of antibiotic-free LB medium to a centrifuge tube under sterile conditions, shake and culture at 28°C for 2-3 hours to recover the bacteria.

The recovered bacterial liquid was cultured on the medium containing Kan and Rif for 2-3 days. A single colony was picked for colony PCR, and the results are shown in Figure 6. The results showed that the plant overexpression vector was successfully transformed into GV3101 cells.

Agrobacterium infection of tobacco leaves:

Agrobacterium-positive single colonies were shaken to infect tobacco by leaf disc method. The infected tobacco leaves were cultured in the dark on the MSO medium for 2 days, then transferred to the MS1 medium, and kept in the light until the callus sprouted ( FIG. 7A ). When the shoots reached 2-3 cm, they were excised and transferred to MS2 to induce rooting of the shoots (Fig. 7B). MS2 medium contains Kan, positive seedlings can grow normally, negative seedlings will die. The healthy seedlings were taken out from the MS2 medium, and the root agar was rinsed with tap water, and planted in soil ( FIG. 7C ).

Identification of genetically modified tobacco:

Extraction of genetically modified tobacco DNA:

The DNA of the transgenic tobacco was extracted, and the DNA was electrophoresed with 1% agarose gel to verify the quality of the DNA. The result is shown in FIG. 8 . The gel image shows that there is no protein contamination in the extracted DNA, and there is no fragmentation, and the quality is good, which can be used for PCR.

PCR verification of genetically modified tobacco:

The DNA of the transgenic tobacco was used as a template, and GS1F-1 and GS1R-1 were used as primers for PCR reaction. The PCR products of BnGS1 transgenic tobacco plants are shown in Figure 9 . The length of the BnGS1 gene is 1071bp, and the agarose gel picture shows that the BnGS1 gene has been successfully transferred into tobacco, and the transgenic tobacco seedlings have been successfully constructed.

qRT-PCR Analysis of Transgenic Tobacco:

The transgenic tobacco was analyzed by qRT-PCR to further confirm the accuracy of the transgenic tobacco plants. Taking tobacco Nlt25 gene as an internal reference gene, the relative expression of BnGS1 gene in transgenic plants is shown in Figure 10. The results showed that BnGS1 was expressed in transgenic plants, and the mRNA level of BnGS1 allele could not be detected in wild type.

Transgenic tobacco GUS staining:

Transgenic tobacco tender leaves were taken for GUS staining to verify the expression of the vector in tobacco leaves. The results are shown in Figure 11. GUS staining experiments showed that the BnGS1 gene was strongly expressed in the transgenic plants.

Nitrogen Content Analysis of Transgenic Tobacco:

During the flowering period, the leaves of BnGS1 transgenic and wild-type tobacco plants were taken to measure the nitrogen content. The plants were observed during sampling, and the results are shown in Figure 12. From the pictures, it can be found that compared with the wild type, the plant height of the BnGS1 transgenic tobacco plants is significantly reduced, and the leaves of the transgenic plants are darker. The nitrogen content and crude protein content of leaves of wild-type and BnGS1 transgenic plants are shown in Figure 13 and Figure 14, respectively. The nitrogen content and crude protein content of BnGS1 transgenic tobacco leaves were 4.88% and 30.52%, respectively, and the nitrogen content and crude protein content of wild-type tobacco leaves were 2.29% and 14.32%, respectively. content showed significant differences.

In summary, the functional verification results of nitrogen and crude protein content of BnGS1 gene in ramie showed that overexpression of BnGS1 gene in tobacco for the first time showed that the nitrogen and crude protein content increased significantly by more than 2 times; the use of BnGS1 gene can be used to breed high Crude protein content of forage ramie varieties.

The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.

sequence listing

<110> Institute of Hemp, Chinese Academy of Agricultural Sciences

Application of <120> gene overexpression in increasing plant nitrogen content and/or protein content or breeding high protein plant varieties

<130> MP1904417

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gaccaagctc ccggggacga cagtgaagtc atcttatacc ctcaagcggt gtttaaggat 240

ccattcagga gaggaaacaa tatcctggtg atgtgtgacg cttatacgcc agctggcgag 300

ccgattccga cgaacaaacg acacaatgct gcgaagattt tcagtcaccc tgatgtggtc 360

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Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser

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Claims (10)

1. The application of gene overexpression in increasing the nitrogen content and/or protein content of plants or breeding high-protein plant varieties; The gene has any one of the following nucleotide sequences: I. have the nucleotide sequence of BnGS1; or II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in I; or III. A sequence having at least 80% homology with the nucleotide sequence shown in I or a nucleotide sequence with the same or similar function as the protein expressed by the gene BnGS1 after translation; or IV. The complementary sequence of the sequence shown in I, II or III. 2. application as claimed in claim 1, is characterized in that, described plant is ramie or tobacco. 3. The expression vector is characterized in that it includes an overexpressed gene and a basic vector; The gene has any one of the following nucleotide sequences: I. have the nucleotide sequence of BnGS1; or II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in I; or III. A sequence having at least 80% homology with the nucleotide sequence shown in I or a nucleotide sequence with the same or similar function as the protein expressed by the gene BnGS1 after translation; or IV. The complementary sequence of the sequence shown in I, II or III. 4. The construction method of the expression vector as claimed in claim 3, is characterized in that, the gene is connected to the basic carrier, transformed, picked bacteria, extracted plasmid, and sequenced; The gene has any one of the following nucleotide sequences: I. have the nucleotide sequence of BnGS1; or II. A nucleotide sequence obtained by modifying, substituting, deleting or adding one or more bases to the nucleotide sequence shown in I; or III. A sequence having at least 80% homology with the nucleotide sequence shown in I or a nucleotide sequence with the same or similar function as the protein expressed by the gene BnGS1 after translation; or IV. The complementary sequence of the sequence shown in I, II or III. 5. transform or transfect the host cell of the expression vector as claimed in claim 3. 6. The application of the expression vector as claimed in claim 3 or the host cell as claimed in claim 5 in improving the nitrogen content and/or protein content of plants or in breeding high-protein plant varieties. 7. The application according to claim 6, characterized in that the plant is ramie or tobacco. 8. The method for improving the nitrogen content in plants is characterized in that, the expression vector according to claim 3 is transferred into the competent Agrobacterium, cultivated, picked, and the positive colonies are taken to infect the plants. 9. The method for improving the protein content in plants is characterized in that, the expression vector according to claim 3 is transferred into the competent Agrobacterium, cultivated, picked, and the positive colonies are taken to infect the plants. 10. The breeding method of high-protein plants, characterized in that, the expression vector according to claim 3 is transferred into the competent Agrobacterium, cultivated, picked, and the positive colonies are taken to infect the plants.