Application of tobacco zeatin glucosyltransferase gene in regulation and control of germination time of tobacco seeds

Abstract

The invention discloses an application of a tobacco zeatin glucosyltransferase gene in regulating and controlling the germination time of tobacco seeds. The gene is NtZOG, the nucleotide sequence of the gene is as shown in SEQ ID NO. 1, and the amino acid sequence of the gene is as shown in SEQ ID NO. 2. Research finds that tobacco zeatin glucosyltransferase NtZOG gene knockout is carried out on wild tobacco K326, a tobacco mutant with the NtZOG gene knockout is constructed, and by comparing the germination conditions of the wild tobacco K326 and the mutant, it is found that the germination rate of the mutant is remarkably lower than that of the wild tobacco when the mutant develops for 4.5 days and is reduced to about 50%, and it can be seen that the mutant has a significant effect on improving the yield of the tobacco zeatin glucosyltransferase gene NtZOG gene knockout of the tobacco zeatin glucosyltransferase gene NtZOG. The tobacco zeatin glucosyltransferase gene NtZOG has an important regulation effect on tobacco seed germination, the germination time of tobacco can be regulated by regulating the expression of the gene, and the tobacco zeatin glucosyltransferase gene NtZOG has a potential application prospect in the field of tobacco planting.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an application of a tobacco zeatin glucosyltransferase gene in regulating the germination time of tobacco seeds. Background Art

[0002] Seed vigor is a key factor in agricultural production, affecting the germination and growth of crops. Zeatin is an important plant growth regulator. Its O-glycosylation modification is widely present in plant tissues and plays an important role in regulating the level of active zeatin. Zeatin glucosyltransferase (ZOG) can convert zeatin into an O-glycosylated form, thereby affecting its activity. Reports on zeatin glucosyltransferase in other crops mainly focus on its involvement in regulating plant growth and development and stress resistance. There are few studies on the molecular mechanism of zeatin glucosyltransferase (ZOG) regulating seed germination, especially on the effect of zeatin glucosyltransferase gene in tobacco on tobacco germination. Summary of the invention

[0003] The purpose of the present invention is to provide a use of a tobacco zeatin glucosyltransferase gene NtZOG in regulating tobacco seed germination, to provide a reference for regulating tobacco germination time, and to have application prospects in the field of tobacco planting.

[0004] In order to achieve the above object, the present invention provides a new use of tobacco zeatin glucosyltransferase gene NtZOG, which can delay the germination time of tobacco by reducing the expression of the NtZOG gene; wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

[0005] The present invention also provides an application of a tobacco zeatin glucosyltransferase gene NtZOG in the field of tobacco planting, and the germination time of tobacco can be delayed by reducing the expression of the NtZOG gene; wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

[0006] Preferably, the reduction of NtZOG gene expression is achieved by constructing a NtZOG gene knockout vector, and then transferring the obtained knockout vector into tobacco to obtain a NtZOG gene knockout tobacco mutant.

[0007] Preferably, the above-mentioned knockout vector is selected from pHSbdcas9i vector.

[0008] Preferably, the nucleotide sequence of the target sequence targeted by the above knockout vector is shown as SEQ ID NO.3 or SEQ ID NO.4.

[0009] Preferably, the above tobacco mutant is constructed by using a knockout vector through Agrobacterium transformation.

[0010] The present invention also provides an application of a tobacco zeatin glucosyltransferase gene NtZOG in promoting early germination of tobacco seeds. By constructing a tobacco strain overexpressing the gene, the germination time of the tobacco strain can be advanced. The nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

[0011] The present invention also provides an application of a tobacco zeatin glucosyltransferase gene NtZOG in preparing an early-germinating tobacco strain. An early-germinating tobacco strain is obtained by constructing an overexpression vector of the gene NtZOG and transferring the overexpression vector into tobacco, thereby obtaining a fast-germinating tobacco strain.

[0012] The present invention has the following advantages:

[0013] The present invention isolates and clones the NtZOG gene from tobacco, and proves for the first time that the gene is involved in the regulation of tobacco seed germination by constructing a CRISPR / Cas9 mutant. Reducing the expression of the gene leads to delayed tobacco seed germination, which in turn indicates that increasing the expression of the gene has the potential to promote early tobacco germination, and has reference and guiding significance in the study of the mechanism of tobacco germination.

[0014] The invention provides a basis for screening tobacco varieties with fast germination, and also provides important gene resources for improving tobacco germination speed, which is of great significance to production and has potential application value in the field of tobacco production and planting. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 The figure shows the expression of the tobacco NtZOG gene at different times of seed imbibition in the present invention.

[0016] Figure 2 This is the germination phenotype of the tobacco NtZOG mutant material of the present invention. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0018] Note: The experimental methods in the following examples are conventional methods unless otherwise specified, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples are all commercially available unless otherwise specified.

[0019] Experimental Example 1 Analysis of tobacco NtZOG gene expression

[0020] The seeds of wild-type tobacco K326 were selected and imbibition was treated for 0, 12, 24, 36, and 48 hours. After the treatment, the seeds were frozen with liquid nitrogen and quickly ground into powder, and the powder samples were stored at -80°C. The experiment was repeated 3 times.

[0021] RNA from each sample was extracted using the TransZol Plant kit (Transgen, www.transgen.com); II Reverse Transcriptase system (Vazyme Biotech Co., Ltd) kit was used to reverse transcribe into cDNA, and the cDNA was used as a template to analyze the expression of tobacco zeatin glucosyltransferase gene NtZOG by fluorescence quantitative PCR. The nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. The specific sequence is shown below. The primer sequence for fluorescence quantitative PCR detection of NtZOG was designed, and its upstream primer nucleotide sequence is shown in SEQ ID NO.9, and its downstream primer nucleotide sequence is shown in SEQ ID NO.10. The tobacco internal reference gene EF1A primers were used, and its upstream primer nucleotide sequence is shown in SEQ ID NO.11, and its downstream primer nucleotide sequence is shown in SEQ ID NO.12. The expression of tobacco NtZOG gene at different times of seed imbibition is shown in the following figure. Figure 1 As shown, the results showed that the expression level of the NtZOG gene showed a gradually downward trend during the seed development and swelling process. It can be seen that with the development of the seeds, the expression level of the NtZOG gene gradually decreased.

[0022] Nucleotide sequence of NtZOG gene (SEQ ID NO.1):

[0023]

[0024] The amino acid sequence encoded by the NtZOG gene (SEQ ID NO.2):

[0025] MAKTSNLENHDQEKVAVVMVPLPAQGHLNQLLHLSRLISAYHLPLHYVGTTTHTRQAKIRVQGWDPHSISNIHFHEFSTPSCEIPPPNPQSSTKFPSQLMPSFHATCHLRDPITSLLRE LARCNKRVIVIYDSLIAWVLQDAPFIANVECYSFRSISALNIHSLALEFAGKSTEPNLPSIEGCFTQEFSEFSRVQTEFRELVNSGDLYNSCYEIEGLYLDLLAKEKSASHKQWAVGPLN PVIPYDKKDSNKRHKCLEWLDKQEPNSVIFVSFGTTTSLSDNEIEELAIGLEQRQQKFIWVLRDADKGDIFTGDVRKVELPKGYEERVIGRGLIVRDWAPQLEILGHLSTGGFMSHCGW NSCMESISMGVPIIAWPMHSDQPRNAFLVTNVLKIGVVLKDWALREELVTSVMVEECVKRLMDSVEGDKMRERAVELRQSVVDGGGSHKEMDSFITHITR*; NtZOG gene fluorescence quantification primer F (SEQ ID NO.9):

[0026] TTCGGATCAGCCAAGAAAATGCG;

[0027] NtZOG gene fluorescence quantitative primer R (SEQ ID NO.10):

[0028] TCTGAGTGCCCAATCCTTCAGC;

[0029] Tobacco internal reference gene EF1A primer F (SEQ ID NO.11):

[0030] AGCTTCACCACCAGGTCATC;

[0031] Tobacco internal reference gene EF1A primer R (SEQ ID NO.12):

[0032] AGAACGCCTGTCAATTCTTGG.

[0033] Experimental Example 2 Construction of tobacco NtZOG gene mutant

[0034] According to the sequence of the target gene, the gDNA target was screened by conventional methods, and two gDNA targets were selected, which were recorded as target 1 and target 2, respectively. The nucleotide sequences were shown in SEQ ID NO.3 and SEQ ID NO.4, respectively. The amplification primers F and R were designed based on the target sequence. The nucleotide sequence of amplification primer F was shown in SEQ ID NO.5, and the nucleotide sequence of amplification primer R was shown in SEQ ID NO.6. The amplification primers were universal for the two targets, target 1 was on amplification primer F, and target 2 was on amplification primer R. The specific sequences are as follows. The PCR product containing the target was amplified using Vazyme P520 (Vazyme Biotech Co., Ltd), the PCR product was purified and recovered, and the purified and recovered PCR product was constructed into the pHSbdcas9i vector using T4 ligase.

[0035] The obtained pHSbdcas9i vector plasmid containing the target gene NtZOG was transformed into Agrobacterium; the Agrobacterium carrying the transformation plasmid was transformed into the wild-type tobacco K326 variety, and the homozygous mutant screening primers were used for PCR amplification and sequencing, and compared with the wild type to screen the homozygous mutants, wherein the upstream primer sequence of the screening primers used is shown in SEQ ID NO.7, and the downstream primer sequence is shown in SEQ ID NO.8, and the specific sequence is as follows. After identifying the transfected tobacco mutants, two tobacco gene NtZOG mutants Ntzog-1 and Ntzog-2 were obtained.

[0036] gDNA target sequence 1 (SEQ ID NO.3):

[0037] AGGTGGAATTTCACAAGAT;

[0038] gDNA target sequence 2 (SEQ ID NO.4):

[0039] TGCTTGACGGGTGTGAGTGG;

[0040] Amplification primer F (SEQ ID NO.5):

[0041] CAGTGGTCTCATGCAAGGTGGAATTTCACAAGATGGTTTTAGAGCT AG;

[0042] Amplification primer R (SEQ ID NO.6):

[0043] CAGTGGTCTCAAAACCCACTCACACCCGTCAAGCATG;

[0044] Homozygous mutant screening primer F (SEQ ID NO.7):

[0045] GCAACGCTCTGTCATCGTTACAAT;

[0046] Homozygous mutant screening primer R (SEQ ID NO.8):

[0047] GCGATTAAGTTGGGTAACGCCAGGG.

[0048] Experimental Example 3 Phenotypic Analysis of Tobacco NtZOG Gene Mutant Materials

[0049] The germination test was conducted using the constructed NtZOG CRISPR / Cas9 mutants Ntzog-1 and Ntzog-2, and the wild-type tobacco K326 variety. The specific method is as follows: each time, healthy and full seeds were selected, placed in a culture dish, 8 mL of distilled water was added, and cultured at 25°C under light / dark conditions for 12 hours each. Germination was allowed to proceed for 4.5 days under water conditions, and the germination rates at different times were counted. The test was repeated 3 times. The germination phenotypes of various seeds are shown in Figure 2 As shown in A, the germination rate was quantitatively counted, and the results are shown in Figure 2 As shown in B and C in Figure 4.5, the results showed that at 4.5 days, the germination rate of the Ntzog mutant was significantly reduced; the germination rate of the wild-type K326 (WT) was 85%, the germination rate of the mutant Ntzog-1 was 31%, and the germination rate of the mutant Ntzog-2 was 35%. Compared with the wild-type seeds, the seedlings of the seeds with the mutation of the Ntzog gene were reduced by 50%. It can be seen that this gene has an important regulatory role in tobacco seed germination, and the reduction of the expression of this gene can significantly delay the germination of tobacco seeds.

[0050] According to the conventional knowledge in the art, combined with the above conclusions, it can be concluded that reducing the expression of this gene can reduce the germination rate of tobacco seeds at 4.5 days, delaying the germination time. The expression of this gene is inversely proportional to the germination rate of tobacco seeds, so it is concluded that by increasing the expression of this gene, the seeds can be promoted to germinate in advance. Based on this, by constructing a mutant that overexpresses the tobacco NtZOG gene, the tobacco mutant can be promoted to germinate in advance.

[0051] In summary, the present invention discloses an application of a tobacco zeatin glucosyltransferase gene in regulating the germination time of tobacco seeds. By knocking out the tobacco zeatin glucosyltransferase gene NtZOG in wild tobacco K326, a tobacco mutant with the NtZOG gene knocked out was constructed. The germination rate of the mutant was significantly lower than that of the wild tobacco at 4.5 days of development, and was as low as about 50%. It can be seen that the tobacco zeatin glucosyltransferase gene NtZOG has an important regulatory effect on the germination time of tobacco seeds. By controlling the expression of the gene, the germination time of tobacco can be significantly delayed, and it has potential application prospects in the field of tobacco planting.

[0052] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

[0053]

[0054]

[0055]

[0056]

[0057]

Claims

1. A new use of tobacco zeatin glucosyltransferase gene NtZOG, characterized in that: By reducing the expression of the NtZOG gene, the germination time of tobacco can be delayed; wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.

1.

2. An application of a tobacco zeatin glucosyltransferase gene NtZOG in the field of tobacco planting, characterized in that: By reducing the expression of the NtZOG gene, the germination time of tobacco can be delayed; wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.

1.

3. The use according to claim 2, characterized in that: The expression of the NtZOG gene is reduced by constructing a NtZOG gene knockout vector, and then transferring the knockout vector into tobacco to obtain a tobacco mutant with the NtZOG gene knocked out.

4. The new application according to claim 3, characterized in that: The knockout vector is selected from pHSbdcas9i vector.

5. The use according to claim 3, characterized in that: The nucleotide sequence of the target sequence targeted by the knockout vector is shown in SEQ ID NO.3 or SEQ ID NO.

4.

6. The use according to claim 3, characterized in that: The tobacco mutant is constructed by using the Agrobacterium transformation method to construct the knockout vector.

7. An application of tobacco zeatin glucosyltransferase gene NtZOG in promoting early germination of tobacco seeds, characterized in that: By constructing a tobacco strain that overexpresses the gene, it can be used to advance the germination time of the tobacco strain, wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.

1.

8. Application of a tobacco zeatin glucosyltransferase gene NtZOG in preparing an early-germination tobacco strain, characterized in that: An early-germination tobacco strain is obtained by constructing an overexpression vector of the gene and transferring the overexpression vector into tobacco, wherein the nucleotide sequence of the NtZOG gene is shown in SEQ ID NO.1.

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