CN113278639A

CN113278639A - Tobacco NUDIX hydrolase gene and application thereof

Info

Publication number: CN113278639A
Application number: CN202110574079.9A
Authority: CN
Inventors: 蒋佳芮; 曾婉俐; 刘欣; 李雪梅; 阴耕耘; 米其利; 翟妞; 许力; 杨文武; 邓乐乐
Original assignee: China Tobacco Yunnan Industrial Co Ltd
Current assignee: China Tobacco Yunnan Industrial Co Ltd
Priority date: 2021-05-25
Filing date: 2021-05-25
Publication date: 2021-08-20
Anticipated expiration: 2041-05-25
Also published as: CN113278639B

Abstract

The invention relates to a tobacco NUDIX hydrolase gene and application thereof, wherein a base sequence is shown as SEQ ID No. 1. In the present application, the preliminary study on the tobacco NUDIX hydrolase NtNUDX14 found that it is related to the anabolism of starch in tobacco leaves. The gene is silenced in Nicotiana benthamiana, the content of amylose, amylopectin and total starch in leaves is obviously reduced, and the content of the total starch is reduced by 59.8%. Based on the characteristic, a certain reference can be provided for the quality control of tobacco leaves and the cultivation of new tobacco varieties.

Description

Tobacco NUDIX hydrolase gene and application thereof

Technical Field

The invention belongs to the technical field of tobacco genetic engineering, and particularly relates to a tobacco NUDIX hydrolase gene and application thereof.

Background

Tobacco is an important economic crop, and the tobacco quality and safety of the tobacco are always the focus of attention of researchers. Starch is an important energy storage substance in tobacco leaves and is an important component of carbohydrate substances in the tobacco leaves. The starch content in the mature fresh tobacco leaves is as high as about 40 percent, and most of starch is degraded into reducing sugar after modulation, but part of starch is remained in the tobacco leaves. Starch has great influence on the quality of tobacco leaves, and the existing research considers that the impure gas and the starch content of the tobacco leaves are in extremely obvious positive correlation, namely the larger the starch content of the tobacco leaves is, the more the impure gas is. At present, the starch content (mass fraction) of flue-cured tobacco in China is about 4-6%, while the starch content of foreign high-quality flue-cured tobacco is only 1-2%. The excessive high residual quantity of starch in the flue-cured tobacco leaves becomes one of the important factors for restricting the improvement of the quality of the domestic tobacco leaves. Therefore, the research on the gene function influencing the starch content in the tobacco provides theoretical support for the improvement of the quality of tobacco leaves and the genetic improvement of tobacco varieties, and has important significance for improving the quality of tobacco products.

Disclosure of Invention

The invention aims to provide a tobacco NUDIX hydrolase gene and application thereof, which aims to solve the problem that the quality of tobacco products is influenced by overhigh starch content in flue-cured tobacco, thereby laying a foundation for tobacco quality regulation and control and new tobacco variety cultivation.

In order to realize the purpose, the invention is realized by the following technical scheme:

a tobacco NUDIX hydrolase gene has a base sequence shown in SEQ ID NO.1, comprises 813 bases and is named as NtNUDX 14.

Furthermore, the amino acid sequence of the tobacco NUDIX hydrolase gene is shown in SEQ ID NO.2 and consists of 270 amino acid residues.

Further, the PCR amplification preparation method of the tobacco NUDIX hydrolase gene comprises the following steps:

(1) extracting genome and reverse transcribing into cDNA for later use;

(2) designing a primer for PCR amplification, and carrying out PCR amplification, wherein the specific primer sequence is designed as follows:

NtNUDX14-F：5’-GCCACGTTATGGACTTCC-3’，

NtNUDX14-R：5’-GTATGTTCTTTAACCATTGCCTG-3’。

further, when the genome is extracted in the step (1), tobacco variety Honghuadajinyuan leaf is taken as a sample.

The application of the tobacco NUDIX hydrolase gene can obviously reduce the content of amylose, amylopectin and total starch in leaves after the protein expressed by the gene is reduced and is related to the starch content in the leaves of plants.

Furthermore, the starch content in the tobacco leaves is regulated and controlled by regulating the expression level of the tobacco NUDIX hydrolase NtNUDX14 by utilizing a gene silencing technology or a gene overexpression method.

Further, a virus-induced silencing vector, an RNAi interference vector or an overexpression vector containing the NtNUDX14 gene is constructed by a transgenic technology, a transient expression technology or a genome editing technology, the tobacco is transformed, and a new tobacco variety with changed starch content is obtained by screening.

Specific examples thereof include: by using a virus-induced gene silencing (VIGS) technology, the expression of the NtNUDX14 gene is interfered to silence the NtNUDX14 gene, the starch content in the NtNUDX14 gene-silenced plant is obviously reduced, and a new plant variety with reduced starch content is obtained.

The invention has the beneficial effects that:

in the application, preliminary study on a specific tobacco NUDIX hydrolase NtNUDX14 shows that the specific tobacco NUDIX hydrolase NtNUDX14 is highly related to the starch content of tobacco, and the starch content in the tobacco is obviously reduced after the gene is silenced. Based on the characteristic, a certain application basis and reference can be provided for the quality control of tobacco leaves and the cultivation of new tobacco varieties.

Drawings

FIG. 1 is a graph showing the relative expression levels of NtNUDX14 gene-silenced plants compared with control plants;

FIG. 2 is a comparison of starch content in tobacco leaves with virus-induced gene silencing and control tobacco leaves.

Detailed Description

The technical solutions of the present invention are described in detail below by examples, and the following examples are only exemplary and can be used only for explaining and explaining the technical solutions of the present invention, but not construed as limiting the technical solutions of the present invention.

Biological material:

nicotiana benthamiana, a commonly used tobacco material in the field, is used for seedling cultivation in a seedling cultivation pot, seedling division is carried out two weeks after germination, the seedlings are planted in a plastic pot (10cm multiplied by 10cm), and cultivation management such as daily fertilizer and water management is carried out under the dark condition of 16h light/8 h at the temperature of 22 ℃.

The VIGS vector used in the following examples is a viral vector (TRV) derived from tobacco rattle virus, specifically TRV2 (a commonly used vector) carrying kanamycin selection marker and 35S promoter, and TRV2 carrying multiple cloning sites such as EcoR I and BamH I, and can be used to carry and transform foreign genes.

Experimental reagent:

LB liquid medium, 1L content contains: 10g bacterial peptone (bacteriological peptone); 10g sodium chloride (NaCl); 5g of yeast extract (yeast extract) and autoclaved.

YEB liquid culture medium, 1L content contains: 5g beef extract (beef extract); 5g bacterial peptone (bacteriological peptone); 5g sucrose (sucrose); 1g yeast extract (yeast extract); 2mL of 1M magnesium sulfate (MgSO4), autoclaved.

1M 2- (N-morpholine) ethanesulfonic acid (MES) stock: ddH₂The dissolution of the oxygen is carried out,filtering and sterilizing, and storing at-20 ℃ for later use.

200mM Acetosyringone (Acetosyringone, As) stock solution: dimethyl Sulfoxide (DSMO) was dissolved and stored at-20 ℃ until use.

Example 1

The construction process of the cloning and silencing vector of the tobacco NtNUDX14 gene is briefly described as follows in the present example.

(1) Cloning of tobacco NtNUDX14 Gene

According to the previous research on the tobacco genome and the related NtNUDX14 gene, a specific coding sequence is selected as a target segment, and a primer sequence for PCR amplification is designed as follows:

NtNUDX14-F：5’-GCCACGTTATGGACTTCC-3’，

NtNUDX14-R：5’-GTATGTTCTTTAACCATTGCCTG-3’。

the cDNA of tobacco safflower gold leaf (firstly extracting genome, then reverse transcribing into cDNA) is used as template, PCR amplification is carried out to obtain NtNUDX14 gene.

The PCR amplification procedure was: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 53 ℃ for 15s, extension at 72 ℃ for 1min, and after 34 cycles, complete extension at 72 ℃ for 5 min.

And carrying out agarose gel electrophoresis detection on the PCR amplification product, and recovering the electrophoresis product for later use.

(2) Construction of recombinant TRV2-NtNUDX14 vector

Carrying out EcoRI and BamHI double enzyme digestion on the PCR amplification product in the step (1), simultaneously carrying out EcoRI and BamHI double enzyme digestion on an empty vector TRV2, respectively recovering enzyme digestion products, and utilizing T4 DNA ligase to carry out ligation.

The ligation product was transformed into E.coli competent DH 5. alpha. and after the transformation, the transformation product was spread on LB solid medium containing 50mg/L Kan and incubated overnight at 37 ℃.

And selecting positive single colonies, amplifying, and then further performing PCR identification, and ensuring that a correctly constructed recombinant vector TRV2-NtNUDX14 is obtained by combining sequencing verification.

The tobacco NtNUDX14 gene comprises 813 basic groups, and the basic sequence is shown as SEQ ID NO. 1.

The tobacco NUDIX hydrolase protein NtNUDX14 comprises 270 amino acids, and the amino acid sequence is shown as SEQ ID NO. 2.

Example 2

On the basis of example 1, the constructed recombinant TRV2-NtNUDX14 vector is further transformed into a tobacco plant by utilizing an agrobacterium-mediated VIGS technology, and verification analysis is carried out on the phenotype change condition of the related plant, and the specific experimental process is summarized as follows.

(1) Transformation of Agrobacterium

It should be noted that, referring to the operation of example 1 and the prior art, the TRV2-GFP recombinant vector was prepared as a control, and the specific transformation process was:

positive cloning plasmids of TRV2-GFP (vector control) and TRV2-NtNUDX14 are respectively transformed into agrobacterium GV3101 competent cells by an electric shock transformation mode, cultured and screened by a YEB plate containing 50mg/L Kan and 50mg/L Rif, and subjected to inverted culture at 28 ℃ for 2 days, and then screened by colony PCR for agrobacterium carrying target genes.

(2) Preparation of a bacterial solution for transfection

The positive Agrobacterium clones selected in step (1) were cultured overnight at 28 ℃ and 250rpm in 5mL YEB liquid medium (containing 50mg/L Kan and 50mg/L Rif).

50uL of the overnight culture was inoculated into 50mL of YEB liquid medium (containing 50mg/L Kan), and cultured to OD₆₀₀Centrifuging at 4000g for 5min, collecting thallus, resuspending with MMA, and adjusting OD₆₀₀About 1.0.

Left and right after standing at room temperature for about 3 hours, the cells were used as a transfection bacterial solution.

(3) Transient transformation

And (3) taking 3-4w (week) of seedling-age tobacco leaves as an experimental material, injecting the bacterial liquid for transfection prepared in the step (2) into the tobacco leaves by using a 1 mL-specification injector, continuously culturing the injected tobacco in an artificial incubator, and observing the phenotypic change.

Further, the expression condition of NtNUDX14 gene is detected by qRT-PCR, the result is shown in figure 1, and it can be seen that, in the infected plant of TRV2-NtNUDX14, the expression level of NtNUDX14 is obviously reduced, and the qRT-PCR primers are as follows:

NtNUDX14-F：5’-AAGGTTCCTGGCATTGTCTTT-3’，

NtNUDX14-R：5’-TGGTCATCATCCAACATTCCT-3’。

furthermore, the test results of the leaf starch content in the experimental group (TRV2-NtNUDX 14-infected plants) and the control group (TRV 2-GFP-infected plants) were examined (see: the measurement method: the amylopectin-amylose-total-starch content kit (spectrophotometry) of Gradenia organisms), and the results are shown in FIG. 2.

As can be seen from the results in FIG. 2, the amylose, amylopectin and total starch contents in the experimental group are all significantly reduced compared with the control group, and the total starch content is reduced by 59.8%. The method further shows that the content of the plant starch in the tobacco leaves can be regulated and controlled by silencing the NtNUDX14 gene, and further, a certain technical basis can be laid for the regulation and control of the tobacco leaf quality and the cultivation of new tobacco varieties.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

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Claims

1. A tobacco NUDIX hydrolase gene is characterized in that the base sequence is shown as SEQ ID NO. 1.

2. The tobacco NUDIX hydrolase gene according to claim 1, wherein the amino acid sequence of the tobacco NUDIX hydrolase gene is as set forth in SEQ ID No. 2.

3. The tobacco NUDIX hydrolase gene according to claim 1 or 2, wherein the method for preparing the tobacco NUDIX hydrolase gene by PCR amplification comprises the steps of:

(1) extracting genome and reverse transcribing into cDNA for later use;

NtNUDX14-F：5’-GCCACGTTATGGACTTCC-3’，

NtNUDX14-R：5’-GTATGTTCTTTAACCATTGCCTG-3’。

4. the tobacco NUDIX hydrolase gene according to claim 3, wherein the leaf of Honghuadajinyuan of tobacco variety is used as a sample when the genome is extracted in step (1).

5. Use of a tobacco NUDIX hydrolase gene according to any of the preceding claims 1 to 4 wherein the gene expresses a protein which is related to the starch content of the leaves of the plant and wherein the reduction of the protein expression results in a significant reduction of the amylose, amylopectin and total starch content of the leaves.

6. The use of the tobacco NUDIX hydrolase gene according to claim 5, wherein the starch content of tobacco leaves is regulated and controlled by regulating the expression level of the tobacco NUDIX hydrolase gene using a gene silencing technique or a gene overexpression method.

7. The use of the tobacco NUDIX hydrolase gene according to claim 6, wherein a virus-induced silencing vector, an RNAi interference vector, an overexpression vector or a genome editing vector containing the tobacco NUDIX hydrolase gene is constructed by a transgenic technique, a transient expression technique or a genome editing technique, the tobacco is transformed, and new varieties of tobacco with varying starch content are screened.