CN106834301B

CN106834301B - Sabina vulgaris induction gene CML9(Q6-1) for regulating plant nitrogen nutrition and alkali stress and application thereof

Info

Publication number: CN106834301B
Application number: CN201611115698.7A
Authority: CN
Inventors: 张�林; 祁智; 杨红艳; 杨佳; 刘亚玲; 王召明; 苑峰; 金悦
Original assignee: Inner Mongolia M Grass Ecological Environment Group Co Ltd
Current assignee: Mengcao Ecological Environment Group Co Ltd
Priority date: 2016-12-07
Filing date: 2016-12-07
Publication date: 2020-11-13
Anticipated expiration: 2036-12-07
Also published as: CN106834301A

Abstract

The invention relates to a gene CML9(Q6-1) which can regulate plant nitrogen nutrition and alkali stress induction and is derived from a strong xerophyte Sabina vulgaris (Sabina vulgaris) in northwest of inner Mongolia. The gene coded protein has calcium ion binding site. Through the preparation of materials, gene cloning and the subsequent construction of plant expression vector, the gene is further introduced into a model plant Arabidopsis wild type through an agrobacterium-mediated floral flocculation infection method, and multiple single-plant gene insertions, purities and T are finally obtained through screening resistant seedlings₃Transgenic plants are generated. The transgenic plant grows better than a wild type plant under the condition of low nitrogen and grows worse than the wild type plant under the condition of alkali stress, and the protein coded by the gene has the function of regulating the nitrogen nutrition and the alkali stress of the plant. The method provides a new direction for cultivating drought-resistant plants by using a genetic engineering technology, and has important significance for molecular breeding work under drought stress.

Description

Sabina vulgaris induction gene CML9(Q6-1) for regulating plant nitrogen nutrition and alkali stress and application thereof

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to a nitrogen nutrition and alkali stress related induction gene CML9(Q6-1) of Sabina vulgaris (Sabina vulgaris) which is a Sabina vulgaris tree species in a northern desertification region of China. The invention selects the tree species sabina vulgaris in the nursery stock base of Mengolia Mongolian grass drought-resistant stock Limited company as an experimental material, obtains the nitrogen nutrition and alkali stress induction genes of the tree species by using an Illumina Solexa transcriptome high-throughput sequencing method, performs bioinformatics analysis on candidate nitrogen nutrition and alkali stress induction genes CML9(Q6-1), and performs phenotype analysis on different elements of the candidate nitrogen nutrition and alkali stress induction genes, thereby more intuitively understanding the physiological functions of the genes.

Background

Sabina vulgaris (Sabina vulgaris), also called juniperus procumbens and juniperus sinkiana, are mainly distributed in inner Mongolia, Shaanxi, Xinjiang, Ningxia, Gansu, Qinghai, etc. The main cultivation bases include Jiangsu, Zhejiang, Anhui, Hunan and the like. The sabina vulgaris can endure wind erosion and sand burying, adapts to arid desert environment for a long time, and is an excellent tree species for wind prevention, sand fixation and water and soil conservation in arid and semiarid regions. The soil is pleased with light, pleased with cool and dry climate, cold-resistant, drought-resistant, barren-resistant, not strict in soil requirement, not waterlogging-resistant and fast in growth in fertile and permeable soil. The strong-vitality evergreen plant has the functions of increasing the weight in northern greening and afforestation, so that the research on sabina vulgaris has important significance for the development and utilization of genes related to drought resistance of wild plants. At present, researches on sabina vulgaris are mainly focused on the aspects of physiological and ecological characteristics, and no report is made on cloning and utilization of drought-resistant genes of sabina vulgaris.

The molecular breeding capability of plants in China is greatly different from that of developed countries, and the stress-resistant genes with independent intellectual property rights are the focus of competition in the field of molecular breeding of plants in the world. For the development and utilization of plant stress-resistant genes, the current research is mainly focused on crops such as model plants arabidopsis thaliana or rice, wheat and the like, and the excavation of abundant stress-resistant genes in wild plants is lacked. The invention relates to a gene CML9(Q6-1) which can screen out nitrogen nutrition and alkali stress induction for a long time in a natural environment, which is obtained by carrying out transcriptome sequencing on Sabina as a first choice tree species in wind prevention and sand fixation in northern desertification areas of China by utilizing a high-throughput sequencing technology of Illumina company.

Disclosure of Invention

The invention aims to clone a related nitrogen nutrition and alkali stress induction gene CML9(Q6-1) by utilizing a molecular cloning technology to finally obtain an expression vector for gene expression, so that the gene is introduced into an arabidopsis wild plant and is expressed in the wild plant, and the function of the gene is further verified by phenotypic analysis.

The implementation scheme of the invention is that sabina vulgaris in nursery stock base of Mengolia Mongolian grass drought-resistant Limited company is selected as an experimental material, a high-throughput sequencing method of Illumina Solexa transcriptome is adopted to identify and analyze the transcriptome sequence, the type and the quantity of Ca2+ binding protein are determined, so that the nucleotide sequence of the sabina vulgaris related nitrogen nutrition and alkali stress induction gene CML9(Q6-1) is obtained, the gene is obtained through a molecular cloning technology and is introduced into Arabidopsis thaliana, finally a transgenic plant is obtained, and the biological function of the gene is known through phenotypic analysis of the transgenic plant.

An object of the invention is to provide a new sabina chinensis drought-resistant related gene, which is named as CML9(Q6-1) and has a sequence of SEQ No. 1.

The invention relates to a gene with higher expression in sabina vulgaris roots, which is named as CML9(Q6-1), the nucleotide sequence of the gene is shown as SEQ NO.1 or SEQ NO.2 in a sequence table, and the coded 67 amino acid sequences are shown as SEQ NO.3 in the sequence table.

In particular, the invention provides isolated polynucleotides comprising one of the following sequences:

(1) SEQ NO.1 or SEQ NO.2 of the sequence Listing;

(2) DNA sequence which has more than 90% of homology with the DNA sequence limited by SEQ NO.1 or SEQ NO.2 in the sequence table and codes the same functional protein;

the above mentioned related polynucleotides also include substitution, deletion and insertion mutants as well as allelic variants, splice variants, fragments, derivatives, etc.

It will be appreciated by those skilled in the art that the isolated polynucleotides described above also include those sequences which have a high degree of homology with the sequence shown in SEQ NO.1 or SEQ NO.2, for example greater than 95%, or 90%, or even 85%; also included are those sequences which hybridize under stringent conditions to the sequences shown in SEQ NO.1 or SEQ NO. 2; or a sequence which is complementary to the sequence of SEQ NO.1 or SEQ NO. 2.

The invention also provides a technical scheme for applying the novel gene CML9(Q6-1) in plant drought-resistant gene engineering.

The invention successfully separates and obtains the anti-related gene CML9(Q6-1) screened in the natural environment for a long time from sabina vulgaris, which provides a new direction for cultivating drought-resistant plants by using a genetic engineering technology and has important significance for molecular breeding work under drought stress. Specifically, one of the embodiments of the present invention is to apply the CML9(Q6-1) gene to plant genetic engineering to improve the viability of plants under drought stress.

Having generally described the invention, the same may be further understood by reference to certain specific examples provided herein which are intended to be illustrative only and not limiting.

Drawings

FIG. 1: the general flow chart of the experiment.

FIG. 2: CML9(Q6-1) five kinds of T₃Purifying and culturing plants respectively in CK and 0 mu M NO₃ ^-And pH7.0, and the phenotype (A) and fresh weight (B) of the leaves under the growth conditions of the three concentration gradients, and the leaves of the transgenic plants at 0 μ M NO can be more clearly seen from the phenotype (A) and the fresh weight (B) of the leaves₃ ^-The growth vigor is better than that of the wild type Col-0, and the transgenic plant grows at the pH of 7.0 less than that of the wild type plant.

FIG. 3: CML9(Q6-1) five kinds of T₃The generation purity and the plant are respectively 0 mu M Ca²⁺The phenotype (A) and fresh weight (B) of leaves under the growth conditions of three concentration gradients of 1. mu.M ABA and 100mM NaCl, and it can be clearly seen that the leaves of the transgenic plants are not different from those of the wild type plants.

FIG. 4: the culture dish was grown in phenotypic analysis.

Detailed Description

Example 1 Sabina sample Collection

In order to maximize the abundance of RNA associated with stress resistance in vivo, the root of sabina vulgaris was sampled in 12 months (2013) in relatively cold weather, and the samples were rapidly stored in liquid nitrogen for later use after collection.

Example 2 construction of expression vector

1. Since the RNA of the sabina plant is difficult to extract, the target gene in the previous stage is synthesized by Nanjing Kinsley company, and the subsequent construction of the expression vector is completed by the gene synthesized by the company. The cloning vector used to synthesize the gene was pUC57 resistant to Amp.

2. Completion of ligation and construction of the vector of interest

The expression vector used in the present invention was pRI 101AN, and the resistance thereof was Kan.

The connection of the target vector is that the synthesized gene fragment and the target vector are simultaneously subjected to 5': sal I3': and (3) carrying out enzyme digestion on the EcoR I, recovering DNA gel, connecting with DNA ligase, then transforming Escherichia coli, and finally obtaining the plasmid of the connected vector gene by colony PCR, plasmid extraction and enzyme digestion verification.

A double enzyme digestion reaction system:

a. and (3) connecting the target fragment with an expression vector:

principle: connecting according to the mol ratio of the target fragment to the expression vector of 3: 1 or 1: 3;

t4DNA ligase (1. mu.l) + buffer (2. mu.l) + 17. mu.l (fragment of interest + expression vector);

ligation was performed overnight at 16 ℃;

b. and (3) transformation: thawing the escherichia coli competence on ice, converting the product connected with the target fragment and the expression vector into the escherichia coli competence again, mixing uniformly, carrying out ice bath for 30 minutes, carrying out heat shock for 90 seconds at 42 ℃, and immediately placing on ice for 2 minutes; adding 500 μ l of nonresistant LB liquid culture medium, standing and culturing for 60 minutes in a shaking bed at 37 ℃ and a constant-temperature incubator at 200rpm or 37 ℃, and then coating the bacterial liquid on a solid LB plate containing corresponding antibiotics (Kan50 mg/ml); the results were observed after culturing at 37 ℃ in an inverted manner overnight.

c. Single colonies on the plates were picked and added to 5ml LB medium containing Kan (50mg/ml) antibiotic and shake-cultured overnight at 37 ℃.

d. Plasmid extraction: and (3) extracting the plasmid by using a TRAN plasmid extraction kit.

1) 2ml of overnight-cultured bacterial suspension was centrifuged at 10000x g for 1 minute, and the supernatant was removed. If the amount of the bacterial liquid is large, the bacterial liquid can be centrifugally collected for many times.

2) And adding 250 mu l of colorless solution RB (containing RnaseA), and shaking to suspend the bacterial sediment without leaving small bacterial lumps.

3) Adding 250 μ l of blue solution LB, gently turning and mixing for 4-6 times to fully crack the thallus to form a blue transparent solution, wherein the color changes from semi-transparent to transparent blue, indicating complete cracking (not longer than 5 minutes).

4) Add 350. mu.l yellow NB and mix gently 5-6 times (color changed from blue to yellow indicating uniform mixing and complete neutralization) until a compact yellow aggregate was formed and let stand at room temperature for 2 minutes.

5) 12000x g were centrifuged for 5 minutes and the supernatant carefully pipetted into the spin column. 12000x g was centrifuged for 1 min and the effluent discarded. If the volume of the supernatant is more than 800. mu.l, the supernatant can be added to the column in several portions, and centrifuged as above to discard the effluent.

6) Then, 650. mu.l of WB solution was added thereto, and the mixture was centrifuged at 12000x g for 1 minute, and the effluent was discarded.

7) 12000x g was centrifuged for 2 min to completely remove the residual WB.

8) The column was placed in a clean centrifuge tube and 30-50. mu.l EB or deionized water (pH > 7.0) was added to the center of the column and allowed to stand at room temperature for 1 minute.

9) 10000x g for 1 min, and the eluted DNA is stored at-20 ℃.

e. Enzyme digestion verification: the restriction enzyme 5' used above was selected successively: sal I3': EcoR I was used to verify the positive cloning plasmid.

Double enzyme digestion reaction system

The incubator is kept at the constant temperature of 37 ℃ and is used for 30 minutes of warm bath. Detecting by 1% agarose gel electrophoresis, and selecting the strain with correct band type for bacteria preservation (40% yellow-cap glycerol).

10. And (3) agrobacterium transformation: transforming agrobacterium by electric transformation method.

1) Taking the agrobacterium infection state, placing the agrobacterium infection state on ice to melt, adding about 5 mu l of plasmid, uniformly mixing, and placing the mixture on ice for 30 minutes;

2) meanwhile, preparing a clean and dry electric rotating cup, and precooling on ice;

3) wiping the surface of the electric rotating cup, adding the agrobacterium with the plasmid into the electric rotating cup, and then putting the electric rotating cup into an electric rotating instrument for electric excitation transformation;

4) adding an lml LB culture solution without antibiotics into the electric rotating cup, repeatedly blowing and sucking the lml LB culture solution into a 2ml centrifugal tube, and restoring the culture in a shaking table at the temperature of 28 ℃ at 200rpm or a constant-temperature incubator at the temperature of 28 ℃ for 1.5 hours;

5) spreading 500 μ l of the bacterial liquid on a solid LB culture medium containing antibiotics (Rif50mg/ml and Kan50mg/ml), and culturing in an inverted incubator at 28 ℃ for 1-2 days until monoclonals appear;

6) selecting a single colony of the positive agrobacterium tumefaciens to shake the bacteria, shaking the bacteria at 28 ℃, and culturing the bacteria for 1 to 2 days at 250 rpm;

11. extracting agrobacterium tumefaciens plasmids: extracting plasmids by using a TRAN plasmid extraction kit;

3) Adding 250 μ l of blue solution 1B, and gently mixing by turning over for 4-6 times to fully lyse the thallus to form a blue bright solution, wherein the color changes from semi-bright to bright blue, indicating complete lysis (not longer than 5 minutes).

6) Then, 650. mu.l of WB solution was added thereto, and the mixture was centrifuged at 12000Xg for 1 minute, and the effluent was discarded.

7) 12000x g was centrifuged for 2 min to completely remove the residual WB.

9) 10000x g for 1 min, and storing the eluted DNA at-20 deg.C

12. Enzyme digestion verification: selecting restriction enzyme 5': sal I3': the extracted plasmid was verified by EcoR I,

double enzyme digestion reaction system

The incubator is kept at the constant temperature of 28 ℃ and is used for 30 minutes. Detecting by 1% agarose gel electrophoresis, analyzing the result, selecting agrobacterium with correct band to preserve glycerol (40% blue cap) for transforming plants.

Example 3 acquisition of transgenic plants

In the embodiment, a floral infection method is adopted to obtain a transgenic plant code CML9 (Q6-1).

1. Activating Agrobacterium which has been correctly preserved (activation is to ensure the activity of the Agrobacterium), adding the activated Agrobacterium into 5ml LB culture solution containing antibiotics (Rif50mg/ml and Kan50mg/ml), shaking overnight at 28 deg.C, placing the shaken solution into a centrifuge, 5000x g, 10min, discarding the supernatant, adding 1ml of an infection solution (5% sucrose, 0.02% silwet77 suspension in water), and suspending the bacteria by gentle aspiration with a rubber-tipped pipette.

2. Selecting three boxes of wild Col-0 plants which have good growth vigor and already bloomed, cutting off the pod formed and the bud which has finished pollination on the plants, sucking the bacteria liquid drop on the bud which does not bloom by using a rubber head dropper, labeling and marking, and infecting once every 2-3 days until all the plants bloom (note: watering the plants one day before each infection).

3. Timely collecting T after the plants are mature₁Generating seeds, sowing the seeds on a resistant solid MQACK culture dish containing antibiotics (Kan50mg/ml) after the seeds are dried for about one week, firstly placing the seeds in a refrigerator at 4 ℃ for vernalization for three days, then culturing the seeds by illumination for about one week, and carrying out T-shaped positive seedlings₁And (4) screening generations.

4. Selecting good-growth T on a resistant culture dish₁Plant generation, soil shifting culture and single plant labeling, and timely collecting T from single plant after mature₂Generating seeds, drying for one week, spreading on resistant culture medium, culturing for several days under illumination, and selecting T with survival-to-death ratio of 3: 1₂Transplanting soil for culturing, and collecting T from single plant after seed is mature₃Seed generation, drying for one week, and mixing₃And (3) sowing the seeds on MQACK culture medium containing glufosinate-ammonium resistance, and selecting full-living homozygous plants for phenotypic analysis.

Example 4 phenotypic analysis of CML9(Q6-1) in different elements

The MQA medium used in this example mainly contains

Macroelements: 1MKNO₃、1MMgSO₄、1MCaCl₂

Trace elements: MS trace (0.5x),

Fe²⁺Salt: MS Fe²⁺Salt (0.5x)

Mn²⁺Salt: MS Mn²⁺Salt (0.5X) and 0.5M MES buffer

Carbon source: 1% sucrose

a. A specific medium protocol is shown in Table 1(100 ml).

Note: 0 μ M Ca²⁺、0μM NO₃ ^-The two ladders1g of 1.0% agarose is added; 1mM Ca²⁺1.2g of 1.2% agar was added in four gradients of 1. mu.M ABA, 100mM NaCl, pH 7.0; KOH or BTP adjusted to pH 5.7 (note: 1mM Ca)²⁺pH7.0 adjusted to pH7.0 with KOH, 1. mu.M ABA was added 0.5. mu.l after autoclaving, when the medium temperature was lowered to a temperature where the hands could touch).

TABLE 1 media protocol for various elements

b. The planting pattern is shown in figure 4.

The seed dibbling process is carried out under aseptic condition, the seeds are firstly disinfected by 75% alcohol (the seed washing time is not more than 30 minutes) before the seeds are dibbled, then the seeds are washed by 100% alcohol under aseptic condition, and are dried on aseptic filter paper together with the alcohol, and after the seeds are completely dried, the seeds are dibbled on a culture medium by using sterile tweezers.

One repetition of each gradient, sealing with sealing film after finishing seeding, vernalizing in a refrigerator at 4 deg.C for three days, placing in a culture chamber at 22 deg.C and 40% RH for fourteen days, observing the phenotype of CML9(Q6-1) and wild type Col-0, collecting root length and fresh weight data and taking photographs, and showing that CML9(Q6-1) is 0 μ M NO relative to wild type Col-0₃ ^-The leaves grow large; under alkaline conditions (pH 7.0) showed small leaves. Meanwhile, CML9(Q6-1) was added at 100mM NaCl, 0. mu.M Ca to wild type Col-0²⁺There was no clear difference in the several concentration gradients, pH 7.0. As shown in detail (fig. 2, fig. 3).

c. Placement of culture dishes

The 6 gradients are divided into five groups

The foregoing examples further illustrate the present invention and are not to be construed as limiting thereof. It is within the scope of the present invention to modify or replace methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention.

Claims

1. Sabina chinensis (A) and (B)Sabina vulgaris ) The nucleotide sequence of the gene CML9 for regulating the nitrogen nutrition of the plant is shown as SEQ ID No. 2.

2. The polypeptide encoded by the gene CML9 of claim 1, which has the sequence shown in SEQ ID No. 3.

3. A gene expression vector comprising CML9 gene of claim 1.