CN111197048B - Grape vyNRT1 gene and encoding protein thereof and application of gene in drought-resistant variety breeding - Google Patents
Grape vyNRT1 gene and encoding protein thereof and application of gene in drought-resistant variety breeding Download PDFInfo
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Abstract
The invention relates to a grape vyNRT1 gene and an encoding protein thereof and application of the gene in drought-resistant variety breeding, belonging to the technical field of plant genetic engineering. In the invention, by utilizing a transgenic technology of a strong promoter (cauliflower mosaic virus 35S promoter) driving principle, an over-expression vector of a Yanshan grape vyNRT1 gene is transferred into Arabidopsis, so that a transgenic Arabidopsis plant is obtained; experiments prove that compared with an arabidopsis plant transformed with an empty vector, the excessive expression of the vyNRT1 gene leads to the accumulation of stress-resistance related substances in transgenic arabidopsis and the expression of drought-resistance related genes, and the drought resistance of the transgenic plant is enhanced. Therefore, the grape vyNRT1 gene and the recombinant expression vector thereof can be used for breeding drought-resistant varieties of plants.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a grape VyNRT1 gene and an encoding protein thereof and application of the gene in drought-resistant variety breeding.
Background
The grapes are cultivated in the western part of the original product of Asia and are distributed in the northern hemisphere in a concentrated manner about 95% of the grapes in the whole world, and the main production areas of China are regions such as Xiao county of Anhui, tulugua, hetian, tobacco stand in Shandong, zhangjia, xuan, changli, dalian, xiong Yue, shenyang, lumiao county of Henan, civil rights, and Ji of Henan. Moisture has an important influence on the yield and quality of grapes in the growth and development process, and the grape industry is easily threatened by water shortage. Under the large background of lack of water resources, the development of drought-resistant grape resources and the research of drought-resistant genes of the grapes have important scientific values and significance for improving the drought resistance of the grapes, cultivating drought-resistant new varieties, saving water and the like.
Absorption and transport of nitrogen plays an important role in adapting plants to drought stress, and plant root systems play a role in NO 3- Is absorbed by (and) and NO 3- Transport in plants is mainly composed of NO 3- Transporter (nitrate transporters, NRTs) mediated. NO found in higher plants 3- Transport proteins mainly include two families of proteins, NRT1 and NRT 2. In the model plant Arabidopsis, more than 50 NRT1 family members have been found, but only 10 of them have been studied for their function, and it is clear that the study of NRT1 family gene function in grape needs further investigation. It is expected to find out the gene related to grape growth and development and resisting abiotic stress, and the gene will provide new thought for genetic improvement of grape stress resistance.
Disclosure of Invention
The invention aims to provide a grape vyNRT1 gene which can increase accumulation of stress-resistance related substances in transgenic plants and expression of drought-resistance related genes and promote drought resistance enhancement of the transgenic plants. The grape vyNRT1 protein coded by the gene can promote the accumulation of stress resistance related substances in transgenic plants, so that the drought resistance of the transgenic plants is enhanced.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a grape gene VyNRT1, wherein the nucleotide sequence of the grape gene VyNRT1 is shown as SED ID NO. 1.
The invention also provides a grape gene VyNRT1 coding protein, the amino acid sequence of the coding protein is shown as SED ID NO.2, and the nucleotide sequence of the coding protein is positioned at the 16 th to 1962 th positions in SED NO. 1.
The invention also provides application of the grape gene VyNRT1 in plant drought-resistant variety breeding.
Preferably, the grape gene VyNRT1 is applied to the breeding of arabidopsis thaliana or grape drought-resistant varieties.
In the invention, by utilizing a transgenic technology of a strong promoter (cauliflower mosaic virus 35S promoter) driving principle, an over-expression vector of a Yanshan grape vyNRT1 gene is transferred into Arabidopsis, so that a transgenic Arabidopsis plant is obtained; experiments prove that compared with an arabidopsis plant transformed with an empty vector, the excessive expression of the vyNRT1 gene leads to the accumulation of stress-resistance related substances in transgenic arabidopsis and the expression of drought-resistance related genes, and the drought resistance of the transgenic plant is enhanced. Therefore, the grape vyNRT1 gene and the recombinant expression vector thereof can be used for breeding drought-resistant varieties of plants.
Drawings
FIG. 1 is a diagram showing analysis of expression of the vyNRT1 gene of a grape in the present invention;
FIG. 2 is a drought resistance identification chart of an arabidopsis plant transformed with the vyNRT1 gene;
FIG. 3 is a graph showing the physiological characteristics of transgenic Arabidopsis plants transformed with the vyNRT1 gene according to the present invention;
FIG. 4 is a diagram showing the analysis of the expression of drought-resistance-related genes in transgenic Arabidopsis plants according to the present invention.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
EXAMPLE 1 analysis of grape vyNRT1 Gene expression
After the Yanshan grape tissue culture seedlings are subcultured for 16 days, the seedlings which are strong and consistent in growth performance are selected for various adversity treatments.
Drought treatment: grape seedlings were pulled out of the medium, placed on filter paper, exposed to conditions of room temperature (32.+ -. 1) C, relative humidity 55%, photoperiod 14 h/10 h darkness, and sampled at 0, 2, 6, 12, 24 h.
And (3) low-temperature treatment: culturing the tissue culture seedling under the conditions of temperature (4+ -1), relative humidity of 75%, light period of 14 h/10 h in dark, and sampling at 0, 2, 6, 12, 24 h.
Salt stress: 20mL 100 mmol/L was added to a flask -1 Is cultured at a temperature of (25.+ -. 1) DEG C, a relative humidity of 75% and a photoperiod of 14 hours/10 hours in the dark, and is sampled at 0, 2, 6, 12, 24 hours.
An equal volume of distilled water was added to the flask as a control for salt stress treatment. Normally cultured tissue culture seedlings served as controls for drought and low temperature treatment.
The Yanshan grape growing in the field for 8-10 years, the grape fruit is taken in the color transferring period, and the root system (first new-born side root), stem (stem segment of 4 th-5 th leaves under the newly-developed leaf), leaf (4 th-5 th leaves under the newly-developed leaf), inflorescence, tendril (1 st branch of the newly-born branch) and other samples are taken in the full bloom period. Extracting total RNA of grape leaf by using plus plant total RNA extraction kit (radix angelicae). The first strand of cDNA was synthesized by ordinary reverse transcription using PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa).
The specific operation steps are as follows: adding the mixture into a PCR tube: random 6mers (50. Mu.M) 1. Mu.L, dNTP mix (10 mM each) 1. Mu.L, total RNA 2. Mu.g, RNase free dH 2 O was added to 10. Mu.L, mixed well and centrifuged transiently to bring the solution to the bottom of the PCR tube. The reaction was carried out on a PCR instrument at 65℃for 5min, and quenched on ice.
Real-time fluorescent quantitative PCR primer is designed according to the VyNRT1 gene sequence,
the forward primer sequence is qRT-PCR-vyNRT1-F:
5'-CCCTTATCCTTCTCATCCCAGT-3' (SEQ ID NO. 3);
the reverse primer sequence is qRT-PCR-vyNRT1-R:
5'-GGAGCTTTATTACCATCTTCACCT-3' (SEQ ID NO. 4).
The VyGAPDH gene is used as an internal reference,
the forward primer sequence is qRT-vyGAPDH-F:
5'-CCCTTGTCCTCCCAACTCT-3' (SEQ ID NO. 5);
the reverse primer sequence is qRT-VyGAPDH-R:
5'-CCTTCTCAGCACTGTCCCT-3' (SEQ ID NO. 6).
Real-time fluorescent quantitative PCR was performed according to TaKaRa
Premix Ex Taq TM II (Perfect Real Time) it is described that this is performed on Bio-Rad IQ5 Real-Time PCR Detection System (Bio-Rad Laboratories, herc. Mu. Les, calif.). 25. Mu.L of reaction system: 1. Mu.L of reverse transcription template; forward and reverse primers were each 1 μl;12.5 mu L
Premix Ex Taq TM (2×); 9 μl of nucleic-free water. The reaction procedure is: 95 ℃ for 30s;40cycles of95 ℃ for 5s;57 ℃ for 30s;72℃for 30s. Results Using 2 -ΔΔC(t) The method was used for analysis.
The result is shown in figure 1, and shows that the VyNRT1 gene is mainly expressed in root system, and secondly has higher expression level in She Li and lower expression level in stems, flowers, fruits and tendrils; 2h after low temperature treatment, vynrt1 transcripts accumulated rapidly, reaching a peak at 6h, followed by gradual decrease; 2h after drought and high salt treatment, vynrt1 transcripts accumulated rapidly and peaked, and then decreased.
Test example 2 acquisition of transgenic plants
The ORF fragment comprising the coding region of the vyNRT1 gene was inserted into a plant over-expression vector and transformed into Agrobacterium. Streaking agrobacterium containing recombinant plant expression vector on LB plate (containing 60mg/L Gent,100mg/L Kan), culturing at 28deg.C for 24 hr; picking up monoclonals and culturing in 10mL LB liquid medium (with corresponding antibiotics added) at 28 ℃ for 24 hours; transferring 5mL of bacterial liquid into 50mL of fresh LB liquid culture medium, and continuously culturing at 28 ℃ until the bacterial liquid OD600 reaches about 0.6; transferring into a centrifugal bottle or a centrifugal tube, centrifuging at 4000rpm for 10min at room temperature, and removing supernatant to collect thalli; resuspended in permeation buffer (0.5 XS, 5% sucrose, 0.03% Silwet L-77 (GE Health)), OD600 was adjusted to 0.8; removing the existing pod on the arabidopsis inflorescence, immersing the inflorescence into the permeate liquid completely for 10-30s (or directly dripping the permeate liquid on the inflorescence by using a liquid transfer device), immediately removing the permeate liquid on the arabidopsis leaves or stems, putting the plant flat in a tray, covering the tray with a plastic film, taking down the film after 24 hours, and continuously culturing in a greenhouse; to increase the conversion efficiency, infection was again carried out after 7 days by the same method; the transformed Arabidopsis plants are normally managed, and seeds are harvested when the pods are white. And screening the plants by kanamycin to obtain the VyNRT1 transgenic plants and transformed empty vector plants. Transgenic plants and empty vector plants were grouped and named, with empty vector groups named EV and VyNRT1 transgenic arabidopsis plants were grouped into 3 groups named oe# 1, oe# 2, oe# 3, respectively.
Test example 3 drought resistance identification of transgenic Arabidopsis plants
After the VyNRT1 transgenic plants and the arabidopsis thaliana transformed with the empty vector (EV expression) were grown on the MS medium for 7 days, they were transferred to a nutrition pot, and normally watered for 20 days to grow into robust seedlings. And then stopping watering the arabidopsis seedlings, namely performing drought treatment until obvious desiccation and wilting symptoms appear on part of arabidopsis plant leaves on the 7 th day. And then rehydrating all plants, and observing the growth condition of the plants after 48 hours.
The phenotype of the arabidopsis plants before and after drought treatment and after rehydration is recorded by photographing, the result is shown in figure 2, and compared with the arabidopsis transformed with an empty vector, the drought resistance of the arabidopsis plants OE# 1, OE# 2 and OE# 3 transformed with the vyNRT1 gene is obviously enhanced as shown in figure 2.
Test example 4 analysis of physiological and Biochemical characteristics of transgenic Arabidopsis plants
Determination of the Water loss Rate: after the VyNRT1 transgenic plant and the transformation empty vector plant grow normally for 3 weeks, about 0.2g rosette leaves are taken for water loss rate measurement. The collected rosette leaves were placed on dry filter paper, and the Fresh Weight (FW) of the leaves was measured every 10min until the water loss rate measurement was completed when 50min was measured. The ratio of the water loss per measurement to the fresh weight measured for the first time was taken as the water loss rate.
Determination of electrolyte leakage rate (conductivity): the leaves were placed in a centrifuge tube, the volume was set to 10mL with super deionized water, and the conductivity of the solution was measured after shaking for 1 hour at room temperature and recorded as C1 before boiling. The solution was then boiled in boiling water for 10min together with the leaves, and the electrical conductivity was measured after isothermal cooling to room temperature and was noted as C2. The ratio of C1 to C2 (C1/C2) was taken as the relative electrolyte leakage value.
The detection result is shown in figure 3, (A) the expression level of the VyNRT1 gene in the transgenic arabidopsis plant is detected; (B) Survival statistics of VyNRT1 gene arabidopsis plants after 18d drought treatment; (C) relative water loss rate of arabidopsis thaliana leaves transformed with VyNRT1 gene; (D) Relative conductivity of VyNRT1 transgenic arabidopsis plants after 18d drought treatment. The graph shows that the expression level of the VyNRT1 gene in the transgenic arabidopsis plant is higher, and the survival rate is obviously improved relative to that of the plant transformed with the empty vector; the water loss rate and conductivity in transgenic VyNRT1 transgenic arabidopsis plants were significantly reduced relative to transformed empty vector plants.
Test example 5 transgenic drought-resistance-related Gene expression analysis of Arabidopsis thaliana
And extracting the total RNA of transgenic arabidopsis leaves after drought treatment by using a plus plant total RNA extraction kit. PrimeScript for ordinary reverse transcription II 1st Strand cDNA Synthesis Kit (TaKaRa) first strand cDNA was synthesized. The specific operation steps are as follows: adding the mixture into a PCR tube: random 6mers (50. Mu.M) 1. Mu.L, dNTP mix (10 mM each) 1. Mu.L, total RNA 2. Mu.g, RNase free dH 2 O was added to 10. Mu.L, mixed well and centrifuged transiently to bring the solution to the bottom of the PCR tube. The reaction was carried out on a PCR instrument at 65℃for 5min, and quenched on ice. The expression of drought-resistant related genes AtCOR15A, atERD and AtRD29A, atP CS1 genes in transgenic Arabidopsis plants is detected by taking Arabidopsis AtActin as an internal reference gene.
The primers designed are as follows:
qRT-AtActin-F:5'-CGGTGGTTCTATCTTGGCATC-3' (SEQ ID NO. 7);
qRT-AtActin-R:5'-GTCTTTCGCTTCAATAACCCTA-3' (SEQ ID NO. 8);
qRT-AtCOR15A-F:5'-CAGCGGAGCCAAGCAGAGCAG-3' (SEQ ID NO. 9);
qRT-AtCOR15A-R:5'-CATCGAGGATGTTGCCGTCACC-3' (SEQ ID NO. 10);
qRT-AtERD15-F:5'-CCAGCGAAATGGGGAAACCA-3' (SEQ ID NO. 11);
qRT-AtERD15-R:5'-ACAAAGGTACAGTGGTGGC-3' (SEQ ID NO. 12);
qRT-AtRD29A-F:5'-GTTACTGATCCCACCAAAGAAGA-3' (SEQ ID NO. 13);
qRT-AtRD29A-R:5'-GGAGACTCATCAGTCACTTCCA-3' (SEQ ID NO. 14);
qRT-AtP5CS1-F:5'-CGACGGAGACAATGGAATTGT-3' (SEQ ID NO. 15);
qRT-AtP5CS1-R:5'-GATCAGAAATGTGTAGGTAGC-3' (SEQ ID NO. 16).
Real-time fluorescent quantitative PCR was performed according to TaKaRa
Premix Ex Taq TM II (Perfect RealTime) it is described that this is performed on Bio-Rad IQ5 Real-Time PCR DetectionSystem (Bio-Rad Laboratories, herc. Mu. Les, calif.). 25. Mu.L of reaction system: 1. Mu.L of reverse transcription template; forward and reverse primers were each 1 μl; 12.5. Mu.L->
Premix Ex Taq TM (2×); 9 μl of nucleic-free water. The reaction procedure is: 95 ℃ for 30s;40cycles of95 ℃ for 5s;57 ℃ for 30s;72℃for 30s. Results Using 2 -ΔΔC(t) The method was used for analysis.
The detection result is shown in fig. 4, and it can be seen from fig. 4 that under drought conditions, compared with the transformation of an empty vector arabidopsis thaliana, the expression level of the drought-resistance related gene in the transgenic arabidopsis thaliana plant is obviously increased, which indicates that the wild grape VyNRT1 gene in the invention can increase the accumulation of the stress-resistance related substances in the transgenic plant and the expression of the drought-resistance related gene, and promote the enhancement of the drought resistance of the transgenic plant.
From the above embodiments, the invention provides a grape gene VyNRT1 and application thereof in drought-resistant breeding. In the invention, by utilizing a transgenic technology of a strong promoter (cauliflower mosaic virus 35S promoter) driving principle, an over-expression vector of a Yanshan grape vyNRT1 gene is transferred into Arabidopsis, so that a transgenic Arabidopsis plant is obtained; experiments prove that compared with an arabidopsis plant transformed with an empty vector, the excessive expression of the vyNRT1 gene leads to the accumulation of stress-resistance related substances in transgenic arabidopsis and the expression of drought-resistance related genes, and the drought resistance of the transgenic plant is enhanced. Therefore, the grape vyNRT1 gene and the recombinant expression vector thereof can be used for breeding drought-resistant varieties of plants.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Sequence listing
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<213> Artificial sequence (Artificial Sequence)
<400> 6
ccttctcagc actgtccct 19
<210> 7
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
cggtggttct atcttggcat c 21
<210> 8
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
gtctttcgct tcaataaccc ta 22
<210> 9
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 9
cagcggagcc aagcagagca g 21
<210> 10
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 10
catcgaggat gttgccgtca cc 22
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 11
<210> 12
<211> 19
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 12
acaaaggtac agtggtggc 19
<210> 13
<211> 23
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 13
gttactgatc ccaccaaaga aga 23
<210> 14
<211> 22
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 14
ggagactcat cagtcacttc ca 22
<210> 15
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 15
cgacggagac aatggaattg t 21
<210> 16
<211> 21
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 16
gatcagaaat gtgtaggtag c 21
Claims (1)
1. The application of the over-expressed grape gene VyNRT1 in breeding of plant drought-resistant varieties is that the plant drought-resistant varieties are arabidopsis, the nucleotide sequence of the grape gene VyNRT1 is shown as SED ID NO.1, and the amino acid sequence of the grape gene VyNRT1 is shown as SED ID NO. 2.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202010020692.1A CN111197048B (en) | 2020-01-09 | 2020-01-09 | Grape vyNRT1 gene and encoding protein thereof and application of gene in drought-resistant variety breeding |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010020692.1A CN111197048B (en) | 2020-01-09 | 2020-01-09 | Grape vyNRT1 gene and encoding protein thereof and application of gene in drought-resistant variety breeding |
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| Publication Number | Publication Date |
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| CN111197048A CN111197048A (en) | 2020-05-26 |
| CN111197048B true CN111197048B (en) | 2023-04-28 |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101535483A (en) * | 2004-12-16 | 2009-09-16 | 赛乐斯股份有限公司 | Modulating plant nitrogen levels |
| CN104774850A (en) * | 2015-04-03 | 2015-07-15 | 华中农业大学 | BnNRT1-3 gene overexpression for improving rape nitrogen utilization rate |
| US20160010101A1 (en) * | 2013-03-13 | 2016-01-14 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over- expressing maize functional low-affinity nitrate transporters in transgenic maize |
-
2020
- 2020-01-09 CN CN202010020692.1A patent/CN111197048B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101535483A (en) * | 2004-12-16 | 2009-09-16 | 赛乐斯股份有限公司 | Modulating plant nitrogen levels |
| US20160010101A1 (en) * | 2013-03-13 | 2016-01-14 | Pioneer Hi-Bred International, Inc. | Enhanced nitrate uptake and nitrate translocation by over- expressing maize functional low-affinity nitrate transporters in transgenic maize |
| CN104774850A (en) * | 2015-04-03 | 2015-07-15 | 华中农业大学 | BnNRT1-3 gene overexpression for improving rape nitrogen utilization rate |
Non-Patent Citations (2)
| Title |
|---|
| "Responses of grapevine rootstocks to drought through altered root system architecture and root transcriptomic regulations";Kubilay Yildrim 等;《Plant Physiology and Biochemistry》;20180331;第127卷;摘要、第3.4节、第4.1节、表2 * |
| "茶树硝态氮转运蛋白NRT1.1基因的克隆及表达分析";杨亦扬 等;《茶叶科学》;20161231;第36卷(第5期);第505-512页 * |
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| CN111197048A (en) | 2020-05-26 |
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