Application of RIE1 gene in delaying plant leaf senescence and improving adversity resistance

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a RIE1 gene in delaying plant leaf senescence and improving adversity resistance, and a coding sequence of the RIE1 gene and corresponding amino acid sequences of the RIE1 gene are shown as SEQ NO.1 and SEQ NO.2. A phenotype observation experiment result shows that compared with a wild type, a 48-day-old RIE1 overexpression strain has an obvious late decline phenotype; a saline-alkali stress experiment result shows that saline-alkali stress can obviously inhibit germination of wild type control seeds, but the germination rate of an RIE1 overexpression material is not influenced; saline-alkali stress can significantly inhibit the growth of main roots of wild control seedlings, but the inhibition degree of the growth of the main roots of RIE1 overexpression materials is obviously reduced, which indicates that the RIE1 overexpression materials are more tolerant to saline-alkali stress. The overexpressed RIE1 gene provided by the invention can obviously delay the aging of transgenic plants and obviously improve the tolerance of the transgenic plants to saline-alkali stress, and is expected to be applied to the cultivation of new varieties for delaying the aging of plant / crop leaves and improving the stress resistance.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to RIE1 The application of genes in delaying plant leaf senescence and improving stress resistance. Background Art

[0002] The transmission of senescence signals and the pathways of stress response intersect with each other, forming a complex regulatory network. The generation of this cross-adaptability involves changes in the ultra-anatomical structure of leaves, osmotic regulating substances, reactive oxygen scavenging systems, plant hormones, photosynthetic systems, and heat shock protein components. Senescence is the last stage of leaf development and is jointly regulated by environmental and developmental signals and changes in endogenous hormone levels. The onset and development of leaf senescence significantly affects the source-sink transport of nutrients in the plant, and is closely related to crop yield and quality.

[0003] Environmental adversity is also an important factor that restricts the growth, distribution and yield of crops. For example, under alkaline stress, high pH values ​​can cause changes in the osmotic potential of plants, imbalances in ion concentrations, and damage to cell membranes, which can seriously disrupt the balance of plant nutrition and metabolism, reduce photosynthetic efficiency, and even lead to plant death.

[0004] Therefore, it is of great scientific value and practical significance to explore the regulatory mechanisms of plant anti-aging and stress resistance, isolate and identify the key node genes of the cross-regulation between the two, and cultivate new anti-aging and stress-resistant varieties. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the related art. To this end, the purpose of the present invention is to provide RIE1 The application of genes in delaying plant leaf senescence and improving stress resistance.

[0006] In order to achieve the purpose of the invention, the technical solution adopted by the present invention is: RIE1 The application of genes in delaying plant leaf senescence and improving stress resistance, RIE1 The coding sequence of the gene is shown in SEQ NO. 1. The nucleotide sequence from the 5' end to the 3' end of SEQ NO. 1 is as follows:

[0007] Further, RIE1 The amino acid sequence of the RIE1 protein encoded by the gene is shown in SEQ NO. 2: MGELRRYRFKAMEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMESLGDLTMEQKLISEEDLNSVSRTSGSMSSYSSDSTAARDQHAPLLRP RHDGSFSSSSSARPTALAVLLGRITGHRAPSMLVRETAARALEERRIDWGYSKPVVAADILWNAALVLASAVMLVGTVEERPNEPIRVWICVYGLQCLFHVVLVWSEYWRRN STRRARDLESYDHEDYNIEYDYEQDSDDNSTTYSFVKRCESINTVISFIWWIIGFYWVVEGGDKLLGEAPNLYWLSVIFLAIDVFFAVFCVVLACLVGIALCCCLPCIIALLY AVAGTEGVSEAELGVLPLYKFKAFHSNEKNITGPGKMVPIPINGLCLATERTLLAEDADCCICLSSYEDGAELHALPCNHHFHSTCIVKWLKMRATCPLCKYNILKGTTDQS.

[0008] Furthermore, the application includes application in plant breeding.

[0009] Furthermore, the application in plant breeding includes 35S:6Myc-RIE1 Expression vector construction.

[0010] Further, 35S:6Myc-RIE1 The expression vector was constructed by PCR amplification using wild-type Arabidopsis cDNA as a template.

[0011] Furthermore, the primers used for PCR amplification were RIE1-6myc-F and RIE1-6myc-R ;in, RIE1- 6MYC-F The nucleotide sequence is shown in SEQ NO. 3, RIE1-6myc-R The nucleotide sequence is shown in SEQ NO. 4; The nucleotide sequence from the 5' end to the 3' end of SEQ NO. 3 is as follows: GTATCTAGAACTAGTGGATCCATGTCATCGTATTCT.

[0012] The nucleotide sequence from the 5' end to the 3' end of SEQ NO. 4 is as follows: GGGCCCCCCCTCGAGGTCGACTCAAGATTGATCAGT.

[0013] Further, 35S:6Myc-RIE1 The double restriction enzyme digestion system was used for PCR amplification in the construction of the expression vector.

[0014] Furthermore, the endonuclease used in the double enzyme digestion system is an endonuclease Bam HI and endonuclease Sal I.

[0015] Further, including transformation, by Agrobacterium-mediated transformation 35S:6Myc-RIE1 Expression vector.

[0016] Further, it also includes impregnation, using conversion 35S:6Myc-RIE1 Agrobacterium infection of Arabidopsis thaliana inflorescences with the expression vector.

[0017] The above one or more technical solutions in the embodiments of the present invention have at least one of the following technical effects: The results of the senescence phenotype observation experiment showed that compared with the wild-type control, the 48-day-old RIE1 The overexpression strains had an obvious late senescence phenotype; the results of the saline-alkali stress experiment showed that saline-alkali stress significantly inhibited the germination of wild-type control seeds, but RIE1 The germination rate of the overexpression material was not affected; saline-alkali stress significantly inhibited the growth of the taproot of the wild-type control seedlings, but RIE1 The degree of inhibition of taproot growth in the overexpression material was significantly reduced, indicating that it is more tolerant to saline-alkali stress. RIE1 The gene can significantly delay the aging of transgenic plants and significantly improve their tolerance to salt-alkali stress, and is expected to be widely used in delaying the aging of plant / crop leaves and breeding new varieties with improved stress resistance.

[0018] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the present invention or the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1The 5 provided in Example 1 of the present invention 35S:6Myc-RIE1 Immunoblotting of exogenous RIE1 protein in independent transgenic lines (lines 11, 18, 23, 25, and 30) and their wild-type control (WT), with actin used as a loading control.

[0021] Figure 2 The 5 provided in Experimental Example 1 of the present invention 35S:6Myc-RIE1 Plant phenotypes of independent transgenic lines (lines 11, 18, 23, 25, and 30) and their wild-type controls (WT) at 48 days of growth in culture soil.

[0022] Figure 3 The experimental example 2 of the present invention provides RIE1 Transgenic Arabidopsis thaliana ( RIE1ox ) and its wild-type control (WT) under saline-alkali stress and normal growth conditions every 12 hours for 96 hours.

[0023] Figure 4 The experimental example 3 of the present invention provides RIE1 Transgenic Arabidopsis thaliana ( RIE1ox ) and its wild-type control (WT) under saline-alkali stress and normal growth conditions. DETAILED DESCRIPTION

[0024] In order to make the purpose, technical scheme and advantages of the present invention clearer, the technical scheme of the present invention will be clearly and completely described below in conjunction with the drawings in the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without creative work are within the scope of protection of the present invention. The following embodiments are used to illustrate the present invention, but cannot be used to limit the scope of the present invention.

[0025] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0026] Example 1 one, RIE1 The gene's coding sequence (CDS), amino acid sequence, and primer sequence used for amplification.

[0027] The nucleotide sequence of RIE1 CDS, i.e., the sequence from 5' to 3' end of SEQ NO. 1 is as follows:

[0028] The amino acid sequence of RIE1 protein, SEQ NO. 2, is as follows: MGELRRYRFKAMEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMEQKLISEEDLNEMESLGDLTMEQKLISEEDLNSVSRTSGSMSSYSSDSTAARDQHAPLLRP RHDGSFSSSSSARPTALAVLLGRITGHRAPSMLVRETAARALEERRIDWGYSKPVVAADILWNAALVLASAVMLVGTVEERPNEPIRVWICVYGLQCLFHVVLVWSEYWRRN STRRARDLESYDHEDYNIEYDYEQDSDDNSTTYSFVKRCESINTVISFIWWIIGFYWVVEGGDKLLGEAPNLYWLSVIFLAIDVFFAVFCVVLACLVGIALCCCLPCIIALLY AVAGTEGVSEAELGVLPLYKFKAFHSNEKNITGPGKMVPIPINGLCLATERTLLAEDADCCICLSSYEDGAELHALPCNHHFHSTCIVKWLKMRATCPLCKYNILKGTTDQS.

[0029] Primers RIE1-6myc-F The nucleotide sequence, i.e., SEQ NO. 3, is as follows from the 5' end to the 3' end: GTATCTAGAACTAGTGGATCCATGTCATCGTATTCT.

[0030] Primers RIE1-6myc-R The nucleotide sequence, i.e., SEQ NO. 4, is as follows from the 5' end to the 3' end: GGGCCCCCCCTCGAGGTCGACTCAAGATTGATCAGT.

[0031] two, RIE1 Creation and phenotypic analysis of overexpressing Arabidopsis thaliana.

[0032] Phenotypic identification plays a vital role in the study of plant gene function, which can help us understand the function of the target gene and its role in plant growth, development and environmental adaptation. Phenotype is a direct reflection of gene expression and function. By observing and analyzing the phenotype of plants, we can provide support for the function of the target gene, and thus provide new ideas for plant breeding and agricultural production.

[0033] 1. 35S:6Myc-RIE1 Construction of expression vector.

[0034] Primers RIE1-6myc-F and RIE1-6myc-R PCR, using wild-type Arabidopsis cDNA as template, PCR amplified RIE1 The target fragment of the full-length gene coding sequence.

[0035] Among them, the primer sequence SEQ NO. 3 GGATCC The sequence is Bam HI restriction site, primer sequence SEQNO.4 GTCGAC The sequence is Sal I restriction enzyme cutting site.

[0036] The PCR amplification system was: 2×KOD One™ PCR premix (TOYOBO) 25 μL, ddH2O 22 μL, RIE1-6myc-F (10 μmol / L)1 μL, RIE1-6myc-R (10 μmol / L) 1 μL, template DNA 1 μL. PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles; 72℃ final extension for 10 min; 4℃ storage.

[0037] After PCR amplification, agarose gel electrophoresis was performed and the target fragment was recovered by gel extraction using a gel recovery kit (Axygen).

[0038] Using restriction enzymes from Takara Bam HI and Sal I double restriction enzyme vector pCAMBIA1307 , the enzyme digestion reaction system is: vector plasmid 20 μL, ddH2O 3 μL, 10× Q Cut Buffer 3 μL, Bam HI and Sal I 2 μL each, digest at 37℃ for 1 h. After electrophoresis detection, use Axygen's gel recovery kit to recover the large vector fragment.

[0039] Among them, large fragment expression vector refers to a vector that can carry large DNA fragments and express them in host cells. Large fragment expression vectors can be of the type of plasmids, viral vectors, artificial chromosomes, etc.

[0040] The target fragment and the vector large fragment were connected by homologous recombination technology using the seamless cloning kit of Beyotime Company to obtain 35S:6Myc-RIE1Expression vector construction. The ligation system was: 2X Seamless Cloning Mix 5 μL, PCR amplification target fragment product 3 μL, enzyme-cut vector 2 μL, react at 50℃ for 15 min and place on ice to complete the ligation.

[0041] The ligation product was transformed into E. coli competent DH5ɑ by heat shock method, spread on LB solid plate containing 50 μg / mL kanamycin, and cultured at 37℃ overnight. RIE1-6myc-F and RIE1-6myc-R PCR identification was performed, positive clones were shaken, plasmids were extracted using a plasmid extraction kit, and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification to obtain the correct 35S:6Myc-RIE1 Expression vector.

[0042] 2. Screening and identification of transgenic Arabidopsis thaliana.

[0043] The constructed vector 35S:6Myc-RIE1 Transform GV3101 Agrobacterium. The specific process is as follows: add 1 μg DNA to 200 μL Agrobacterium competent cells, place on ice for 30 min, freeze with liquid nitrogen for 1 min, warm at 37 °C until thawed, add 1 mL YEP liquid medium to the tube, and culture at 28 °C for 4 h. Collect the bacteria by centrifugation and spread on LB plates containing 50 μg / mL kanamycin, 25 μg / mL gentamicin, and 25 μg / mL rifampicin, and place them in an incubator at 28 °C and culture them upside down. Pick a single colony and use primers to RIE1-6myc-F and RIE1-6myc-R Perform PCR identification; shake the positive clones identified by PCR until OD 600 When the concentration is greater than 1.0, it is used for infection of Arabidopsis inflorescence.

[0044] The specific process of inflorescence infection is: centrifuge the bacterial solution, collect the precipitate, and then resuspend the bacteria with the prepared transformation solution, wherein the composition of the transformation solution is: 10 mM 1 / 2MS medium, 2.5 mM 2-morpholineethanesulfonic acid (4-Morpholineethanesulfonic acid, abbreviated as MES, 5% sucrose, 1 mg / mL 6-benzylaminopurine (abbreviated as 6-BA). Before infecting Arabidopsis, 50 mL / L of polysiloxane polyether (SilwetL-77) was added, and the inflorescence part of Arabidopsis was fully contacted with the bacterial solution. After the infection was completed, it was first cultured in the dark for 24 hours, and then cultured under normal conditions until the end of the growth cycle. After harvesting the seeds, hygromycin selection medium was used to screen and combine genomic PCR to identify T1 generation seeds. The positive plants obtained by soil culture screening were collected. After maturity, T2 generation seeds were collected for hygromycin resistance screening, and single copy strains were identified according to the segregation ratio of Mendel's law of inheritance until T3 generation single copy homozygous strains were obtained.

[0045] The specific method of genomic PCR is: sampling, extracting genomic DNA, 6Myc-F and RIE1-464-R The PCR amplification system was as follows: 10 μL of 2×Taq PCR Mix (GenStar), 8 μL of ddH2O, 6Myc-F (10 μmol / L) 0.5 μL, RIE1-464-R (10 μmol / L) 0.5 μL, template DNA 1 μL. PCR amplification program: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 30 cycles; 72℃ final extension for 10 min; 4℃ storage.

[0046] 6MYC-F The nucleotide sequence is shown below: 5′-CGACGGTATCGATTTAAAGC-3′; RIE1-464-R The nucleotide sequence is shown below: 5′-ATCACTGTCTTGTTCATAAT-3′; The protein level detection method of the T3 generation single-copy homozygous strains was as follows: the total plant protein of different single-copy homozygous strains was extracted with protein extraction buffer (50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.5 M EDTA (pH 8.0), 1% Triton X-100 and EDTA-free protease inhibitor cocktail), and the accumulation level of 6Myc-RIE1 protein was detected by SDS-PAGE electrophoresis combined with western blot immunoblotting, and anti-Myc antibody (Proteintech; 16286-1-AP) was used, and anti-actin antibody (Abmart; M20009L) was used as a loading control. The accumulation level of RIE1 protein in each single-copy homozygous strain of the T3 generation was detected by immunoblotting using anti-Myc antibody to detect 5 35S:6Myc-RIE1 The accumulation level of exogenous RIE1 protein in independent transgenic lines (lines 11, 18, 23, 25, and 30), with actin protein as a loading control, was identified as follows Figure 1 shown.

[0047] Experimental Example 1 The specific process of aging phenotype observation experiment is as follows: Will 35S:6Myc-RIE1 Transgenic Arabidopsis and wild-type control seeds were sterilized with 1% (v / v) NaClO for 2-3 min, washed with sterile distilled water 6-8 times, and spread on normal 1 / 2 MS medium. After two days of dark treatment, they were placed in an Arabidopsis incubator for culture. The culture conditions were 20°C-22°C long day (16 h light and 8 h dark), and the light intensity was 90 μmol·m −2 ·s −1 ~110 μmol·m −2 ·s −1 , relative humidity 60%~70%. Then, Arabidopsis seedlings grown on 1 / 2 MS medium for 7 to 10 days with the same growth and development status were transferred to the culture soil, and the phenotypes of the plants at different growth stages were recorded by taking photos. Figure 2 As shown, compared with the wild-type control (WT), 35S:6Myc-RIE1 Leaf senescence of independent transgenic lines (lines 11, 18, 23, 25, and 30) was significantly delayed.

[0048] Experimental Example 2 The specific process of the salt-alkali stress germination experiment is as follows: Will 35S:6Myc-RIE1After sterilization, transgenic Arabidopsis and wild-type WT control seeds were planted in normal 1 / 2 MS medium and 1 / 2 MS medium containing 8 mM NaHCO3, and placed in an Arabidopsis incubator for two days in the dark at 4°C (i.e., stratification treatment). Germination rates were counted every 12 h. Microsoft Excel was used to calculate the germination rates of different materials, and the mean and standard error were calculated. Figure 3 As shown in Figure 2, the results showed that salinity stress significantly inhibited WT seed germination, but RIE1 The germination rate of the overexpression material was not significantly affected.

[0049] Experimental Example 3 The main root growth experiment under saline-alkali stress, the specific process is as follows: After the transgenic Arabidopsis and wild-type control seeds were sterilized, they were plated on 1 / 2 MS and 1 / 2 MS vertical plates containing 2.5 mM NaHCO3, placed in the dark at 4°C for two days, and then grown vertically in an Arabidopsis incubator for five days. The root length of Arabidopsis was counted. Microsoft Excel was used to count the germination rates of different materials, and the average and standard error were calculated. The results are shown in Figure 4 As shown, A is a phenotypic photo of main root growth, B is a statistical chart of main root length, RIE1 Overexpression of Arabidopsis thaliana ( RIE1ox ) had no significant difference in root length between the wild type (WT) and the control on normal medium, but on medium containing 2.5 mM NaHCO3, their root length was significantly longer than that of the wild type control. This result shows that salt-alkali stress significantly inhibits the growth of the taproot of the wild type control seedlings, but RIE1 The degree of inhibition of primary root growth in overexpressing Arabidopsis was significantly reduced, and the phenotype showed that it was more tolerant to salt-alkali stress.

[0050] Leaf senescence is the final stage of leaf development. Various biological and abiotic stresses in nature can cause plant leaf senescence, such as drought, high temperature, high salt, pathogen infection and nutrient deficiency. The overexpression RIE1 The gene can significantly delay the aging of transgenic plants and significantly improve their tolerance to salt-alkali stress, and is expected to be used in delaying plant / crop leaf aging and improving stress resistance breeding.

[0051] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: RIE1 The coding sequence of the gene is shown in SEQ NO.

1.

2. The method according to claim 1 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: Said RIE1 The amino acid sequence of the RIE1 protein encoded by the gene is shown in SEQ NO.

2.

3. The method according to claim 2 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: Said applications include applications in plant breeding.

4. The method according to claim 3 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: The application in plant breeding includes 35S:6Myc-RIE1 Expression vector construction.

5. The method according to claim 4 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: 35S:6Myc-RIE1 The expression vector was constructed by PCR amplification using wild-type Arabidopsis cDNA as a template.

6. The method according to claim 5 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: The primers used for PCR amplification were RIE1-6myc-F and RIE1-6myc-R ;in, RIE1-6myc-F The nucleotide sequence is shown in SEQ NO. 3, RIE1-6myc-R The nucleotide sequence is shown in SEQ NO.

4.

7. The method according to claim 5 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: 35S:6Myc-RIE1 The double restriction enzyme digestion system was used for PCR amplification in the construction of the expression vector.

8. The method according to claim 7 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: The endonuclease used in the double enzyme digestion system is endonuclease Bam HI and endonuclease Sal I.

9. The method according to claim 4 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: Including transformation, through transformation to Agrobacterium-mediated transformation 35S:6Myc-RIE1 Expression vector.

10. The method according to claim 9 RIE1 The application of the gene in delaying plant leaf senescence and improving stress resistance is characterized in that: Also includes impregnation, using transformation 35S:6Myc-RIE1 Agrobacterium infection of Arabidopsis thaliana inflorescences with the expression vector.

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