CN114790459B - Application of Maize ZmPRA1C1 Gene in Improving Heat Stress Resistance of Plants - Google Patents

Abstract

The invention provides the application of the corn ZmPRA1C1 gene in improving plant heat stress resistance. The present invention isolates and clones the gene ZmPRA1C1 from corn, connects the CDS sequence of ZmPRA1C1 to the expression vector pCAMBIA3300 driven by the Ubi promoter, obtains the overexpression vector of the ZmPRA1C1 gene of corn through genetic engineering technology, and thus obtains the The overexpression lines were screened to finally obtain the maize ZmPRA1C1 gene overexpression homozygous lines. The experimental results show that the gene can make the model plant maize have good heat stress resistance, specifically in that it can not only increase the fresh weight of the aboveground part of the seedlings under heat stress, but also reduce the damage degree of maize cells. The invention confirms the potential application value of the corn ZmPRA1C1 gene in improving the heat tolerance of the corn through experiments, and lays a good theoretical and application foundation for using the gene to breed heat-resistant corn varieties.

Description

Application of Maize ZmPRA1C1 Gene in Improving Heat Stress Resistance of Plants

technical field

The invention belongs to the technical field of plant genetic engineering, and in particular relates to the application of corn ZmPRA1C1 gene in improving plant heat stress resistance.

Background technique

Temperature is one of the key physical parameters affecting life on Earth. Thus, almost all organisms have evolved signaling pathways to sense slight changes in ambient temperature and adjust their metabolism and cellular functions to prevent heat-related damage. Heat stress can adversely affect all aspects of plant growth, development, reproduction and yield. Since plants are organisms fixed in the soil and cannot escape heat, they mainly change the metabolism in the body through heat acclimation to prevent damage caused by heat. If the heat in the body exceeds the tolerance level of the plant, the plant activates specific cells or tissues Programmed cell death, leading to leaf loss, flower wilting, fruit reduction, and even the death of the entire plant. During the period from 1964 to 2007, drought and extreme high temperature reduced the global grain production by 10.1% and 9.1% respectively, among which the extreme drought weather caused a total loss of about 1.82 billion US dollars (approximately equal to the global corn and wheat production in 2013), The extreme high temperature disaster caused a total of about 1.19 billion US dollars in losses.

Due to the large number of PRA1 families, the exact function of most plant PRA1 has not been elucidated. Generally speaking, the function of PRA1 is mainly exerted by interacting with downstream Rab proteins. In rice, the authors discovered the plant Yip homologue OsPRA1, which is an integral membrane protein localized in the vacuolar precursor, which has the molecular characteristics of the typical Yip/PRA1 protein family, has two hydrophobic domains, and can mediate Membrane fusion. The ability to interact with downstream OsRab7 plays an important role in targeting OsRab7 into the tonoplast membrane through vacuolar precursors during vacuolar trafficking, which is critical for vacuolar trafficking in plant cells. In addition, OsPRA1 can also interact with OsVamp3 protein, which may be involved in the process of vesicle fusion, but the author did not conduct a more in-depth study on this phenomenon. In Arabidopsis, Lee et al. studied the effect of AtPRA1.B6 on the anterograde transport of proteins targeted to various endomembrane systems. Overexpression of AtPRA1 resulted in varying degrees of inhibition of the anterograde transport of different proteins mediated by coat protein complex II vesicles, including vacuolar proteins, secretory proteins, and plasma membrane proteins, while the transport of Golgi-localized proteins was not affected. In addition, the authors found that AtPRA1.B6-mediated inhibition of anterograde transport occurs in the endoplasmic reticulum, and the expression level of this protein is affected by 26S proteasome-mediated proteolysis. AtPRA1.B6 is a negative regulator of vesicle-mediated coat protein complex II in the endoplasmic reticulum. Similar to the above studies, PRA1.F4 mainly functions in Golgi protein transport. The author first studied the phenotype of overexpressed mutants of this gene. The mutants showed short plant development, short roots, and sensitivity to salt stress; overexpressed The number of secondary roots and root hairs was higher than that of the wild type, which also showed a salt-sensitive phenotype. In addition, the mutants of this gene are defective in vacuolar transport, while overexpression inhibits the outward transport of a series of proteins in the Golgi apparatus, including vacuolar proteins, plasma membrane proteins, trans-Golgi proteins, etc., but also Not all types of Golgi export proteins are inhibited, such as secreted proteins. Based on the above, the authors believe that this gene is crucial for the function of the Golgi apparatus.

In addition to studies of Arabidopsis PRA1 function, in tomato, PRA1A interacts with LeEIX2, a pattern recognition receptor in tomato that activates microbe/pathogen-associated molecular patterns (MAMP/PAMP) to trigger immunity against pathogen progression reaction. Overexpression of SlPRA1A can significantly reduce LeEIX2 endosomal localization and LeEIX2 protein level. Thereby reducing the innate immune response to EIX. Inhibition of vacuole function can inhibit the reduction of LeEIX2 protein level mediated by SlPRA1A, and SlPRA1A can transport LeEIX2 to the vacuole for degradation.

Contents of the invention

For the situation of above-mentioned prior art, the object of the present invention is to provide the application of corn ZmPRA1C1 gene in improving plant heat stress resistance, the present invention is isolated and cloned into a Prenylated Rab Acceptor 1 (PRA1) family gene from corn, and will It is named ZmPRA1C1, and the ZmPRA1C1 gene overexpression vector is obtained through this gene, and then a ZmPRA1C1 gene overexpression homozygous strain is obtained, and the overexpression homozygous strain has good heat stress resistance.

In order to achieve the above-mentioned purpose of the invention, the present invention adopts the following technical solutions to achieve:

The invention provides a gene ZmPRA1C1 for improving the heat tolerance of corn, characterized in that: its nucleotide sequence is shown in SEQ ID No: 1, its CDS sequence is shown in SEQ ID NO: 2, and the amino acid of the protein encoded by it is The sequence is shown in the sequence SEQ ID NO:3. The primer sequences for amplifying the ZmPRA1C1 gene are:

Upstream primer 5'-GGATCCATGTCCAAGTACGGCACCATTC-3';

Downstream primer 5'-GAGCTCTCAGTGCGACGGCTGCTG-3'.

The present invention also provides the application of the ZmPRA1C1 gene in regulating the heat resistance phenotype of plant seedlings.

Plant overexpression homozygous lines containing the maize ZmPRA1C1 gene have significantly lower plant wilting degree under heat stress than wild-type lines.

Further, the ion leakage rate of the overexpression homozygous line of the plant containing the maize ZmPRA1C1 gene is significantly lower than that of the wild type line under heat stress.

Further, the overexpression homozygous lines containing the maize ZmPRA1C1 gene have significantly higher shoot fresh weight under heat stress than wild-type lines.

Further, the overexpression vector includes pCAMBIA3300-ZmPRA1C1, which is obtained by connecting the corn ZmPRA1C1 gene to the Ubi promoter in the vector plasmid after double digestion of the recombinant plasmid.

Further, the recombinant plasmid includes pMD19-T-ZmPRA1C1, which is obtained by amplifying the product of the maize ZmPRA1C1 gene, recovering, purifying and cloning it into a cloning vector.

Compared with the prior art, the advantages and technical effects of the present invention are: the present invention overexpresses the ZmPRA1C1 gene through genetic engineering technology, then obtains the overexpression vector, transforms the vector into strains, and obtains the ZmPRA1C1 gene overexpression through screening. The expression of homozygous lines can significantly improve the ability of plants to resist heat stress. Overexpression of the ZmPRA1C1 gene not only increases the fresh weight of the aboveground parts of plants under heat stress, but also reduces the ion leakage rate of plants under heat stress, reduces the degree of wilting of plants, and reduces the degree of heat damage of plants, which is more conducive to the growth of plants under high temperature. growth and development in the environment. The present invention confirms for the first time through experiments that the corn ZmPRA1C1 gene can improve the heat stress of plants, and in view of the application of the ZmPRA1C1 gene in heat stress, it can be considered that the gene has potential application value for improving the heat stress of plants, and the present invention also provides The use of ZmPRA1C1 gene to breed high temperature resistant crop varieties has laid a good theoretical and application foundation.

Description of drawings

Figure 1A is a schematic diagram of the constructed expression vector and restriction sites. Wherein, the expression vector is pCAMBIA3300, the promoter is Ubi, and the restriction sites at both ends of the target gene ZmPRA1C1 are BamHI and SacI, respectively.

Fig. 1B is the identification of positive seedlings of transgenic lines; No. 1 swimming lane is wild-type B104, and the other swimming lanes are transgenic lines.

Fig. 2A is the transcription level of ZmPRA1C1 gene in B104 and Ubi::ZmPRA1C1 transgenic lines detected by RT-qPCR.

Fig. 2B is western blot detection of ZmPRA1C1 protein levels in B104 and Ubi::ZmPRA1C1 transgenic lines.

Fig. 3A is a graph showing the phenotypes of the transgenic line Ubi::ZmPRA1C1 and wild-type B104 grown in the substrate for 2 weeks under normal conditions and after heat stress treatment.

Fig. 3B is the data statistics of the fresh weight of the above-ground parts of the transgenic line Ubi::ZmPRA1C1 and the wild-type B104 grown in the substrate for 2 weeks under normal conditions and after heat stress treatment, and a significant difference was found.

Figure 3C shows the statistics of the ion leakage rates of the transgenic line Ubi::ZmPRA1C1 and wild-type B104 grown in the matrix for 2 weeks under normal conditions and after heat stress treatment, and a significant difference was found.

Detailed ways

The technical solutions of the present invention will be further described in detail below in conjunction with the accompanying drawings and specific embodiments. The experimental methods not specified in the following examples are generally in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer; the reagents or materials not specified in detail are all commercially available products. In the present invention, the above-mentioned expression vector is introduced into the model plant maize cells, and the introduction methods are well known to those skilled in the art, and these methods include but are not limited to: Agrobacterium-mediated transformation method, gene gun method, electric shock method, ovary injection method wait. In addition, what the present invention adopts is the prior art in this field.

In the present invention, the total RNA is first obtained from corn, and the first strand of cDNA is obtained by reverse transcription, and then used as a template, and the obtained cDNA is subjected to high-fidelity enzyme amplification with specific primers corresponding to ZmPRA1C1.

The extraction and purification of corn RNA can directly adopt existing technology, also can adopt the technology described in the present invention; The synthesis of cDNA first strand is also the same situation, also can adopt existing technology or kit, but above-mentioned two All preferably adopt the specific technique described in the present invention.

Embodiment 1: Obtaining of transgenic strains

(1) Sequence analysis, cloning and vector construction of maize ZmPRA1C1 gene

The present invention utilizes the corn genome database website (https://www.maizegdb.org) to find the gene ZmPRA1C1, the genome sequence is 3529bp in full length (sequence shown in SEQ ID NO.1), and the CDS sequence is 582bp (sequence shown in SEQ ID NO.1). shown in ID NO.2), the protein encoded by the gene has a size of 193 amino acids (sequence shown in SEQ ID NO.3).

According to the CDS sequence of the found maize ZmPRA1C1 gene, primers were designed and cloned. The cloning method was as follows:

1. Extraction of RNA: The total RNA of corn was extracted using the All-Purpose Plant RNA Extraction Kit (Cat. No. CW2598) of Kangwei Century Company.

(1) Take 0.1-0.2 g of corn tissue and grind it into powder in liquid nitrogen, add 500 μL of Buffer RLS, and immediately vortex to mix;

(2) Centrifuge at 12000rpm for 2min at 4°C;

(3) Take the supernatant and put it in a filter column (Spin Columns FS), centrifuge at 12000rpm at 4°C for 1min, absorb the supernatant and transfer it to a new centrifuge tube;

(4) Add 250 μL of absolute ethanol, mix well, transfer the obtained solution to an adsorption column (Spin Columns RM), centrifuge at 12,000 rpm for 1 min at 4°C, and discard the waste liquid;

(5) Add 350 μL of Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm for 1 min at 4°C, and discard the waste liquid;

(6) Prepare DNase I solution: Take 52 μL RNase-Free Water, add 8 μL 10×ReactionBuffer and 20 μL DNase I, mix well, and prepare 80 μL DNaseI solution;

(7) Add 80 μL DNase I solution to the adsorption column, and let stand at 20-30°C for 15 minutes;

(8) Add 350 μL Buffer RW1 to the adsorption column, centrifuge at 12,000 rpm at 4°C for 1 min, and discard the waste liquid;

(9) Add 500 μL Buffer RW2 to the adsorption column, centrifuge at 12,000 rpm at 4°C for 1 min, and discard the waste liquid;

(10) Repeat step (9);

(11) Centrifuge at 12,000 rpm for 2 minutes at 4°C;

(12) Put the adsorption column into a new RNase-Free centrifuge tube, add 30 μL RNase-Free Water dropwise to the middle of the adsorption membrane, leave it at room temperature for 1 min, centrifuge at 12,000 rpm for 1 min, and store the obtained RNA solution in a -80°C refrigerator .

2. Synthesis of the first strand of reverse-transcribed cDNA: The extracted RNA was dissolved and the RNA concentration was measured, and then reverse-transcribed using the TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix Reverse Transcription Kit.

Take 5 μg of total RNA, add 10 μL of 2× reaction buffer, 1 μL of primer oligo dT (0.5 μg/μL), 1 μL of reverse transcriptase, 1 μL of degenomic enzyme, replenish water to 20 μL, incubate at 42°C for 30 minutes, and the enzyme will disappear at 85°C Live for 5 minutes.

3. Cloning of ZmPRA1C1 gene:

ZmPRA1C1 gene primer sequence:

Upstream primer 5'- GGATCC ATGTCCAAGTACGGCACCATTC-3'; (SEQ ID NO.4);

Downstream primer 5'- GAGCTC TCAGTGCGACGGCTGCTG-3'; (SEQ ID NO.5);

Wherein, the underlined place is the restriction site, the restriction site of the upstream primer is BamHI, and the restriction site of the downstream primer is SacI.

Use Phanta Max Super-Fidelity DNA Polymerase high-fidelity enzyme (Norwegian Zan Company, product number P505) for amplification. The reaction system is: 2×Phanta Max Buffer 25 μL, deoxyribonucleic acid (dNTP) 1 μL, upstream primer 2 μL, downstream primer 2 μL, Add 1 μL of Phanta Max Super-Fidelity DNA Polymerase, 1 μL of cDNA template, and make up to 50 μL with water.

The PCR reaction conditions are: pre-denaturation at 95°C for 3 minutes;

Denaturation at 95°C for 15 seconds, annealing at 53°C for 15 seconds, extension at 72°C for 30 seconds, a total of 37 cycles;

Extend at 72°C for 5 minutes;

Keep warm at 16°C.

After the reaction, agarose gel electrophoresis was performed. After the target band was detected, the gel was cut and recovered. The gel recovery method was performed according to BioTeke's Quick Agarose Gel DNA Recovery Kit (Cat. No. DP1722).

The end of the high-fidelity enzyme amplification product is blunt-ended, and polyA needs to be added before T-A cloning. The reaction system is as follows: 1.5 μL of 10× reaction buffer, 1.2 μL of deoxyribonucleic acid (dNTP), 0.15 μL of Taq enzyme, and gel recovery product Make up 15 μL. Then react at 72°C for 30 minutes.

4. Take the above 4 μL tailed product and connect it to the pMD19-T cloning vector. The operation steps are carried out according to the product pMD19-T manual of TaKaRa Company, and then the ligated product is transformed into E. coli TOP10 strain by heat shock method, and the LB Grow overnight on plates. Pick a single white colony and streak it on the LB plate for colony PCR. The reaction system is as above, and select the positive colony in the LB liquid medium overnight.

5. Plasmid DNA extraction: Plasmid DNA was extracted using Kangwei Century High Purity Plasmid Small Extraction Kit (CW0500A).

6. Sequence determination: This work was carried out at Sangon Bioengineering (Shanghai) Co., Ltd.

7. Construction of the expression vector: use BamHI and SacI enzyme digestion and sequencing to correct the plasmid with the ZmPRA1C1 gene and the pCAMBIA3300 empty vector with the Ubi promoter (Fig. 1A), after digestion at 37°C for one hour, carry out agarose gel After electrophoresis, the correct band was excised for gel recovery, and the gel recovery product was ligated with T4 DNA ligase from Thermo Fisher Scientific, and the ligation product was transformed into TOP10 strain, and grown overnight on an LB plate containing kana. Pick a single white colony and streak it on the LB plate, carry out colony PCR, and select the positive colony in LB liquid medium overnight. Plasmid DNA extraction: use Kangwei Century High Purity Plasmid Small Extraction Kit to extract plasmid DNA and identify it by enzyme digestion. The overexpression vector of Ubi::ZmPRA1C1 was constructed.

8. Take 2.5 μL of the plasmid to transform Agrobacterium EHA105.

(2): Obtaining and screening positive seedlings of transgenic maize overexpressing ZmPRA1C1

1. Obtaining positive seedlings of transgenic maize overexpressing ZmPRA1C1

The EHA105 Agrobacterium carrying the Ubi::ZmPRA1C1 overexpression vector was used to transform maize tissue by Bomeixingao Biotechnology Company.

2. Screening of transgenic positive seedlings

(1) The maize seed after a single plant receives infection is the T0 generation.

(2) Select 20 seeds of the T0 generation to cultivate on the substrate, extract the genome of the transgenic line, detect the transgenic line by PCR, select positive seedlings in the field, and the seed received by a single plant is the T1 generation.

(3) Select 20 seeds in each T1 generation fruit ear to cultivate on the substrate, extract the genome of the transgenic strain, detect the transgenic strain with PCR, select positive seedlings in the field, and the seed received by a single plant is the T2 generation .

(4) Select 20 seeds in each T2 generation fruit ear to cultivate on the substrate, extract the genome of the transgenic strain, and detect the transgenic strain with PCR, and the T2 strains of the previous generation that are all positive strains are homozygous. Seeds of transgenic lines, the results are shown in Figure 1B. Seeds of homozygous transgenic lines were used in all the following experiments.

Example 2: Identification of ZmPRA1C1 expression in transgenic lines

According to the design requirements of qRT-PCR primers, gene-specific primers were designed using software such as Beacon Designer 7 and Primer Premier 5.0. Select the SYBR Green Design option, create a file, input the sequence, run the BLASTsearch sequence and Template structure search tools, and run the Primer search tool to select the optimal primer sequence (first ensure the specificity of the primers and then consider avoiding the influence of the template structure). The primers were synthesized at Shanghai Sangon Bioengineering Service Co., Ltd. and purified by PAGE. The primer sequences are as follows:

qRT-ZmPRA1C1-F: CCCCGTCTCCCCTCATCGTAT; (SEQ ID NO. 6)

qRT-ZmPRA1C1-R:GTGAGGAGGAGCAGGACGA; (SEQ ID NO.7)

Application qRT-PCR special 96-well plate (Axygen, the United States) and high light transmittance sealing film (Axygen, the United States), fluorescent quantitative PCR instrument Icycler real-time PCR system (Bio-Rad, the United States) for qRT-PCR analysis, each Each sample was repeated 3 times. The reverse transcription product is used as a template, and the reaction system refers to the instructions of SYBR Green Realtime PCR Master Mix (QPK-201). The reaction conditions are as follows:

(1) 95.0°C for 60s; (2) 95.0°C for 10s; (3) 58.0±5.0°C for 10s; (4) 72.0°C for 15s; (5) PlateRead; (6) Incubate at 65°C for 20s; (7) Melting curve From 65°C to 95°C, read every 0.5°C, hold 1s; (8) End; where (2) (3) (4) 50-60cycles.

Mix multiple samples for the first amplification, check whether the primers are available, and verify the specificity of primer amplification according to the melting curve. A single peak is considered specific amplification. If there are double peaks, adjust the annealing temperature and the amount of primers appropriately. If still unable to amplify specifically, redesign primers. Use the mixed template to dilute sequentially at 10-fold concentration, co-dilute 4 times, and use 5 concentrations of samples to construct a relative standard curve to verify the amplification efficiency of all primers and whether the target sequence has a linear amplification relationship within this concentration range.

Using tubulin as an internal reference, adjust the concentration of each template so that the difference between the internal reference Ct values is less than 2. Each gene was amplified with an internal reference at the same time. Under the default conditions, the Ct value was read, and each sample was repeated three times. The data analysis was performed using the double standard curve method. The average expression level and relative deviation were calculated and plotted with Excel. At the same time, 2 ^-ΔΔCt was used to roughly calculate the relative expression level with the internal reference to determine the expression abundance of the gene. Relative expression was calculated as fold change=2- ^△△CT , where △△CT=(C _T-gen - _{C T-tubulin} ) _treatment- (C _T-gene - _{C T-tubulin} ) _control . The results showed ( FIG. 2A ), that the ZmPRA1C1 gene was expressed at a higher level in the overexpressed homozygous lines OE-1 and OE-2.

In addition, this study also used the anti-ZmPRA1C1 antibody to detect the expression of ZmPRA1C1 in B104, OE-1, and OE-2 strains by western blot experiments. The results are shown in Figure 2B. At the protein level, the two The protein content of ZmPRA1C1 in the overexpression lines was significantly higher than that of the control B104. In summary, the ZmPRA1C1 gene showed a higher level of expression in the overexpression homozygous lines OE-1 and OE-2, so this strain was selected Department of follow-up research.

Example 3: The effect of the maize ZmPRA1C1 gene on the phenotype of seedlings under heat stress

(1): Cultivation of overexpressed ZmPRA1C1 transgenic maize

Corn seeds were sterilized with 70% ethanol for 5 minutes, then sterilized with 15% sodium hypochlorite for 5 minutes, and finally rinsed with sterile ddH ₂ O for 5-7 times, and the sterilized aseptic seeds were planted in the substrate and placed in a long-term room at 25°C. Grow in a sunlight incubator for 14 days. The long-day conditions were 16h light/8h dark at 25°C.

(2): Phenotype observation of ZmPRA1C1 transgenic lines under heat stress

Select 30 B104 wild-type, OE-1, and OE-2 corn seedlings with the same growth in (1), and carry out heat stress treatment at 45°C for 5.5 hours in a heat incubator at the same time, observe the growth status of the seedlings before and after the treatment, and take pictures , the results are shown in Figure 3A. Under normal conditions, the phenotypes of the three have no significant difference. After heat stress treatment, B104 maize seedlings have already appeared in a state of wilting. Compared with B104, two overexpressed ZmPRA1C1 transgenes The growth status of the strains was relatively good, and the degree of wilting was low.

(3): Fresh weight statistics of aerial parts of overexpressed ZmPRA1C1 transgenic lines under heat stress

Select 30 B104 wild-type, OE-1, and OE-2 corn seedlings with the same growth in (1), and carry out heat stress treatment at 45°C for 5.5 hours in a heat incubator at the same time, and count the aerial parts before and after heat stress treatment Fresh weight, the results are shown in Figure 3B. Under normal conditions, there was no significant difference in the fresh weight of the aerial parts of the three, all about 1g. After the heat stress treatment, the fresh weight of the aerial parts of B104 corn seedlings decreased by about 50%. It shows that the plant biomass is seriously reduced. Compared with B104, the fresh weight of above-ground parts of the two overexpressed ZmPRA1C1 transgenic lines remained about 80% of that of the untreated lines, and the biomass decreased to a lesser degree.

(4): Statistics on ion leakage rate of transgenic lines overexpressing ZmPRA1C1 under heat stress

Select 30 B104 wild-type, OE-1, and OE-2 corn seedlings with the same growth in (1), and carry out heat stress treatment at 45°C for 5.5 hours in a heat incubator at the same time, and count the seedling ions before and after heat stress treatment. Leakage rate, the results are shown in Figure 3C. Under normal conditions, there is no significant difference in the ion leakage rate among the three, which are all about 8%. After the heat stress treatment, the ion leakage rate of B104 corn seedlings increased significantly When it reaches about 20%, it means that the cells are obviously damaged by heat stress. Compared with B104, the ion leakage rate of the two overexpressed ZmPRA1C1 transgenic lines was significantly increased to about 14%, and the degree of damage was significantly lower than that of B104.

All the evidence above shows that the ZmPRA1C1 gene can significantly improve the heat stress resistance of plant seedlings, and alleviate the heat stress challenge suffered by the seedlings.

The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art can still understand the foregoing embodiments. Modifications are made to the technical solutions described, or equivalent replacements are made to some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions claimed in the present invention.

sequence listing

<110> Shandong Agricultural University

<120> Application of Maize ZmPRA1C1 Gene in Improving Heat Stress Resistance of Plants

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atcacgtgag ctgtgtttgt gtttgatcga tcgcttatca aagattctgc tgctttttta 2760

attcatgacc cccccccccc ccccccccaa ataatgggtt tagacattca tgttgagcac 2820

atcattcaca cttgaatctt ccaaagaggg atacaatgga ctgatatgga cacagattct 2880

ccacttgttc ttctcatgtt ggtaaattca attatgagac aaataaatca aagtcaatta 2940

tgagaaaaaa atagcacaag tggctgggag cttcaaaatt ggaatacgca atggcctgca 3000

gttctaatca atctgctagt atctcttttt ctgttttctc gctaagttta atactcagca 3060

aaccaatttt gattttgaag actgagaaaa atgctatgcg tgtcttgcag gatctgaatg 3120

tggccgttct gcgccttggc tgtgatccta ttctgtactc cctgtttccg agttcctcca 3180

gctgcgcgag ctgggccatc cccatctcat ctgcgatgaa aaacctgtcc ctgttggttg 3240

tgattcacct gctgccggtt ccatgggaag ttttggatcc agcgacgacg cagggctgtt 3300

aaacttgttc tccacgccca tttctgtggt acgtttaaag cgtctcgttc acgtgtatca 3360

aatccggact gaagtctagt gtacattttg agttggagaa atgtgatgcc agggacctgc 3420

gttcagattt tatcgacgcc atgcatgtag gcaagggatg ctgatatttc agacatggtt 3480

ttattctacg tggtgtctgg gagcctggga agaaatctcg ttttgcttg 3529

<210> 2

<211> 582

<212>DNA

<213> Artificial Sequence

<400> 2

atgtccaagt acggcaccat tcccacctcc tcctccgcgg gcggagggcc cgtgcccctc 60

ggcggcgcct ccccgctcga tttcatctcc cgcgccaagg ctcggggcgc ctcggcgctg 120

gcgacgcgcc ggccctggcg cgagctcgtg gacgtccacg ccgtcggcct gcccccgagc 180

ctcggcgacg cgtacctccg cgtgcgcgcc aacctcgccc acttcgccat gaactatgcc 240

atcgtcgtcc tcgtcgtcgt cttcctctcc ctcctctggc accccgtctc cctcatcgta 300

ttcctcgtct gcatgcttgc ctggctcgtc ctctacttcc tccgcgacga gcccctcgtc 360

ctcttcggcc gcgtcgtcgc cgacggctac gtcctcgccg tgctcgccgt cgtcacgctc 420

gtcctgctcc tcctcaccga cgccaccgcc aacatcctct cctcgctgct catcggcctc 480

gtgctcgtcc tcgtccacgc cgcgctgcac aaggcggagg acaacgccgc cgacgaggct 540

gaccgctggt acgcgccggt gtcacagcag ccgtcgcact ga 582

<210> 3

<211> 193

<212> PRT

<213> Artificial Sequence

<400> 3

Met Ser Lys Tyr Gly Thr Ile Pro Thr Ser Ser Ser Ser Ala Gly Gly Gly

1 5 10 15

Pro Val Pro Leu Gly Gly Ala Ser Pro Leu Asp Phe Ile Ser Arg Ala

20 25 30

Lys Ala Arg Gly Ala Ser Ala Leu Ala Thr Arg Arg Pro Trp Arg Glu

35 40 45

Leu Val Asp Val His Ala Val Gly Leu Pro Pro Ser Leu Gly Asp Ala

50 55 60

Tyr Leu Arg Val Arg Ala Asn Leu Ala His Phe Ala Met Asn Tyr Ala

65 70 75 80

Ile Val Val Leu Val Val Val Phe Leu Ser Leu Leu Trp His Pro Val

85 90 95

Ser Leu Ile Val Phe Leu Val Cys Met Leu Ala Trp Leu Val Leu Tyr

100 105 110

Phe Leu Arg Asp Glu Pro Leu Val Leu Phe Gly Arg Val Val Ala Asp

115 120 125

Gly Tyr Val Leu Ala Val Leu Ala Val Val Thr Leu Val Leu Leu Leu

130 135 140

Leu Thr Asp Ala Thr Ala Asn Ile Leu Ser Ser Leu Leu Ile Gly Leu

145 150 155 160

Val Leu Val Leu Val His Ala Ala Leu His Lys Ala Glu Asp Asn Ala

165 170 175

Ala Asp Glu Ala Asp Arg Trp Tyr Ala Pro Val Ser Gln Gln Pro Ser

180 185 190

His

Claims (5)

1. The application of the ZmPRA1.C1 gene in regulating the heat tolerance phenotype of corn seedling stage, characterized in that: the nucleotide sequence of the ZmPRA1.C1 gene is shown in SEQ ID No: 1, and the CDS sequence is shown in SEQ ID NO: 2 As shown, the amino acid sequence of the protein encoded by it is shown in the sequence SEQ ID NO:3; The maize homozygous line overexpressing the ZmPRA1.C1 gene has significantly lower plant wilting degree under heat stress than the wild-type line. 2. the application of the ZmPRA1.C1 gene according to claim 1 in regulating and controlling the heat tolerance phenotype of corn seedling stage, it is characterized in that, the corn homozygous line that overexpresses described ZmPRA1.C1 gene is under heat stress The ion leakage rate was significantly lower than that of the wild-type strain. 3. the application of the ZmPRA1.C1 gene according to claim 1 in regulating and controlling the heat tolerance phenotype of corn seedling stage, it is characterized in that, the corn homozygous line that overexpresses described ZmPRA1.C1 gene under heat stress The fresh weight of aerial parts was significantly higher than that of wild-type lines. 4. the application of the ZmPRA1.C1 gene according to claim 1 in regulating and controlling the heat tolerance phenotype of corn seedling stage, it is characterized in that, the carrier of described overexpression comprises pCAMBIA3300-ZmPRA1.C1, and it passes recombinant plasmid through double After digestion, the ZmPRA1.C1 gene is connected to the Ubi promoter in the vector plasmid to obtain it. 5. the application of the ZmPRA1.C1 gene according to claim 4 in regulating the heat tolerance phenotype of corn seedling stage, it is characterized in that, said recombinant plasmid comprises pMD19-T-ZmPRA1.C1, and it is by amplifying said The product of the ZmPRA1.C1 gene is recovered and purified and then cloned into a cloning vector to obtain it.