CN117820451A

CN117820451A - Sunflower transcription factors HaNAC84 and HaNAC146 protein and application thereof

Info

Publication number: CN117820451A
Application number: CN202410028380.3A
Authority: CN
Inventors: 曾幼玲; 刘豫新; 李文辉; 黄启秀; 雷中华; 向理军; 李强; 侯献飞; 张玲玲
Original assignee: Cash Crop Research Institute Of Xinjiang Academy Of Agricultural Sciences Xinjiang Uygur Autonomous Region Sugar Beet Improvement Center Of Xinjiang Uygur Autonomous Region Cotton Research Institute; Xinjiang University
Current assignee: Cash Crop Research Institute Of Xinjiang Academy Of Agricultural Sciences Xinjiang Uygur Autonomous Region Sugar Beet Improvement Center Of Xinjiang Uygur Autonomous Region Cotton Research Institute; Xinjiang University
Priority date: 2024-01-09
Filing date: 2024-01-09
Publication date: 2024-04-05

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to sunflower transcription factors HaNAC84 and HaNAC146 proteins and application thereof. The invention aims to solve the technical problem of digging gene resources which can resist adversity stress and promote better growth of plants. The technical scheme of the invention is that the sunflower transcription factor HaNAC84 protein has an amino acid sequence shown as SEQ ID No. 3; the invention also provides a sunflower transcription factor HaNAC146 protein, and the amino acid sequence of the protein is shown as SEQ ID No. 6. Overexpression of HaNAC84 and HaNAC146 is effective in improving stress tolerance and growth in plants. The high-quality candidate gene is provided for cultivating new varieties of crops with high yield, salt tolerance and drought resistance.

Description

Sunflower transcription factors HaNAC84 and HaNAC146 protein and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to sunflower transcription factors HaNAC84 and HaNAC146 proteins and application thereof.

Background

Oil sunflower (Helianthus annuus l.) has high edible and economic value. Compared with other oil crops, the sunflower has certain drought resistance, barren resistance and salt and alkali resistance, and does not contend with grain, so that the sunflower is mostly planted on drought barren lands and even used as pioneer crops for improving the salt and alkali lands.

Plants are susceptible to extreme environmental effects during the critical stages of seed development, seedling growth and flowering. The sunflower has strong tolerance to stress and can be used as a candidate gene material for searching not only the stress resistance but also the yield improvement. NAC (NAM/ATAF/CUC) is one of the largest families of transcription factors in plants, and serves as the primary regulator of stress adaptation signals and regulation mechanisms to regulate plant development, senescence, morphogenesis, and abiotic stress resistance. There is a balance between stress tolerance and plant growth. At present, few genes which have stress resistance and can increase yield are excavated in crops. Such as overexpression of OsDREB1A, atDDF and AtNCED6, would compromise beneficial agronomic traits by metabolic or hormonal perturbation. Arabidopsis overexpressing ATAF1 enhances tolerance to drought stress, but leads to dwarfing of primary roots, increasing sensitivity to ABA, salt and oxidative stress. Similarly, rice overexpressing ATAF homologous gene OsNAC6 increased tolerance to high salinity, but inhibited the growth of transgenic rice. These results indicate that there is an urgent need to explore gene resources that are capable of both withstanding stress and promoting better plant growth.

Stable genetic transformation of plants is a common technical means for studying gene function, however, obtaining stably transformed transgenic plants is generally long-lasting, costly and technically demanding. Transient transformation systems are complementary to stable genetic transformation. To date, most plants do not establish regeneration systems and stable genetic transformation systems, which are major limiting factors in studying plant gene function, and therefore, the use of efficient transient transformation systems is necessary for identifying the gene function of this species and for conducting molecular biological studies.

Disclosure of Invention

The invention aims to solve the technical problem of digging gene resources which can resist adversity stress and promote better growth of plants.

The technical scheme of the invention is that the sunflower transcription factor HaNAC84 protein has an amino acid sequence shown as SEQ ID No. 3.

Further, the nucleotide sequence of the coding HaNAC84 protein is shown as SEQ ID No.1 or SEQ ID No. 2.

The technical scheme of the invention is that the sunflower transcription factor HaNAC146 protein has an amino acid sequence shown in SEQ ID No. 6.

Further, the nucleotide sequence of the coding HaNAC84 protein is shown as SEQ ID No.4 or SEQ ID No. 5.

The invention also provides application of the sunflower transcription factor HaNAC84 protein in regulating and controlling the stress tolerance of plants.

The invention also provides application of the sunflower transcription factor HaNAC146 protein in regulating and controlling the stress tolerance of plants.

Further, the plant is sunflower or arabidopsis.

Wherein the stress is high salt or drought.

Specifically, the regulation is positive regulation.

Further, the upregulation is such that either the HaNAC84 protein or the HaNAC146 protein is overexpressed.

The invention also provides application of the sunflower transcription factor HaNAC84 protein in regulating and controlling plant growth.

The invention also provides application of the sunflower transcription factor HaNAC146 protein in regulating and controlling plant growth.

The growth conditions comprise plant height, plant type, root system, leaf size, number of branches for bolting a single plant, number of seeds or seed size.

Further, the plant is sunflower or arabidopsis.

Specifically, the regulation is positive regulation.

The invention also provides a method for improving the stress resistance of plants, which constructs a HaNAC84 protein or HaNAC146 protein over-expression vector, and transforms plants by transferring the vector into agrobacterium tumefaciens.

In particular, the plant is sunflower.

Further, the conversion is as follows: selecting four days old sunflower seedlings by water planting method, and transforming the concentration of Agrobacterium in the invasion solution to OD ₆₀₀ The value of 0.8, the components of the agrobacteria invasion solution are 7% sucrose, 100 mu M acetosyringone and 50mM CaCl ₂ 0.1mg/L ascorbic acid, 20 mu M5-azacytidine and 0.02% SILWETL-77, the infection time of seedlings in agrobacterium infection solution is 2-4 h, and then the seedlings are grown for 3d in a dark environment.

The invention has the beneficial effects that: haNAC84 and HaNAC146 are transcription factors from sunflower NAC family, transient sunflower transformation seedlings with RNA interference and transgenic HaNAC84 and HaNAC146 arabidopsis with heterologous expression of single copy homozygous are obtained by agrobacterium tumefaciens mediated transformation and stable transformation system of arabidopsis, respectively, and the overexpression of HaNAC84 and HaNAC146 is found to make the transient sunflower transformation seedlings and transgenic arabidopsis show better growth phenotype than those of the control group under high salt and drought stress in seed germination stage, seedling stage and adult plant stage, and the transient sunflower transformation seedlings with RNA interference show salt intolerance and drought resistance phenotype; under normal growth conditions, over-expression of HaNAC84 and HaNAC146 results in transgenic arabidopsis plants that have better and higher growth and seed yield than wild-type arabidopsis plants. The high-quality candidate gene is provided for cultivating new varieties of crops with high yield, salt tolerance and drought resistance.

Drawings

FIG. 1, schematic diagram of HaNAC84 and HaNAC146 overexpression and interference vector construction. (a) a schematic diagram of the HaNAC84 and HaNAC146 overexpression vector construction; (B) HaNAC84 and HaNAC146 interfering vector construction schematic; (C) A flow chart for constructing stem-loop RNAi constructs using inverse overlap extension PCR (IR-PCR). And (3) injection: caMV 35S Pro is a CaMV 35S promoter, Ω is a 5' -untranslated region Ω sequence for enhanced expression, E9 is a terminator, haNAC84 stem loop RNAi transcript is a transcript of the HaNAC84 stem-loop RNAi structure, and HaNAC146 stem loop RNAi transcript is a transcript of the HaNAC146 stem-loop RNAi structure; primers and templates is primer and template, sense Primers is Sense marker connecting primer S1/2, linker is GUS linker Sequence, target Sequence is 3' UTR region Sequence of Target gene, antisense Primers is antisense marker connecting primer A1/2, L1/2 is GUS linker primer; first round of PCR is the first round of PCR reaction, antisense orientation is the antisense direction, sense orientation is the sense direction; second round of PCR is a second round of PCR reaction, terminal Primers are used as end Primers, three fragment OE-PCR for 8cycles are used for carrying out overlap extension PCR on three fragments, 8 times of cycles are carried out, add T1+T2 Primers for 35cycles are used for adding T1+T2 Primers, 35 times of cycles are carried out, and T1/2 is an end connection primer; cloning step is a Cloning step, sacI, pstI is a restriction enzyme site introduced to clone RNAi structure into pCAMBIA2300 vector; stem-loop RNAi transcript is Stem loop RNAi transcript, "a, b, c, a ', b', c ', x, y, z, x', y ', z', t1/2" is an auxiliary indicator for ease of understanding the construction process.

FIG. 2, expression of two genes in sunflower seedlings transiently transformed with the overexpression vector of the HaNAC84 and HaNAC146 genes, the interference vector and the empty vector and phenotypes before and after treatment with 350mM NaCl salt stress, 400mM Mannitol drought stress. (A) Phenotype of sunflower seedlings before and after salt and drought stress treatment; (B) Relative expression of the HaNAC84 gene in sunflower seedlings prior to salt stress treatment; (C) Relative expression of the HaNAC146 gene in sunflower seedlings prior to salt stress treatment. And (3) injection: control is a Control condition, salt is a Salt stress condition, and Drright is an Drought stress condition; OE is sunflower seedlings transformed with the over-expression vector, RNAi is sunflower seedlings transformed with the interference vector, EV is sunflower seedlings transformed with the empty vector; haNAC84 relative expression is the relative expression of HaNAC84, and HaNAC146 relative expression is the relative expression of HaNAC 146.

FIG. 3, growth phenotype of HaNAC84 transgenic Arabidopsis in seedling stage under salt, drought stress. (A) The phenotype of transgenic Arabidopsis and wild type stress 5d under 300mM NaCl; (B) Phenotype of 12d and rehydration 3d of transgenic arabidopsis and wild type natural drought treatment; (C) The survival rate of transgenic arabidopsis and wild type natural drought treated and rehydrated is counted for 30 seedlings and repeated for three times for each strain; and (3) injection: WT is wild type Arabidopsis, OE13, OE15 are HaNAC84 transgenic Arabidopsis single copy homozygous lines; control is a Control condition, salt is a Salt stress condition, drright is an Drought stress condition, rewarming is a rehydration condition, and survivin rate is Survival rate.

FIG. 4, salt, drought stress growth phenotype of Hanac146 transgenic Arabidopsis thaliana seedling stage (A) phenotype of transgenic Arabidopsis thaliana and wild type stress 5d at 300mM NaCl; (B) Phenotype of 12d and rehydration 3d of transgenic arabidopsis and wild type natural drought treatment; (C) The survival rate of transgenic arabidopsis and wild type natural drought treated and rehydrated is counted for 30 seedlings and repeated for three times for each strain; and (3) injection: WT is wild type Arabidopsis, OE8 and OE9 are HaNAC146 transgenic Arabidopsis single copy homozygous lines; control is a Control condition, salt is a Salt stress condition, drright is an Drought stress condition, rewarming is a rehydration condition, and survivin rate is Survival rate.

FIG. 5, observations made during growth of HaNAC84 transgenic Arabidopsis. (a) transgenic plants and wild type aerial phenotype; (B) transgenic plants and wild-type whole plant phenotypes; (C) plant leaf phenotype; (D) a kerb phenotype; (E) seed phenotype, scale = 200 μm; (F) Dry weight; (G) Plant height; (H) The number of branches of bolting of a single plant (Number of bolting branches per plant); (I) number of pods per plant (Siliques per plant); 120 seedlings are counted for each strain in the data; (J) The Silique length (Silique size), each strain counts the length of 100 siliques; (K) Seed number in individual siliques (Seed number per silique), each strain counted the seed number in 100 siliques; (L) Seed length (Seed); (M) Seed width (Seed width), each strain measured the length and width of 100 seeds; (N) thousand Seed weight (Seed weight per 1000 seeds), 10 thousand Seed weight per strain were counted; and (3) injection: WT is wild type Arabidopsis, OE13, OE15 are HaNAC84 transgenic Arabidopsis single copy homozygous lines.

FIG. 6, observations made during growth of HaNAC146 transgenic Arabidopsis. (a) transgenic plants and wild type aerial phenotype; (B) transgenic plants and wild-type whole plant phenotypes; (C) plant leaf phenotype; (D) a kerb phenotype; (E) seed phenotype, scale = 200 μm; (F) Dry weight; (G) Plant height; (H) The number of branches of bolting of a single plant (Number of bolting branches per plant); (I) number of pods per plant (Siliques per plant); 120 seedlings are counted for each strain in the data; (J) The Silique length (Silique size), each strain counts the length of 100 siliques; (K) Seed number in individual siliques (Seed number per silique), each strain counted the seed number in 100 siliques; (L) Seed length (Seed); (M) Seed width (Seed width), each strain measured the length and width of 100 seeds; (N) thousand Seed weight (Seed weight per 1000 seeds), 10 thousand Seed weight per strain were counted; and (3) injection: WT is wild type Arabidopsis, OE8, OE9 are HaNAC146 transgenic Arabidopsis single copy homozygous lines.

Detailed Description

The invention is further elucidated by the following detailed description of specific embodiments, without being limited thereto, by way of example only.

The experimental methods in the following examples are all conventional methods unless otherwise specified, the experimental materials used are all conventional biochemical reagents and medicines unless otherwise specified, the experiments are repeated for more than three times, and the results are averaged. The specific experimental conditions are not specified, and are either conventional conditions well known to those skilled in the art or conditions suggested by the manufacturer. The strain used: the agrobacterium tumefaciens GV3101, the escherichia coli DH5 alpha and the vector pBI121 are common products which are commercially available, and the vector pCAMBIA2300 is modified in the laboratory.

EXAMPLE 1 cloning of sunflower transcription factor genes HaNAC84 and HaNAC146 and construction of plant overexpression and interference vectors

By performing a whole genome analysis to identify 150 members of the sunflower NAC transcription factor family, 47 other plant species of abiotic stress resistant NAC and all HaNAC protein sequences were used to construct phylogenetic trees, haNAC84 and HaNAC146 being clustered close to most resistant NAC, respectively, and expression of HaNAC84 and HaNAC146 in sunflower roots and leaves being significantly responsive to a variety of abiotic stresses and related hormones. The two NAC members were thus candidate for further investigation of their abiotic stress resistance and role in growth development.

The plant expression vector adopts the pCAMBIA2300 modified in the laboratory, the screening marker of prokaryotic cells of the vector is kanamycin resistance, the screening marker of plant cells is NPTII gene, the vector is provided with a 35S promoter, a 5 '-untranslated region omega sequence for enhancing expression and an E9 terminator, and the expressed gene is inserted between the 5' -untranslated region omega sequence and the E9 terminator.

Construction of plant overexpression vector (fig. 1A): specific primers were designed based on the sunflower target gene sequence fragments known from Ensemblplants genome database (http:// plants. Ensembl. Org /), haNAC84 and HaNAC146 fragments were amplified from the cDNA of oil sunflower (early-short-large-head backbone parent), recovered, TA cloned, transformed, screened, PCR amplified and restriction identified, and the correct sequences of HaNAC84 and HaNAC146 were obtained by sequencing. The clone was constructed on the pCAMBIA2300 vector by double cleavage.

Construction of the overexpression vector primer (5 '-3'):

SEQ ID No.7HaNAC84 ORF-F：GAGCTCATGGGTTTACCGGTTACC；

SEQ ID No.8HaNAC84 ORF-R：CTGCAGTCACTGTCTAAAGCCCAAAAC；

SEQ ID No.9HaNAC146 ORF-F：GAGCTCATGACTTGTGATAATTTGGAGTTGC；

SEQ ID No.10HaNAC146 ORF-R：CTGCAGTCACATCTGAAAGGGCTTT。

construction of interference vector (fig. 1B): RNA interference vectors were prepared according to the method reported by Pawloski et al 2005 (Pawloski L C, deal R B, mckiney E C, et al, introduced repeat PCR for the rapid assembly of constructs to induce RNA interference [ J ]. Plant & Cell Physiology 2005,46 (11): 1872-1878.), i.e., using reverse overlap extension PCR (IR-PCR) to clone the 3' untranslated region (UTR) of the gene of interest as an interference fragment according to the principles of the stem-loop method, and GUS gene fragment was cloned as a "loop" from the pBI121 vector. Starting from the second nucleotide after the stop codon of the gene of interest, extending down to the 3' UTR, sequences were amplified from each of HaNAC84 and HaNAC 146. The two ends of the sense and antisense fragments were differentially labeled by IR-PCR (primers see table 1) to assemble stem regions in each RNAi construct. Two pairs of 3' UTR sequences were combined with GUS linker by overlap extension PCR to assemble the final products of HaNAC84 RNAi and HaNAC146 RNAi, respectively. The constructed target gene interference structure is connected to the pCAMBIA2300 vector by a double enzyme digestion method. The empty vector used hereinafter is the pCAMBIA2300 empty vector.

The IR-PCR procedure (FIG. 1C) was as follows: starting in the first round of PCR, three separate DNA fragments were created: an antisense oriented target gene sequence, a sense oriented identical gene sequence, and a beta-Glucosidase (GUS) linker sequence. In the first amplification, IR-PCR relies on differential labeling of target gene sequences in sense and antisense orientation with different primer sequences. When the three fragments are mixed together to begin the second round of PCR reaction, their complementary 3' ends are annealed and extended by overlap extension PCR (Overlap extension PCR, OE-PCR) using each other as templates to form one integral fragment. The stem-loop full length structural product was then amplified from the mixture using terminal primer T1/2 and the RNAi structure was cloned into the pCAMBIA2300 vector expressing the stem-loop RNA product.

TABLE 1 interfering vector construction primers

Note that: restriction enzyme sites are underlined, linker sequences are underlined, and target gene sequences are bolded.

SEQ ID No.1 sunflower HaNAC84 Gene full length

TGAAAAAAAAAAAAAAGAAATTCATAACTCTTGAATTCTTTAACAACAATCTTCCTTCTCCTTTCCCAGAAAAAAAAAACGTGAAAAAAATGGGTTTACCGGTTACCGACCCGATGACCCAATTAAGCTTACCACCTGGTTTCCGGTTTTACCCGACCGATGAGGAACTTCTTGTCCAATATCTTTGCCGGAAAGTTGCCGGTCACGATTTTTCTCTTCAGATTATTGCTGATATTGATTTGTACAAGTTTGATCCATGGCAGCTTCCTAGTAAGTTTGTGTTTTTTTTTTTTTATTTTTTTTATTTTTTATTTTTTTTATTTTTTTTTTAGGTTTTTAGGTATGAATGGCTAATGGTGTAATGTGTTGATGTAGGTAAGGCGATGTTTGGGGAGAAAGAGTGGTATTTTTTTAGCCCGAGAGATCGGAAGTATCCGAATGGGTCTAGACCGAATAGAGTAGCCGGGTCGGGTTACTGGAAGGCTACCGGAACTGATAAGGTGATCACGACCGAGGGACGAAGGGTTGGGATCAAGAAAGCTTTGGTGTTTTATGTTGGTAAAGCCCCTAAAGGAAATAAAACTAATTGGATCATGCATGAGTATAGGTTATCGGATCCCCAGAGGAAAACCGGTAGCTCCAGGGTAAGTCACCATTTCATCGTTCTTTTTATCATTTAAATGGTCTACCTTGTTTCCAAACTTTGGAGCAGCAATAAGGTTGCTCTCATGTCATCTGAGCAGGCAATAGATATCTTCATAGAAGATATGGTTAGACCTGTTTAAAGTACATAATGTGATTTTTTACTTTTTTTTAAATAAATAAAATTGTGAGGTTAGGATTCATCATTTATTTTTCTTGGTACATAAAATATAAAACTGTGTTATAGACATGAGATATCAAGTAGTTAAACGAGTTGTATTCATATTTAATATTTGTGTTATATGCATCGTTAGAGACATGTTAAATTGATAAGACATGGTTTGTCAGCCTAAGTACTAGGGTCATGGGCTCATACCTGACCCTGTGCATGCAGAGCCGGTGCTTCGGGCTTGCCTTTATAGTATGTTAATCTTTATTTTTCTTTTGAAAAAAATGTTAATGTGTTTTTTATGTTGTGCAGTTGGATGAATGGGTGCTATGTCGAATTTACAAGAAGAACTCAAGTGCACAAAAAACCATTTCTGGCGGTCAAACAACCGAACAGAGCCACGGCTCGCCATCATCATCATCCTCTCAATTCGACGATGTTCTTGAGTCACTACCCGAGATACAAGACCGGTGCTTCAACTTACCAAGGGTTAATTCCATAAAAACCTTCCAACAAGAAGACCAAAAGCTAAATCTCCAAAAGTTCGATTCGGGCAACTACGACTGGGCCAGCATCGCCTCATTCGGGTTGCCCGAACCCGTTGTTCAAGATCCACTACCACAACCCACAATGAATGGTGGTTCCTCAATGGCACCAATATACACTATGGACACCAAGTTTGGAAGATCAGTAGAAGATGAAGTTCAAAGTGGAATCAGAAGCCAACGGGTGGATCATCCGGGCTACTTTCAATCGAACTTGAGCCCGTTTGGTCATAGCCAGAGTGTATCGAACACGATCGACCCGTTTGGTATTAGGTACCCGACCCAACAAGGGGTTTTGGGCTTTAGACAGTGAAAGGGTGGCGTGTAAATATATGATGATTCTCTGGGGGGTTAGTTGTAGAGTCAAGGATTGTACGAGGGGTGGAGAACAATATTGTTTTAGATTTAACATGTTATGGAAATGAAATGGCGTATTCAACGCAATTTTGTGTATGATCTTTGACCCGATGTTATCTATACGAAAAGTAAGTTTACATTTAACA

SEQ ID No.2 coding sequence of sunflower Hanac84 gene

ATGGGTTTACCGGTTACCGACCCGATGACCCAATTAAGCTTACCACCTGGTTTCCGGTTTTACCCGACCGATGAGGAACTTCTTGTCCAATATCTTTGCCGGAAAGTTGCCGGTCACGATTTTTCTCTTCAGATTATTGCTGATATTGATTTGTACAAGTTTGATCCATGGCAGCTTCCTAGTAAGGCGATGTTTGGGGAGAAAGAGTGGTATTTTTTTAGCCCGAGAGATCGGAAGTATCCGAATGGGTCTAGACCGAATAGAGTAGCCGGGTCGGGTTACTGGAAGGCTACCGGAACTGATAAGGTGATCACGACCGAGGGACGAAGGGTTGGGATCAAGAAAGCTTTGGTGTTTTATGTTGGTAAAGCCCCTAAAGGAAATAAAACTAATTGGATCATGCATGAGTATAGGTTATCGGATCCCCAGAGGAAAACCGGTAGCTCCAGGTTGGATGAATGGGTGCTATGTCGAATTTACAAGAAGAACTCAAGTGCACAAAAAACCATTTCTGGCGGTCAAACAACCGAACAGAGCCACGGCTCGCCATCATCATCATCCTCTCAATTCGACGATGTTCTTGAGTCACTACCCGAGATACAAGACCGGTGCTTCAACTTACCAAGGGTTAATTCCATAAAAACCTTCCAACAAGAAGACCAAAAGCTAAATCTCCAAAAGTTCGATTCGGGCAACTACGACTGGGCCAGCATCGCCTCATTCGGGTTGCCCGAACCCGTTGTTCAAGATCCACTACCACAACCCACAATGAATGGTGGTTCCTCAATGGCACCAATATACACTATGGACACCAAGTTTGGAAGATCAGTAGAAGATGAAGTTCAAAGTGGAATCAGAAGCCAACGGGTGGATCATCCGGGCTACTTTCAATCGAACTTGAGCCCGTTTGGTCATAGCCAGAGTGTATCGAACACGATCGACCCGTTTGGTATTAGGTACCCGACCCAACAAGGGGTTTTGGGCTTTAGACAGTGA

SEQ ID No.3 amino acid sequence of sunflower Hanac84 protein

MGLPVTDPMTQLSLPPGFRFYPTDEELLVQYLCRKVAGHDFSLQIIADIDLYKFDPWQLPSKAMFGEKEWYFFSPRDRKYPNGSRPNRVAGSGYWKATGTDKVITTEGRRVGIKKALVFYVGKAPKGNKTNWIMHEYRLSDPQRKTGSSRLDEWVLCRIYKKNSSAQKTISGGQTTEQSHGSPSSSSSQFDDVLESLPEIQDRCFNLPRVNSIKTFQQEDQKLNLQKFDSGNYDWASIASFGLPEPVVQDPLPQPTMNGGSSMAPIYTMDTKFGRSVEDEVQSGIRSQRVDHPGYFQSNLSPFGHSQSVSNTIDPFGIRYPTQQGVLGFRQ

Nucleic acid sequence of full length of SEQ ID No.4 sunflower HaNAC146 gene

CAAAACCACCCTCAAGAAGAACAACCTCGAACCAAGCCTTCTTATCCAAGCAATTTCTCTCTCCTGCTCGTTCATCGAAGCGACGTTTTCATGTTCCAGATGCAATCTTCCACGTGTTCATTATACCTTTATATATATCTCTATACCTTGCTCTAAACCTCACTCTGCTGAAAACATTTTGGATAACATACAACTTTACATTAACAAGCTTTTCTTTGACTTTATTATTACAAGGAACAAGTTGTTGGTTTTGTGGAATGACTTGTGATAATTTGGAGTTGCCTCCCGGGTTTAGGTTTCACCCGACGGATGATGAGTTAGTTATGCATTATTTGTGTCGGAAATGCGCGTCTCAGCCGATTTCGGTCCCGATTATTGCTGAGATTGATCTGTATAAGTTCGATCCCTGGCAGCTTCCTGAAATGGCTTTGTATGGAGAAAAGGAGTGGTACTTCTTCTCACCGAGAGATAGGAAGTACCCGAATGGATCACGACCGAATAGAGCGGCTGGAACGGGTTATTGGAAGGCCACGGGAGCGGATAAACCGATCGGGAAACCGAAACCGGTTGGGATTAAGAAGGCGCTTGTGTTCTACGCCGGAAAAGCTCCAAGAGGGGTGAAAACCAATTGGATAATGCACGAGTATCGTCTTGCTAATGTTGATCGATCCGCCGGCAAACGGAACAACAATCTTAGGTTAGATGATTGGGTATTATGTCGAATATACAACAAGAAAGGAACCCTAGAGAAGCACTATGTCAATGATAATCAATCATCAGAAATGGAGGTTGAAACGAAACCCAAGATTACCCAAAATACCCCTGCACCCGGGGTACATCCACCGGCGTCAGTACCACATCACGTCATGAATGACGTGTTTCATTTCGATACGTCAGAATCAGTTCCGACGTTACATACGGATTCAAGTCCTGAACACGAAAGAGAGGTCCAAAGTGACCCGAAACAAAATGATGTTGGGTTTAGTTACATGGACCCGTTTCAAGATGACGGTTTTACCCCTCAATCGCAATACTATAACGACTTTCAACTGTCCCCGTTACAGGACATGTTTATGTACACACCAAAGCCCTTTCAGATGTGAAAACTCTTTAAGTTTCGTTTGTAGTTGTACAATGGTTTATGGGCAAGGTACATATTGCCATAGAAGATGTGAAGGAATTTGGGCCCATTTAGAACATATTATCAAGTTGAAATGGGTGACAAGTTGAAGGGCAAAATGGGTACAGTTTTGTTATGGGTCAAGGAGCAAGTGGGGGTGTGGATAGAGTAGTGATGGGCAAATTGCCATAGATGCAAGAAATCTTGATGTTGGCATGATTTTCCCATTGTAGTTAGTAGAACACAAATAACACTCTTAGATGTGTAATTTGATGAAGGAAATGAACTATTAATGTGTTGGTTTCTATGGTCA

SEQ ID No.5 coding sequence of sunflower HaNAC146 gene

ATGACTTGTGATAATTTGGAGTTGCCTCCCGGGTTTAGGTTTCACCCGACGGATGATGAGTTAGTTATGCATTATTTGTGTCGGAAATGCGCGTCTCAGCCGATTTCGGTCCCGATTATTGCTGAGATTGATCTGTATAAGTTCGATCCCTGGCAGCTTCCTGAAATGGCTTTGTATGGAGAAAAGGAGTGGTACTTCTTCTCACCGAGAGATAGGAAGTACCCGAATGGATCACGACCGAATAGAGCGGCTGGAACGGGTTATTGGAAGGCCACGGGAGCGGATAAACCGATCGGGAAACCGAAACCGGTTGGGATTAAGAAGGCGCTTGTGTTCTACGCCGGAAAAGCTCCAAGAGGGGTGAAAACCAATTGGATAATGCACGAGTATCGTCTTGCTAATGTTGATCGATCCGCCGGCAAACGGAACAACAATCTTAGGTTAGATGATTGGGTATTATGTCGAATATACAACAAGAAAGGAACCCTAGAGAAGCACTATGTCAATGATAATCAATCATCAGAAATGGAGGTTGAAACGAAACCCAAGATTACCCAAAATACCCCTGCACCCGGGGTACATCCACCGGCGTCAGTACCACATCACGTCATGAATGACGTGTTTCATTTCGATACGTCAGAATCAGTTCCGACGTTACATACGGATTCAAGTCCTGAACACGAAAGAGAGGTCCAAAGTGACCCGAAACAAAATGATGTTGGGTTTAGTTACATGGACCCGTTTCAAGATGACGGTTTTACCCCTCAATCGCAATACTATAACGACTTTCAACTGTCCCCGTTACAGGACATGTTTATGTACACACCAAAGCCCTTTCAGATGTGA

Amino acid sequence of protein encoded by SEQ ID No.6 sunflower HaNAC146 gene

MTCDNLELPPGFRFHPTDDELVMHYLCRKCASQPISVPIIAEIDLYKFDPWQLPEMALYGEKEWYFFSPRDRKYPNGSRPNRAAGTGYWKATGADKPIGKPKPVGIKKALVFYAGKAPRGVKTNWIMHEYRLANVDRSAGKRNNNLRLDDWVLCRIYNKKGTLEKHYVNDNQSSEMEVETKPKITQNTPAPGVHPPASVPHHVMNDVFHFDTSESVPTLHTDSSPEHEREVQSDPKQNDVGFSYMDPFQDDGFTPQSQYYNDFQLSPLQDMFMYTPKPFQM

Example 2 tolerance assay of sunflower transient transformed seedlings overexpressing Hanac84, hanac146 and RNA interference under high salt and drought stress

(1) Acquisition of transient transformed seedlings of sunflower overexpressing Hanac84, hanac146 and RNA interference

The growth status, seedling age, ascorbic acid in the agrobacteria infesting solution for transformation, sucrose concentration and transformation infestation time of sunflower seedlings (early short-and-large-headed backbone parents) were adjusted by experiments. Agrobacterium infection solution is prepared by transforming the plant expression vector pBI121 with the Agrobacterium strain GV3101, and sunflower seedlings are soaked in the infection solution, so that the GUS gene on pBI121 is transferred into the sunflower seedlings. The method is characterized in that the condition of each condition in the transient transformation system is identified by detecting GUS dyeing condition and GUS gene expression quantity in transient transformation seedlings so as to obtain the sunflower transient transformation system with high gene expression efficiency.

The final determined sunflower transient transformation optimization system was: selecting four days old sunflower seedlings by water planting method, and transforming the concentration of Agrobacterium in the invasion solution to OD ₆₀₀ The agrobacteria infesting solution had a value of about 0.8 and a composition of 7% sucrose, 100. Mu.M acetosyringone, 50mM CaCl ₂ 0.1mg/L ascorbic acid, 20 mu M5-azacytidine and 0.02% SILWETL-77, the infection time of seedlings in agrobacterium infection solution is 2-4 h, and then the seedlings are grown for 3d in a dark environment. The sunflower transient transformation system with high gene expression efficiency and complete and uniform expression tissue can be obtained by using the method. And (3) transiently transforming sunflower seedlings by using the constructed pCAMBIA2300 over-expression vector, interference vector and empty vector according to an optimized method.

(2) High salt and drought stress tolerance detection of sunflower transient transformed seedlings overexpressing HaNAC84, haNAC146 and RNA interference

Firstly, the expression quantity of the HaNAC84 and the HaNAC146 genes in the transient transformed sunflower seedlings is identified by using qRT-PCR technology to determine the target genes which are successfully over-expressed or interfered, and HaActin is taken as an internal reference gene. By 2- ^ΔΔCT The relative expression levels of the genes were calculated by the method and the transformation empty vector was normalized to one. Gene expression levels were measured using the Duncan test (p<0.05 For evaluation, the different lowercase letters are indicated at p<There was a significant difference in the 0.05 level. The data were both plotted and analyzed using Graphad prism5.0 and SPSS (IBM SPSS statistic) software.

Transiently transformed seedlings were tested for tolerance under high salt and drought stress. The high salt treatment condition is that 350mM NaCl is added into 1/2 Hoagland nutrient solution, and the treatment time is 72 hours; the drought stress treatment was carried out by adding 400mM Mannitol to 1/2 Hoagland nutrient solution for 72 hours. Water treatment was used as a control group.

qRT-PCR reaction primer (5 '-3'):

SEQ ID No.25HaNAC84-F:ATTCGGGCAACTACGACTGG；

SEQ ID No.26HaNAC84-R:TGACCAAACGGGCTCAAGTT；

SEQ ID No.27HaNAC146-F:CTCCGAGAGGGGTCAAAACC；

SEQ ID No.28HaNAC146-R:GGGTGGAGGGGTATTTTGGG；

SEQ ID No.29HaActin-F:TCAACGTTCCCGCCATGTAT；

SEQ ID No.30HaActin-R:GACCACTGGCATAGAGGGAAAG。

as shown in FIG. 2, after gene transformation is performed by using a sunflower transient transformation system, qRT-PCR technology is used to detect the expression of a target gene in seedlings and the phenotypic identification of drought stress. The result is that the expression levels of HaNAC84 and HaNAC146 in seedlings transformed with the over-expression vector are significantly increased compared to those of seedlings transformed with the empty vector, while the expression levels of HaNAC84 and HaNAC146 in seedlings transformed with the interfering vector are significantly decreased. This indicates that transient transformation of HaNAC84 and HaNAC146 in sunflower seedlings was successful and that subsequent experiments were possible.

Before stress treatment, there is no obvious difference between the growth states of sunflower seedlings transformed with over-expression and interference vectors and sunflower seedlings transformed with no load, after 72 hours of salt treatment, the growth states of seedlings transformed with Hanac84 and Hanac146 over-expression vectors are better than those of the empty vectors, and the growth states of seedlings transformed with interference vectors are worst. Likewise, after 72h of drought stress treatment of seedlings, the overexpressed and disturbed sunflower seedlings also exhibited results consistent with the growth status of the salt-treated seedlings of each type.

The above results indicate that overexpression of HaNAC84 and HaNAC146 confers salt and drought tolerance to sunflower seedlings, while their interference increases the sensitivity of sunflower seedlings to salt drought stress.

EXAMPLE 3 obtaining transgenic Arabidopsis thaliana

(1) Transformation of Agrobacterium tumefaciens GV3101

And (3) transforming the successfully constructed vector containing the target gene into an agrobacterium competent cell (GV 3101) by utilizing a liquid nitrogen freeze thawing method, randomly picking single agrobacterium colonies on a solid culture medium plate, inoculating the single agrobacterium colonies into a YEB+Rif+Kana liquid culture medium for shake culture, extracting plasmids in a bacterial liquid after culturing for a certain time, and carrying out PCR amplification by taking the plasmids as a template, wherein the positive agrobacterium transformants are identified correctly and then carrying out subsequent experiments.

(2) Agrobacterium-mediated inflorescence infection method for stable genetic transformation of Arabidopsis thaliana

PCR identification of the correct Agrobacterium Strain was grown to OD in YEB liquid Medium containing Kana and Rif ₆₀₀ 0.8 to 1.0. Agrobacterium cells were collected by centrifugation at 4000rpm for 20min and resuspended in MS liquid medium to prepare an infection resuspension. Selecting Arabidopsis plants with good growth conditions and petals which are not yet opened, cutting off the horns (growing for about 5 weeks), and placing inflorescences into infection heavy suspension for gene transformation. And bagging the plants, and placing the plants with the bags removed in a normal growth environment for growth after the plants are subjected to dark treatment for 24 hours.

(3) Acquisition of stably transformed Arabidopsis thaliana single copy homozygous lines

T after harvesting Arabidopsis inflorescence infection ₁ And (3) replacing seeds, taking a proper amount of seeds, sowing the seeds in an MS+kana solid culture medium, and carrying NPTII genes in a pCAMBIA2300 vector, wherein the pCAMBIA2300 vector has kanamycin resistance, so that positive seedlings can normally grow in the kanamycin culture medium. And (5) selecting seedlings with green true leaves and longer roots, transplanting the seedlings into flower soil, and culturing in a greenhouse. And (5) when the plant grows to be mature, harvesting the seeds of the single plant. Harvesting T from individual plants ₂ And (3) replacing seeds, sowing a proper amount of seeds into an MS+Kana flat plate, observing the resistance separation ratio of the seeds to kanamycin, taking single copy of a strain line meeting a ratio of 3:1, transplanting green seedlings into flower soil, and culturing normally. T (T) ₃ The seeds are sown on a 1/2MS+Kana plate, and the seedlings are homozygotes. Obtaining stable transgenic plants T ₃ And (3) replacing.

Example 4 identification of salt drought stress resistance of transgenic Arabidopsis thaliana overexpressing HaNAC84 and HaNAC146

(1) Plant material

Select T ₃ The homozygous single copy transgenic lines of the generation Hanac84 OE13, hanac84 OE15, hanac146OE 8 and Hanac146OE9 were subjected to functional identification of salt stress. The wild type Arabidopsis thaliana is Columbia type (Col-0).

(2) Identification of resistance of seedling stage Hanac84 and Hanac146 transgenic Arabidopsis to salt drought stress

Sterilizing Arabidopsis seeds with sodium hypochlorite and 75% ethanol for 5min, seeding sterilized Arabidopsis seeds into 1/2MS culture medium, culturing for 14d, transplanting into flowerpot containing matrix (perlite:vermiculite:flower soil=1:1:3), and irrigating once every 3d to ensure soil wetting.

Arabidopsis thaliana, which had been transferred to soil and grown normally for about 4 weeks, was subjected to 300mM NaCl stress treatment for 5 to 7 days, and phenotypic changes were observed.

Drought stress treatment is to stop irrigating Arabidopsis thaliana for about 12d, and corresponding stress phenotypes are recorded; the water was then re-hydrated for 3d and recovery phenotypes and survival assays were recorded.

(3) Data analysis

Survival was assessed using the Duncan test (p < 0.05), with different lowercase letters indicating significant differences at the p <0.05 level. The data were both plotted and analyzed using Graphad prism5.0 and SPSS (IBM SPSS statistic) software.

The results are shown in FIG. 3 and FIG. 4, and under normal growth conditions, both the HaNAC84 and HaNAC146 transgenic Arabidopsis thaliana and the wild type exhibited healthy growth states; under salt stress conditions, haNAC84 and HaNAC146 overexpressed arabidopsis thaliana exhibited better growth status than wild-type, with fewer leaf wilting, whereas wild-type arabidopsis thaliana generally exhibited leaf wilting, yellowing. After 12d of natural drought, the wild arabidopsis has the phenomenon of a large number of leaves drying, and the growth conditions of transgenic arabidopsis with over-expression of Hanac84 and Hanac146 are better; after rehydration for 3d, the survival rate of wild type arabidopsis thaliana (around 50%) is also significantly lower than that of two transgenic arabidopsis thaliana (up to more than 70%).

From the above experiments, it was determined that HaNAC84 and HaNAC146 are capable of increasing the resistance of transgenic arabidopsis thaliana to salt stress and drought stress.

EXAMPLE 5 growth observations of sunflower Hanac84 and Hanac146 transgenic Arabidopsis

(1) Planting of Arabidopsis thaliana

Seeding sterilized Arabidopsis seeds (Col-0, haNAC84 OE13 and HaNAC84 OE15, haNAC146OE 8 and HaNAC146OE 9) into 1/2MS culture medium, germinating and growing for 14d, transplanting about 120 seedlings of each strain into flowerpot (perlite: vermiculite: flower soil=1:1:3), and observing the growth condition.

(2) Observation of Arabidopsis growth conditions during vegetative and reproductive growth and seed size and yield determination

The main statistical indexes in the plant growth process are plant height, single plant bolting branch number, leaf size, single plant horn number, seed number in single horn, horn length, thousand grain weight, seed length and width. Seed size was measured by first placing it under a microscope for observation and then measuring its length and width with Image J software (National Institutes of Health, usa).

(3) Data analysis

All data were evaluated using the Duncan test (p < 0.05), with different lowercase letters indicating significant differences at the p <0.05 level. The data were both plotted and analyzed using Graphad Prism5.0 and SPSS (IBM SPSS statistic) software.

As a result, as shown in fig. 5 and 6, it was found that both transgenic arabidopsis were grown more strongly than the wild type by observing the growth conditions of both transgenic arabidopsis and the wild type under the normal growth environment. In contrast, the plant height of both transgenic arabidopsis thaliana is significantly higher than that of the wild type, and the root system is more developed than that of the wild type; the leaves of the transgenic plants are larger, and the number of branches of bolting of a single plant is obviously higher than that of a wild plant; the number of seeds in a single pod in a transgenic arabidopsis plant is significantly greater than that in the wild type and the resulting seeds are also significantly greater than that in the wild type, whereby the thousand seed weight of the seeds of both transgenic arabidopsis plants is also significantly higher than that in the wild type.

The above results indicate that overexpression of the HaNAC84 and HaNAC146 genes not only promotes plant growth but also increases seed yield.

Claims

1. The sunflower transcription factor Hanac84 protein is characterized in that: the amino acid sequence is shown as SEQ ID No. 3.

2. The sunflower transcription factor HaNAC84 protein of claim 1, wherein: the nucleotide sequence of the coding HaNAC84 protein is shown as SEQ ID No.1 or SEQ ID No. 2.

3. The sunflower transcription factor Hanac146 protein is characterized in that: the amino acid sequence is shown as SEQ ID No. 6.

4. A sunflower transcription factor HaNAC146 protein according to claim 3, characterized in that: the nucleotide sequence of the coding HaNAC84 protein is shown as SEQ ID No.4 or SEQ ID No. 5.

5. Use of the sunflower transcription factor HaNAC84 protein of claim 1 or 2, or the sunflower transcription factor HaNAC146 protein of claim 3 or 4, for modulating stress tolerance in plants.

6. Use of the sunflower transcription factor HaNAC84 protein of claim 1 or 2, or the sunflower transcription factor HaNAC146 protein of claim 3 or 4, for regulating plant growth.

7. The use according to claim 5, characterized in that: the stress is drought or high salt.

8. The use according to claim 6, characterized in that: the growth conditions comprise plant height, plant type, root system, leaf size, number of branches for bolting a single plant, number of seeds or seed size.

9. The use according to claim 6, characterized in that: the plant is sunflower or Arabidopsis thaliana.

10. The use according to claim 6, characterized in that: the regulation is positive regulation; further, the upregulation is such that either the HaNAC84 protein or the HaNAC146 protein is overexpressed.