CN107987141B

CN107987141B - Application of corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification

Info

Publication number: CN107987141B
Application number: CN201810090650.8A
Authority: CN
Inventors: 张举仁; 王保海; 王洁敏; 李朝霞
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-01-30
Filing date: 2018-01-30
Publication date: 2022-10-14
Anticipated expiration: 2038-01-30
Also published as: CN107987141A

Abstract

The invention discloses an application of a corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification, which is to clone the ZmNF-YA1 gene from corn, recombine the gene into a plant expression vector in a sense or antisense form or an RNAi structural form, and introduce the ZmNF-YA1 gene into a plant by adopting a transgenic technology; by detecting the transgenic expression and carrying out stress resistance measurement on the plants, transgenic plants and descendants with improved or reduced stress resistance are screened out, and a new germplasm with application value in plant breeding is created. The ZmNF-YA1 gene is used for participating in the regulation of plant stress resistance through gene expression regulation, and has important significance for cultivating high-yield transgenic crops.

Description

Application of corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification

Technical Field

The invention belongs to the field of bioengineering breeding of crops, and particularly relates to an application of a corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification; namely a scheme and application for changing the stress resistance of the corn by constructing a transgenic overexpression structure and a transgenic way.

Background

NF-Y (nuclear factor-Y) is Sub>A transcription factor compound commonly existing in eukaryotes, consists of 3 different subunits (NF-YA/CBF-B, NF-YB/CBF-A and NF-YC/CBF-C), and regulates the expression of Sub>A target gene by acting on promoters of other regulatory factors. The complete NF-Y transcription complex binds to the CCAAT motif in the target gene promoter, regulating transcription of the target gene. Wherein NF-YA is a subunit specific to DNA sequence, and is combined on a core pentamer nucleotide CCAAT motif (motif) of a gene promoter region. The NF-Y complex can function as a transcriptional activator or repressor, and its binding to DNA and transcriptional regulatory activity are also regulated by other transcription factors, which act by interacting with NF-Y subunits.

In yeast and mammals, each NF-Y subunit is encoded by a single gene, which has multiple splicing patterns and multiple post-translational modifications. In mammals, the physiological functions of NF-Y complexes and their involvement in regulatory pathways include a variety of molecular biochemical processes, such as endoplasmic reticulum stress, DNA damage and repair, cell cycle regulation, and the like. However, in plants, each NF-Y subunit is encoded by a gene family, and thus the NF-Y complex has diversity consisting of different subunits. In Arabidopsis, the NF-YA subunit is encoded by 10 genes, the NF-YB subunit is encoded by 13 genes, and the NF-YC subunit is encoded by 13 genes. The rice has at least 11 NF-YA genes, 12 NF-YB genes and 8 NF-YC genes. In corn, 14 NF-YA genes, 18 NF-YB genes and 18 NF-YC genes are found at present. GUS expression analysis is carried out on 36 NF-Y subunit genes of arabidopsis thaliana, and each subfamily member is found to have a complex and diverse expression pattern, so that the function diversification of different NF-Y complexes formed by the family members is suggested, and the expression of various genes can be regulated.

The existing work shows that the NF-Y three subunit family members are all involved in the regulation and control of plant growth and development. LEAFY COTYLEDON1 induces vegetative cells to develop into embryos in Arabidopsis, and two NF-Y subunit genes (AtLEC 1/AtNF-YB 9) regulate embryogenesis and seed maturation. AtNF-YB9 and LEC1-LIKE (L1L/NF-YB 6) regulate the development of Arabidopsis embryos by inducing the expression of genes related to embryo formation and cell differentiation. Multiple NF-YA subunit genes in Arabidopsis are expressed in embryos, such as NF-YA1, YA2, A3, A4, A6, A7, A8, and A ^] . Over-expressing NF-YA1, A5, A6, or A9 strains are hypersensitive to ABA during seed germination, and male gamete formation, embryogenesis, seed morphology and germination are affected. These over-expressed NF-Y materials can form somatic embryos directly from vegetative organs. NF-YA3 and-A8 showed the highest expression levels from globular embryos to torpedo embryos. nf-ya3nf-ya8 double mutant is lethal to the embryo, but nf-ya3 and nf-ya8 single mutants do not show significant phenotypic changes, and the two genes are presumed to be functionally redundant in the early embryogenesis process of Arabidopsis thaliana.

In addition to affecting plant embryogenesis and seed maturation, NF-Y transcription factors also regulate vegetative or/and reproductive growth of plants. Overexpression of AtNF-YB2 promotes primary root elongation by stimulating cell division and cell elongation. In the bean model plant Zygophyllaceae, mtHAP2-1 is expressed in the root nodule meristematic region and plays an important role in the differentiation of root nodule cells. Research on overexpression of kidney bean PvNF-YC1 and interference of the gene expression through RNAi reveals that NF-Y plays an important role in the nodule organ formation process. In green skewers, yu et al found that an NF-YC protein regulated the growth direction of pollen tubes by interacting with PwFKBP 12.

Work has reported that NF-Y regulates plant drought tolerance. Respectively overexpress AtNF-YB1 and a corn homologous gene ZmNF-YB2 thereof in arabidopsis thaliana and corn, and the growth vigor and the survival rate of transgenic plants are superior to those of non-transgenic plants under drought conditions. Chip detection results show that the expression change of AtNF-YB1 does not affect dehydration-response element binding proteins (ABA-dependent drought-resistant pathways), and suggest that AtNF-YB1 may act through an ABA signal-independent drought-resistant pathway. Multiple work suggests that NF-Y interacts with bZIP, while bZIP proteins participate in ABA signaling pathways.

The AtNF-YA5 gene is up-regulated by drought, osmotic stress and salt stress. Transcriptional regulation of AtNF-YA5 is regulated by an ABA-dependent mechanism, and miR169 is involved in post-transcriptional modification of the AtNF-YA 5. Under drought conditions, miR169 down-regulates expression, and the process depends on ABA.35S miR169 and nf-ya5 mutant plants are more sensitive to drought stress. AtNF-YA5 regulates and controls the size of stomata of guard cells, and AtNF-YA5 also improves the drought tolerance of plants by activating the expression of stress response genes in other cells, such as oxidative stress response related genes. Besides AtNF-YA5, other AtNF-YA genes such as YA2, YA3, YA7 and YA10 can be overexpressed to improve the drought tolerance of plants ^[148] . The expression level of 11 wheat NF-Y genes under the drought condition is obviously different from that under the normal condition, wherein 8 genes are subjected to drought down-regulation expression and 3 genes are subjected to up-regulation expression. One NF-YA gene (OsHAP 2E) of rice is a target of miR169, is induced to express by salt stress and can be regulated and controlled by an ABA-dependent pathway. OsNF-YA7 is induced and expressed by drought stress, but the expression does not respond to ABA. The drought tolerance of rice plants over-expressing OsNF-YA7 is improved. Analysis of the OsNF-YA7 promoter revealed 3 DRE/CRT elements regulated by the ABA-independent drought-regulated transcription factor OsDREB 2. OsNF-YA7 is therefore presumed to regulate drought tolerance in plants primarily through an ABA-independent pathway. OsNF-YA reduction in drought stress2 and the drought tolerance of the osnf-ya2 insertion deletion mutant is improved, which indicates that the gene is possibly a negative regulatory factor of drought in rice. Overexpression of a wheat NF-YA gene TaNF-YA10-1 in arabidopsis thaliana can improve drought tolerance of plants and promote growth of root systems and plants, but the TaNF-YA10 overexpression plants are more sensitive to salt stress, which indicates that the gene has different regulating functions in responding to drought stress and salt stress.

There have been several works reporting that NF-Y transcription factors regulate plant responses to heat and cold stress. The cold stress resistance of plants can be improved by over-expressing AtNF-YA2 or AtNF-YC1 in arabidopsis thaliana. The result of transcriptome analysis shows that some abiotic stress response genes in the AtNF-YA2 overexpression plant are expressed in a down-regulation mode, but the mechanism that the down-regulation of the genes can improve the cold resistance of the AtNF-YA2 overexpression plant is not clear. AtNF-YC1 may regulate the anti-freeze stress via a pathway dependent on C-REPEAT BINDING FACTORS (CBFs).

The increasing impact of heat stress on crop yield is a subject of research that is currently being focused on in plant science. Sato et al found that DREB2A interacted with DNA POLYMERASE II SUBUNIT B3-1 (DPB 3-1/NF-YC 10), especially under heat stress. DPB3-1/NF-YC10 can form a trimer with NF-YA2 and NF-YB3 to regulate the expression of DREB2A mediated heat stress related genes. Overexpression of DPB3-1/NF-YC10 increases the expression level of HEAT stress response gene HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA 2), while the expression level of HSFA2 is down-regulated in NF-YC10 mutant ^[158] . The overexpression of NF-YC10 does not affect the growth of arabidopsis thaliana plants, and the overexpression of arabidopsis thaliana AtNF-YC10 in rice can improve the heat resistance of rice plants without affecting the growth and development of the plants. NF-YC10 belongs to a single source group, is highly conserved among terrestrial plants, and thus can be a candidate gene for creating heat stress resistant crops.

NF-Y transcription factors are also involved in Endoplasmic Reticulum (ER) stress responses. In Arabidopsis, NF-YC2, NF-YA4, NF-YB3 and bZIP28 form a transcription complex, so as to up-regulate the expression of genes related to endoplasmic reticulum stress, prevent the accumulation of misfolded and unfolded proteins in the endoplasmic reticulum and relieve the reduction of protein synthesis. Although the analysis results of the bioinformatics and the chip data show that the expression of NF-Y gene family members is regulated and controlled by various stresses, the research on the specific stress resistance function and mechanism of the plant is less.

Among many reports, the expression patterns of the NF-YA gene family of plants are spatio-temporally specific. Expression analysis of Arabidopsis NF-YA family members in different tissues shows that certain members have common organ expression patterns, such as AtNF-YA4 and AtNF-YA7, which are similar in leaf and flower expression patterns, and the functional redundancy of the members is suggested. Zhang et al systematically identified 50 ZmNF-Y genes and analyzed their expression patterns by using chip data to obtain that some ZmNF-Y family members exhibit specific expression in vegetative organs and reproductive organs, and that there are differences in the responses to biotic stress and abiotic stress among different members. By respectively inoculating pathogenic bacteria fusarium moniliforme, head smut bacteria and lawn anthrax bacteria and analyzing the response of ZmNF-Y to biological stress, zmNF-YA3, zmNF-YA8 and ZmNF-YA12 are found to be up-regulated and ZmNF-YA1 is down-regulated. Luan Mingda and the like perform subcellular localization analysis on 7 ZmNF-YAs regulated by mature miR169, and the ZmNF-YAs are all located in cell nuclei, but lack the capability of transcriptional activation. Analyzing the expression patterns of ZmNF-YA and miR169 responding to adversity stress, finding that the expression of ZmNF-YA and miR169 responds to PEG, ABA and salt stress in the corn leaves, but the expected negative correlation relationship does not occur between the ZmNF-YA and the miR 169. At corn roots, short-term stress (0-48 hours) caused the expression of miR169 to be down-regulated, while long-term stress (15 days) up-regulated the expression of miR169, and most ZmNF-YA showed opposite expression changes to miR169 during short-term stress. When the stress response is carried out, the expression level of miR169 is in a positive correlation with the elongation of the corn root, while ZmNF-YA14 is in a negative correlation with the elongation of the root, namely the miR169/ZmNF-YA14 regulation module possibly participates in the regulation process of the corn root responding to the stress. In addition, the over-expression of ZmNF-YA14 can up-regulate the expression quantity of peroxidase-related genes, actively eliminate the accumulation of active oxygen in cells and enhance the resistance of corn to salt stress.

The number of corn NF-Y family genes is large, and a corn genome sequence is searched to find that 36 corn NF-YA family possible members, 28 NF-YB members and 25 NF-YC members exist. However, the functional research of the NF-Y family genes is in the beginning stage, and only research reports that the drought tolerance of plants is improved by over-expression of the corn ZmNF-YB2 gene and patents applied by Mengshan company are seen in the search. The specific application of the corn nuclear factor gene ZmNF-YA1 in the plant stress resistance modification is rarely reported.

Disclosure of Invention

Aiming at the current research situation, the invention provides an application of a corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification. The ZmNF-YA1 gene is used for participating in the regulation of plant stress resistance through gene expression regulation, and has important significance for cultivating high-yield transgenic crops.

The application of the corn nuclear factor gene ZmNF-YA1 in plant stress resistance modification is to identify and clone a ZmNF-YA1 sequence from corn, construct a fusion gene by using a full-length or partial sequence, connect a promoter of the fusion gene in front of a target gene coding frame (in a sense form) or an RNAi structure, insert the fusion gene into a plant expression vector, and introduce a recombinant gene into a plant cell by adopting a transgenic technology to obtain a transgenic plant; by detecting the transgene expression and carrying out character identification on the plants, the transgenic plants with obviously changed target characters and descendants thereof are screened out, and new germplasm and new varieties with application prospects in plant breeding are created. The specific scheme is as follows:

the application of a corn nuclear factor gene ZmNF-YA1 in the plant stress resistance modification is characterized in that: cloning ZmNF-YA1 gene from corn, recombining the gene into a plant expression vector in a sense or antisense form or an RNAi structure form to form a fusion gene; introducing the recombinant gene into a plant by using a transgenic technology; by detecting the transgenic expression and carrying out stress resistance measurement on the plants, screening out transgenic plants and descendants with improved or reduced stress resistance, and creating a new germplasm with application value in plant breeding; wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNA is shown as SEQ ID No.1, and the coded amino acid sequence thereof is shown as SEQ ID No. 2.

The corn nuclear factor gene ZmNF-YA1 is applied to the plant stress resistance modification: the ZmNF-YA1 gene has a cDNA form or a genome gene form, and a coding sequence thereof is inserted into a plant expression vector in a sense form, an antisense form or an RNAi structural form to construct a fusion gene; the promoter is a stress inducible promoter or a constitutive promoter.

Wherein: the plant stress resistance comprises drought resistance, salt resistance or heat resistance; the plant is corn, wheat or bluegrass.

Expression analysis of ZmNF-YA1 Gene

In the study of drought-tolerant mechanism of corn, drought-tolerant inbred line Q319 and drought-sensitive inbred line 65232 are used as materials, chip hybridization technique is adopted to compare the influence of seedling stage drought treatment (18% PEG solution is used for irrigating sand-cultured 3-leaf stage corn seedlings, and 12h, 24h and 48h after treatment and 24h after recovery watering) on transcriptome of different genotypes, and a batch of transcription factors with different variation trends in different genotypes are selected. Then, the expression intensity of these genes was verified by quantitative RT-PCR technique. From the genes, zmNF-YA1/GRMZM2G000686 were selected for study. Under the condition of proper growth, the expression abundance of ZmNF-YA1 in the roots is greatly higher than that of leaves. In roots, the expression quantity of the drought-tolerant inbred line Q319 is 2.5 times that of the drought-sensitive inbred line 65232; in leaves, Q319 and 65232 are expressed in similar amounts, about 1/4 of 65232 roots. When the drought-tolerant inbred line Q319 is treated for 24 hours under osmotic stress, the expression abundance is reduced to about 70% before treatment, and the expression amount is remarkably increased in 65232 roots and is higher than that of Q319; in the leaves, Q319 and 65232 changed in opposite directions, i.e., Q319 rose significantly and 65232 dropped to a level less than 1/10 of that in the pre-treated roots. When the root is treated by osmotic stress for 48 hours, the expression abundance of ZmNF-YA1 in the root is slightly increased in Q319 compared with that in 24 hours, and 65232 is obviously reduced and is slightly higher than the level before treatment; in leaves, the expression abundance of ZmNF-YA4 of Q319 is greatly increased compared with that of 24h, which is about 4 times that before treatment, and 65232 only shows a small increase. The abundance of transcripts of 24h rehydration, zmNF-YA1 in 65232 and Q319 roots is almost equal, and is slightly higher than the expression level of 65232 roots before treatment; in leaves, Q319 expression was reduced to a lower abundance than before treatment, whereas 65232 expression was slightly more abundant and more abundant than Q319. That is, the expression of ZmNF-YA1 is osmotic stress suppressed in Q319 roots and osmotic stress induced in Q319 leaves; whereas in 65232 roots it was osmotic stress induced and in 65232 leaves it was osmotic stress inhibited. I.e., they have significant differences in osmotic stress responses and may be associated with their drought resistance.

Production of transgenic maize

Respectively fusing the coding frame of the ZmNF-YA1 gene with a dehydration stress induced promoter Prd29A, recombining the coding frame into a plant expression vector to construct a vector pCambia1300-Prd29A, zmNF-YA4-PCaMV35S, bar, transferring the T-DNA region of the plasmid into a corn backbone selfing line by adopting an agrobacterium-mediated method or a gene gun bombardment method, transplanting the transformed seedling into a living state, bagging and selfing to obtain seeds. The progeny of the transformed plant is detected by herbicide screening and molecular biological detection methods (PCR, southern blotting and RT-PCR) to obtain the transgenic plant. Meanwhile, according to the ZmNF-YA1 gene specific sequence, an RNAi structure is constructed and recombined into a plant expression vector for corn genetic transformation, and a transgenic corn plant is obtained. Through selfing and molecular identification of 3 successive generations, transgenic homozygous lines were generated. Under normal cultivation conditions, the transgenic corn plant is normal in shape and growth and development, the expression strength of an over-expression strain ZmNF-YA1 is obviously higher than that of an un-transgenic plant, and the expression strength of the ZmNF-YA1 of a RNAi structure-converted strain is obviously lower than that of the un-transgenic plant.

If the gene gun bombardment method is adopted, young embryos (1.0-1.5 mm) of inbred line plants of corn (Zea mays L.) are inoculated on an induction culture medium 9-15 days after the inbred pollination, the culture is carried out for 4-6 weeks to obtain crisp and light yellow II-type callus, and then the subculture is carried out once every 10-15 days by using a subculture medium. The obtained II type callus is used as receptor material for genetic transformation.

The gene gun pellet is prepared by a conventional method. Weighing gold powder with the size of 1.0 mu m, washing by 70% ethanol, standing for 15 minutes, and centrifuging to remove supernatant; thoroughly washed with sterile water 3 times, and then stored in 50% sterilized glycerin (final concentration of 60mg/ml micro-bomb) for later use. When in use, the gold powder was broken by vortexing for 5 minutes, and 5. Mu.l of plasmid T-DNA (1. Mu.g/. Mu.l) and 50. Mu.l of 12.5M CaCl were added in this order ₂ 20. Mu.l of 0.1M spermidine, the sample is added with vortexing. Then, continuing the swirlStanding for 2-3 min and 1 min. Centrifuging, removing supernatant, adding 70% ethanol, and standing. Then centrifuging and removing supernatant, then resuspending by using absolute ethyl alcohol, sampling and adding on a micro-elastic carrier. The dosage of the micro-bullet is 0.5mg per bullet. A culture dish of 9cm diameter was poured with 0.4cm thick medium and then the callus was placed in high density in the dish and bombarded once per dish. And (3) obtaining bombardment parameters: the distance between the breakable wafer and the carrier is 2.5cm, the distance between the carrier and the blocking net is 0.8cm, and the micro-bullet flying distance is 6-9 cm. Other parameters are valued according to the instruction. And (3) after bombardment, restoring and culturing the material in the dark for 3 days, and then transferring the material into a new culture medium with unchanged components for culturing for 3 weeks to ensure that the transferred target gene is fully expressed. The material is transferred to a medium with a selection agent (e.g. 0.1% herbicide glufosinate) for screening. Three generations of the screen were screened consecutively, 15 days each. And eliminating sticky dead tissue blocks during subculture. Transferring the screened resistant callus to a culture medium without a screening agent, recovering and culturing for 1 generation under illumination for 16 hours/day, and transferring the resistant callus to a differentiation culture medium to differentiate seedlings. And (3) rooting the plantlet generated by the callus in a rooting culture medium, transplanting the strong plantlet into a flowerpot, planting the strong plantlet into a field when the strong plantlet grows to be about 10cm high, and selfing for fructification.

Character detection and utilization of transgenic corn plant

For the transgenic homozygous plant obtained by continuous bagging selfing subculture, the normal growth and development of the transgenic plant under normal cultivation conditions are firstly determined, and then the resistance difference of the transgenic plant and the control plant under stress conditions is analyzed.

Heat resistance test: transgenic homozygous maize plants grown at 28 ℃ (light, 13 h/d)/22 ℃ (dark, 11 h/d) were transferred to 36 ℃ (light) for 2h of growth followed by a 4-day continuous heat treatment at 39 ℃ (light 13h/d, dark 11 h/d) and then restored to growth at 28 ℃. After heat treatment, the non-transgenic control (wt) died almost completely. Compared with the non-transgenic plants, the meadow bluegrass of the RNAi structural strain line subjected to heat stress treatment has obviously improved heat resistance, almost all the non-transgenic plants die, and the damage of the transgenic suppression expression strain line is not obvious, namely the heat resistance is obviously superior to that of the control.

Cold resistance test: transgenic homozygous maize plants grown at 28 ℃ (light, 13 h/d)/22 ℃ (dark, 11 h/d) were grown for 1 day sequentially at 16 ℃ (light 13 h/d), 10 ℃ (light 13 h/d), then continuously cold treated at 4 ℃ for 3 days (light 13 h/d), and then restored to growth at 28 ℃. In cold treatment, the non-transgenic control (wt) grows slowly, and is obviously smaller than that of a transgenic line after treatment; after being treated for 1 day at 4 ℃, a few transgenic over-expression plants and non-transgenic control leaves are damaged, the leaf tips are always wilted, and the corn plants with the RNAi structure are sensitive to cold damage and are seriously damaged; after 3 days of treatment at 4 ℃, the transgenic overexpression lines are recovered for 1 day, the damage degree of the transgenic overexpression lines is obviously lower than that of the control lines, part of the transgenic overexpression lines only have leaf tip wilting, the transgenic RNAi structure corn plants are damaged heavily, and the seedlings almost die.

Drought tolerance test: mature seeds of homozygous lines which are fully dried and respectively transformed with ZmNF-YA1 gene and RNAi structure or antisense form thereof are sown in a soil tray and are placed under proper conditions for growth. And stopping watering the plants in the 4-leaf stage, carrying out drought treatment for one week, then recovering watering, observing the growth conditions and survival rates of the plants, and determining the drought resistance of the plants. The result shows that the drought resistance of the plant is reduced no matter the ZmNF-YA1 gene expression is reduced in the seedling stage or the jointing stage, and the over-expression of the ZmNF-YA1 gene obviously improves the drought resistance of the plant.

The ZmNF-YA1 gene, the RNAi structure or antisense form of the gene, corn and non-transgenic contrast inbred line seeds are sown in flowerpots and fields, drought stress treatment is carried out in a seedling stage female and male ear development stage (9-13 leaf stage), a flowering and pollination stage and a filling stage respectively, namely watering and rain-drenching prevention are controlled, the relative water content of soil is kept at about 55-60 percent, the duration is 15 days, transgenic plant character observation and physiological index detection are carried out, open pollination is carried out, fruit clusters are harvested and seeds are tested. The drought resistance of the ZmNF-YA 1-transgenic overexpression strain is obviously higher than that of the non-transgenic control and RNAi-transgenic structure or in an antisense form, so that the damage symptoms of the strain are light, the strain can recover and grow quickly after stress is relieved, and the economic characters such as the yield of single-plant grains are obviously better than that of the non-transgenic control and RNAi-transgenic structure; the drought resistance and the grain yield of the plants with the converted RNAi structure or antisense form are obviously inferior to those of the plants without the transgenic control. The resistance test and plant screening of transgenic Poa pratensis plants are described in examples 3 and 4.

Through a resistance detection test, the drought tolerance, the heat resistance and the cold tolerance of a stress-induced promoter-initiated transgenic overexpression plant are obviously improved compared with those of a non-transgenic control. On the basis, test results in multiple aspects are integrated, excellent transgenic plants are selected for bagging selfing and homozygous, the combining ability is measured, and the transgenic inbred line with the same combining ability as the donor inbred line or with improved combining ability is selected for the breeding of the corn single hybrid.

Detailed Description

Example 1: zmNF-YA1 gene transferred maize drought and salt tolerant inbred line

1) The receptor system is established by using backbone selfing lines used in agricultural production in China as materials, such as Zheng 58, chang 7-2, DH4866 and the like. Soaking the seeds in 70% ethanol for 8 min, soaking in 0.1% mercuric chloride for 8-12 min, and washing with sterile water for 3-5 times. The seeds are shaken continuously during the sterilization process to ensure complete surface sterilization. After sterilization, seeds are put into a sterile triangular flask for germination, and a small amount of sterile water is put into the flask under the dark condition (25-28 ℃) for 1-2 days. After germination (white emergence) the seeds were germinated in the dark on modified MS medium. When the embryo bud extends for 3-4 cm, the coleoptile and 2-3 young leaves are peeled off, and the growth cone at the top of the stem tip is exposed.

2) The corn stem tip transformation and plant regeneration construct a fusion gene (in a sense form) carrying the complete sequence of a corn nuclear factor gene ZmNF-YA1, and the transgenic ZmNF-YA1 is started by a stress inducible promoter RD29A/B or CaMV35RNA promoter or a corn Ubiquitin promoter. And recombining the fusion gene into a T-DNA zone, wherein the T-DNA zone has a plant herbicide resistance gene bar, and the Agrobacterium tumefaciens strain Mini-Ti plasmid is used to obtain a genetic transformation vector.

Agrobacterium tumefaciens (e.g., LBA4404, etc.) harboring a binary vector (Mini- -Ti plasmid harboring a selection agent resistance gene and a ZmNF-YA1 gene) was shake-cultured at 28 ℃ at a shaking rate of 110rpm (rev/min) in an antibiotic-added LB medium (containing, per liter of medium, tryptone 10g, yeast extract 5g, naCl 10g, pH 7.0, autoclaving) to place the bacteria in a logarithmic growth phase. Then, the mixture was centrifuged at 3000rpm for 10 minutes, and the supernatant was discarded. The strain is washed with a liquid seed germination medium (the components of the seed germination medium are reduced by half and agar powder is removed) with the concentration of 1/2, and then the strain is centrifugally collected. Then the thallus is suspended by a liquid modified MS culture medium with the concentration of 1/2 added with 100 mu mol/l of acetosyringone (As), and diluted by 5-20 times for transformation. Pouring the bacteria liquid into a culture dish with the diameter of 4.5 cm, inclining the culture dish, soaking the aseptic seedlings with the stem tip growth cone exposed in the bacteria liquid, and treating for 8-12 minutes under the atmospheric pressure of 0.5 multiplied by 105 Pa. And then sucking the impregnated bud tips with sterile filter paper, placing the germinated seeds on an improved MS culture medium, and culturing for 2-3 days in the dark at the culture temperature of 22-24 ℃. The sterile shoots were then incubated under scattered light for 2 days. Transplanting the aseptic seedlings after the illumination culture into a flowerpot paved with upper vermiculite and lower loam, and covering the top of the plant with vermiculite. Then the plant is grown under natural illumination, the daily temperature is 22-28 ℃, the night temperature is 15-21 ℃, and 1/2 of the improved MS culture medium inorganic salt is irrigated every other day.

3) Resistance detection and selection utilization of transgenic plants

After the transformed plant has grown 3 leaves, spraying 0.12% aqueous solution of glufosinate herbicide to make the plant drop. Untransformed control plants stopped growing after 4 days post-spray and started dying after 9 days. After spraying, some individuals of the transformed plants have similar changes to those of the control plants, and other individuals of the transformed plants continue to grow and have insignificant changes. When the survival plants grow to 5 leaves, the plants are planted in the field, and the plants are bagged and selfed to form seeds. Taking the leaves of the transplanted survival plants to carry out molecular biological detection to determine the transgenic plants. Then the transgenic plant (T0) is bagged, selfed and fruited. T1 seeds from different T0 plants are sowed in a greenhouse or a field with a protective facility, 0.18 percent of herbicide glufosinate-ammonium aqueous solution is sprayed, and the resistance of the plants is observed. And (4) continuously bagging and selfing herbicide-resistant plants screened from the T1 generation, and continuously performing molecular biological identification and resistance detection on filial generations of the herbicide-resistant plants. Through several generations of selfing homozygosis and resistance detection and selection, the transgenic corn homozygosis line is finally obtained. In drought resistance identification and selection, a transgenic plant line is respectively planted in a flowerpot, a greenhouse and a field, drought stress treatment is carried out in a 3-leaf stage, an elongation stage and a flowering early stage, a emasculation and powder scattering stage and a grain filling stage, the change of physiological parameters and grain yield are measured, and a plant line which has strong drought resistance, is remarkably increased in grain yield compared with a non-transgenic control material and is slightly improved in grain yield with a receptor inbred line under a proper cultivation condition is screened. The strain can be used for preparing corn drought-resistant herbicide-resistant hybrid seeds.

Sowing seeds of the homozygous transgenic line in a sand-filled flowerpot, watering with an aqueous solution of 0.5% or 0.7% NaCl, watering after emergence with a 1/3MS medium inorganic salt solution added with 0.5% or 0.7% NaCl, counting the emergence rate, the necrosis degree of leaves, the survival rate of the plants at 5-leaf stage, and selecting the transgenic line with excellent salt tolerance for inbreeding line breeding and new variety breeding.

Example 2: zmNF-YA1 gene transfer to create heat-resistant maize inbred line

1. Obtaining transgenic corn plant

Seed germination, plasmid construction and genetic transformation of the elite inbred line of maize were as in example 1. After the transformed plant grows 3 leaves, spraying herbicide

(Hoechst Schering agrEvo GmbH, containing herbicide glufosinate ammonium) aqueous solution with concentration of 9.6 ml-10.8 ml

Preferably, the plant drops. Untransformed control plants stopped growing after 4 days post-spray and started dying after 9 days. After the transformed plants are sprayed, some individuals have similar changes to the control plants, and other individuals continue to grow and have insignificant changes. When the survival plants grow to 5 leaves, the plants are planted in the field, and the plants are bagged and selfed to form seeds.

2. Transgenic plant homozygous and progeny analysis

10.8ml for T1 generation plants growing to 3 leaves

Treating with water solution, and observing and counting the individual proportion of resistance and sensitivity; detecting the exogenous gene by PCR technology, and counting the division of the exogenous gene in the filial generation plantAnd (4) separation ratio. Transplanting the survival plants to a field, bagging and selfing. The T2 generation plants are subjected to southern blotting verification by detecting exogenous genes by adopting a PCR technology except bagging self-seeding, and the transgenic expression strength is detected by adopting an RT-PCR technology. For the selected transgenic line, the change of net photosynthetic rate of the plant at different temperatures (28-39 ℃) and light intensities is measured, and the yield and biomass of the individual plant are measured and compared with the non-transgenic plant. After selecting excellent transgenic strains, observing and comparing the corn yield characters under the field cultivation condition, and selecting high-yield and high-light-efficiency strains to enter a heat resistance identification test and a corn breeding test.

3. Transgenic line heat resistance identification test

Seedling stage heat resistance test: transgenic homozygous maize plants grown at 28 ℃ (light, 13 h/d)/22 ℃ (dark, 11 h/d) were transferred to 36 ℃ (light) for 2h of growth followed by a 4-day continuous heat treatment at 39 ℃ (light 13h/d, dark 11 h/d) and then restored to growth at 28 ℃. After heat treatment, almost all of the non-transgenic control (wt) died, and only individual strains in the transgenic strains have small difference with the non-transgenic control, but the heat resistance of most strains is remarkably superior to that of the control, wherein partial transgenic strains are not obviously damaged, namely the primarily selected transgenic heat-resistant strains.

Heat resistance test at pollination and grouting initial stage: the seeds of the initially selected transgenic heat-resistant strain are sown in a greenhouse and grown to a pollination period under a proper growth condition, then the greenhouse is grown for 7 days under the temperature of 36 ℃ (illumination, 13 h/d)/30 ℃ (dark, 11 h/d), then the seeds are grown for 7 days under the temperature of 38 ℃ (illumination, 13 h/d)/30 ℃ (dark, 11 h/d), and the number of green leaves and the number of fruit clusters are counted after 20 days. And observing parameters such as development condition of the fruit clusters, seed setting rate, hundred grain weight and the like after harvesting, and screening out a transgenic line with excellent heat resistance.

Example 3: creation of drought and salt tolerant bluegrass by transferring ZmNF-YA1 gene

The stem tip of seedling of different varieties of Poa pratensis L, such as prize, new George, rugby-A, midnight, etc. is used as test material to induce the base part to callus and expand, cut and expand tissue, and set on the subculture and clump culture medium to induce the generation of clumpy buds, which continuously proliferate on the subculture and clump culture medium to provide transgenic receptor material. The exogenous gene is introduced into receptor cell by means of agrobacterium mediating process and transformed cell and plant are obtained through selection. In the agrobacterium-mediated genetic transformation, the transformation frequency is effectively improved by adopting the treatment of a surfactant Silwet L-77 and the reduced pressure treatment, and an efficient poa annua transgenic technical system with small restriction on genotype is established. The specific operation is as follows.

Preparing various culture mediums

Basic culture medium: for improving MS culture medium (inorganic salt of MS culture medium, thiamine hydrochloride 10.0mg L) ^-1 1.0mg L of pyridoxine hydrochloride ^-1 Nicotinic acid 1.0mg L ^-1 Glycine 2.0mg L ^-1 Inositol 100.0mg L ^-1 Biotin 0.05mg L ^-1 Casein hydrolysate 500mg L ^-1 ) Sucrose 30g L ^-1 Agar powder 6.5g L ^-1 And pH is 5.8-6.0. Is used for seed germination. Removing agar powder from the liquid culture medium.

Induction medium: minimal medium supplemented with different combinations of plant growth regulating substances (hormones). For most Poa annua genotypes, the concentration of 2,4-D is 0.01-1.0mg.L ^-1 The concentration of 6-benzylpurine (6-BA) or kinetin or zeatin is 0-5mg ^-1 . Solid media were used. The optimum hormone concentration varies within this range depending on the genotype of the culture material.

Subculture and clumping medium: solid culture medium, adding plant growth regulating substances of different combinations into the minimal medium, wherein the concentration of 2,4-D is 0-0.2mg L ^-1 The concentration of 6-BA or kinetin or zeatin is 0-3mg L ^-1 And varies according to the Poa annua genotype.

Rooting culture medium: adding 0.002-2.0mg L into the minimal medium ^- 1 Naphthalene Acetic Acid (NAA) or indolebutyric acid (IBA) or indoleacetic acid (IAA), and adopting a solid culture medium for rooting or strong seedling culture of rootless seedlings. The optimum hormone concentration varies within this range depending on the genotype of the culture material.

Seed sterilization and germination and clumping bud induction

70% wine for seedsSterilizing for 4-5 min, adding 0.2% mercuric chloride for 10-15 min, and washing with sterile water for 3-5 times. The seeds are shaken continuously during the sterilization process to ensure thorough surface sterilization. The sterilized seeds are sown on wet sterile filter paper, and the seeds are put in a culture dish for germination under the dark or low-light condition at the temperature of 20-28 ℃. Cutting stem tip in inducing culture medium (adding 2,4-D0.2 mg L) within 10-15 days ^-1 ，6-BA 2mg L ^-1 ) And (3) performing upper culture to induce the basal part to expand, wherein the expansion rate is 100%. Culturing in inducing culture medium for 20 days, cutting stem tip base part swelling tissue (diameter 1-3 mm), transferring into subculture and clumping culture medium (taking 2,4-D0.1 mg L ^-1 ，6-BA 2mg L ^-1 ) And (3) inducing cluster buds to grow, wherein most of the expanded base parts differentiate the cluster buds after 15-20 days, the cluster forming rate is about 75%, and the number of each cluster bud is 15-20. The clump buds are divided and then transferred to a subculture and clump forming culture medium, the clump buds rapidly proliferate, and the subculture is generally carried out once in 15 days. If a larger seedling is to be obtained, the subculture and clumping medium is removed, 2,4-D is removed, and 2mg of L-16-BA is reserved. Culturing the in vitro shoot tip, the expanded plant at the base of the shoot tip and the clumped bud block under the illumination of 500-1000Lx (14 h/d) at the temperature of 24 +/-2 ℃ to regenerate a complete plant.

Genetic transformation of Poa pratensis

ZmNF-YA1 Gene cloning and fusion Gene construction and recombination of transformed plasmids As in example 1 Agrobacterium tumefaciens (e.g., AGL0 and LBA 4404) with binary vector (Mini-Ti plasmid carrying plant selection marker Gene) were shake-cultured at 28 ℃ with shaking rate of 170-180rpm (rpm) in LB medium (per liter: tryptone 10g, yeast extract 5g, naCl 1 g, pH 7.0, autoclaving) supplemented with antibiotics to keep the bacteria in logarithmic growth phase (OD. Sup.) (OD. Sup. - ₆₀₀ = 0.4-0.6). Then, the mixture was centrifuged at 3000rpm for 10 minutes, and the supernatant was discarded. The mycelia were washed with a 1/2 concentration of the liquid minimal medium and collected by centrifugation. Suspending the bacteria in a liquid minimal medium with a concentration of 1/2, diluting the suspension by 5-20 times, and adding 100 mu mol L of acetosyringone (As) ^-1 And surfactant Silwet L-77 (0.1-0.3% concentration) for transformation.

Cutting cluster buds of Poa pratensis to expose the growth point of bud tip, dip-dyeing in the bacterial liquid, and adding 0.95-0.10 atmosphere (0.95X 10) ⁵ Pa--1×10 ⁴ Pa), and dip dyeing for 4-8 minutes. Then using sterile filter paper to suck out bacterial liquid, transferring cluster bud blocks to subculture and cluster culture medium for co-culture for 2-3 days, then transferring to 250mgL medium added with antibiotic cefadriamycin (Cefotaxime) ^-1 Or carbenicillin (Carb) 500mgL ^-1 The medium of (3) is cultured in the dark to inhibit the growth of the bacteria. The clumpy buds gradually recover to grow on the antibacterial culture medium, and are transferred to a screening culture medium containing a selective agent for screening after 10 to 15 days. Screening for 3 generations continuously, each generation for 15-20 days. The selection agent resistance gene bar or the antibiotic resistance gene hpt (hygromycin phosphotransferase gene). Then transferring the resistant tissue blocks to a subculture and clumping culture medium to differentiate seedlings. The latter grows in rooting medium. Transplanting the rooted plantlets into a flowerpot, irrigating once every 5 days with 1/2 inorganic salt solution of a basic culture medium, and irrigating once every other day. Taking leaves of the transplanted survival plantlets for molecular biological detection. The transformation rate (number of shoots producing transgenic plants/number of shoots infected with Agrobacterium x 100) was around 2-5% as analyzed by PCR and Southern blotting. The induced basal enlargement is more sensitive to light. At low 2,4-D concentration (<0.1mg.L-1), light intensity>The root is easy to differentiate into clumps when the illumination is 1000Lx, and the root is difficult to grow when the illumination is weak (500 Lx). At a more appropriate concentration of 2,4-D, the light intensity is about 1000Lx for good growth (the enlarged tissue is yellowish green), clumping is easier than 500Lx (the enlarged tissue is biased towards white), and the number of clumps per clump is also higher. When the concentration is 2,4-D, the influence of the illumination intensity is not great, the expansion is large, the rooting is not easy, but the clumping rate is low. This stage should be incubated under high light at a suitable concentration of 2,4-D to facilitate later clumping. 5-8 days before subculture of the cluster buds under the condition of light intensity>1000Lx, the tissue is easy to root, which is not favorable for bud differentiation, and once the cluster buds are formed, the differentiation is easy to be promoted when the illumination is strong. Therefore, in the first 5-8 days after subculture, clumped buds are cultured under weak light (about 500 Lx), and when several buds grow, the buds are placed under illumination intensity>The number of buds in each cluster can reach 15-25 when the buds are cultured under 1000 Lx.

Determination of the concentration of the screening agent

The aqueous herbicide solution was filter sterilized and then added (at a medium temperature of less than 50 ℃) to the induction medium from which the casein hydrolysate was removed. The concentration of glufosinate-phosphine is 0.1%, 0.15%, 0.2% and 0.25% respectively, namely 4 gradient concentrations. Cutting the induced cluster buds into single buds, culturing in culture medium containing chlorsulfuron of different concentration, and inoculating more than 100 plantlets in each selective culture medium. Subculture every 15 days for three consecutive screenings. And determining the proper screening dose of the herbicide for the seedlings of different varieties according to the survival rate after screening. For example, the concentration of glufosinate of the variety prize is 0.2%, the survival rate of the plantlet is more than 10% when the concentration is continuously screened for 3 generations. The transgenic PCR detection of the survival plantlet is generally positive.

Plantlet transplantation and transgenic plant screening

The plantlets are induced to root on a suitable rooting medium. When the root is 2-3cm long, culturing the plantlet in natural light for 1-2 days, removing sealing film, hardening the plantlet for 1-2 days, transplanting in flowerpot (soil below the flowerpot and vermiculite on the upper part) in the early morning or evening, watering 1/2 basic culture medium inorganic salt solution once in 5 days, and watering once in 3 days. The growth temperature of the transplanted seedlings is 15-25 ℃. When the seedlings grow to 4-5 tillers, irrigating water, continuously drying for more than 1 month, recovering watering after the leaves of the plants completely wither for one week, and selecting the plants which recover quickly after rehydration for cloning and propagating. Transplanting part of clone seedlings into a flowerpot filled with loam, excessively irrigating 0.3% -0.5% NaCl aqueous solution when the plants grow to about 5 tillers, continuously irrigating for 3 days to make the soil salinity reach the set concentration (not higher than 0.5% NaCl), maintaining for 1-3 months, selecting the survival plants, and cloning. Meanwhile, the cloned seedlings of the transgenic plants are repeatedly screened by spraying the herbicide glufosinate-ammonium aqueous solution with constant concentration. The strains with excellent salt resistance and drought resistance and glufosinate resistance are subjected to space isolation or bagging so as to be seeded under the isolation condition.

The harvested transgenic plant seeds are respectively sowed in flowerpots, herbicide glufosinate-butyl with the concentration of 0.25% is sprayed in the 4-5 leaf stage, the surviving plant plants are subjected to salt tolerance and drought tolerance detection again, excellent salt-tolerant and drought-tolerant transgenic stable plants are screened out from the plants, and the plants can be used for environmental greening after propagation.

Example 4: creation of Heat-resistant grassland Poa pratensis by transformation of ZmNF-YA1RNAi Structure or antisense form

The ZmNF-YA1 antisense form or RNAi structure is respectively fused with a dehydration stress induced promoter Prd29A, and then recombined into a plant expression vector to transform the Poa pratensis, thereby obtaining a transgenic plant. The later is transplanted to survive and grows to 5-6 tillering stages under natural conditions, 0.25% glufosinate solution is sprayed, the surviving plants are propagated through asexual cloning, and transgenic molecule detection is carried out. The obtained transgenic early-maturing grass plants are subjected to 2-generation molecular identification and single plant homozygous, and transgenic homozygous strains are generated. The transgenic plants are taken as materials, and are respectively subjected to heat stress treatment and cold stress treatment, so that the difference of the stress resistance of the transgenic plants and the stress resistance of the control plants is determined. The heat treatment method is as follows.

After transplanting the transgenic early-maturing grass plants for 2 months, a large amount of tillers are generated. And (3) selecting the transgenic annual bluegrass plants which are orderly and consistent, transplanting the transgenic annual bluegrass plants to flowerpots and paper cups, mowing after the plants grow for 2 months, and measuring the heat resistance when the new leaves grow to about 10 cm. When in treatment, the materials are put into a climatic chamber, the photoperiod is controlled to be 12h/d, and the relative humidity is controlled to be 70 percent. In the 40 ℃ heat stress treatment, the temperature was varied in a gradient of 30 ℃ (2 h) → 32 ℃ (2 h) → 34 ℃ (2 h) → 36 ℃ (2 h) → 38 ℃ (2 h) → 40 ℃ (3 d) → 23 ℃ (3 d). The 46 ℃ heat stress treatment was varied in a gradient of 30 ℃ (2 h) → 32 ℃ (2 h) → 34 ℃ (2 h) → 36 ℃ (2 h) → 38 ℃ (2 h) → 40 ℃ (3 h) → 46 ℃ (5 h) → 23 ℃ (7 d). The growth conditions of the material were observed during the treatment, including degree of wilting, survival rate, leaf color change, inter-line variation, etc. Meanwhile, relevant physiological parameters including chlorophyll content, membrane damage and chlorophyll fluorescence are measured before treatment, treatment at 40 ℃ for 2h, 12h and 3d, treatment at 46 ℃ for 5h and treatment at 10d, and measurement of physiological indexes is carried out according to modern plant physiology experimental guidelines (Shang Zhang city main code). Fv/Fm values were determined half an hour after dark adaptation.

And (3) when the short-term heat stress treatment is carried out at 46 ℃, different bluegrass plants are treated at 46 ℃ for 5 hours and then placed at 23 ℃ for recovery culture, the growth conditions of the plants in different treatment periods are observed, and relevant physiological indexes such as chlorophyll content, membrane damage, chlorophyll fluorescence change and the like are measured. After the treatment at 46 ℃, the ZmNF-YA1 gene expression reducing strain is better than a control strain, and all the strains survive after growing for 10 days at 23 ℃, but the growth potential difference is large. The non-transgenic plants have poor growth potential, are seriously damaged by heat stress and are slowly recovered; the ZmNF-YA1 gene expression reducing plant has the advantages of fast recovery, more tillering, strong growth vigor and long new leaves, shows that the plants are slightly damaged in heat stress and have good heat resistance. During the heat treatment, the blade Fv/Fm tends to decrease and gradually increases during the recovery period. The malondialdehyde content of different Poa annua strains in the first 2 days of heat treatment and recovery is increased, and the non-transgenic control is obviously higher than that of the ZmNF-YA1 gene expression-reduced plant, which shows that the degree of membrane lipid peroxidation of the cell of the latter plant is low. During heat stress treatment and recovery, the ion extravasation rate of the non-transgenic plant is obviously higher than that of a plant line of the non-transgenic plant for reducing ZmNF-YA1 gene expression, the membrane damage presents the maximum value when the plant line is recovered for 2 days, and the latter has the plasma membrane stability which is obviously higher than that of a contrast in the heat stress treatment and recovery period, which shows that the heat resistance of the plant line is obviously improved by reducing the ZmNF-YA1 gene expression.

In the long-time heat stress treatment at 40 ℃, after 1 day of treatment, the plants have mild wilting and have little difference; after 3 days of treatment, the wilting degree of the plants of different strains is obviously different, the plants which are not transgenic have heavier wilting, the strains which reduce the ZmNF-YA1 gene expression have obviously improved heat resistance, straight leaves and less water loss. When the recovery treatment is carried out, the non-transgenic control dies completely, so that the heat resistance of the ZmNF-YA1 gene expression strain is reduced, and the survival rate of part of the strain is higher than 70%. The damage to the plants caused by the stress treatment at 40 ℃ for 3 days is far more than that caused by the stress treatment at 46 ℃ for 5 hours, namely, the long-time heat stress treatment can cause the damage which is difficult to repair to the bluegrass cells.

The plant line with obviously improved heat resistance compared with the contrast is cloned and propagated, and 0.2 percent of herbicide glufosinate solution (the dosage is 3 times of that of field weeding) is sprayed, and the plant with weak resistance is eliminated. And (4) performing isolated pollination on the other plants, and mixing and harvesting seeds according to strains for breeding new varieties.

Different transgenic lines from the same variety also show obvious difference in heat tolerance, which is probably due to different integration sites and expression intensity of exogenous genes in transgenic plants. The plant recovery rate and the survival rate after stress treatment and the measured values of physiological indexes in the stress treatment and recovery process are analyzed and judged, the short-term heat stress treatment at 46 ℃ is relatively suitable for primary selection materials, the period is short, and the screening effect is good. The heat stress treatment at 40 ℃ for 3 days or more can effectively screen out a transgenic strain with excellent heat resistance, and the transgenic strain can be directly used for breeding new varieties of heat-resistant early-maturing bluegrass.

Sequence listing

<110> Shandong university

<141> 2018-1-29

<160> 2

<170> PatentIn Version 3.5

<210> 1

<211> 1270

<212> cDNA

<213> Artificial sequence

<220>

<221> corn

<222>（1）…（1270）

<223> nucleotide sequence of ZmNF-YA1 gene cDNA

<400> 1

agagatagga aagggcccaa cagctcaaca gaaaagccaa gcaaaggctg ctgcatactg 60

gaaggccctc tgtctgtgtg cgagcgcaag agaaagggag tcagagagag agagagaggg 120

aggagacctt gcagaggagc gaagcaagca aggtgggaaa gaggcagcag caagggcggc 180

gggctgccgg aaggggaaca tgctccctcc tcatctcaca gagaatggcg cggtgatgat 240

tcagtttggc catcagatgc ctgattacga ctccccggct acccagtcaa ccagtgagac 300

gagccatcaa gaagcgtctg gaatgagcga agggagcctc aacgagcata ataatgacca 360

ttcaggcaac cttgatgggt actcgaagag tgacgaaaac aagatgatgt cagcgttatc 420

cctgggcaat ccggaaacag cttacgcaca taatccgaag cctgaccgta ctcagtcctt 480

cgccatatca tacccatatg ccgatccata ctacggtggc gcggtggcag cagcttatgg 540

cccgcatgct atcatgcacc ctcagctggt tggcatggtt ccgtcctctc gagtgccact 600

gccgatcgag ccagccgctg aagagcccat ctatgtcaac gcgaagcagt accacgctat 660

tctccggagg agacagctcc gtgcaaagct agaggcggaa aacaagctcg tgaaaagccg 720

caagccgtac ctccacgagt ctcggcacct gcacgcgatg aagagagctc ggggaacagg 780

cgggcggttc ctgaacacga agcagcagcc ggagtccccc ggcagcggcg gctcctcgga 840

cgcgcaacgc gtgcccgcga ccgcgagcgg cggcctgttc acgaagcatg agcacagcct 900

gccgcccggc ggtcgccacc actatcacgc gagagggggc ggtgagtagg gagccccgac 960

actggcaact catccttggc ttatcagcga ttcgactcgg ctctccctcg tctgaaactg 1020

aactctctgc aactactgta actgtaacta aactgggtgt gcccggattg gcggtcgttc 1080

tgttctacta ctagtacctg ctacgcgtcg ttgggttggg tctggactag agagcgtgct 1140

ggttctttga tgaacttggc tggacttgag ggtgttgact agcgcgaagc tgagttccat 1200

gtaaaacttt tgcttcaaga ccgatgactg gcggcataat aagtagcagt aataaccatt 1260

cttctgtgtc 1270

<210> 2

<211> 249

<212> PRT

<213> Artificial sequence

<220>

<221> corn

<222>（1）…（249）

<223> amino acid sequence encoded by ZmNF-YA1 gene

<400> 2

MLPPHLTENG AVMIQFGHQM PDYDSPATQS TSETSHQEAS GMSEGSLNEH NNDHSGNLDG 60

YSKSDENKMM SALSLGNPET AYAHNPKPDR TQSFAISYPY ADPYYGGAVA AAYGPHAIMH 120

PQLVGMVPSS RVPLPIEPAA EEPIYVNAKQ YHAILRRRQL RAKLEAENKL VKSRKPYLHE 180

SRHLHAMKRA RGTGGRFLNT KQQPESPGSG GSSDAQRVPA TASGGLFTKH EHSLPPGGRH 240

HYHARGGGE 249

Claims

1. The application of the over-expression corn nuclear factor gene ZmNF-YA1 in cultivating drought-tolerant corn or bluegrass; wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNA is shown as SEQ ID No.1, and the coded amino acid sequence thereof is shown as SEQ ID No. 2.

2. The application of reducing the expression of a corn nuclear factor gene ZmNF-YA1 in cultivating heat-resistant meadow bluegrass; wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNA is shown as SEQ ID No.1, and the coded amino acid sequence thereof is shown as SEQ ID No. 2.