CN116622761A - Application of corn auxin response protein IAA15 - Google Patents

Abstract

The application discloses application of a corn auxin response protein IAA15 or a gene for encoding the corn auxin response protein IAA15, and the gene or the protein can be overexpressed in a plant body to improve salt tolerance and/or drought tolerance of the plant. The research on the gene function of the auxin related gene ZmIAA15 in plants such as tobacco and corn can provide a certain theoretical basis for genetic improvement of plants and a new gene for cultivating new drought-resistant and salt-resistant varieties.

Description

Application of corn auxin response protein IAA15

Technical Field

The application relates to a new application of early auxin response protein indoleacetic acid, in particular to an application of corn auxin response protein IAA15.

Background

Auxin (Auxin) is one of the earliest discovered plant hormones, one of the most important plant hormones involved in plant growth and development, and its chemical nature is indole-3-acetic acid (IAA), which has very wide activity. Auxin/indole-3-acetic acid (Aux/IAA) is an early auxin response gene family, and the encoded protein belongs to early auxin response proteins, can be specifically combined with auxin response factors (auxin response factor, ARF), participates in the expression regulation of a large number of genes downstream of auxin signals, and plays an important role in regulating auxin signal transduction.

The Aux/IAA gene family is present in plants such as soybean (Glycine max), arabidopsis (Arabidopsis thaliana), wheat (Triticum aestivum), rice (Oryza sativa), and maize (Zea mays), and the biological functions of the Aux/IAA gene vary from plant to plant, such as root development, flower organ formation, fruit development, and the like.

The Aux/IAA protein is found to be involved in a number of auxin signal transduction and plays a key role in the growth and development of plants. Such as: ghAUX1 participates in interaction of IAA and GA in cotton, can regulate expression of gene downstream of GA synthesis pathway, and inhibit growth of cotton seedling ^[1] . In Arabidopsis, most Aux/IAA genes exhibit similar phenotypes during plant growth and development. For example, atIAA3 is involved in hypocotyl elongation, leaf morphogenesis, lateral root formation, and root hair formation, while AtIAA16 is associated with apical dominance, plant size, mutants of AtIAA16 show smaller rosettes, shorter root hairs, and fewer lateral roots than wild type ^[2,3] . Overexpression of the blueberry Aux/IAA gene VcIAA27 in Arabidopsis leads to auxin-related defects, such as leaf curl down, dwarfing, etc ^[4] . Overexpression of the apple Aux/IAA gene MdIAA18 in Arabidopsis leads to a compact phenotype, and the MdIAA gene may be involved in vegetative and reproductive growth of apples ^[5] . SiIAA3 controls multiple processes of auxin signaling in tomato, including leaf morphogenesis, floral organ development, setting and development ^[6] . The strain with SiIAA17 gene down-regulated expression shows bigger fruits and thicker pericarps ^[7] The method comprises the steps of carrying out a first treatment on the surface of the SiIAA27 shows reduced levels of gene expression in plants involved in chlorophyll synthesis ^[8,9] . In rice, osIAA1 is induced by auxin and plays an important role in rice photoresponse and coleoptile elongation ^[10]

The Aux/IAA gene family is huge, the functions of the Aux/IAA genes in different plants are different, and the functions of the different Aux/IAA genes in the same plant are also different, so that the functions of the different Aux/IAA genes or proteins in the family are one of the hot research directions in the field of plant bioscience.

With the ever-expanding population and over-utilization of land, the earth's ecosystem is being gradually changed, and even in conservative situations, future climate change including further increases in global average air temperature, severe drought in some areas, extreme drought and extreme high temperatures are increasingly frequent and severe. Soil salinity is one of the most important abiotic stresses that hinder plant growth and development, ultimately resulting in reduced crop yield. Crop losses due to soil salinity pose an increasing threat to modern agriculture, and it is estimated that more than 6% of the total world land area is affected by salt, with over 1200 ten thousand hectares being irrigated land, pose a serious threat to irrigated agriculture. In addition to the elevated high salt groundwater levels and increased evaporation due to drought, excessive use of fertilizers and soil amendments, improper drainage and seawater intrusion, excessive irrigation and climate change further lead to increased soil salinization. Therefore, creating new drought-resistant and salt-tolerant crop germplasm and improving the agricultural yield of saline-alkali soil are important to global grain safety.

Reference to the literature

【1】 Wang Li, wang Lifeng, zhao Panpan, et al, aux/IAA family gene GhAGux 1 silencing promotes cotton seedling growth [ J ]. Henan agricultural science, 2019,48 (06): 46-51+80.

【2】GOH T,KASAHARA H,MIMURA T,et al.Multiple AUX/IAA-ARF modules regulate lateral root formation:the role of Arabidopsis SHY2/IAA3-mediated auxin signalling[J].Philosophical Transactions of the Royal Society B-Biological Sciences,2012,367(1595):1461-1468.

【3】WEI S,CHEN Y,HOU J,et al.Aux/IAA and ARF Gene Families in Salix suchowensis:Identification,Evolution,and Dynamic Transcriptome Profiling During the Plant Growth Process[J].Front Plant Sci,2021,12:666310.

【4】HOU Y,LI H,ZHAI L,et al.Identification and functional characterization of the Aux/IAA gene VcIAA27 in blueberry[J].Plant Signal Behav,2020,15(1):1700327.

【5】WANG L,XU K,LI Y,et al.Genome-Wide Identification of the Aux/IAA Family Genes(MdIAA)and Functional Analysis of MdIAA18 for Apple Tree Ideotype[J].Biochem Genet,2019,57(5):709-733.

【6】CHAABOUNI S,JONES B,DELALANDE C,et al.Sl-IAA3,a tomato Aux/IAA at thecrossroads of auxin and ethylene signalling involved in differential growth[J].J Exp Bot,2009,60(4):1349-62.

【7】SU L,BASSA C,AUDRAN C,et al.The auxin Sl-IAA17 transcriptional repressor controls

fruit size via the regulation of endoreduplication-related cell expansion[J].Plant Cell Physiol,2014,55(11):1969-76.

【8】UETAR,ABE C,WATANABE T,et al.Rapid breeding of parthenocarpic tomato plants usingCRISPR/Cas9[J].Scientific Reports,2017,7.

【9】SU L Y,BASSA C,AUDRAN C,et al.The Auxin Sl-IAA17 Transcriptional RepressorControls Fruit Size Via the Regulation of Endoreduplication-Related Cell Expansion[J].

Plant and Cell Physiology,2014,55(11):1969-1976.

【10】THAKUR J K,TYAGI AK,KHURANA J P.OsIAA1,an Aux/IAA cDNA from rice,and

changes in its expression as influenced by auxin and light[J].DNA Res,2001,8(5):193-203.

Disclosure of Invention

Aiming at the problems in the prior art, the application aims to provide the application of the maize auxin response protein IAA15.

In order to achieve the above purpose, the present application adopts the following technical scheme:

in a first aspect the present application provides the use of the maize auxin response protein IAA15 or a gene encoding the same for increasing stress tolerance in plants, including salt tolerance and/or drought tolerance.

In the use according to the first aspect of the present application, the plant may be maize, tobacco, rice etc., as a preferred embodiment the plant is tobacco, in particular it may be a seed of tobacco or a tobacco plant.

In the use according to the first aspect of the present application, as a preferred embodiment, the zea mays auxin response protein IAA15 is selected from any one of the following:

(a) A protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1;

(b) A protein derived from (a) having the same activity as the protein of (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a).

The protein (b) and the protein (a) which have the same activity as the protein (a), namely are derived from the protein (a), have at least the activity of improving the salt tolerance and/or drought tolerance of plants.

In the use according to the first aspect of the present application, the gene encoding the protein may be a gene encoding the protein of the above (a) or a gene encoding the protein of the above (b), and as a preferred embodiment, the gene encoding the protein has a base sequence as shown in SEQ ID NO.2 of the sequence Listing.

In the use of the first aspect of the present application, as a preferred embodiment, the improvement of plant stress tolerance is achieved by over-expressing the auxin response protein IAA15 in plants.

In the use of the first aspect of the present application, as a preferred embodiment, the improvement of plant stress tolerance is achieved by modulating plant physiological indicators including superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA), wherein the superoxide dismutase and peroxidase levels are up-regulated and the malondialdehyde levels are down-regulated.

In a second aspect, the application provides a method of increasing stress resistance in a plant comprising: introducing a gene encoding a maize auxin response protein IAA15 into said plant such that the maize auxin response protein IAA15 is overexpressed in the plant; the stress resistance includes salt and/or drought tolerance.

In the method of the second aspect of the present application, as a preferred embodiment, the introduction of the gene encoding the maize auxin response protein IAA15 into the plant is introduction of a recombinant vector containing the gene encoding the maize auxin response protein IAA15 into the plant. More preferably, the construction of the recombinant vector comprises ligating a gene encoding the maize auxin response protein IAA15 to the multiple cloning site of the plant binary expression vector pCAMBIA1300, thereby forming the recombinant vector pCAMBIA 1300-zmbia 15. Further, the gene encoding the maize auxin response protein IAA15 was ligated to the multiple cloning site of the plant binary expression vector pCAMBIA1300 by XbaI and BamHI cleavage sites.

In the method of the second aspect of the present application, as a preferred embodiment, the gene encoding the maize auxin response protein IAA15 may be a gene encoding the protein of the above (a) or a gene encoding the protein of the above (b), and as a preferred embodiment, the gene encoding the protein has a base sequence as shown in SEQ ID NO.2 of the sequence Listing.

In the method of the second aspect of the application, the plant may be maize, tobacco, rice or the like, as a preferred embodiment the plant is tobacco, in particular it may be a seed of tobacco or a tobacco plant.

In a third aspect, the present application provides a use of the maize auxin response protein IAA15 or a gene encoding the same for promoting plant growth by increasing expression of the growth genes ROT3 and/or AN3 in plants.

In the use according to the third aspect of the present application, the plant may be maize, tobacco, rice or the like, and as a preferred embodiment the plant is tobacco, in particular, may be a seed of tobacco or a tobacco plant.

In the use according to the third aspect of the present application, as a preferred embodiment, the zea mays auxin response protein IAA15 is selected from any one of the following:

(a) A protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1;

(b) A protein derived from (a) having the same activity as the protein of (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a).

The protein (b) and the protein (a) which have the same activity as the protein (a), namely are derived from the protein (a), have at least the activity of promoting the growth and development of plants.

In the use according to the third aspect of the present application, the gene encoding the protein may be a gene encoding the protein of the above (a) or a gene encoding the protein of the above (b), and as a preferred embodiment, the gene encoding the protein has a nucleotide sequence shown in SEQ ID NO.2 of the sequence Listing.

In the use of the third aspect of the present application, as a preferred embodiment, the promotion of plant growth is achieved by over-expressing the maize auxin response protein IAA15 in plants, thereby increasing expression of the growth and development genes ROT3 and/or AN3 in plants.

In a fourth aspect, the present application provides a method of promoting plant growth, comprising: introducing a gene encoding a maize auxin response protein IAA15 into the plant to over-express the maize auxin response protein IAA15 in the plant, thereby increasing expression of the auxin response genes ROT3 and/or AN3 in the plant.

In the method of the fourth aspect of the present application, as a preferred embodiment, the introduction of the gene encoding the maize auxin response protein IAA15 into the plant is introduction of a recombinant vector containing the gene encoding the maize auxin response protein IAA15 into the plant. More preferably, the construction of the recombinant vector comprises ligating a gene encoding the maize auxin response protein IAA15 to the multiple cloning site of the plant binary expression vector pCAMBIA1300, thereby forming the recombinant vector pCAMBIA 1300-zmbia 15.

In the method of the fourth aspect of the present application, as a preferred embodiment, the gene encoding the maize auxin response protein IAA15 may be a gene encoding the protein of the above (a) or a gene encoding the protein of the above (b), and as a preferred embodiment, the gene encoding the protein has a base sequence as shown in SEQ ID NO.2 of the sequence Listing.

In the method of the fourth aspect of the application, the plant may be maize, tobacco, rice or the like, as a preferred embodiment the plant is tobacco, in particular it may be a seed of tobacco or a tobacco plant.

The method of introducing the gene encoding the maize auxin response protein IAA15 into the plant of the application may be any method feasible in the art, such as the gene gun method, the Agrobacterium-mediated method, etc.

Compared with the prior art, the application has the following beneficial effects:

1. the application uses corn inbred line Chang 7-2 as material to clone ZmIAA15 gene, biological information analysis shows that ZmIAA15 belongs to AUX-IAA superfamily, is an early auxin response gene family, the length of open reading frame sequence coded by ZmIAA15 gene is 663bp, codes 220 amino acids, the relative molecular weight of coded protein is 23.48KD, and theoretical isoelectric point is 7.77.

2. ZmIAA15 is expressed in different tissues and organs of corn, wherein the expression level in roots is highest, and the stems and leaves are the next. The expression of zmbia 15 is affected by salt and drought stress, and it is speculated that zmbia 15 may be involved in the defense responses against drought and salt stress.

3. Further, the inventor constructs a plant binary expression vector pCAMBIA1300-ZmIAA15 containing ZmIAA15 genes, and utilizes agrobacterium to infect the tobacco to obtain a ZmIAA15 gene over-expression tobacco strain. The results show that: compared with the wild type, the over-expression of the ZmIAA15 gene in the raw tobacco obviously promotes the germination rate of tobacco seeds under mannitol and NaCl treatment, which indicates that the drought resistance and salt tolerance of the over-expressed transgenic tobacco in the seed germination period are stronger than those of the wild type tobacco.

4. For tobacco T ₃ The transgenic strain and the wild type are subjected to 350mmol/L NaCl treatment and 20% PEG6000 drought treatment, salt stress and drought stress treatment are found, the leaf wilting of wild type tobacco is more obvious compared with ZmIAA15 over-expressed tobacco, the phenotypic characters of thick stem, fresh weight on the ground, leaf area, fresh weight under the ground, root length and the like are measured, and the damage degree of the over-expressed strain after being subjected to stress is found to be obviously smaller than that of the wild type. After stress, physiological indexes including superoxide dismutase (SOD), peroxidase (POD), malondialdehyde (MDA) and relative water content are measured, and the content of the protective enzymes POD and SOD in the transgenic strain is found to be increased remarkably, the MDA content is reduced remarkably, so that the transgenic strain is further proved to have stronger resistance to salt and drought.

In conclusion, the ZmIAA15 gene can enhance the drought resistance and salt tolerance of tobacco plants. The research on the gene function of the auxin related gene ZmIAA15 in plants such as tobacco and corn can provide a certain theoretical basis for genetic improvement of plants and a new gene for cultivating new drought-resistant and salt-resistant varieties.

Drawings

FIG. 1 is an electrophoretogram of ZmIAA15 gene clone, wherein M is a 2000bp marker band, 1-4: amplifying the product bands.

FIG. 2 shows the amino acid sequence alignment of ZmIAA15 amino acid sequence homologous genes.

FIG. 3 shows the relative expression levels of ZmIAA15 gene in different tissues and organs of corn, and is prepared by: "+": significant difference (P < 0.05), "x": the differences were very pronounced (P < 0.01).

FIG. 4 is an analysis of expression of the 48h ZmIAA15 gene for drought stress and salt stress for Chang 7-2, A, B, C: drought stress and salt stress treat the expression level of ZmIAA15 genes of corn in three leaf stages of 0h, 12h, 24h, 36h and 48 h.

FIG. 5 is PCR identification of pMD19T-ZmIAA15 plasmid colonies, annotated: m: takara DL 2000DNAmarker;1-4: PCR identification of pMD19T-ZmIAA15 plasmid colonies.

FIG. 6 is a plant expression vector pCAMBIA1300-ZmIAA15 plasmid PCR identification (A) and double cleavage verification (B), wherein A: m: takara DL 2000 DNAmaroker, 1-5: screening positive clone PCR, taking bacterial liquid as a template, and taking a strip as positive clone; b: m: takara DL 15000DNA marker;1-4: double cleavage results of recombinant plasmid.

FIG. 7 shows the results of PCR amplification of T0 transgenic benthonic tobacco lines (A), hygromycin screening (B) and the relative expression levels of ZmIAA15 in T3 transgenic benthonic tobacco (C), wherein FIG. 7A: m: takara DL 2000DNA marker; CK, wild-type benthonic tobacco; OE1 to OE3: screening Miao Ben smoke generation PCR results by converting T0 generation into ZmIAA 15; fig. 7B: CK, wild-type benthonic tobacco; OE1 to OE3: screening Miao Ben to generate smoke by converting T3 generation into ZmIAA 15; fig. 7C: WT is wild type benthamiana, OE1, OE2, OE3 are three transgenic lines T3 generation ZmIAA15.

FIG. 8 is an analysis chart of gene expression related to the growth and development of the T3 generation ZmIAA15 gene-transferred smoke, wherein A: LNG1, B: ROT3, C: AN3.

FIG. 9 is a result of the phenotypic effects of salt stress and drought stress treatment on wild type and transgenic T3 generation tobacco germination, wherein A: control group, MS medium; b: salt treatment, 120mmol/L NaCl; c: drought treatment, 200mmol/L D-mannitol; d: and (5) calculating germination percentage statistics.

FIG. 10 is a graph showing the results of phenotypic identification of one month old wild type and T3 generation ZmIAA15 transgenic benthonic plants after salt stress, wherein A: treating the tobacco of one month old for 7 days with 350mmol/L NaCl and clear water; b: plant height after salt treatment for 7d; c: the stems are thick after salt treatment for 7d; d: leaf area after salt treatment 7d; e: the fresh weight on the ground after salt treatment for 7d; f: underground fresh weight after salt treatment for 7d. "*": p <0.05, "x": p <0.01.

FIG. 11 is a comparison of root length after salt stress of a month old wild type and T3 generation ZmIAA15 transgenic benthonic plants, wherein A: the root system scanning picture of the wild type benthonic tobacco of one month old is treated for 7 days by 350mmol/L NaCl; B-D: the transgenic strains OE1, OE2 and OE3 with the age of one month are subjected to treatment for 7 days by 350mmol/L NaCl, and root system scanning pictures are obtained; e: total root length is compared. "*": p <0.05, "x": p <0.01.

FIG. 12 is a physiological index identification after salt stress of a month-old wild type and T3 generation ZmIAA15 transgenic benthonic plant, wherein A: POD activity after salt treatment 7d; b: SOD activity after 7d of salt treatment; c: MDA content after salt treatment for 7d; d: relative water content after salt treatment 7d. "*": p <0.05, "x": p <0.01.

FIG. 13 is a phenotypic characterization after PEG6000 stress of a one month old wild type and T3 generation ZmIAA15 transgenic primary tobacco plant, wherein A: t3, 20% peg6000 of 1 month old tobacco and 7d of clear water treatment; b: plant height after PEG6000 treatment; c: the stem after PEG6000 treatment is thick; d: leaf area after PEG6000 treatment; e: ground fresh weight after PEG6000 treatment; f: underground fresh weight after PEG6000 treatment. "*": p <0.05, "x": p <0.01.

FIG. 14 is a comparison of root length after drought stress for a month old wild type and T3 generation ZmIAA15 transgenic primary tobacco plants, wherein A: root system scanning pictures after 7d of treatment of 20% PEG6000 of wild type benthonic cigarettes of one month old; B-D: root system scanning pictures after 7d treatment of transgenic lines OE1, OE2 and OE3 with 20% PEG 6000; e: total root length is compared. "*": p <0.05, "x": p <0.01.

FIG. 15 is a physiological index identification after natural drought stress of a month-old wild type and T3 generation ZmIAA15 transgenic primary tobacco plant, wherein A: POD activity after 7d of natural drought treatment; b: SOD activity after 7d of natural drought treatment; c: MDA content after 7d of natural drought treatment; d: relative water content after 7d of natural drought treatment. "*": p <0.05, "x": p <0.01.

Detailed Description

The application will be further illustrated with reference to specific examples. It should be understood that these examples are only for the purpose of the present application and are not intended to limit the scope of the present application. It is to be understood that various changes and modifications may be made by those skilled in the art after reading the disclosure herein, and that such equivalents are intended to fall within the scope of the application as defined by the appended claims.

The experimental materials and reagent sources used in the embodiment of the present application are as follows:

the corn inbred line Chang 7-2 and the wild type tobacco (Nicotiana benthamiana) are provided by Qingdao university crop breeding laboratories, and the two experimental materials are also conventional commercial products and can be obtained through commercial channels.

Strains: coli (Escherichia coli) competent cells DH5 a, agrobacterium (Agrobacterium rhizogenes) competent cells GV3101 were all purchased from the monograph company.

Plasmid: the intermediate vector pMD19-T was purchased from Takara, and the plant expression vector pCAMBIA1300 was supplied from Shandong university and was also purchased from Shanghai-associated Bioengineering Co.

The kit comprises: RNA extraction kit (TAKARA), plasmid extraction kit (Vazyme), purification recovery kit (Vazyme), physiological and biochemical index detection kit, and the like.

Primers used in the following examples were designed by software Primer Premier5 and synthesized by catalpa biotechnology limited, qingdao, qinghao, and shown in table 1.

TABLE 1 primer sequences

EXAMPLE 1 cloning of the maize ZmIAA15 Gene

(1) cDNA template synthesis

RNA from the roots of maize inbred line Chang 7-2 trefoil stage seedlings was extracted using TaKaRa Plant RNA Extraction Kit kit and then reverse transcribed into cDNA, the reverse transcription system being shown in Table 2 below. The preparation of the system is carried out on an ice box, and the reaction is carried out in a PCR instrument after the system is fully and uniformly mixed, and the reaction procedure is as follows: 37 ℃ C.: 15min,85℃:5s,4 ℃.

TABLE 2 reverse transcription system

(2) Amplifying the ZmIAA15 gene of corn by using the cDNA template obtained in the step (1)

The PCR amplification system is shown in Table 3 below, and the reaction steps are shown in Table 4 below.

TABLE 3PCR reaction System

TABLE 4PCR reaction procedure

The gel electrophoresis diagram of the PCR amplified product is shown in figure 1, the amplified product is a single band of about 663bp, the band is clear and consistent with the size of a target gene, and the base sequence is shown in a sequence table SEQ ID NO.2 after sequencing.

Example 2 bioinformatics analysis of maize ZmIAA15 Gene

Analysis of the conserved regions of the zmbia 15 gene by the conserved functional region analysis program CDs (Conserved Domains) of NCBI (National Coalition Building Institute) showed that the zmbia 15 gene belongs to the AUX-IAA superfamily and is an early auxin response gene family.

Sequence analysis of the ZmIAA15 gene by Bioxm software shows that the sequence length of an open reading frame (Open Reading Frame, ORF) encoded by the ZmIAA15 gene is 663bp, the base sequence is shown as SEQ ID NO.2 of the sequence table, 220 amino acids are encoded (GenBank: ONM 29537.1), and the amino acid sequence is shown as SEQ ID NO.1 of the sequence table. SEQ ID NO.1 (maize auxin response protein IAA 15)

MSVETERSSTESSGASGLDFEDTALTLTLRLPGSAPSAAAAAAASLSLSSSSSSAFPDPDRKR

ASSDADPGRSSPLAASSDAAPAPKARVVGWPPVRSYRKNALADAAGSSKAAKFVKVAVDG

APYLRKVDLQAYAGYDQLLRALQDKFFSHFTIRKFADDERKLVDAVNGTEYVPTYEDKDG

DWMLVGDVPWKMFVETCQRLRLMKGSEAVNLAPRAAR

SEQ ID NO.2 (corn ZmIAA15 Gene)

ATGTCGGTGGAGACGGAGCGGAGCTCCACCGAGTCCTCCGGGGCTTCCGGGCTCGACT

TCGAGGACACCGCGCTCACGCTCACCCTCCGCCTCCCGGGCTCCGCGCCTTCCGCCGCC

GCCGCCGCCGCGGCTTCCCTGTCCCTCTCCTCCTCGTCGTCCTCCGCCTTCCCCGACCCC

GACCGCAAGCGCGCCTCCTCCGACGCTGACCCCGGCCGCTCCTCCCCGCTCGCCGCGTC

CTCCGACGCTGCACCGGCACCCAAGGCTCGTGTGGTGGGCTGGCCGCCGGTGAGGTCG

TACCGCAAGAACGCGCTCGCCGACGCCGCGGGCTCCAGCAAGGCCGCCAAGTTCGTCA

AGGTGGCCGTCGACGGCGCGCCCTACCTGCGGAAGGTGGACCTGCAGGCGTACGCCGG

CTACGACCAGCTGCTCCGCGCGCTCCAGGACAAGTTCTTCTCCCACTTCACCATCAGGA

AGTTCGCCGACGACGAGAGGAAGCTGGTGGACGCGGTGAACGGGACGGAGTACGTAC

CCACGTACGAGGACAAGGATGGCGACTGGATGCTCGTCGGCGACGTCCCCTGGAAGAT

GTTCGTGGAAACCTGCCAGCGCCTTCGTCTGATGAAAGGTTCAGAGGCCGTGAACTTG

GCACCAAGAGCCGCCCGATGA

The ZmIAA15 gene is predicted and analyzed by ProtParam (https:// web. Expasy. Org/protParam /) on-line software, and the result shows that the protein coded by the ZmIAA15 gene has a molecular formula of C1029H1629N293O326S5, a relative molecular weight of 23.48kDa, a theoretical isoelectric point of 7.77, a total number of positive charge residues (Arg+Lys) of 29, a total number of negative charge residues (Asp+Glu) of 28, and an instability coefficient of 48.07, and belongs to an unstable protein.

The homologous gene of the ZmIAA15 gene in tobacco is found through NCBI website, the amino acid sequence of the DNAMAN is compared with the amino acid sequence of the homologous gene of tobacco, the similarity is found to be 47.81%, and the comparison result is shown in figure 2.

Example 3 analysis of expression of ZmIAA15 Gene in maize

(1) Tissue-specific analysis of ZmIAA15 Gene expression

Taking corn inbred line Chang 7-2 as a test material, and taking roots, stems and leaves of corn seedlings in the trefoil period under normal management for tissue-specific expression analysis.

The method for obtaining cDNA of each sample is the same as in example 1, then real-time fluorescence quantitative PCR reaction is carried out by taking the obtained cDNA as a template, qRT-ZmIAA15-F and qRT-ZmIAA15-R primers are adopted to amplify target gene ZmIAA15, and meanwhile, corn internal reference gene primers, namely action-F and action-R, are adopted to amplify internal reference gene action, the reaction system is shown in the following table 5, and the reaction steps are shown in the following table 6. And (3) obtaining the relative expression quantity of the target gene ZmIAA15 according to the correction of the expression quantity of the reference gene.

(2) Analysis of expression of ZmIAA15 under salt stress and drought stress

The maize inbred line Chang 7-2 is used as a test material, and is normally managed, and salt stress and drought stress treatment are carried out in the trefoil period. The test set up two treatments and controls: (1) control CK (normal management); (2) 500ml, 150mmol/L NaCl treatment; (3) 500ml, 20% PEG6000 treatment. The test was repeated three times, three plants were repeated for each treatment, and samples were taken at 0h, 12h, 24h, 36h, 48h of the treatment to determine the expression level of the maize ZmIAA15 gene under drought stress and salt stress. The method for obtaining cDNA of each sample was the same as in example 1, and then a real-time fluorescent quantitative PCR reaction was performed using the obtained cDNA as a template, and the method for obtaining the relative expression amount of the gene ZmIAA15 in each sample, as well as the PCR reaction system and the reaction conditions, were as described in example (1).

The real-time fluorescent quantitative PCR reaction system is shown in the following Table 5, and the reaction steps are shown in the following Table 6.

TABLE 5 Real Time PCR reaction System

TABLE 6 Real Time PCR reaction procedure

Data analysis adopts 2 ^-ΔΔCt The DPS software performs data diversity analysis, microsoft Excel 2007 performs data sort and mapping analysis relative to quantitative analysis methods.

As shown in FIG. 3, the results of the part (1) of this example show that ZmIAA15 gene is expressed in the corn root in the highest amount, and that root RNA can be used as a template for the subsequent gene cloning.

The results of the part (2) of this example are shown in fig. 4, and the real-time fluorescence quantitative PCR method is used to detect the expression levels of roots, stems and leaves of the ZmIAA15 gene in trefoil-stage corn under drought (20% PEG 6000) and salt conditions (150 mmol/LNaCl), and the results indicate that the expression levels of the ZmIAA15 gene in the corn are induced by drought stress and salt stress, and the expression levels are different in roots, stems and leaves, and the expression levels of the ZmIAA15 gene in the roots, stems and leaves in drought stress are in a trend of decreasing after decreasing, and the expression levels of the ZmIAA15 gene in the roots and stems in salt stress are in a trend of decreasing after increasing and then decreasing, and the expression levels in the leaves are in a trend of decreasing after increasing. And the expression of the ZmIAA15 gene of the corn in stems and She Li is up-regulated by stress, which shows that the gene is regulated by drought and salt stress and has a certain drought resistance and salt tolerance function.

EXAMPLE 4 construction of plant expression vector pCAMBIA1300-ZmIAA15

4.1 amplification of maize Gene ZmIAA15 see example 1.

4.2 recovery of amplified products and addition of A tail

And (3) recovering and purifying the amplified product in the step (1) by adopting a Norpraise company gel recovery kit, adding an A tail, reacting for 30min at 72 ℃ by using a PCR reaction system shown in the following table 7, and purifying and recovering again after the reaction is finished.

TABLE 7PCR reaction System

4.3 construction of intermediate vector pMD19T-ZmIAA15

(1) And constructing a pMD19T-ZmIAA15 vector by taking the product of the last step as a template, wherein the reaction system is shown in the following table 8, and reacting for 30min at 16 ℃ in a PCR instrument after fully and uniformly mixing to obtain a connection product.

TABLE 8 construction of pMD19T-ZmIAA15 System

(2) Transformation of DH5 alpha competent cells

a. Inserting competent cells taken out from a refrigerator at-80 ℃ into ice for 5min, adding a connecting product after fungus blocks are melted, and standing in ice for 25min after light mixing.

b, adjusting the temperature of the water bath kettle to 42 ℃, carrying out heat shock on competent cells for 45 seconds, rapidly putting back on ice, standing for 2 minutes, and recording that shaking is not needed.

c. 700. Mu.l of LB (sterile medium without antibiotics) was added to the centrifuge tube, and after thorough mixing, resuscitated at 37℃for 60min at 200 rpm.

The cells were collected by centrifugation at 5000rpm for 1min, 100. Mu.l of the supernatant was left to gently blow the resuspended pellet, and the pellet was spread on LB medium containing antibiotics (Amp).

f. The medium was inverted and placed in an incubator at 37℃overnight for cultivation.

(3) Screening of intermediate vector pMD19T-ZmIAA15

White single colonies are selected through blue and white spot screening, and bacterial liquid PCR verification is carried out. The reaction system is shown in Table 9 below, and the reaction conditions are shown in Table 4.

TABLE 9PCR reaction System

The result of PCR amplification is shown in FIG. 5, and a single band of about 663bp in size was amplified. And sending the bacterial liquid with the correct PCR result to a company for sequencing and carrying out subsequent experiments.

(4) Identification of the intermediate vector pMD19T-ZmIAA15

a. The intermediate vector pMD19T-ZmIAA15 plasmid is extracted from bacterial liquid, and specific operation steps are shown in a specification (Vazyme FastPure Plasmid Mini Kit kit).

b. The pMD19T-ZmIAA15 plasmid was subjected to double digestion with restriction endonucleases Xba I and BamHI, and reacted at 37℃for 3-5 hours under the reaction conditions shown in Table 10 below for 3-5 hours.

Table 10pMD19T-ZmIAA15 plasmid cleavage reaction System

Positive clones were screened for sequencing by company.

4.4 construction of plant expression vector pCAMBIA1300-ZmIAA15

(1) Cleavage of the Positive cloning plasmid with the pCAMBIA1300 vector

The positive cloning plasmid and the plant expression vector pCAMBIA1300 were digested with XbaI and BamHI, respectively, and reacted at 37℃for 3-5 hours, and the reaction system was as in Table 10.

(2) Enzyme cutting product recovery

The target fragment of 663bp of recovered product was ligated with the cut pCAMBIA1300 expression vector, and the reaction system was as shown in Table 11 below.

TABLE 11 construction of pCAMBIA1300-ZmIAA15 plasmid System

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(3) Transformation of DH5 alpha competent cells

For specific transformation steps, see example 4.3 (2), white colonies were then picked, transformed colonies were verified and positive plasmids were sent to the company for sequencing.

The plasmid PCR verification results are shown in FIG. 6A, wherein lanes 1-5 use bacterial liquid as a template, and all the lanes have bands, which shows that lanes 1-5 are positive clones. FIG. 6B shows the results of double digestion (XbaI and BamHI) of recombinant positive plasmid pCAMBIA1300-ZmIAA15, with lanes 1-4 having two bands, respectively, with clear and correct size, indicating successful construction of plant expression vector pCAMBIA1300-ZmIAA15.

EXAMPLE 5 genetic transformation of ZmIAA15 Gene

5.1 transformation and post-transformation culture methods

1. Planting of the smoke

Taking a proper amount of seeds into a centrifuge tube, and performing the following operations in an ultra-clean workbench:

(1) Soaking in 75% alcohol for 1min.

(2) 2% NaClO soaked for about 10min.

(3) The sterilized water was washed 3 times.

(4) And (5) airing on the filter paper.

(5) Uniformly dispersing on MS culture medium with a blade or a gun head, transplanting into small pots containing nutrient soil after seeds grow for 7-8 days, transplanting one plant in each pot, and culturing in a tissue culture type artificial climate chamber.

2. Positive plasmid pCAMBIA1300-ZmIAA15 prepared in example 4 transformed GV3101 competent cells

Transformation of Agrobacterium was accomplished according to the instructions for GV3101 competence.

3. The leaf disc method was used to infect the leaf of the present smoke obtained in step 1 using agrobacterium of step 2, see literature (Liu Suhong, leaf disc method was used to establish the genetic transformation system [ D ] for salt mustard (Thellungiella halophila), university of eastern shan, 2005.).

4. The infected tobacco leaves are placed in an MS culture medium containing cephalosporin (with the final concentration of 2000 mu L/L), hygromycin (with the final concentration of 200 mu L/L), 6BA (with the final concentration of 3000 mu L/L) and NAA (with the final concentration of 200 mu L/L) to induce callus, the obtained callus is placed in an MS culture medium containing cephalosporin (with the final concentration of 2000 mu L/L) and hygromycin (with the final concentration of 200 mu L/L) to carry out bud growth culture, the screened buds are cut out and placed in a 1/2MS culture medium containing cephalosporin (with the final concentration of 2000 mu L/L), hygromycin (with the final concentration of 200 mu L/L) and NAA (with the final concentration of 100 mu L/L) to carry out rooting culture, tobacco plants with young roots are grown out seedling training, and finally transplanted into nutrient soil to be placed in a artificial climate tissue culture chamber to carry out normal culture.

5.2ZmIAA15 transgenic Bensheng cigarette screening and molecular detection

And shearing tobacco seedling leaves which are subjected to hygromycin screening and normally grown, extracting DNA of the seedling leaves, and carrying out molecular detection of T0 generation positive seedling lines, wherein ZmIAA15-F and ZmIAA15-R are adopted as primers for PCR amplification. As a result, as shown in FIG. 7A, three positive seedlings of T0 generation ZmIAA15 were obtained in total, and the three positive seedlings were all positive seedlings obtained by successful transformation, and were designated as OE1, OE2 and OE3, and nucleotide sequences with a fragment length of 663bp were amplified in the transgenic lines OE1 to OE 3. Three transgenic lines and wild type were used as subsequent study materials.

After two selection and planting, the T3-transformed ZmIAA15 gene strain was grown in MS medium containing hygromycin as shown in FIG. 7B. As can be seen from FIG. 7B, the transgenic lines OE1-OE3 grew better than the wild-type benthonic CK.

Extracting RNA of the T3 generation ZmIAA15 gene and wild tobacco leaves, and performing reverse transcription after extraction. The expression level of ZmIAA15 gene in transgenic tobacco was detected by fluorescent quantitative PCR using wild-type tobacco as control and endogenous genes of tobacco (amplification primers shown in NbEF1a-F and NbEF1a-R in Table 1), and the results of the fluorescent quantitative PCR reaction were shown in FIG. 7C. As can be seen from FIG. 7C, the relative expression levels of the ZmIAA15 gene in the transgenic lines OE1-OE3 were all higher than that of WT, and the relative expression levels of the ZmIAA15 gene in OE2 and OE3 were all significantly higher than that of OE1.

Example 6 Effect of overexpression of ZmIAA15 Gene in Bensheng cigarette on expression of genes involved in promoting plant growth and development

To investigate whether overexpression of zmbia 15 gene in this smoke promotes expression of plant growth-related genes, the inventors found plant growth-related genes LNG1 and ROT3 related to leaf length and plant growth-related gene AN3 related to leaf width. DNA of wild type tobacco and transgenic strain OE1-OE3 leaves is extracted, and corresponding target genes are amplified by taking sequences SEQ ID NO.13-18 in Table 1 as primers, and the result is shown in figure 8, wherein the expression quantity of ROT3 and AN3 in transgenic raw tobacco is obviously higher than that of wild type, which indicates that the over-expression of ZmIAA15 gene promotes the growth and development of transgenic tobacco and improves the expression of genes related to the growth and development.

Example 7 test of ZmIAA 15-transformed tobacco response to salt stress and drought stress

7.1 determination of the Effect of salt stress and drought stress on seed germination Rate

In order to explore the capability of ZmIAA15 gene to resist salt stress and drought stress in the growth and development process of tobacco, the sprouting of T3 generation transformed ZmIAA15 gene in the germination period of the raw tobacco is firstly tested, and NaCl and mannitol are used for simulating salt treatment and drought treatment, and the method comprises the following steps: in an ultra clean bench, the MS medium (FIG. 9A), the MS medium containing 120mmol/LNaCl (FIG. 9B) and the MS medium containing 200mmol/L D-mannitol (FIG. 9C) were divided into four parts, and seeds of 3 lines of wild type benthamine and ZmIAA15 transgenic benthamine were sown respectively, 25 grains of each line, 3 replicates of each line, and the germination number was recorded every day to determine germination percentage during germination period.

As a result of the test, referring to fig. 9, under the control MS medium, the germination rate of three strains over-expressed by zmbia 15 gene and wild-type benthamiana is 100% (fig. 9A); whereas the germination rate of wild-type benthonic tobacco under 120mmol/L NaCl treatment is significantly lower than that of the over-expressed strain tobacco (FIG. 9B); the germination rate of wild-type benthames under 200mmol/L mannitol stress was slightly higher than that of salt treatment, but was still significantly lower than that of overexpressed transgenic tobacco (fig. 9C); according to statistics of germination conditions of tobacco seeds under three treatments of MS culture medium, 200mmol/L mannitol and 120mmol/L NaCl, it can be seen that the germination rate of transgenic over-expressed tobacco under mannitol and salt stress is extremely higher than that of wild type benthonic tobacco (figure 9D). Therefore, the drought resistance and salt tolerance of the three ZmIAA15 gene over-expression lines in the germination period are all obviously higher than those of wild tobacco, and further the fact that the ZmIAA15 gene is over-expressed in the tobacco can improve the tolerance of tobacco seeds in the germination period to salt and drought.

7.2 determination of the Effect of salt stress on young Nicotiana benthamiana seedlings of one month old

The young tobacco seedlings of the first month are treated as follows: salt stress is carried out by pouring 350mmol/L NaCl, the pouring amount is 1L each time, 7 days of continuous pouring are carried out, and the phenotype identification, the relative water content measurement of the leaves, the Malondialdehyde (MDA) content measurement, the Peroxidase (POD) and the superoxide dismutase (SOD) activity measurement are carried out after 7 days of treatment. And meanwhile, a control group is arranged, and wild tobacco and an over-expression strain in the control group are normally managed.

Method for measuring relative water content of blade

Taking 0.5g (Wf) of tobacco leaves, immersing the tobacco leaves in distilled water for 8 hours, taking out the leaves, measuring the weight Wt, deactivating enzyme at 105 ℃ in a drying box, and drying at 75 ℃ to constant weight Wd.

Blade relative water content (%) = (Wf-Wd)/(Wt-Wd) ×100%

Malondialdehyde (MDA) content determination method

0.2g of raw tobacco leaves are weighed, 2mL of 10% trichloroacetic acid (TCA) is added into a mortar, fully ground and uniformly mixed, the centrifugal machine is used for centrifugation at 5000rpm for 10min, 0.4mL of supernatant is taken and mixed with 0.4mL of thiobarbituric acid (TBA) which is newly prepared, the water bath is used for 100 ℃ for 15min, the cooling is carried out, and the light absorption values at 600nm, 532nm and 450nm are measured by centrifugation.

MDA content (μmol/g) =CV/(1 000W)

C: MDA concentration (mu mol/L)

V: volume of extractive solution (mL)

W: fresh weight of plant (g)

Peroxidase (POD) and superoxide dismutase (SOD) activity assays (purchased with the solebao assay kit).

Phenotypic results see fig. 10, wherein fig. 10A shows that both wild-type tobacco and over-expressed strains in the control group grow normally, leaf wilting appears at the lower part of tobacco of each strain of wild-type tobacco and zmiia 15 transgenic over-expressed after NaCl treatment, and the phenotypic characteristics of the transgenic zmiia 15 natural tobacco and the wild-type natural tobacco salt treated are measured, and as shown in fig. 10B-F, the plant height (B), stem thickness (C), leaf area (D), fresh weight on the ground (E) and fresh weight on the ground (F) of the transgenic zmiia 15 natural tobacco are significantly higher than those of the wild-type natural tobacco, which indicates that the overexpression of the zmiia 15 gene in the natural tobacco increases the resistance of the plant to salt stress.

The total root length was measured using a root system scanner, as shown in fig. 11, and the number of roots of tobacco in each strain of zmbia 15 transgenic overexpression (fig. 11B-D) was significantly greater than that of wild-type benthonic tobacco (fig. 11A), and the total root length of all strains was reduced after salt treatment compared to fresh water irrigation, but the total root length of transgenic strains was significantly higher than that of wild-type benthonic tobacco (fig. 11E), so that overexpression of zmbia 15 gene in benthonic tobacco significantly promoted plant root growth under salt stress.

The salt stress can generate a large amount of active oxygen, so that adverse effects are generated on plant bodies, the damage of the salt stress on plant membranes can be deduced through analysis of SOD and POD activities and MDA content and relative water content, the SOD and POD enzyme activities and relative water content of each strain of the over-expressed tobacco after the salt stress is 7d are all obvious or extremely obvious higher than those of wild type tobacco, the MDA content is extremely obvious lower than those of the wild type tobacco (figure 12), and the transgenic over-expressed tobacco has obviously higher resistance to the salt stress than those of the wild type tobacco.

7.3 determination of the Effect of drought stress on one month old Bensheng tobacco seedlings

In order to further explore the role of the zmbia 15 gene in tobacco under drought stress, the present application performed the following treatments on young tobacco seedlings of the same age as one month: drought stress treatment with 20% PEG6000 was performed by watering 1L per day for 7 days. Phenotyping, leaf relative moisture determination, malondialdehyde (MDA) content determination, peroxidase (POD) and superoxide dismutase (SOD) activity determination were performed 7 days after treatment. And meanwhile, a control group is arranged, and wild tobacco and an over-expression strain in the control group are normally managed. Leaf relative moisture determination, malondialdehyde (MDA) content determination, peroxidase (POD) and superoxide dismutase (SOD) activity determination methods are described in section 7.2.

Phenotypic results see fig. 13, where fig. 13A shows that PEG6000 treated overexpressed tobacco wilts slower than wild-type, upper leaf wilts more than overexpressed tobacco; the post-PEG 6000 treatment over-expressed tobacco had significant or very significant above-ground fresh weights compared to the wild type, whereas the fresh water-irrigated control had only OE3 significantly higher than the wild type (fig. 13B, E). By measuring the tobacco phenotype data after PEG6000 treatment, the ZmIAA15 gene can be preliminarily judged to have a certain resistance to simulated drought stress.

The total root length was measured using a root system scanner, as shown in fig. 14, the number of root systems of tobacco in each strain of zmbia 15 transgenic overexpression (fig. 14B-D) was significantly greater than that of wild-type benthonic tobacco (fig. 14A), and clear water was used as a control, and it can be seen that the total root length of all strains was affected after salt treatment, but the total root length of transgenic strains was still significantly higher than that of wild-type benthonic tobacco (fig. 14E), so that overexpression of zmbia 15 gene in benthonic tobacco significantly promoted plant root growth under drought stress.

A large amount of active oxygen can be generated similarly under the simulated drought stress, the membrane structure of the plant is damaged, further adverse effects are generated on the plant body, and the MDA content, POD, SOD enzyme activity and relative water content are analyzed, so that the great damage to the plant caused by the simulated drought stress can be estimated. POD, SOD enzyme activity and relative water content of each strain of the over-expressed tobacco after PEG6000 stress for 7d are all extremely higher than those of wild type tobacco, MDA content of the over-expressed strain is extremely lower than that of the wild type tobacco (figure 15), which shows that the resistance of the transgenic over-expressed tobacco to drought stress is extremely higher than that of the wild type tobacco.

Claims (10)

1. Use of zeatin response protein IAA15 or a gene encoding zeatin response protein IAA15 for increasing stress tolerance, including salt and/or drought tolerance, in a plant.

2. A method of increasing stress resistance in a plant comprising: introducing a gene encoding a maize auxin response protein IAA15 into said plant such that the maize auxin response protein IAA15 is overexpressed in the plant; the stress resistance includes salt and/or drought tolerance.

3. The use according to claim 1 or the method according to claim 2, wherein the plant is tobacco.

4. The use according to claim 1 or the method according to claim 2, wherein the maize auxin response protein IAA15 is selected from any one of the following:

(a) A protein consisting of an amino acid sequence shown in a sequence table SEQ ID NO. 1;

(b) A protein derived from (a) having the same activity as the protein of (a) by substituting, deleting or adding one or more amino acids in the amino acid sequence defined in (a).

5. The method according to claim 4 or claim 4, wherein the gene encoding the maize auxin response protein IAA15 is a gene encoding the protein of claim 4 (a) or a gene encoding the protein of claim 4 (b).

6. The use according to claim 5 or the method according to claim 5, wherein the gene encoding the maize auxin response protein IAA15 has a base sequence as shown in the sequence table SEQ ID No.2.

7. The method according to claim 1 or claim 2, characterized in that,

said improvement in plant stress tolerance is achieved by over-expressing said maize auxin response protein IAA15 in a plant; and/or the number of the groups of groups,

the improvement of plant stress resistance is achieved by adjusting plant physiological indexes including superoxide dismutase, peroxidase and malondialdehyde, wherein the content of the superoxide dismutase and the peroxidase is up-regulated, and the content of the malondialdehyde is down-regulated.

8. The method of claim 2, wherein said introducing a gene encoding the zein response protein IAA15 into said plant is introducing a recombinant vector comprising a gene encoding the zein response protein IAA15 into said plant.

9. The method of claim 8, wherein the construction of the recombinant vector comprises ligating a gene encoding a maize auxin response protein IAA15 to a multiple cloning site of a plant binary expression vector pCAMBIA1300, thereby forming a recombinant vector pCAMBIA 1300-zmbia 15.

10. The method of claim 9, wherein the gene encoding the maize auxin response protein IAA15 is ligated to the multiple cloning site of the plant binary expression vector pCAMBIA1300 by xbai and bamhi cleavage sites.