CN111118020B - WRI3/4 gene, cloning method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a cyperus esculentus WRI3/4 gene, a cloning method and application thereof. The nucleotide sequence of the cyperus esculentus WRI3/4 gene is shown as SEQ ID No. 1; the coded amino acid sequence is shown as SEQ ID No.2, or the coded amino acid sequence is an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown as SEQ ID No.2 and expresses the same function. The clone method of the cyperus esculentus WRI3/4 gene comprises the following steps: taking cyperus esculentus as a material, designing a degenerate primer according to a WRI gene conserved segment sequence of a higher plant, amplifying the WRI gene conserved segment of the cyperus esculentus, and then carrying out 5 '-RACE and 3' -RACE to obtain a5 'end sequence and a 3' end sequence of the cyperus esculentus; and designing full-length sequence primers according to the 5 'end sequence and the 3' end sequence of the cyperus esculentus, and amplifying to obtain the WRI3/4 gene of the cyperus esculentus.

Description

WRI3/4 gene, cloning method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a cyperus esculentus WRI3/4 gene, a cloning method and application thereof.

Background

Cyperus esculentus, also known as tiger nut, iron water chestnut, underground walnut, is an annual tuber plant of the family Cyperaceae suitable for growing in sand. The cyperus esculentus is drought-resistant, waterlogging-resistant, barren-resistant and saline-alkali-resistant, can grow in sandy soil, white serous soil, saline-alkali soil, black soil, barren hillsides and other soil places, can be planted no matter the soil fertility is high or low, and is most suitable for sandy loam and potassium-favored fertilizers. Therefore, the development and utilization of the drought-resistant gene of the cyperus esculentus has high research value in the practical application of drought-resistant plant cultivation.

Disclosure of Invention

In view of the above, the invention provides a cyperus esculentus WRI3/4 gene, a cloning method and an application method thereof in cultivation of plant drought-resistant germplasm.

The invention provides a cyperus esculentus WRI3/4 gene, wherein the nucleotide sequence of the cyperus esculentus WRI3/4 gene is shown as SEQ ID No.1, the coding amino acid sequence thereof is shown as SEQ ID No.2, or the coding amino acid sequence thereof is an amino acid sequence which has homology of more than 90% with the amino acid sequence shown as SEQ ID No.2 and has the same expression function.

The invention also provides a recombinant vector containing the cyperus esculentus WRI3/4 gene.

The invention also provides a transformant containing the recombinant vector.

The invention also provides a cloning method of the cyperus esculentus WRI3/4 gene, which comprises the following steps:

taking cyperus esculentus as a material, designing a degenerate primer according to a WRI gene conserved segment sequence of a higher plant, amplifying the WRI gene conserved segment of the cyperus esculentus, and then carrying out 5 '-RACE and 3' -RACE to obtain a5 'end sequence and a 3' end sequence of the cyperus esculentus; and designing full-length sequence primers according to the 5 'end sequence and the 3' end sequence of the cyperus esculentus, and amplifying to obtain the WRI3/4 gene of the cyperus esculentus.

Further, the degenerate primer sequence designed according to the sequence of the conserved segment of the higher plant WRI gene is as follows:

degenerate FP：5‘-GGTTCGAGGCCCACYTNTGGGAYAA；

degenerate RP：5‘-TCGATGGCGGCCADRTCRTANGC；

the full-length sequence primer designed according to the sequences of the 5 'end and the 3' end of the cyperus esculentus is as follows:

WRI3/4full length FP：5‘-TCCCCTCTCGTTTTAGCTCTCCTTCATTTC；

WRI3/4full length RP：5‘-GGCAAGCAGTGGTATCAACGCAGAGTAC。

the cyperus esculentus WRI3/4 gene provided by the invention can be applied to cultivation of plant drought-resistant germplasm.

Further, the method for cultivating the drought-resistant germplasm of the plant by utilizing the cyperus esculentus WRI3/4 gene comprises the following steps:

s1, constructing a vector: carrying out enzyme digestion and connection on the cyperus esculentus WRI3/4 gene and a plant expression vector plasmid, transforming escherichia coli, screening and identifying recombinants, and obtaining an overexpression vector;

s2, plant transgene: transforming a plant to be cultivated by utilizing agrobacterium with an overexpression vector plasmid, harvesting seeds of T1 generation, carrying out PCR identification after the seeds of T1 generation germinate, carrying out selfing on positive plants to obtain seeds of T2 generation, carrying out PCR identification after the seeds of T2 generation germinate, and carrying out selfing on the positive plants to obtain homozygous T3 generation transgenic seeds.

Further, in step S2, the specific process of vector construction is as follows: the cyperus esculentus WRI3/4 gene and pCambia 2300-35S-nos vector plasmid are subjected to enzyme digestion and ligation by utilizing PstI and SalI, escherichia coli DH5 alpha is transformed, and recombinants are screened and identified to obtain an overexpression vector pCambia 2300-35S-CeWRI 3/4-nos.

Further, the agrobacterium is agrobacterium EHA 105.

Further, the plant to be cultivated includes, but is not limited to, arabidopsis thaliana.

Further, in step S2, the sequences of the primers for PCR identification are:

NPTII FP：5‘-CCGGCCGCTTGGGTGGAGAGG；

NPTII RP：5‘-CGCCCAATAGCAGCCAGTCCCTTC。

the technical scheme provided by the invention has the beneficial effects that: the invention provides a cyperus esculentus WRI3/4 gene, and the cyperus esculentus WRI3/4 gene has important research significance for cultivating plant drought-resistant germplasm. The cultivated transgenic plant enhances the content of wax on the surface of the plant by promoting the expression of fatty acid and wax synthesis genes, thereby improving the drought resistance.

Drawings

FIG. 1 is a diagram of the evolutionary relationship of the double AP2 domain genes of different plants.

FIG. 2 is a schematic representation of the identification of transgenic Arabidopsis thaliana using NPTII primers.

FIG. 3A are seedlings of 10 day old Wild Type (WT) and transgenic Arabidopsis lines B1 and K2 under no PEG stress (top) and 14 day old Wild Type (WT) and transgenic Arabidopsis lines B1 and K2 under 5% PEG stress (bottom); FIG. 3B is a schematic representation of the fresh weight change of wild type and transgenic Arabidopsis lines; FIG. 3C is a schematic representation of the change in the length of the main root of wild type and transgenic Arabidopsis lines.

FIG. 4 is a schematic representation of the plant morphology of Wild Type (WT) and transgenic lines B1, K2.

FIG. 5 is a graph showing the wilting frequency (left) of Wild Type (WT) and transgenic lines B1, K2 after 15 days of water cut off, and the revival frequency (right) of Wild Type (WT) and transgenic lines B1, K2 after 22 days of water cut off and 1 day of water restoration, respectively.

FIG. 6 is a diagram showing the results of fluorescent quantitative PCR of Wild Type (WT) and transgenic Arabidopsis lines A5, A7, B1, K2 fatty acids and wax synthesis-related genes.

FIG. 7 is a diagram showing physiological indicators after water-break stress of Wild Type (WT) and transgenic Arabidopsis lines A5, A7, B1 and K2.

FIG. 8 is a graph showing the results of waxy composition and content in leaves of Wild Type (WT) and transgenic lines B1 and K2.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, embodiments of the present invention will be further described with reference to the accompanying drawings.

The embodiment of the invention provides a cyperus esculentus WRI3/4 gene, the nucleotide sequence of which is shown in SEQ ID No.1, and the corresponding coding amino acid sequence of which is shown in SEQ ID No.2, or the coding amino acid sequence of which is an amino acid sequence which has more than 90 percent of homology with the amino acid sequence shown in SEQ ID No.2 and expresses the same function.

The gene cloning steps of the cyperus esculentus WRI3/4 gene are as follows: taking a cyperus esculentus strain hubu-1 stored in a laboratory as a material, designing a degenerate primer according to a sequence of a WRI gene conserved segment of a higher plant, and amplifying the WRI gene conserved segment of the cyperus esculentus; carrying out 5 '-RACE and 3' -RACE on the basis to obtain the 5 'end and 3' end sequences of the cyperus esculentus; and designing a full-length sequence primer according to the 5 'end sequence and the 3' end sequence of the cyperus esculentus, and amplifying to obtain a WRI gene full-length sequence of the cyperus esculentus.

Wherein, the degenerate primer sequence designed according to the sequence of the conserved segment of the WRI gene of the higher plant is as follows:

degenerate FP：5‘-GGTTCGAGGCCCACYTNTGGGAYAA；

degenerate RP：5‘-TCGATGGCGGCCADRTCRTANGC；

the 5' -RACE primer sequence is: 5' -CGGCGGCTTCTTCTTCTGTTGCGTATG;

the 3' -RACE primer sequence is as follows: 5' -TTGGGATAAGAACAGTTGGAATGAGAC;

the full-length sequence primer designed according to the sequences of the 5 'end and the 3' end of the cyperus esculentus is as follows:

WRI3/4full length FP：5‘-TCCCCTCTCGTTTTAGCTCTCCTTCATTTC；

WRI3/4full length RP：5‘-GGCAAGCAGTGGTATCAACGCAGAGTAC。

sequence analysis:

the obtained full-length sequence of the cyperus esculentus WRI gene is subjected to conserved domain analysis through an NCBI website (https:// www.ncbi.nlm.nih.gov/Structure/cdd /), and the gene is found to have 2 AP2/ERF domains and belongs to an AP2 family gene; further, the MEGA7.0 software is used for carrying out cluster analysis on the AP2 family genes of different plants, and the obtained cyperus esculentus WRI gene is found to be clustered with WRI3 and WRI4 (see figure 1), so that the cyperus esculentus WRI3/4 gene is named as Ce WRI 3/4.

The cyperus esculentus WRI3/4 gene can be used for cultivating drought-resistant germplasm of plants, and the specific steps are as follows:

step S1, constructing a vector: carrying out enzyme digestion on a cyperus esculentus WRI3/4 gene and a pCambia 2300-35S-nos vector plasmid preserved in a laboratory by using PstI and SalI, connecting, transforming escherichia coli DH5 alpha, screening and identifying a recombinant to obtain an overexpression vector pCambia 2300-35S-CeWRI 3/4-nos; the primer sequence of the vector construction is as follows:

vector construction FP：5‘-ATAGGATCCTCCCCTCTCGTTTTAGCTCTCCTTCATTTC；

vector construction RP：5‘-ATACTGCAGGGGCAAGCAGTGGTATCAACGCAGAGTA；

step S2, plant transgenosis: transforming arabidopsis thaliana by using agrobacterium EHA105 with pCambia 2300-35S-CeWri3/4-nos plasmid through an inflorescence dip-dyeing method, harvesting T1 generation seeds, carrying out PCR identification on T1 generation seeds after germination by using NPTII primers, carrying out selfing on positive plants to obtain T2 generation seeds, carrying out PCR identification on T2 generation seeds after germination by using the NPTII primers, and carrying out selfing on the positive plants to obtain homozygous T3 generation transgenic arabidopsis thaliana seeds.

In step S2, the NPTII primer sequences for PCR identification are:

NPTII FP：5‘-CCGGCCGCTTGGGTGGAGAGG；

NPTII RP：5‘-CGCCCAATAGCAGCCAGTCCCTTC。

FIG. 2 is a schematic diagram showing the identification of transgenic Arabidopsis thaliana using NPTII primer, in FIG. 2, M represents DL2000 Marker; 1 is a positive control; 2-7 are transgenic arabidopsis plants; 8 is wild type Arabidopsis thaliana.

The drought resistance identification, the related gene expression identification, the drought resistance physiological and biochemical index detection and the leaf wax content determination are carried out on the obtained T3 generation transgenic arabidopsis seeds, and the specific processes are as follows:

1. and (3) drought resistance identification:

seeds of wild type and transgenic arabidopsis T3 generations were sown on 1/2 MS medium with/without 5% PEG6000 added for germination, and the length of the main root was measured 2, 4, 6, 8 days after seed germination. Growth differences between wild-type arabidopsis (WT) and transgenic arabidopsis strains (B1, K2) were not significant under non-stress (CK) conditions (see fig. 3A). However, on the medium containing 5% PEG6000, wild-type arabidopsis (WT) had only 2 very small yellowing true leaves, with severely inhibited main root growth and significantly reduced lateral root numbers, whereas transgenic arabidopsis (B1, K2) seedlings had 4 larger true leaves, with no significant inhibition of main root elongation and lateral root growth (see fig. 3A). Major root length (see fig. 3B) and fresh weight measurements (see fig. 3C) also show that the transgenic lines are more resistant to PEG-simulated drought stress than the wild type.

In FIG. 3, DAG is a short hand for Day after germination, showing that after germination, FIG. 3A is a 10-Day-old wild-type (WT) and transgenic Arabidopsis lines B1 and K2 seedlings under PEG-free stress (top) and 14-Day-old wild-type (WT) and transgenic Arabidopsis lines B1 and K2 seedlings under 5% PEG-stress (bottom); FIG. 3B is the fresh weight change of Wild Type (WT) and transgenic Arabidopsis lines (A5, A7, B1, K2); FIG. 3C is the major root length variation of Wild Type (WT) and transgenic Arabidopsis lines (B1, K2); p <0.01, P < 0.05.

After 2 weeks of seed germination, Arabidopsis seedlings were transferred to nutrient soil-filled trays for growth and watered once every 3 days. Drought resistance of arabidopsis seedlings grown under normal conditions is identified by water cut. And (4) counting the wilting frequency 15 days after water cut off, carrying out rehydration treatment 22 days after water cut off, and counting the recovery frequency 1 day after water cut off. Prior to water-break drought stress, Wild Type (WT) and transgenic arabidopsis thaliana (B1, K2) grew similarly (see fig. 4). After water cut off for 15 days, the wilting frequency of wild plants reaches 63.6-81.8%, while the wilting frequency of transgenic lines is only 8.2-25% (see fig. 4-5). After 22 days of water cut and 1 day of rehydration, the reactivation rate of the wild plants is 18.1-36.3%, and the reactivation rate of the transgenic plants is 58.3-63.6% (see fig. 4-5); FIG. 4 shows the plant morphology of Wild Type (WT) and transgenic lines B1, K2, FIG. 5 shows the wilting frequency (left) of Wild Type (WT) and transgenic lines B1, K2 after 15 days of water cut off, and the revival frequency (right) of Wild Type (WT) and transgenic lines B1, K2 after 1 day of water cut off; p < 0.01.

2. And (3) related gene expression identification:

wild type arabidopsis (WT) and transgenic arabidopsis T3 plants of 2 weeks size were treated with water/5% PEG6000 for 10 hours, RNA was extracted, and fluorescence quantitative PCR was performed on fatty acid and wax synthesis-related genes. The results found that prior to PEG treatment, the genes PIPK- β 1(At5g52920), BCCP2(At5g15530) and PDHE1 α (At1g01090) associated with fatty acid synthesis did not differ significantly between wild type and transgenic lines; after 10 hours of treatment with 5% PEG, the expression of the above-mentioned genes was slightly reduced in the wild type but increased by 130-.

The genes LACS1(At2g47240), WSD1(At5g37300), KCS1(At1g01120), CER1(At1g02200) and CER4(At4g33790) associated with wax synthesis in the transgenic lines were 10-160%, 50-140%, 30-70%, 50-270% and 90-130% higher than the wild type, respectively, before PEG treatment (see FIGS. 6D-H).

The expression level of the gene in the wild type after PEG treatment is slightly reduced compared with that before the PEG treatment; however, in the transgenic lines, the above genes were increased by 130-fold, 300%, 30-80%, 20-80%, 10-110%, 80-140% respectively as compared with those before treatment (see FIGS. 6D-H).

Fig. 6 shows the fluorescent quantitative PCR results for Wild Type (WT) and transgenic arabidopsis strains a5, a7, B1, K2 fatty acids and wax synthesis related genes, # P <0.01, # P < 0.05.

Fatty acid and wax synthesis related genes are shown in table 1:

table 1: fatty acid and wax synthesis related gene

3. Detecting drought resistance physiological and biochemical indexes:

after 2 weeks of water-cut and drought treatment, the contents of soluble sugar, free proline and malonaldehyde in leaves of wild arabidopsis thaliana respectively reach 27.1mg/g (fresh weight, the same below), 69.4 mug/g and 5.3mmol/g, while in transgenic lines, the contents are respectively only 14.3-29.9% (see fig. 7A), 26.9-50.6% (see fig. 7B) and 36.5-45.5% (see fig. 7C) of wild type, which indicates that the drought damage of the transgenic lines is far lower than that of the wild type.

Fig. 7 is physiological indices of Wild Type (WT) and transgenic arabidopsis strains a5, a7, B1 and K2 after water-break stress,. P < 0.01.

4. Leaf wax content determination:

the wax in Arabidopsis leaves is mainly composed of alkane and primary alcohol, and 68% of the total wax is contained in C29 and C31 in the alkane, and C31 is mainly contained in the alkane (see FIGS. 8A-B). The total wax in the transgenic lines, C26, C28, and C30 in the primary alcohol, and C27 and C31 in the alkanes were all significantly higher than the wild type. C33 in alkanes, C32 in primary alcohol, in both wild type and transgenic lines did not differ significantly between wild type and transgenic lines (see FIGS. 8A-C). C29 in alkanes was significantly higher in transgenic line B1 than wild type, but there was no significant difference between transgenic line K2 and wild type (see fig. 8A).

Figure 8 is waxy composition and content in leaves of Wild Type (WT) and transgenic lines B1 and K2, P < 0.05.

The features of the embodiments and embodiments described herein above may be combined with each other without conflict.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims (5)

1. The application of the cyperus esculentus WRI3/4 gene in cultivating plant drought-resistant seeds is characterized in that the nucleotide sequence of the cyperus esculentus WRI3/4 gene is shown as SEQ ID No. 1; the coded amino acid sequence is shown as SEQ ID No.2, and the method for cultivating the plant drought-resistant seeds by utilizing the cyperus esculentus WRI3/4 gene comprises the following steps:

s1, constructing a vector: carrying out enzyme digestion and connection on the cyperus esculentus WRI3/4 gene and a plant expression vector plasmid, transforming escherichia coli, screening and identifying recombinants, and obtaining an overexpression vector;

s2, plant transgene: transforming a plant to be cultivated by using agrobacterium tumefaciens with an overexpression vector plasmid, wherein the plant to be cultivated is arabidopsis thaliana, harvesting seeds of T1 generation, carrying out PCR identification after the seeds of T1 generation germinate, carrying out PCR identification after the seeds of T2 generation are germinated in positive plants, carrying out PCR identification after the seeds of T2 generation germinate, and carrying out selfing on the positive plants to obtain homozygous T3 generation transgenic seeds.

2. The use according to claim 1, wherein the agrobacterium is agrobacterium EHA 105.

3. The use of claim 1, wherein in step S2, the primer sequences for PCR identification are:

NPTII FP：5‘-CCGGCCGCTTGGGTGGAGAGG；

NPTII RP：5‘-CGCCCAATAGCAGCCAGTCCCTTC。

4. the use according to claim 1, wherein the cloning method of the cyperus esculentus WRI3/4 gene comprises the following steps: taking cyperus esculentus HUBU-1 as a material, designing a degenerate primer according to a WRI gene conserved segment sequence of a higher plant, amplifying the WRI gene conserved segment of the cyperus esculentus, and then carrying out 5 '-RACE and 3' -RACE to obtain a5 'end sequence and a 3' end sequence of the cyperus esculentus; and designing full-length sequence primers according to the 5 'end sequence and the 3' end sequence of the cyperus esculentus, and amplifying to obtain the WRI3/4 gene of the cyperus esculentus.

5. The use of claim 4, wherein the degenerate primer sequence designed based on the sequence of the conserved segment of the higher plant WRI gene is:

degenerate FP：5‘-GGTTCGAGGCCCACYTNTGGGAYAA；

degenerate RP：5‘-TCGATGGCGGCCADRTCRTANGC；

the full-length sequence primer designed according to the sequences of the 5 'end and the 3' end of the cyperus esculentus is as follows:

WRI3/4 full length FP：5‘-TCCCCTCTCGTTTTAGCTCTCCTTCATTTC；

WRI3/4 full length RP：5‘-GGCAAGCAGTGGTATCAACGCAGAGTAC。

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