CN107384936B

CN107384936B - ThRap2.1 gene influencing flooding resistance of sequoia intermedia and application thereof

Info

Publication number: CN107384936B
Application number: CN201710613030.3A
Authority: CN
Inventors: 宣磊; 华建峰; 王芝权; 杨颖�; 范文才; 於朝广; 殷云龙
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2017-07-25
Filing date: 2017-07-25
Publication date: 2019-12-27
Anticipated expiration: 2037-07-25
Also published as: CN107384936A

Abstract

The invention discloses a ThRap2.1 gene influencing flooding resistance of a piece of Central African fir and application thereof, wherein a nucleotide sequence of the ThRap2.1 gene is shown as SEQ ID NO. 1. The invention transfers the ThRap2.1 gene into the poplar which is a woody plant, the transgenic poplar which excessively expresses the ThRap2.1 has slower growth rate and obviously shortened internode, the expression level of the ThRap2.1 gene in the transgenic plant is 600 times higher than that of the control poplar by 300 times, and the expression level of the ThRap2.1 gene is increased by thousands of times under the water flooding stress. The ThRap2.1 gene is an important gene of plants responding to water logging stress and has important application value in the field of forest genetic engineering.

Description

ThRap2.1 gene influencing flooding resistance of sequoia intermedia and application thereof

Technical Field

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a ThRap2.1 gene influencing flooding resistance of Taxus chinensis and application thereof.

Background

China has abundant resources of mudflat land and wetland and has great potential in developing greening and forestation of water-affected lands. Develop and combineThe popularization of the research of the flooding-resistant tree species is beneficial to improving the land resource utilization rate of coastal areas and wetlands in China, and is an important means for solving the problems of flood disasters, reduced biodiversity, restoration of wetland construction and the like of coastal areas and river zones in China. China fir (Zhongshan fir)Taxodiumhybrid 'zhongshanshashan') is a superior hybrid clone of the genus larch cultivated by the plant research institute of Chinese academy of sciences of Jiangsu province, has obvious heterosis and has strong flooding resistance. In recent years, the method plays an important role in constructing wetland ecosystems in Yunnan pond, Anhui nest lake, Chongqing three gorges reservoir area and other areas. Wherein, the Chinese fir 406 (Taxodium. mucronatum ♀×TaxodiumThe flooding resistance of the distichum is outstanding, and indoor simulation tests, multi-year regional tests, physiological studies and transcriptomic studies prove that the taxus chinensis 406 is an ideal material for researching the flooding resistance of woody plants.

Earlier in other model plants, such as Arabidopsis, rice, it has been shown that ethylene transcription factor (ERF) family members are closely related to plant abiotic stress, with class VII members (ERF-VIIs) of ERF transcription factors being demonstrated to be the major transcription factors regulating hypoxia-related gene expression and responding to flooding stress. ERF-VIIs transcriptionally activate downstream genes in a cascade amplification mode, convert extracellular signals into intracellular signals, and promote plants to generate a series of adaptation mechanisms, such as glycolysis acceleration, aeration tissue formation, oxygen transportation rate acceleration and the like. The ERF-VIIs transcription factor regulates the expression of hypoxia stress related genes through an ethylene signal path, thereby influencing the stress resistance reaction of plants to flooding stress. VII ERFs in Arabidopsis comprise RAP2.12, RAP2.2, RAP2.3, HRE1 and HRE2, ERF-VIIs in rice have Sub1A, SK1 and SK2, wherein RAP2.12 and Sub1A are considered as key genes for regulating and controlling flooding-resistant traits of Arabidopsis and rice, flooding-resistant capability of Arabidopsis can be remarkably improved by over-expressing RAP2.12 and Sub1A genes, and a rice plant becomes sensitive to flooding stress due to gene knockout or silencing of Sub1A genes.

Disclosure of Invention

The purpose of the invention is as follows: aiming at the defects in the prior art, the invention aims to provide a ThRap2.1 gene influencing the flooding resistance of the sequoia intermedia. Another object of the present invention is to provide the use of the ThRap2.1 gene affecting the flooding resistance of the said Taxus chinensis.

The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

a ThRap2.1 gene influencing flooding resistance of the Chinese fir has a nucleotide sequence and an amino acid sequence coded by the nucleotide sequence shown in SEQ ID No. 1.

The vector containing the ThRap2.1 gene influencing the flooding resistance of the sequoia intermedia.

The ThRap2.1 gene influencing the flooding resistance of the taxus chinensis is applied to plant breeding.

The ThRap2.1 gene influencing the flooding resistance of the taxus chinensis is applied to the flooding resistance of plants.

Recently, the subject group of the inventor discovers that a large number of genes with obvious differential expression exist in a phytohormone signal conduction pathway-ethylene signal conduction pathway by mining the water logging stress transcriptome data result of the taxus chinensis 406, so that the key genes of the taxus chinensis ERF-VIIs flooding resistance are screened and identified, the deep research on the expression mode and the biological function is carried out, and the method has important significance for the abundance of gene resources of the taxus chinensis trees and the flooding resistance molecular breeding of woody plants.

The invention uses the tissue culture seedling root of the sequoia intermedia 406 as the material, clones the ThRap2.1 full-length gene of the sequoia intermedia by the RACE technology。Meanwhile, an excessive expression vector pH35GS-ThRap2.1 is constructed by adopting a channel cloning technology, the gene is positioned behind a promoter P35S, under the drive of a promoter P35S, ThRap2.1 is efficiently expressed in poplar, and the growth rate of a transgenic plant is slowed down along with the occurrence of phenotypic variation, internodes are obviously shorter than that of a control poplar, and the plant is dwarfed.

Has the advantages that: compared with the prior art, the ThRap2.1 gene is transferred into the poplar which is a woody plant, the growth rate of the transgenic poplar which excessively expresses the ThRap2.1 is reduced, internodes are obviously shortened, the expression level of the ThRap2.1 gene in the transgenic plant is 600 times higher than that of the control poplar by 300-fold, and the expression level of the ThRap2.1 gene is increased by thousands of times under flooding stress. The ThRap2.1 gene is an important gene of plants responding to water logging stress and has important application value in the field of forest genetic engineering.

Drawings

FIG. 1 is a schematic representation of a plant overexpression vector pH35 GS;

FIG. 2 is a comparison graph of the overall phenotype of over-expressed ThRap2.1 transgenic poplar and non-transgenic poplar, wherein the left part of the graph is non-transgenic poplar and the right part of the graph is transgenic poplar;

FIG. 3 shows the difference in gene expression between over-expressed ThRap2.1 transgenic plants and non-transgenic plants;

FIG. 4 is a diagram of the gene expression change of the transgenic plant over-expressing ThRap2.1 under water-logging stress.

Detailed Description

The present invention will be further described with reference to the following specific examples. In the following examples, the procedures not described in detail are all routine biological experimental procedures, and can be performed with reference to molecular biology experimental manuals, published journal literature, and the like.

EXAMPLE 1 cloning of ThRap2.1 Gene by RACE technique

1) Extraction of RNA and inversion of cDNA

The root of the Taxus cuspidata 406 tissue culture seedling is used as a material, and the extraction of total RNA of the Taxus cuspidata is carried out by using RNeasy Plant Mini Kit (QIAGEN company), and the operation steps are improved because the root contains more secondary metabolites. Before RNA extraction, all gun heads, centrifuge tubes and mortar are soaked overnight in 0.1% DEPC water, autoclaved for 40min and dried. All solutions should be made up with 0.1% DEPC water. Determination of total RNA concentration and purity was performed with NanoDrop 1000. Total RNA was separated by 1.0% agarose gel electrophoresis, and one RNA with better integrity should observe three clear bands, 5.8SRNA was slightly darker, and 28SRNA band brightness was about twice as bright as 18SRNA, indicating that RNA was not degraded, and then reverse transcribed by using the SMARTer PCR cDNA Synthesis Kit, Advantage 2 PCR Kit from Clontech.

2) Obtaining of target fragment of ThRap2.1 gene

Screening Unigene sequences of ThRap2.1 according to the sequencing result of the epistaxis transcriptome, and amplifying a target fragment of ThRap2.1 gene by using specific primers designed by Oligo6, wherein forward primers of the target fragment of ThRap2.1 are as follows: 5'-AAATGGGTTTCAGAAGTCAGGG-3', 5'-ACACCATGATTGATTTTCTTGG-3' are provided. And (3) PCR reaction system: TakaraLA Taq (5U/. mu.L) 0.5. mu.L, 10 × LA PCR Buffer (Mg)²⁺Free)5.0μL，MgCl₂5.0. mu.L (25 mM), 8.0. mu.L dNTP mix (2.5 mM reach), 2.0. mu.L Forward Primer (10. mu.M), 2.0. mu.L Reverse Primer (10. mu.M), 1.0. mu.L cDNA template, 26.5. mu.L Milli-Q Water. The reaction procedure is as follows: 3min at 94 ℃; 30cycles at 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 2 min; 6 min at 72 ℃; forever at 4 ℃. Sequencing the product to obtain the ThRap2.1 gene target fragment sequence shown in SEQ ID NO. 3.

2) Amplification of 3'RACE fragment and 5' RACE fragment of ThRap2.1 gene

3'RACE and 5' RACE primers are respectively designed according to the target fragment of the ThRap2.1 gene, and SMARTer RACE 5 '/3' Kit is used for carrying out the amplification of the 3'RACE and 5' RACE of the gene.

Designing a primer: gene-specific primers (GSPs) should meet the following requirements: the length is 23-28 nt to ensure specific annealing; the GC content is between 50 and 70 percent; tm is more than or equal to 65 ℃; the 3' end of the common primer sequence provided by the kit is as non-complementary as possible. CDS primer sequences provided in the kit: CDS1 = 5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3', CDS 2= 5'-CTAATACGACTCACTATAGGGC-3'.

Generating RACE-Ready cDNA: buffer Mix was prepared for the cDNA synthesis reaction at the following volumes. To ensure sufficient volume, 1 more reaction was prepared. The following reagents were mixed, spun briefly in a microcentrifuge, and left at room temperature for subsequent experiments. 4.0. mu.L of 5XFirst-Strand Buffer, 0.5. mu.L of DTT (100mM), 1.0. mu.L of dNTPs (20mM), Total Volume: 5.5. mu.L.

The following reagents were added to separate microcentrifuge tubes: preparation of 5' -RACE-Ready cDNA: 1-10. mu.L of RNA, 1. mu.L of 5' -CDS Primer, 0-9. mu.L of Sterile H2O, 1. mu.L of SMARTER II A OIIGONUCLEOTIDE, Total Volume: 12 μ L. Preparation of 3' -RACE-Ready cDNA: 1-11. mu.L of RNA, 1. mu.L of 3' -CDS Primer, 0-10. mu.L of Sterile H2O, Total Volume: 12 μ L. The components were mixed, spun briefly in a microcentrifuge, incubated at 72 ℃ for 3min and cooled at 42 ℃ for 2 min. After cooling, the tube was centrifuged at 14,000 Xg for 10 sec to collect the reagents at the bottom of the tube. The following solutions were added and mixed in the following order on ice: mu.L of RNase Inhibitor (40U/. mu.L), 2.0. mu.L of SMARTSCRIBE reverse transcriptase (100U), 5.5. mu.L of Buffer Mix prepared at room temperature in the early stage, and the total volume of the final 5 '-RACE-Ready cDNA and the 3' -RACE-Ready cDNA were 20. mu.L. Gently sucking and mixing, centrifuging briefly to collect components at the bottom of the tube, incubating at 42 ℃ for 90 min, heating at 70 ℃ for 10min, and diluting the second cDNA synthesis reaction product to 100 mu L by using Tricine-EDTA Buffer. The cDNA samples were stored at-20 ℃ for up to three months.

5 '-RACE PCR and 3' -RACE PCR reactions: PCR Master Mix was prepared in the following volumes. Please add an additional amount of reaction to ensure sufficient volume. 5' -RACE PCR System: 2 μ L of 5' -RACE-Ready cDNA, 15.5 μ L of PCR-Grade H₂O, 25.0. mu.L of 2X SeqAmp Buffer, 1. mu.L of SeqAmp DNA Polymerase, 5' GSPs Primer 1: 5'-CTGTTACTGGGTCCAAAAGCCAA-3' (for the first round of reaction), SDS1 Primer (for the first round of reaction), 5' GSPs Primer 2: 5'-CACCAGCAACACCATGATTGATTTT-3' (for the second round of reaction) SDS2Primer (for the first round of reaction), Total Volume: 50 μ L. 3' -RACE PCR System: 2 μ L of 3' -RACE-Ready cDNA, 15.5 μ L of PCR-Grade H₂O, 25.0. mu.L of 2X SeqAmp Buffer, 1. mu.L of SeqAmp DNA Polymerase, 3' GSPs Primer 1: 5'-TGGTGGTTGTAAGGTGAGTAAGG-3' (for the first round of reaction), SDS1 Primer (for the first round of reaction), 3' GSPs Primer 2: 5'-GGCACTTGTTGGCTTTTGGACCCAGTAACAGT-3' (for the second round of reaction) SDS2Primer (for the first round of reaction), Total Volume: 50 μ L. Mix gently. The PCR procedure was nested PCR with two rounds of amplification, the first round was touchdown PCR: 94 ℃ for 30sec, 72 ℃ for 2min, 5 cycles; 94 ℃ for 30 sec; 30sec at 70 ℃, 3min at 72 ℃, 5 cycles; 94 deg.C 30sec, 68 deg.C 30sec, 72 deg.C 3min20 cycles; forever at 4 ℃. The first round was normal PCR: 3min at 94 ℃; 30cycles at 94 ℃ for 30sec, 56 ℃ for 30sec, 72 ℃ for 2 min; 6 min at 72 ℃; forever at 4 ℃.

Gel purification is carried out by using a NuceloSpin Gel and PCR Clean-Up Kit, a target fragment is cut, the target fragment is recovered by using a Gel cutting Kit, the Gel cutting Kit is connected to a PMD19-T simple Vector of takara, the competence of Top10 escherichia coli of takara is transformed, screening is carried out by an LB screening culture plate containing Amp, and the screened monoclonal colony is subjected to PCR detection and then sent to the Kingsler company for sequencing and identification.

Splicing 3'RACE and 5' RACE products obtained by sequencing by using Bioedit software to obtain a target gene containing 1048bp of basic groups, determining high homology of the target gene and a rice/arabidopsis thaliana Rap2.1 gene by Blast, and naming the target gene as ThRap2.1 gene, wherein the nucleotide sequence of the target gene is shown as SEQ ID No.1, the coded protein comprises a complete coding reading frame of 732bp, and the amino acid sequence of the coded protein is shown as SEQ ID No. 2.

Example 2 construction of plant expression vector for ThRap2.1 Gene

Transferring the target gene segment to a target expression vector by using Gateway directional cloning technology, wherein the target expression vector comprises a BP reaction part and an LR reaction part. The carrier used in this example is shown in FIG. 1.

1) The purpose of the BP reaction was to transfer the gene fragment to an entry vector, reaction: 10-20ng of vector ThRap2.1 ORF fragment, 1 muL of Salt solution, 1 muL of pCRTM8/GW/TOPOTM vector (entry vector), adding nucleic-free Water to the total volume of 6 muL, gently mixing, reacting at 22 ℃ for 60min, and transferring to ice; and (3) transforming the reaction product of 6 mu L in the previous step into TOP10 competent cells, coating the competent cells on an LB screening culture plate, selecting a single clone to perform bacterial liquid PCR detection, wherein the primers are a gene specific upstream primer and a T7 primer on a carrier, and the detected positive clone is further subjected to sequencing verification.

2) The LR reaction aims at re-cloning a target gene recombined into a portal vector into the target vector, and the reaction system comprises: 100ng of plasmid after purification of an entry vector, 1.5 mu L of a target vector (100 ng/. mu.L), 2 mu L of LR clone II enzymemix, 1 × TE (pH8.0) to the total volume of 8 mu L, vortex, then carrying out short-time centrifugation, carrying out warm bath at 25 ℃ for 1h, adding 1 mu L of LProteinase K solution, mixing uniformly, and carrying out warm bath at 37 ℃ for 10min to stop the reaction; and (2) transforming Top10 competent cells by taking 2 mu L of LR reaction products, coating the competent cells on an LB screening culture plate, selecting a single clone to carry out PCR detection on bacterial liquid, wherein the primers are ThRap2.1 gene specific upstream primers and 35s primers on a carrier, and further sequencing and verifying the detected positive clone. After verification, the recombinant vector after LR reaction is recovered and purified, and the expression vector of the target gene is obtained and stored at-20 ℃ for later use.

Example 3 genetic transformation of ThRap2.1 Gene

The ThRap2.1 gene is transformed into agrobacterium by a liquid nitrogen freeze-thawing method, and then the hybrid populus davidiana is transformed by a stem section method. The method comprises the following operation steps: adding at least 100ng of recovered and purified expression vector into EHA105 competent cells, gently mixing, ice-bathing for 30min, quickly freezing for 1min with liquid nitrogen, heat shocking for 3min at 37 ℃, rapidly placing on ice for 1-2min, adding 800 μ L of LB culture medium, recovering for 3h at 28 ℃, 100rmp, centrifuging for 3min at 4000rmp, sucking off 800 μ L of LB culture medium, mixing the rest bacteria liquid uniformly, smearing on LB plate containing antibiotics, performing inverted culture for 30-48h at 28 ℃, detecting positive clone by PCR, expanding and propagating the colony of the positive clone, inoculating into 120mL of LB liquid culture medium containing corresponding antibiotics, culturing for about 24h with bacteria shaking (220 rmp) at 28 ℃ until OD is reached₆₀₀The value is about 0.5, the bacterial liquid is divided into 50mL centrifuge tubes, 1400rcf is centrifuged for 10min, the thalli is collected, a certain volume of MS (without adding cane sugar) solution is used for resuspending the thalli to the OD600 value of 0.5, the final concentration of acetosyringone (As) is 20 MuM, the bacterial liquid is gently oscillated (90 rmp) for 45min at 25 ℃, the tissue culture seedlings of the populus deltoids with basically consistent growth potential and 4-6 weeks are selected, the middle upper stem section is taken, the leaves and the axillary buds are trimmed off, the stem section is rapidly cut into 1-1.5cm stem sections by a blade, the stem sections are transferred into the prepared impregnation liquid, the stem sections are gently oscillated for 30min at 25 ℃ (250 mL triangular flask and 90 rmp), the stem sections are taken out, the residual bacterial liquid is sucked out, the stem sections are transferred into MS1 differentiation culture plates without antibiotics for dark culture for 48h, the stem sections are transferred into MS1 culture plates containing the antibiotics after bacteria washing, the stem sections are transferred into 2 for screening culture plates, the stem sections are transferred into resistance culture plates after bacteria are subjected to elongation, when adventitious, when the resistant adventitious bud is elongated and cultured in the stemWhen the seedlings grow to about 1cm in the medium, the seedlings are cut off and transferred to 1/2MS culture plates containing antibiotics for rooting screening of resistant plants, so that complete plants are obtained, and the seedlings of the resistant plants are cut on a 1/2MS culture medium for phenotype observation of cuttings. After 4W of growth, as can be seen from FIG. 2, the overexpression of ThRap2.1 has a certain influence on the phenotype of the plants, and compared with the control plants, the growth speed of the transgenic plants is obviously reduced, the internode variation occurs, and the internode length is obviously shorter than that of the control plants.

Example 4 molecular detection of transgenic plants

And (3) carrying out real-time quantitative detection on the transgenic ThRap2.1 poplars growing to 4W on the screening culture medium and the control poplars tissue culture seedlings. Meanwhile, a top-submerged experiment is adopted, and sampling is carried out after 1W for real-time quantitative molecular detection. The real-time quantitative primer is designed by adopting Oligo6.6 software, the length of an amplification product is 123bp, the Tm value of the primer is 60 ℃, and the sequence of the primer is as follows: qRap2.1F: 5'-CTTCCTTAAATTTCCCT-3' q Rap2.1R: 5'-TGGCATGGTCCACAG-3' are provided. The reference gene of the real-time quantitative PCR adopts the screened APRT gene APRT-F: 5'-TCCACAGGTTCTTGAATCGCT-3', APRT-R: 5'-TGACTTGAGCCTCATTCGCTC-3' are provided. qRT-PCR was performed using the Analitik Jena qTOWER2.2 (Germany) system. Following the protocol of the SYBR Green kit (Rox), the amplification procedure was: 2min at 55 ℃ and 10min at 95 ℃; 40 cycles of 95 ℃ for 15 s and 60 ℃ for 1 min. The melting curve was then generated by setting 60 ℃ to 95 ℃. Each sample was subjected to 3 technical replicates, and 20 μ L of the reaction system contained: mu.L of diluted cDNA (dilution ratio 1: 3, concentration of diluted cDNA of about 350 ng. mu.L)⁻¹) 10 μ L FastStart Universal SYBR Green Master (Rox), 6 pmol upstream primer, 6 pmol downstream primer and 6.8 μ L ddH₂And O. Relative expression amount of the genes was in accordance with 2^-ΔΔCtThe method performs the calculation. The test data were statistically analyzed and plotted by the SPSS 19.0 software. The real-time quantification results are shown in fig. 3: the gene expression level of the 9 plants with phenotypic variation is 302-655 times higher than that of the control plants as shown in FIG. 3, and the gene over-expression vector sequence and the plant genome sequence are preliminarily judged to be fused. After 1W of the flooding experiment, the substrate is inverted as shown in FIG. 4As the plants still grow well, real-time quantitative detection is carried out again, and the expression quantity of the ThRap2.1 gene in the transgenic plants is found to be increased to 600 times of that of 300-.

SEQUENCE LISTING

<110> institute of plant of Chinese academy of sciences of Jiangsu province

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Claims

1. A ThRap2.1 gene influencing the flooding resistance of the sequoia intermedia has a nucleotide sequence shown in SEQ ID NO. 1.

2. The ThRap2.1 gene of claim 1, wherein the encoded protein sequence is shown in SEQ ID No. 2.

3. A vector comprising the ThRap2.1 gene affecting flooding resistance of said Taxus chinensis as claimed in claim 1.

4. The use of the ThRap2.1 gene affecting flooding resistance in an Ascendens intermedia as claimed in claim 1 in plant breeding.

5. The use of the ThRap2.1 gene affecting flooding resistance of Taxus chinensis as claimed in claim 1 in plant flooding resistance.

6. A host bacterium containing ThRap2.1 gene affecting the flooding resistance of said Taxus chinensis as claimed in claim 1.