CN113755510B

CN113755510B - Encoding soybean FtsH metalloprotease gene GmFtsH25 and application thereof

Info

Publication number: CN113755510B
Application number: CN202111183353.6A
Authority: CN
Inventors: 喻德跃; 王莉; 杨宇明; 黄方
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2021-10-11
Filing date: 2021-10-11
Publication date: 2023-05-12
Anticipated expiration: 2041-10-11
Also published as: CN113755510A

Abstract

The invention discloses a gene GmFtsH25 for encoding soybean FtsH metalloprotease and application thereof. The coding region (CDS) nucleotide sequence of the GmFtsH25 is shown as SEQ ID NO.1, the amino acid sequence of the coding protein is shown as SEQ ID NO.2, and the transgenic plant with the over-expression GmFtsH25 is created by using a soybean cotyledonary node transformation method mediated by agrobacterium tumefaciens. The invention discloses application of a gene GmFtsH25 for encoding soybean FtsH metalloprotease, and the gene is found to be capable of remarkably improving net photosynthetic rate of a transgenic plant by over-expression compared with a control plant. Therefore, the gene disclosed by the invention can be used as a target gene to be introduced into plants, and the photosynthesis capacity of transgenic plants is regulated, so that the gene has important significance in cultivating new soybean varieties with high light efficiency.

Description

Encoding soybean FtsH metalloprotease gene GmFtsH25 and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and relates to a gene GmFtsH25 for encoding soybean FtsH metalloprotease and application thereof.

Background

Photosynthesis is called "the most important chemical reaction on earth", and uses solar energy and inorganic CO ₂ And H is ₂ The organic matters formed by O not only provide necessary energy for the vital activities of plants, but also are almost all forms of lifeMicroorganisms, plants, animals, and humans) depend on the premise of survival, development, and prosperity. For plants, about 90% -95% of the dry matter is derived from photosynthetic products, and it is an important approach to increase crop yield by increasing the light energy utilization efficiency of crops, and many studies have been proved to be viable at present: successful inhibition of the photorespiration of C3 crops, such as by artificial design and modification of plant photorespiration processes, results in increased yield of C3 crops (South et al, 2019); in addition, there are also control of stomatal switches using optogenetics to increase biomass by a factor of 2.2 (Papanatsiou et al, 2019); the novel path for synthesizing the nuclear source D1 protein is created manually, so that the yield increase of the rice reaches 8.1% -21.0% (Chen et al 2020). As C3 crops, the low photosynthetic efficiency of the soybean is an important intrinsic factor for limiting the increase of the yield of the soybean, so that genetic engineering means are utilized to modify photosynthetic related regulating genes and analyze biological functions of the soybean, and theoretical basis is provided for revealing the mechanism of the efficient absorption, transmission and conversion processes of light energy and improving the utilization efficiency of the soybean light energy and further improving the yield of the soybean.

FtsH is a membrane-associated ATP-dependent Zn metalloprotease complex having an AAA domain and a Zn-containing sequence ²⁺ Bound H-E-x-x-H motif (Ito and Akiyama, 2005). Terrestrial plants have multiple FtsH orthologs. 12 FtsH homologs were found in arabidopsis, of which 9 were located in chloroplasts and 3 were located in mitochondria (Sakamoto et al, 2003). Mutations in VAR1 (AtFtsH 5) or VAR2 (AtFtsH 2) lead to typical leaf mottle phenotypes and sensitivity to light inhibition, and AtFtsH8 and AtFtsH1 have been found to act as dimers with AtFtsH2 and AtFtsH5, respectively, together with participation in photodamage degradation of the PSII reaction center D1 protein (Sakamoto et al, 2003). In addition, it has been reported that seeds from which AtFtsH12 mutants were knocked out fail to develop normally, and that a phenomenon of arrest of development occurs early (Meinke et al, 2008). It can be seen that FtsH proteases regulate plant growth in a number of ways, and that it is unexpected what biological function a particular FtsH protease has. At present, few researches on the proteins in soybeans are reported, so that the FtsH gene is researched in soybeans to analyze that the FtsH gene is regulatedThe biological function of soybean in growth and development has important significance.

Disclosure of Invention

The invention aims to disclose an application of a gene GmFtsH25 for encoding soybean FtsH metalloprotease in regulating soybean photosynthesis, the gene can be used as a target gene to be stably transformed into soybean, and the net photosynthetic rate of the soybean is improved by over-expression of GmFtsH25 so as to provide a theoretical basis for soybean high-light-efficiency breeding.

The aim of the invention is realized by the following technical scheme:

the soybean FtsH metalloprotease gene GmFtsH25 has a CDS nucleotide sequence of: SEQ ID NO.1.

The soybean FtsH metalloprotease gene GmFtsH25 has the amino acid sequence: SEQ ID NO.2.

The recombinant expression vector for encoding the soybean FtsH metalloprotease gene GmFtsH25 is overexpressed.

As a preferred embodiment of the present invention, the recombinant expression vector is obtained by inserting the soybean FtsH metalloprotease gene GmFtsH25 into the overexpression vector pBA 002.

The application of the soybean FtsH metalloprotease gene GmFtsH25 in regulating the photosynthesis of soybean is that the recombinant plasmid containing the FtsH metalloprotease gene GmFtsH25 is introduced into target plant soybean by using an agrobacterium tumefaciens-mediated soybean cotyledonary node transformation method to obtain a transgenic soybean plant with the over-expressed GmFtsH25, and the photosynthetic efficiency of the transgenic plant is obviously improved compared with that of a control plant.

The recombinant expression vector disclosed by the invention is applied to improving the net photosynthetic rate of transgenic soybean offspring.

The invention has the beneficial effects that:

the invention finds that the photosynthesis efficiency can be regulated by over-expressing the GmFtsH25 gene in soybean. Measurement of photosynthetic efficiency of over-expressed transgenic plants and control plants revealed that the expression level was 1000. Mu. Mol m ^-2 s ^-1 The net photosynthetic rate of transgenic plants was significantly higher than the control under optical density conditions, while transgenic plants were foundThe phenotype of the plants in the seedling stage is also superior to that of the control plants. Therefore, the achievement of the invention can be applied to the expression of the GmFtsH25 gene regulated by biotechnology, and has important significance for revealing the efficient transfer and transformation of soybean light energy and the efficient breeding of soybean light energy.

The GmFtsH25 plant expression vector can be used for transforming plant cells or tissues by using Ti plasmids, ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electric conduction, agrobacterium mediation and other conventional biological methods, and regenerating plants by using plant tissue culture technology. The transformed host can be monocotyledonous plants such as rice, wheat, corn, etc., or dicotyledonous plants such as soybean, cucumber, tomato, alfalfa, etc.

Drawings

FIG. 1pBA002-GmFtsH25 overexpression vector map. And (3) injection: gmFtsH25 CDS: the coding region of the target gene GmFtsH 25; caMV 35S promoter: a cauliflower mosaic virus (CaMV) 35S promoter; NOS terminator: nopaline synthase gene terminators; NOS master: a nopaline synthase gene promoter; bar: a phosphinothricin acetyltransferase gene; e9terminator: a ribulose-1, 5 bisphosphate carboxylase E9 gene small subunit 3' end sequence;

SacI: restriction enzyme cutting site of SacI; mluI: mluI restriction enzyme cleavage site. LB T-DNA repeat: T-DNA left border; RB T-DNA repeat: T-DNA right border; primer F: an upstream primer for amplifying a target gene GmFtsH 25; primer R: a downstream primer of the target gene GmFtsH25 is amplified. The vector was used for subsequent transgene transformation.

FIG. 2 relative expression levels of GmFtsH25 in Jack tissues. The expression pattern of GmFtsH25 in different tissues of plants was verified using fluorescent quantitative qRT-PCR with cDNA of the root, stem, leaf, flower, 15 and 45 day pod and seed of Jack soybean material as template.

Fig. 3T ₁ And (5) verifying the result of PCR of the generation positive transgenic soybean plants. And (3) injection: wherein the upper gel discharging diagram is Bar detection strip, and the lower gel discharging diagram is target fragment specific detection strip. M1 represents a 2000bp Marker, band composition (from bottom to top): 100bp,250bp,500bp,750bp,1000bp,2000bp; m2 represents 5000bp Marker, band composition (from bottom to top): 100bp,250bp,500bp,750bp,1000bp,2000bp,3000bp,5000bp; lane 1 is positive plasmid, lane 2 is negative control, lane 3 is blank control, lanes 4-7 are line 7, lanes 8-11 are line 10, and lanes 12-17 are line 12..

FIG. 4 phenotype map of GmFtsH25 over-expressed transgenic plants. And (3) injection: jack stands for the receptor control material, OE-7, OE-10 and OE-12 respectively for three GmFtsH25 overexpressing transgenic plants.

FIG. 5 net photosynthetic rate of GmFtsH25 overexpressing transgenic plants. And (3) injection: jack stands for the receptor control material, OE-7, OE-10 and OE-12 respectively for three GmFtsH25 overexpressing transgenic plants.

Detailed Description

The terms used in the present invention generally have meanings commonly understood by those of ordinary skill in the art unless otherwise indicated.

The present invention will be described in further detail with reference to the following specific preparation examples and application examples. It should be understood that these examples are intended to illustrate the invention and are not intended to limit the scope of the invention in any way.

In the following examples, various processes and methods, which are not described in detail, are conventional methods well known in the art. The primers used are all indicated at the first occurrence, and the same primers used thereafter are all identical to the first indicated ones.

The methods used in the examples described below are conventional methods unless otherwise specified.

Example 1 tissue expression of the Soybean GmFtsH25 Gene in Jack Material

(1) Designing a primer, extracting RNA, reversing cDNA:

total RNA from each tissue of soybean Jack was extracted with a plant total RNA extraction kit (DP 419, tiangen) and the integrity of the RNA was checked by 1% agarose gel electrophoresis. cDNA Synthesis reference

1st Strand cDNA Synthesis Kit kitThe operation is described in (2). The primers were designed as follows:

SEQ ID NO.3: gmFtsH25 qPCR primer F '5-CGACTGTGTTTGTGAGTGTGC-3'

SEQ ID NO.4: gmFtsH25 qPCR primer R '5-ACTCTGATTCCTGCAACCTACTAC-3'

SEQ ID NO.5: tubulin qPCR primer F '5-GGAGTTCACAGAGGCAGAG-3'

SEQ ID NO.6: tubulin qPCR primer R '5-CACTTACGCATCACATAGCA-3'

(2) The real-time fluorescence quantitative PCR comprises the following specific steps:

step one: each tissue cDNA sample obtained in the above (1) was diluted 10-fold for qPCR reaction, and a reaction solution (20. Mu.l system) was prepared according to ChamQ Universal SYBR qPCR Master Mix: 10. Mu.l 2X ChamQ Universal SYBR qPCR Master Mix, primers F and R each 0.4. Mu.l, 5. Mu.l cDNA template, 4.2. Mu.l ddH ₂ The O was replenished to a volume of 20. Mu.l.

Step two: reaction in Bio-RAD CFX96 ^TM The reaction is carried out on a Real-Time System fluorescence quantitative instrument, and the reaction program is set as follows: pre-denaturation at 95 ℃ for 30sec; then 95 ℃ for 10sec and 60 ℃ for 30sec, and 40 cycles are carried out; then, dissolution profile collection was performed at 95℃for 15sec,60℃for 60sec, and 95℃for 15 sec.

Step three: the data was analyzed using EXCEL. The relative expression level of the candidate gene in soybean leaves was analyzed by comparing CT (Cycle Threshold) values, and the expression level was expressed relative to 2 according to the formula ^-△△CT (△△CT＝(CT _Target -CT _Tubulin ) _genotype -(CT _Target -CT _Tubulin ) _calibrator ) The relative expression level of GmFtsH25 in each tissue was calculated. The results show that: gmFtsH25 is expressed in roots, stems, leaves, flowers, pods and seeds, wherein the expression level in leaves is the highest.

Example 2 cloning of the Soybean GmFtsH25 Gene and construction of plant overexpression vector

(1) CDS fragments were amplified by high-fidelity polymerase chain reaction using the cDNA of soybean Jack leaves obtained in example 1 as a template. The primers were designed as follows:

SEQ ID NO.7: primer F'5-GGATCTTCCAGAGAT TACCCACGTTCCAAGTTCCG-3’

SEQ ID NO.8: primer R'5-CTGCCGTTCGACGAT GCTTCGGAGCAATGGTGTCA-3’

(2) The PCR amplification comprises the following specific steps:

step one: the PCR reaction solution (50. Mu.l system) was prepared in the following order: 25 μl 2× Planta Max Buffer, 2 μl each of primers F and R, 1 μl dNTP Mix,2 μl leaf cDNA template, 1 μl Super-Fidelity DNA Polymerase,17 μl ddH ₂ The O was replenished to a volume of 50. Mu.l.

Step two: the reaction was performed on a BIO-RAD PTC-200 type PCR apparatus, and the reaction procedure was set as follows: pre-denaturation at 95℃for 3min; then, the temperature is 95 ℃ for 15sec, the temperature is 58 ℃ for 15sec, the temperature is 72 ℃ for 2min for 15sec, and the total time is 35 cycles; then thoroughly extending for 5min at 72 ℃; preserving at 4 ℃.

Step three: the PCR product is recovered and then connected to pUC19-T vector (Novain), transformed E.coli TOP10, ampicillin (Amp) screening, shaking and sequencing through recombination, and the sequence is shown as SEQ ID NO.1.

(3) Construction of plant overexpression vectors

Designing homologous recombination joint primer SEQ ID NO.9: OE-F '5-GGGCCCAGGCCTACGCGTATGTTACCCTTAGAGCATCA-3' and SEQ ID NO.10: OE-R '5-TCGGGGAAATTCGAGCTCTCAGGAACCTGTCTGCGATG-3', using the T vector containing GmFtsH25 gene fragment obtained in the above (2) as a template, amplifying GmFtsH25CDS fragment with recombinant linker, introducing GmFtsH25 gene into an expression vector pBA002 by homologous recombination technology to construct a pBA002-GmFtsH25 over-expression vector (shown in figure 1), and transferring the vector into Agrobacterium tumefaciens strain EHA105 for subsequent transgenic transformation of soybean by freeze thawing. Meanwhile, the expression vector also contains a marker gene Bar, which is a gene separated from streptomyces and used for encoding PPT acetyl transferase (phosphinothricin acetyl-transferase), and the Bar gene has resistance to herbicide glufosinate-ammonium, can be used for positive identification of transgenic plants, and is one of the marker genes with application safety.

Example 3 cultivation of transgenic soybeans overexpressing GmFtsH25 Gene

The method for transforming soybean cotyledonary node mediated by agrobacterium tumefaciens is used for transforming the pBA002-GmFtsH25 over-expression vector into a receptor material Jack, and the specific method is as follows:

(1) Selecting mature, full, no disease spot and no hard and solid clean seeds, arranging the seeds in a culture dish with a single layer of 90 x 15mm, sterilizing the surface of the soybean seeds by a chlorine dry method, and sterilizing the soybean seeds in a fume hood for 6-7 hours. The culture dish is covered and transferred to a sterile super clean bench, the cover of the culture dish is opened, and residual chlorine is removed by blowing with strong wind for 25-40 minutes. Seeding the sterilized seed on germination medium (SG 4) with umbilicus downward, stacking culture dishes, wrapping with preservative film, and culturing in the dark at 24deg.C for 16-24 hr.

(2) Agrobacterium is prepared that infects the cotyledonary node of soybean. 100. Mu.l of the Agrobacterium solution of the pBA002-GmFtsH25 overexpression vector obtained in example 2 was pipetted into 5ml of the YEB liquid medium with the addition of the antibiotic (1/1000), shake-cultured at 28℃and 220rpm for 24-36 hours, and then 0.2-1ml of the saturated bacterial solution was pipetted into 250ml of the YEB liquid medium with the addition of the antibiotic to expand the culture to OD ₆₀₀ =0.85-0.9. The bacterial solutions were packed into two 50ml sterile centrifuge tubes, centrifuged (5000 rpm,10min,25 ℃), colonies were collected, gently blown with 25-50ml liquid co-culture medium (LCCM), and the pellet was resuspended for further use.

(3) And (5) culturing plant tissues. Firstly, cutting a wound at cotyledonary nodes of the swelled soybean seeds by using a surgical knife to obtain explants, and then putting all the explants into prepared infectious microbe liquid, and carrying out shaking infection for 30 minutes at 120-130 rpm. The explants were then removed and placed on sterile absorbent paper to blot residual dye liquor, which was then placed on solid co-culture medium (CCM) with sterile absorbent paper, all in a super clean bench. Stacking the culture dishes, sealing with a preservative film, and culturing in an artificial intelligent incubator at 25 ℃ in the dark for 3-5 days. After sterilization, the explants were inserted onto a Shoot Induction Medium (SIM) with the addition of the screening agent glufosinate, and incubated at 25 ℃ for 14 days under light. And then cutting off residual cotyledons and transferring the cotyledons to a bud elongation culture medium (SEM), culturing for 2-4 weeks at 25 ℃ under light, timely replacing a new SEM culture medium according to actual conditions during the period, cutting off the elongated regenerated plants from roots in time, quickly inserting the elongated regenerated plants into a solid rooting culture medium (SM), taking out the rooting seedlings from the culture medium when the roots grow for about 2-3 cm, cleaning the residual culture medium of the roots, transferring the culture medium into soil, and culturing in a greenhouse.

(4) Positive identification of transgenic plants. After the regenerated plant grows a third new leaf, the upper, middle and lower three layers of leaves are cut by scissors and mixed, and DNA is extracted according to the instruction of the Shanghai Pudi biotechnology Co., ltd. Then, PCR amplification is carried out by using the DNA sample as a template and using a 2X Rapid Taq Master Mix (Northenzan) kit instruction, including the identification of the target gene GmFtsH25 and the screening gene Bar at the DNA level, and specific fragment amplification primers including the target gene GmFtsH25 are as follows: SEQ ID NO.11:35S-F '5-GACGCACAATCCCACTATCC-3'; SEQ ID NO.12: OE-R '5-TCGGGGAAATTCGAGCTC TCAGGAACCTGTCTGCGATG-3'. The screening gene Bar specific amplification primers are as follows: SEQ ID NO.13: bar-F '5-CGAGACAAGCACGGTCAACTT-3'; SEQ ID NO.14: bar-R '5-AAACCCACGTCATGCCAGTTC-3'. The band of interest was then detected by 1% agarose gel electrophoresis (see FIG. 3). If 2480bp target gene fragment and 360bp Bar gene fragment are amplified, the transgenic plant is positive. The single plant of the positive plant is harvested and planted until T ₂ And (5) identifying homozygous transgenic plants from the generation to obtain GmFtsH25 gene overexpression plants.

Example 4, determination of net photosynthetic Rate of transgenic plants overexpressing the GmFtsH25 Gene

Three stable transgenic plants overexpressing the GmFtsH25 gene were identified according to the plant transgenic method described in example 3 and designated OE-7, OE-10 and OE-12, respectively. To determine the growth phenotype and its photosynthetic associated traits of the overexpressed transgenic plants, acceptor material Jack and three overexpressed transgenic soybean materials were grown under greenhouse conditions and net photosynthetic rate was determined when the plants were grown to seedling stage (as in fig. 4), as follows: the net photosynthetic rate (Pn) of photosynthetic associated phenotypic trait data was determined using an LI-6400XT portable photosynthesis measurement system (LiCor Inc, lincoln, NE, USA). Selecting a growth promoting healthPhenotype determination of photosynthetic related traits by reverse trilobate of Kangma Soybean plant, and illumination intensity of 1000. Mu. Mol m ^-2 s ^-1 3 strains were determined per strain, 3 times per strain and averaged as independent phenotype values.

Analysis of variance was performed on the phenotypic data using the EXCEL table. The comparison result of the net photosynthetic rate of the transgenic plant and the control material shows that: the net photosynthetic rates of all three GmFtsH25 gene overexpressing transgenic plants were significantly increased compared to the control material (fig. 5). Therefore, gmFtsH25 plays an important role in regulating the photosynthesis efficiency of soybean.

Sequence listing

<110> Nanjing agricultural university

<120> a gene GmFtsH25 encoding soybean FtsH metalloprotease and use thereof

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gctggtacaa agtcatctga acaaggagaa acagtcactt ttgctgatat tgctggtgtt 960

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cagttgctca ctgagatgga tgggtttgat agcaattctt cagtgattgt tcttggagct 1380

actaatcggt cagatgtctt ggatcctgca cttcgccggc caggaagatt tgatcgtgta 1440

gttatggtag aagcacctga tagaattgga agagaagcca tcctaaaagt tcatgtttcc 1500

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ggttttactg gagcagatct tgcaaaccta gtaaatgagg ctgctttact ggctgggaga 1620

caaaataaag ttgtggtgga gaaacttgat ttcatccaag ctgtagaaag atctatagct 1680

ggcatagaga agaagactgc caagttgcga ggaagtgaga aggctgtagt tgcacgacat 1740

gaggctggtc atgctgtagt aggtactgca gttgcaaagc ttcttcctgg acagccacgt 1800

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Leu Leu Pro Ser Gln Thr Gly Ser

785 790

<210> 3

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ggatcttcca gagattaccc acgttccaag ttccg 35

<210> 4

<211> 35

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

ctgccgttcg acgatgcttc ggagcaatgg tgtca 35

<210> 5

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cgactgtgtt tgtgagtgtg c 21

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

actctgattc ctgcaaccta ctac 24

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

ggagttcaca gaggcagag 19

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

cacttacgca tcacatagca 20

<210> 9

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

gggcccaggc ctacgcgtat gttaccctta gagcatca 38

<210> 10

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

tcggggaaat tcgagctctc aggaacctgt ctgcgatg 38

<210> 11

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

gacgcacaat cccactatcc 20

<210> 12

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

tcggggaaat tcgagctctc aggaacctgt ctgcgatg 38

<210> 13

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

cgagacaagc acggtcaact t 21

<210> 14

<211> 21

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

aaacccacgt catgccagtt c 21

Claims

Encoding soybean FtsH metalloprotease gene shown in SEQ ID NO.1GmFtsH25Use in increasing the net photosynthetic rate of transgenic soybean progeny.
2. Overexpression of the Gene encoding soybean FtsH metalloprotease shown in SEQ ID No.1GmFtsH25The use of a recombinant expression vector of (2) in increasing the net photosynthetic rate of progeny of a transgenic soybean.
3. The use according to claim 2, characterized in that said overexpression of the gene coding for soybean FtsH metalloprotease as shown in SEQ ID No.1GmFtsH25The recombinant expression vector of (a) encodes the soybean FtsH metalloprotease geneGmFtsH25Is inserted into an overexpression vector pBA 002.