CN108220302B - Soybean GmCYS20 gene and application thereof - Google Patents
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Abstract
The invention discloses a cysteine protease inhibitor GmCYS20 gene participating in regulating symbiotic nodulation of leguminous plants, which is 291bp in length, is positioned at 8475315-8478532 site of a chromosome 20 of a soybean genome and contains 2 exons and 1 intron. And discloses the application of the gene in regulating and controlling the number of root nodules of leguminous plants (Lotus japonicus, Astragalus sinicus, alfalfa, soybean, peanut and the like). The invention separates a cysteine protease inhibitor gene GmCYS20 from soybean root tips by using a reverse transcription PCR technology, and confirms that the gene is a cysteine protease inhibitor gene; meanwhile, the gene is proved to participate in and positively regulate the nodulation of leguminous plants by an overexpression genetic transformation method. The gene GmCYS20 of the soybean cysteine protease inhibitor is proved to be regulating symbiotic nodulation of leguminous plants for the first time, and the gene has an application prospect in the symbiotic nitrogen fixation of the leguminous plants.
Description
Technical Field
The invention relates to the technical field of plant genetic engineering, in particular to a cysteine protease inhibitor GmCYS20 gene participating in regulating symbiotic nodulation of leguminous plants and application thereof.
Background
Cystatins are widely present in the body, and are proteins that specifically bind to cysteine proteases and inhibit their proteolytic activity. Cystatin proteins are first reported to participate in the regulation of plant development processes, including the strict control of protein degradation in plants, and the participation in physiological processes such as the deposition, regulation and transportation of storage proteins. In addition, cystatin protein can delay the leaf senescence process by regulating the activity of cysteine protease. In recent years, studies have found that the cystatin gene plays an important role in improving the resistance of plants to abiotic stress. The rice cystatin gene OC-I influences the stress resistance of plants by regulating the activity of cysteine protease. The arabidopsis AtCYS4 and AtCYS5 genes are subjected to high-temperature induced expression, and the over-expression of the genes enhances the high-temperature resistance of arabidopsis. The apple MpCYS2, MpCYS4 and MpCYS5 genes respectively improve the resistance of the transgenic arabidopsis thaliana to drought, ABA and salt stress. The wild soybean cystatin protein GsCPI14 participates in alkali stress reaction by interacting with calcium-binding receptor protein kinase GsCBRLK. In addition, the growth inhibition of plant cystatin protein on harmful insects such as Lepidoptera and Coleoptera, and the participation of cystatin in the defense process of plants against pathogenic bacteria invasion have been studied more deeply.
In conclusion, a large number of evidences indicate that the plant cystatin gene plays an important role in regulating plant development, improving abiotic stress and pest resistance of plants and the like. But few reports about the function research of the genes in the symbiotic nodulation process of leguminous plants are reported. The invention separates and identifies 1 soybean cystatin gene GmCYS20 which is up-regulated and expressed by rhizobium induction, and after the gene is over-expressed in the lotus japonicus of leguminous plants, the nodulation number is obviously increased, thereby obviously enhancing the nitrogen fixation capacity of leguminous plants, therefore, the gene has important application value in agriculture and ecology.
Disclosure of Invention
The invention aims to provide a cysteine protease inhibitor GmCYS20 gene participating in regulating symbiotic nodulation of leguminous plants, the sequence of the gene is a nucleotide sequence shown as SEQ ID NO. 1, the length of the gene is 291bp, the gene is positioned at the 8475315-8478532 site of a chromosome 20 of a soybean genome, and the gene contains 2 exons and 1 intron.
The sequence of the protein coded by the cysteine protease inhibitor GmCYS20 gene is an amino acid sequence shown in SEQ ID NO. 2.
Still another object of the present invention is to provide an application of the cystatin GmCYS20 gene involved in the regulation and control of symbiotic nodulation of leguminous plants in the regulation and control of the number of root nodules of leguminous plants (Lotus japonicus, Astragalus sinicus, alfalfa, soybean, peanut, etc.).
The purpose of the invention is realized by the following technical scheme:
a preparation method of a cysteine protease inhibitor GmCYS20 gene involved in regulating symbiotic nodulation of leguminous plants comprises the following steps: after the surface of the cultivated soybean W82 seed is sterilized, the hilum is laid on the sterile wetting filter paper downwards, and dark culture is carried out at 28 ℃ until germination. Collecting fresh soybean root tip tissue, and quickly freezing by liquid nitrogen. Total RNA from soybean root tip tissue was extracted according to the instructions of RNA extraction kit (Invitrogen, USA). First strand cDNA was obtained according to the instructions of the reverse transcription kit of TIANGEN. Primers F-GmCYS20 and R-GmCYS20 are designed according to the sequence of soybean GmCYS20 gene (Glyma.20g045500) published by https:// www.soybase.org/search/website, and target genes of GmCYS20 are amplified by PCR. And recovering the target fragment, connecting the target fragment with a T vector, and sending the T vector to Nanjing Kingsry company for sequencing to obtain the full length of the gene. The length of the cysteine protease inhibitor gene GmCYS20 is 291bp, is positioned at 8475315-8478532 site of the soybean genome No. 20 chromosome, and contains 2 exons and 1 intron. Sequence analysis shows that the CDS region of the GmCYS20 gene codes for 97 amino acid residues, the isoelectric Point (PI) of the protein is 5.83, the N terminal has no signal peptide, and the three-dimensional structure comprises 2 alpha helices and 6 beta folds. GmCYS20 belongs to the III cystatin protein and contains 4 common conserved domains of GG, LARFAVE, QVVGG and SW. The GmCYS20 gene and any DNA of interest or homologous DNA thereof can be obtained by amplifying genome, mRNA and cDNA by using PCR (polymerase chain reaction) technology.
The application of cysteine protease inhibitor gene GmCYS20 participating in symbiotic nodulation of leguminous plants in Lotus japonicus, Astragalus sinicus, alfalfa, soybean and peanut is realized by genetic transformation. The invention constructs a plant over-expression vector of a GmCYS20 gene, transfers the plant over-expression vector into agrobacterium LBA1334 by a freeze-thaw method, transfers the plant over-expression vector into Lotus corniculatus of leguminous plants by agrobacterium-mediated hairy root transformation, obtains a positive plant by GUS identification, plants the positive plant in a mixed pot culture of sand and vermiculite 1:1, and inoculates rhizobium. The result shows that the nodulation number of the Lotus corniculatus of leguminous plants is obviously increased after overexpression, which is the process of confirming that the cystatin gene GmCYS20 positively regulates and controls symbiotic nodulation in the Lotus corniculatus for the first time.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention separates a cysteine protease inhibitor gene GmCYS20 from soybean root tips by using a reverse transcription PCR technology, and confirms that the gene is a cysteine protease inhibitor gene by methods of homologous protein multiple sequence comparison, conservative domain prediction and the like; meanwhile, the gene is proved to participate in and positively regulate the nodulation of leguminous plants by an overexpression genetic transformation method. This is the first demonstration that the soybean cystatin gene GmCYS20 is regulating symbiotic nodulation of leguminous plants.
2. Leguminous plants increase the fertility of soil by symbiotic nitrogen fixation of root nodules, but naturally, the number of root nodules in the roots of leguminous plants is limited, so that the fertility of soil is increased by increasing the number of root nodules and further increasing the nitrogen fixation capacity of leguminous plants, the use of chemical fertilizers and the like is effectively reduced, and the leguminous plants have great significance in agriculture and ecology. The invention proves that the gene positively regulates nodulation by an overexpression technology, so that the gene can be overexpressed in leguminous plants by a transgenic means, the number of nodules can be greatly increased, and the nitrogen fixation capability is enhanced, thereby having a certain application prospect in the research of nitrogen fixation mechanisms of leguminous crops and agricultural production.
Drawings
FIG. 1 is an analysis diagram of clone 1A and encoded amino acid sequence 1B of GmCYS20 gene.
FIG. 2 is a schematic diagram of alignment of a protein sequence predicted by GmCYS20 gene and a GmCYS20 homologous protein sequence by using DNMAN software, wherein:
Sequence source:Glycine soja，accession KHN35444.1
Sequence source:Vigna radiata，accession XP_014516943.1
Sequence source:Cajanus cajan，accession XP_020236689.1
Sequence source:Vigna angularis，accession XP_017411678.1
Sequence source:Cicer arietinum，accession XP_012574834.1
Sequence source:Medicago truncatula，accession XP_003599710.2
Sequence source:Lupinus angustifolius，accession XP_019419326.1
Sequence source:Medicago sativa，accession AAZ98791.1
Sequence source:Lotus japonicus，accession AFK41117.10
Sequence source:Arachis hypogaea，accession CBX19819.1
Sequence source:Arachis duranensis accession XP_020995318.1
Sequence source:Arachis ipaensis，Ai:accession XP_016197069.1
FIG. 3 is a schematic diagram of a transgenic plant with overexpression of GmCYS20 gene promoting nodulation of Lotus corniculatus, wherein 3A is the nodulation of the transgenic plant with overexpression compared with a control normal plant; 3B is the comparison of the average number of nodules per plant of the overexpression transgenic plant and the control normal plant.
FIG. 4 is a schematic diagram of detecting the overexpression efficiency of the GmCYS20 gene and the expression level of the symbiotic marker gene in the overexpressed transgenic plant by using Real Time-PCR.
Detailed Description
To further illustrate the technical means adopted by the present invention and the effects thereof, the following detailed description is given with reference to the accompanying drawings and preferred embodiments of the present invention.
Example 1
A preparation method of a cysteine protease inhibitor gene GmCYS20 involved in symbiotic nodulation of leguminous plants comprises the following steps:
after the surface of the W82 soybean seed is sterilized, the hilum is laid down on a sterile wetting filter paper, and dark culture is carried out at 28 ℃ until germination. Collecting fresh soybean root tip tissue, and quickly freezing by liquid nitrogen. Total RNA from soybean root tip tissue was extracted according to the instructions of RNA extraction kit (Invitrogen, USA). First strand cDNA was obtained according to the instructions of the reverse transcription kit of TIANGEN. Primers F-GmCYS20 and R-GmCYS20 (Table 1) are designed according to the sequence of soybean GmCYS20 gene (Glyma.20g045500) published by https:// www.soybase.org/search/website, and a target gene of GmCYS20 (shown in figure 1) is amplified by PCR. And recovering the target fragment, connecting the target fragment with a T vector, and sending the T vector to Nanjing Kingsry company for sequencing verification.
TABLE 1 primers used in the present invention
Note: the underlined region is the sequence of the cleavage site
Analyzing the amino acid sequence encoded by the gene, comprising the steps of: the Blastp tool of NCBI website was used to search for homologous proteins of soybean (Glycine max) GmCYS20 protein (NP _001239817.1), including wild soybean (Glycine soja, KHN35444.1), mung bean (Vigna radiata, XP _014516943.1), pigeon pea (Cajanus cajan, XP _020236689.1), red bean (Vigna angularis, XP _017411678.1), chickpea (citrus arietinum, XP _012574834.1), tribulus lucidus (medical truncata, XP _003599710.2), Lupinus angustifolia (Lupinus angustifolius, XP _019419326.1), alfalfa (medical go sativa, AAZ98791.1), Lotus japonicus (Lotus japonicum, AFK41117.10), cultivated peanut (arabis pogoaea, CBX19819.1), peanut (arabis duensis, XP _020995318.1) and peanut (aristicta sp, 016197069.1) of different species of leguminous. The DNAMAN software was used for multiple sequence alignment analysis of homologous proteins (shown in figure 2).
Example 2
The application of GmCYS20 gene participating in regulating symbiotic nodulation of leguminous plants in regulating the number of root nodules of leguminous plants (Lotus japonicus, Astragalus sinicus, alfalfa, soybean, peanut and the like) comprises the following steps:
in order to research the biological function of GmCYS20 in the Lotus japonicus of leguminous plants, the biological function of GmCYS20 is explored by an overexpression method (shown in figure 3). Mainly relates to the construction and genetic transformation of an overexpression vector, and comprises the following specific implementation steps:
construction of the overexpression fusion vector: primers F-OX and R-OX (shown in Table 1) for introducing enzyme cutting sites EcoR I and Sma I are designed, a target gene with correct sequencing is inserted into a plant expression vector p1302G (Gus gene is used as a screening marker gene), a p1302G-GmCYS20 recombinant plasmid is constructed, and the recombinant plasmid is transferred into agrobacterium rhizogenes A. rhizogenes LBA1334 (the strain is a strain published internationally and is from professor Hongzhance of America) by a freeze-thaw method for hairy root transformation of the Lotus corniculatus. The transformation method of the hairy roots of the Lotus japonicus refers to Lotus japonica Handbook.
The specific implementation steps are as follows:
(1) plant material cultivation: lotus japonicus MG20 seeds were germinated 5 days earlier. Sanding the seeds (lighter than the ordinary germinated seeds) with sand paper, freezing in liquid nitrogen for 1 minute, soaking in 75% (by mass/volume) ethanol for 1 minute, soaking in 5% (effective chlorine concentration) NaClO for 15 minutes, washing with sterile water for 5-6 times, and removing residual NaClO. After the surface of the seeds is disinfected, a little sterile water is left to just submerge the seeds, the seeds are vernalized for 1 day at 4 ℃ under the dark condition, then the seeds are transferred to a solid culture medium without sucrose for MS and cultured for 2 days at 23 ℃ in the dark, and then the seeds are placed in an illumination incubator (16h illumination and 8h darkness) for continuous culture for 2 days at 23 ℃ for standby application.
(2) Strain and plasmid preparation: the strain is A.rhizogenes LBA1334 (spectinomycin resistance), and plasmid containing target gene is introduced into Agrobacterium A.rhizogenes LBA1334 by electrotransformation, and the strain is preserved at-70 deg.C after identification. The preserved strain is taken out for plating (containing plasmid resistance plate) for activation 2 days in advance.
(3) Infection transformation: the first day: in the afternoon, a single colony was selected and inoculated into 5ml of LB medium (containing plasmid resistance), and shake-cultured at 28-30 ℃ for 16-24 hours. The next day: and (3) carrying out amplification culture on the small amount of bacteria inoculated on the previous day, wherein the mass ratio of 1: inoculating at a ratio of 100, and culturing at 28-30 deg.C for about 8 hr under shaking. The mass-cultured bacterial solution was centrifuged at 6000 rpm for 10 minutes, and the cells were collected and resuspended in sterile water so that the OD600 became about 0.8. Cutting off hypocotyl base of seedling, soaking the part with cotyledon with the re-suspended bacteria liquid for 30min, taking out explant, drying with filter paper, and co-culturing in MS culture medium without sucrose.
(4) Co-culturing: co-culturing in dark for 3-5 days. The explants were then transferred to HRE and 300. mu.g mL-1cefotaxime medium for another 10 days during which time hair roots grew out of the hypocotyl incisions.
(5) Positive transgenic root identification: a root segment about 0.5cm long at the root tip is cut and put into GUS dye solution, dark culture is carried out at 37 ℃ overnight, and the blue root is a positive transgenic root.
(6) Hardening and transplanting seedlings: after identification, non-transformed roots were cut off, and the seedlings were put in a dish filled with water (without being covered) to be acclimatized for 1 day, and then transplanted into a flowerpot to be cultured in a light incubator (16h light, 8h dark) at 23 ℃. The base material mixed by vermiculite (vermiculite) and sand (sand) according to the proportion of 1:1 is added in the flowerpot in advance. The nitrogen-free nutrient solution is poured once every 3 days.
(7) And (3) nodulation statistics: rhizobium baenii MAFF303099 (from the laboratory of professor Wu national river, south plant Garden of China, see the genetic resource table) of Chinese academy of sciences is activated on a YMA plate, inoculated into a liquid TY culture medium, and subjected to shaking culture at 28 ℃ for 24-36 h; transferring the cultured rhizobia into a sterile centrifuge tube, centrifuging at 4 ℃ at 7000r/min for 5min, and collecting thalli; washing with sterile Fahraeus nitrogen-free nutrient solution and centrifuging for 2 times; adding nitrogen-free nutrient solution to re-suspend the thallus, and inoculating the thallus to the roots of the crowtoe seedlings. Irrigating nitrogen-free nutrient solution 1 time every day. After 4 weeks of inoculation, statistical photographs were taken of the plant nodulation phenotype. According to the number of nodules of the single plant and the sample size (n), the average number of nodules of the single plant is calculated, the test is repeated twice, and the average value is taken.
(8) And (3) gene expression detection: the transcription level of each gene in the positive hairy roots of the complex plants is detected by using fluorescent quantitative primers of GmCYS20 and nodulation related genes NIN, ENOD40-1 and ENOD40-2 in the table 1, and the Lotus corniculatus UBI gene is used as an internal reference. And (3) carrying out fluorescence quantitative PCR detection according to an operation instruction of a PrimeScript RT reagent Kit of Takara company, and calculating a result according to a relative quantitative method (2-delta Ct). The experiment was repeated 3 times and the experimental data was analyzed using Excel 2007 and SPSS 13.0 software and plotted graphically (shown in figure 4).
The formula of the culture medium and the reagent in the steps is as follows:
1) MS culture medium: 4.3g of MS basal salt mix (Sigma), 0.103g of MS vitamin powder, 0.7-0.8% (mass to volume) of agar powder and ddH2And O is metered to 1000mL and is adjusted to pH 5.8.
2) YMA medium: 10.0g mannitol (or sucrose), 0.4g Yeast Extract, 0.5g K2HPO4，0.2g MgSO4·7H2O，0.1g CaCl2·6H2O, 0.1g NaCl, 4mL Rh microelement liquid, ddH2And (4) metering the volume of O to 1000mL, adjusting the pH value to 6.8-7.0, and sterilizing at 115 ℃ for 20 min.
3) Rh trace element liquid: 5.0g H3BO3，5.0g Na2MoO4，ddH2And O is metered to 1000 mL.
4) Fahraeus nitrogen-free nutrient solution: 0.10g of CaCl2·2H2O，0.12g MgSO4·7H2O，0.10g KH2PO4，0.15g Na2HPO4·12H2O, 1mL of Gibson's microelement liquid, 5mg of ferric citrate, ddH2And O is metered to 1000 mL.
5) Gibson trace element liquid: 2.86g H3BO3，0.22g ZnSO4·7H2O，2.03g MnSO4·4H2O，0.13g Na2MoO4·2H2O，0.08g CuSO4·5H2O，ddH2And O is metered to 1000 mL.
6) GUS dye solution: 100mM sodium phosphate buffer, pH 7.0, 0.1% Triton X-100, 0.1% N-laurylsarcosine, 10mM Na2EDTA, 1mM potassium ferricyanide (K)3Fe(CN)6) 1mM potassium ferrocyanide (K)4Fe(CN)6) And 0.5mg/mL X-GluC.
7) HRE medium: 20X SH-A salt, 20X UM-C vitamin, 10g sucrose, 3mL 1M MES, 0.7-0.8% ((S))Mass to volume ratio) agar powder, ddH2And O is metered to 1000mL and is adjusted to pH 5.8. Sterilizing at 110 deg.C for 30 min.
20X SH-A salt:
dissolving FeSO in 100mL of sterile water respectively4·7H2Dissolving O and NaEDTA and other salts in 700mL of sterile water, mixing to a constant volume of 1000mL, and subpackaging into 50mL tubes for preservation at-20 ℃.
20X UM-C vitamin:
ddH2and O is metered to 1000mL, and each tube is subpackaged with 50mL and stored at-20 ℃.
MES (2- [ N-Morpholino ] ethane-sulfonic acid),1M stock solution:
weigh 29.28g MES (Sigma) in ddH2And (4) metering the volume of O to 150mL, adjusting the volume to pH 5.8, subpackaging into 6mL tubes, and storing at-20 ℃.
Example 3
Example 3 is substantially the same as example 2, except that,
the specific implementation of the plant material culture in the step (1) is that alfalfa is germinated 5 days in advance respectively; the Rhizobium in step (7) was inoculated with Sinorhizobium meliloti 1021 (a strain that has been published internationally, Juan et al, company of development and Stress-Induced Nodule Senesence in medical procedure apparatus, plant physiology, 152:1574-, (2010)).
Example 4
Example 4 is substantially the same as example 2, except that,
specifically, the Rhizobium japonicum strain inoculated in the step (7) is Rhizobium huakuii (the strain is a strain which has been published internationally, and is an antibiotic-specific plant protein protease of Li et al, AsNODF32, and is secreted in the gene expression assay and the gene hybridization activity of the green human gene expression strain.N.phytologist 180: 185-1922008).
Example 5
Example 5 is substantially the same as example 2, except that,
specifically, the inoculation of Rhizobium japonicum with HN01lux in step (7) is carried out (this strain is a strain published internationally, Chenchan et al, and the promotion of nitrogen fixation efficiency of Rhizobium japonicum HN01lux by constitutive nifA, scientific report 44(5) 1999).
Example 6
Example 6 is substantially the same as example 2, except that,
specifically, the rhizobium inoculation in the step (7) is peanut rhizobium Spr (the strain is a strain published internationally, in Jingli and the like, the celB gene marking method is used for researching the inoculation and molybdenum application effects of acid soil peanuts, and the report on plant nutrition and fertilizer, 12(2): 250-.
The leguminous plant (Leguminosae sp) is a dicotyledonous plant, such as an arbor, a shrub, a subshrubb or a herb, and is erected or climbed, and root nodule plants capable of fixing nitrogen are frequently used. The mode belongs to: faba p. About 650 genus, 18000 species, widely distributed all over the world. Chinese 172 genus, 1485 species, 13 subspecies, 153 variety, 16 variant; each province is divided. The family has important economic significance and is one of the important sources of starch, protein, oil and vegetables in human food. The leguminous plant is specifically Lotus japonicus (Lotus japonicus), alfalfa (Medicago truncatula), Astragalus sinicus (Astragalius sinicus), Soybean (Soybean), Peanut (Peanout) and the like.
The above description is only for the specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto, and other modifications or equivalent substitutions made by the technical solution of the present invention by the ordinary skilled in the art should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solution of the present invention.
Sequence listing
<110> Xinyang college of teachers and schools
<120> soybean GmCYS20 gene and application thereof
<141> 2018-03-28
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 294
<212> DNA
<213> Glycine max
<400> 1
atggcagcac ttggtgggaa tcgtgatgtg acaggaagcc agaacagcgt tgagatcgat 60
gctctagctc gctttgctgt tgaagaacac aacaaaaaac agaatgccct tttggagttt 120
gaaaaggtgg taactgcgaa acagcaagtg gtttctggta ccttgtacac catcactttg 180
gaggcaaaag atggtgggca aaagaaggtt tatgaagcca aagtttggga gaagtcatgg 240
ttgaacttca aggaggtgca agagttcaag cttgttggag atgcacctgc atag 294
<210> 2
<211> 97
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<213> Glycine max
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Met Ala Ala Leu Gly Gly Asn Arg Asp Val Thr Gly Ser Gln Asn Ser
1 5 10 15
Val Glu Ile Asp Ala Leu Ala Arg Phe Ala Val Glu Glu His Asn Lys
20 25 30
Lys Gln Asn Ala Leu Leu Glu Phe Glu Lys Val Val Thr Ala Lys Gln
35 40 45
Gln Val Val Ser Gly Thr Leu Tyr Thr Ile Thr Leu Glu Ala Lys Asp
50 55 60
Gly Gly Gln Lys Lys Val Tyr Glu Ala Lys Val Trp Glu Lys Ser Trp
65 70 75 80
Leu Asn Phe Lys Glu Val Gln Glu Phe Lys Leu Val Gly Asp Ala Pro
85 90 95
Ala
Claims (1)
1. The application of a cysteine protease inhibitor GmCYS20 gene involved in regulating symbiotic nodulation of leguminous plants in regulating the number of roots of crowtoe plants; the sequence of the GmCYS20 gene is a nucleotide sequence shown as SEQ ID NO. 1; the sequence of the protein coded by the GmCYS20 gene is an amino acid sequence shown in SEQ ID NO. 2.
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| CN103290040A (en) * | 2012-02-24 | 2013-09-11 | 华中农业大学 | Cysteine protease gene for regulating and controlling root nodule senescence, and preparation method and application thereof |
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| CN103290040A (en) * | 2012-02-24 | 2013-09-11 | 华中农业大学 | Cysteine protease gene for regulating and controlling root nodule senescence, and preparation method and application thereof |
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