CN106834304B - Soybean synaptotagmin gene Gmsyt4-X16 and its application - Google Patents
Soybean synaptotagmin gene Gmsyt4-X16 and its application Download PDFInfo
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Abstract
The invention discloses a kind of soybean synaptotagmin genesGmsyt4‑X16, synaptotagmin gene Gmsyt4-X16 has been cloned in separation in soybean, and constructs plant vector pCAMBIA3301-Gmsyt4‑X16Gmsyt4-X16 carrier is expressed with plant interference is turned, transformation of tobacco plant, it is cultivated together with adjoining tree NC89 to Seedling Stage, the low-temperature treatment 24 hours under the conditions of 6 DEG C, quantitative fluorescent PCR discovery, Gmsyt4-X16 gene relative expression quantity under low temperature stress processing significantly increases, and illustrates that the gene is expressed by induction of chilling stress.
Description
Technical field
The invention belongs to molecular biology field, specific soybean synaptotagmin gene Gmsyt4-X16 and its cultivating
Application in terms of cold resistant plant kind.
Background technique
Synaptotagmin Synaptotagmin is the Secretory vesicles being widely present in nerve and endocrine cell
On protein families, it not only can trigger and adjust target membrane with the fusion of vesica, simultaneously participate in regulation hormone and nerve is passed
The release of matter, additionally it is possible to adjust the transhipment of the protein and film of cell.At present in mammals oneself it is found that the family 16
Kind or more hypotype, their positioning in the cell are had nothing in common with each other, and the regulatory function played is also different.
The Synaptotagmin structure of plant cell is similar with animal, but functional study is unclear.In 2004,
Molly Craxton passes through screening and RT- to the transcription sequence in genome, common sequence database and in cDNA library
The comparison of the transcription sequence obtained in PCR is analyzed, and 6 kinds of Syt genes are had found in arabidopsis, are named as SytA-SytF.2008
Year, the discovery such as Tomokazu Yamazaki, sytl takes part in the calcium dependent plasma membrane reparation under arabidopsis low temperature stress.Freezing
Melt in circulation, plant cell is received from the dehydration induced that freezes, and is melted caused by the hydration of induction and the growth of ice crystal
Mechanical stress (Levitt, 1980), but plasma membrane whether in freeze thawing it is mechanical be punctured it is not clear.It is interesting that in matter
Film nearby has found the ultra micro vesicle that Vesicle fusion can be largely participated under refrigerated environment.The researchs such as Kawamura hair
Existing, while plant Cold exposed obtains frost resistance, sytl is quicklyd increase as a kind of plasmalemma protein.It is added in suspension
After the specific antibody of the C2A structural domain of Sytl, physiology and it is immunohistochemical the results show that with ice-crystal growth generate machinery
The calcium ion that the relevant resistance of stress relies on foreign aid is related with film reparation.The relevant film reparation of Sytl on cellular level influences whole
The frost resistance of a plant.Syt participate in plasma membrane fusion process and plasma membrane reparation is played a role, therefore in its family it is other at
Member also can may compel generation effect to various abiotic ribs are resisted.
Summary of the invention
The purpose of the present invention is the frost resistances to improve plant, and provide a kind of soybean synaptotagmin geneGmsyt4-X16。
Soybean synaptotagmin geneGmsyt4-X16, its base sequence is as shown in sequence SEQ ID NO.1.
A kind of plant vector pCAMBIA3301- Gmsyt4-X16, it is that base sequence is inserted in pCAMBIA3301
The gene as shown in sequence SEQ ID NO.1, and withBarFor selection markers, promoter CaMV35S.
Soybean synaptotagmin geneGmsyt4-X16Application in terms of cultivating cold resistant plant kind.
The present invention provides soybean synaptotagmin genesGmsyt4-X16, cynapse combination has been cloned in separation in soybean
Protein gene Gmsyt4-X16, and construct plant vector pCAMBIA3301- Gmsyt4-X16It is expressed with plant interference is turned
Gmsyt4-X16 carrier, transformation of tobacco plant are cultivated together with adjoining tree NC89 to Seedling Stage, under the conditions of 6 DEG C at low temperature
Reason 24 hours, quantitative fluorescent PCR discovery, Gmsyt4-X16 gene relative expression quantity under low temperature stress processing are significantly increased, are said
The bright gene is expressed by induction of chilling stress, and observation discovery turns plant overexpression Gmsyt4-X16 compared with adjoining tree NC89
The tobacco plant blade of carrier is not apparent from the influence by low temperature, and stem and blade slightly tilted, by 12 hours suitable items
Part renewal cultivation, plant get well state, grow fine.Opposite, turn the cigarette of plant interference expression Gmsyt4-X16 carrier
In low-temperature treatment, there is apparent atrophy, wilting, clot phenomenon to careless plant in rear blade for 24 hours, by 12 hours preference temperatures
Restore, the improvement of wilting phenomenon is unobvious, and plant is cooled stress influence seriously.
Detailed description of the invention
The PCR of Fig. 1 Gmsyt4-X16 gene expands electrophoretogram；Wherein M:DNA standard molecular weight；1-2:PCR is produced
Object；
The simulation of Fig. 2 Gmsyt4-X16 protein three-dimensional structure；
Fig. 3 Gmsyt4-X16 protein system chadogram；
The building of Fig. 4 plant over-express vector；A. the double digestion of plant expression vector pCAMBIA3301；B. target patch
The PCR amplification of section；
The building and verifying of Fig. 5 plant over-express vector；A. double digestion is verified；B. PCR is verified；
The T-DNA plot structure of Fig. 6 plant over-express vector pCAMBIA3301-Gmsyt4-X16；
The double digestion of the building A. plant expression vector pCAMBIA3301 of Fig. 7 plant interference expression vector；B. target
The PCR amplification of segment；
The building and verifying of Fig. 8 plant interference expression vector；A. double digestion is verified；B. PCR is verified；
The T-DNA plot structure of Fig. 9 plant interference expression vector pCAMBIA3301- Gmsyt4-X16-RNAi；
Figure 10 Agrobacterium tumefaciens mediated tobacco genetic transformation；A: the stage is sprouted；B: the stage is co-cultured after dip dyeing；C: leaching
The screening and culturing stage after dye；D: differentiation screening and culturing stage；E: culture of rootage stage；F: transplanting cultivation stage；
Figure 11 turns plant overexpressionGmsyt4-X16The PCR of carrier transformed plant is detected；A: target geneGmsyt4- X16；B: riddled basinsBar, M:DNA molecular weight standard；P: positive control;C: negative control (plant by unconverted soybean
Strain);1-10: transgenic positive plant；
Figure 12 turns plant interference expressionGmsyt4-X16The PCR of carrier transformed plant is detected；A:35S promoter；B:nos
Terminator M:DNA molecular weight standard；P: positive control;C: negative control (unconverted soybean plant strain);1-10: transgenosis
Positive plant；
Performance of Figure 13 difference transformed plant under 6 DEG C of cold treatments；A: suitable condition；B:6 DEG C is handled 24 hours；C: suitable
Suitable condition is restored 12 hours；
Figure 14 Gmsyt4-X16 gene is in the plant for turning plant over-express vector pCAMBIA3301-Gmsyt4-X16
Relative expression quantity；
Figure 15 Gmsyt4-X16 gene is turning plant interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi's
Relative expression quantity in plant；
Figure 16 T2 is detected for Southern blot；M:DNA molecular weight standard；P: positive control;C: negative control (without
The soybean plant strain of conversion);1: gene masculine plant OEA1；2: gene masculine plant OEA2；3: gene masculine plant OEA10；4:
Gene masculine plant IEA3；5: gene masculine plant IEA4；6: gene masculine plant IEA9.
Specific embodiment
The clone of 1 Gmsyt4-X16 gene of embodiment
Soybean (Glycine max) mutant material M18, tobacco (Nicotianatabacum L.) kind NC89, it is above
Material is provided by Jilin Agriculture University's Plant Biotechnology center.
The clone of the cDNA sequence of gene Gmsyt4-X16
The RNAiso kit produced using TAKARA company is from soybean M18 Seedling root tissue extraction total serum IgE, with reversion
The cDNA of record is template, according to RNA-seq sequencing result sequence, designs special primer syt, expands purpose using RT-PCR method
Segment is simultaneously sequenced.
The special primer for being 1062bp according to the Gmsyt4-X16 sequence fragment design amplification length screened early period
Syt-s and syt-as.After PCR is expanded, the nucleic acid bands (Fig. 1) of a treaty 1062bp are obtained, by PCR product cloning
It is sequenced after to PMD18-T carrier.Sequencing result is compared with data in NCBI, does not find and target gene phase one
The known array of cause judges to clone obtained gene tentatively for unknown new gene.It is found by comparing, target gene and sequence
Number homology highest is compared for the sequence of XR_001387984.1, the consistency in 87% coverage level reaches 98%, and with
It is higher that other Glycine max synaptotagmin-4-like gene orders compare similitudes, illustrate target gene with
The homology of Glycine max synaptotagmin-4-like family member is higher, therefore is named as the target gene
Gmsyt4-X16。
The building of the bioinformatic analysis and systematic evolution tree of 2 Gmsyt4-X16 gene of embodiment
It willGmsyt4-X16Gene complete sequence is connected into cloning vector pMD18-T Vector, after recombinant vector is sequenced
Bioinformatic analysis is carried out, sequencing is won Radix Polygalae Bioisystech Co., Ltd by Beijing three and completed.
It utilizes On-line analysis program ORF finder (https: //www.ncbi.nlm.nih.gov/orffinder/)
Possible open reading frame analysis is carried out to new gene sequence, new gene ORF is translated by amino acid by DNAMAN software
Sequence；Protein is predicted using ProtParam software (http//www. web. expasy. org/protparam/)
Sequencing theory parameter；It is constructed using SWISS-MODEL online softwareGmsyt4-X16The protein steric structure of gene；It utilizes
Protscale software (http://web. expasy. org //protscale/prediction albumen hydrophilic and hydrophobic.
All known syt gene orders are downloaded in the website NCBI, protein are carried out by DNAMAN software more
It compares again, analyzes homology, construct systematic evolution tree.
It is analyzed by nucleic acid sequence of the On-line analysis program ORF FINDER to new gene Gmsyt4-X16, and pre-
Survey the position of theoretic code area.Analysis the result shows that, open reading frame on Gmsyt4-X16 gene theory is 687bp,
228 amino acid residues of codified.
It is 26075.68 that ProtParam software, which analyzes its relative molecular weight, and theoretical isoelectric point (PI) is 9.01, positive charge
Total amino acid residues (Arg+Lys) are 30, and negative electrical charge total amino acid residues (Asp+Glu) are 25, and molecular formula is
C1183H1911N303O334S11..Amino acid composition analysis shows: leucine, lysine, isoleucine content be higher to account for 12.3% respectively,
10.1%,9.6%.Overall average hydropathy index Grand average of hydropathicity (GRAVY) is -0.153.It utilizes
The 3D three-dimensional structure diagram (Fig. 2) of 3.70 software building Gmsyt4-X16 of ProMod version, is described as Extended
The stereochemical structure function and feature of Gmsyt4-X16 is illustrated in synaptotagmin-2 in theory.
The amino acid sequence for searching for whole pulse family class syt gene known to plant on the website NCBI, utilizes DNAMAN software
Establish Gmsyt4-X16 protein system chadogram.Using Neighbor-Joining statistic law, according to each Gmsyt4-X16 egg
Evolutionary analysis result is divided into three groups by white sequence homology, and the homology of Protein G msyt4-X16 and soybean Gmsyt4-X8 is most
Height, compared with red bean, mung bean, the trend of evolution of Gmsyt4-X16 albumen and soybean Gmsyt4 family (Glycine max in soybean
Synaptotagmin-4-like) genetic distance is nearest.
The building of 3 plant expression vector of embodiment and genetic transformation
Utilize restriction enzymeBglII andBstEII carries out double digestion to plant expression vector pCAMBIA3301, utilizes
Seamless Assembly Clonng Kit kit obtains cloneGmsyt4-X16It is former in full-length gene replacement 3301
HaveGUSGene.Building withBarFor selection markers, CaMV35S promoter starts target geneGmsyt4-X16's
pCAMBIA3301- Gmsyt4-X16Plant over-express vector.
It utilizesBglII andBstEII restriction enzyme carries out double digestion to plant expression vector pCAMBIA3301, obtains
Size is the 3301 carrier large fragments of 10kb and the gus gene segment of 1841bp；Using primer Sytover to cloning vector
Pmd18-T-Gmsyt4-X16 carries out PCR amplification, obtains the Gmsyt4-X16 gene ORF full length fragment (Fig. 4) of size 687bp,
Gmsyt4-X16 gene ORF full length fragment is connected into plant expression vector pCAMBIA3301 using T4 ligase.
PCR and double digestion identification are carried out to successful plant over-express vector pCAMBIA3301-Gmsyt4-X16 is constructed,
Obtain the Gmsyt4-X16 gene ORF full length fragment 687bp(Fig. 5 being consistent with expected size), it is sequenced, sequencing knot
Fruit is correct, illustrates that plant over-express vector pCAMBIA3301- Gmsyt4-X16 constructs successfully (Fig. 6).
The building of 4 plant interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi of embodiment
Double digestion is carried out to plant expression vector pCAMBIA3301 using BglII and BstEII restriction enzyme, is obtained
Size is the 3301 carrier large fragments of 10kb and the gus gene segment of 1841bp；Using primer sytZ, SSR, sytF respectively to gram
Grand carrier Pmd18-T-Gmsyt4-X16 and Pmd18-T-SSR carries out PCR amplification, obtains the target gene that size is 239bp
Sense fragment, antisense fragments and the size of Gmsyt4-X16 core fragment are that the SSR of 205bp is included sub-piece (Fig. 7), are utilized
Gmsyt4-X16 sense fragment, introne SSR and Gmsyt4-X16 antisense fragments are connected into plant by seamless clone's ligase simultaneously
Expression vector pCAMBIA3301 obtains plant interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi.
To construct successful plant interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi carry out double digestion and
PCR identification.With BglII the and BstEII double digestion plasmid, the sytZ-SSR-sytF interference that a size is about 683bp is obtained
Segment；Respectively to the sense fragment of Gmsyt4-X16 core fragment, antisense fragments, SSR introne, progress specific amplification, obtain
To amplified band be all consistent with expected size, obtain the Gmsyt4-X16 core fragment that size is 239bp sense fragment,
The SSR of 205bp include sub-piece, 239bp Gmsyt4-X16 core fragment antisense fragments (Fig. 8).It is sequenced, is surveyed
Sequence result is correct, illustrates that plant interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi constructs successfully (Fig. 9).
The PCR of 5 transformed plant of embodiment is detected
The genetic transformation of tobacco
Using the CTAB method of improvement, genomic DNA is extracted from tobacco leaf, as template, is with vector plasmid
Positive control is started respectively with Gmsyt4-X16 gene, riddled basins bar and 35S using unconverted plant as negative control
The specific primer (table 1) of son carries out PCR amplification.
Study on Genetic Transformation is carried out to tobacco NC89 using agrobacterium-mediated transformation, obtains and turns with kalamycin resistance
Change 53 plants of plant, 24 plants of transformed plant of transfer plant over-express vector pCAMBIA3301-Gmsyt4-X16-over, turns to plant
29 plants of (see figure 10)s of transformed plant of object interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi.
The preferable overexpression Gmsyt4-X16 gene of growing way and interference expression are randomly selected from regeneration resistant plant
Each 10 plants of the transformed plant of Gmsyt4-X16 gene, leaves genomic DNA is extracted, uses target gene Gmsyt4-X16 and sieve respectively
The specific primer of marker gene bar is selected to carry out PCR detection to the transformed plant for turning plant overexpression Gmsyt4-X16 carrier
(Figure 11) as a result obtains 7 plants of PCR positive strain of overexpression Gmsyt4-X16 gene.
Using 35s promoter and no terminator as special primer, to the transformed plant for disturbing expression Gmsyt4-X16 carrier of becoming a cadre
PCR detection is carried out, 8 plants of PCR positive strain (Figure 12) of interference expression Gmsyt4-X16 gene are as a result obtained.
Influence of the low-temperature treatment to different transformed plant Gmsyt4-X16 gene relative expression quantities
By the tobacco plant for turning plant overexpression Gmsyt4-X16 carrier, turn plant interference expression Gmsyt4-X16 carrier
Tobacco plant and adjoining tree NC89 cultivated under optimum conditions to Seedling Stage, when growing the 5th leaf lobus cardiacus, move to 6 DEG C
Cold Stress treatment is carried out in low temperature incubator, after low-temperature treatment 24 hours, is moved under 25 DEG C of suitable conditions and is carried out renewal cultivation.It sees
The wilting degree of different transformed plant blades is examined, and measures relative expression of the target gene Gmsyt4-X16 in seedlings root
Amount.
By the tobacco plant for turning plant overexpression Gmsyt4-X16 carrier, turn plant interference expression Gmsyt4-X16 carrier
Tobacco plant and adjoining tree NC89 cultivate to Seedling Stage, the low-temperature treatment 24 hours under the conditions of 6 DEG C, observation discovery with
Adjoining tree NC89 is compared, and the tobacco plant blade for turning plant overexpression Gmsyt4-X16 carrier is not apparent from shadow by low temperature
It rings, stem and blade slightly tilt, and by 12 hours suitable condition renewal cultivations, plant got well state, grow fine.
Opposite, turning the tobacco plant of plant interference expression Gmsyt4-X16 carrier, rear blade occurs significantly for 24 hours in low-temperature treatment
Atrophy, wilting, clot phenomenon were restored by 12 hours preference temperatures, and the improvement of wilting phenomenon is unobvious, and plant, which is cooled, coerces shadow
Ring serious (Figure 13).
The root system total serum IgE for extracting PCR positive plant, is dyestuff with SYBR Green I, carries out qRT-PCR detection.Using
Relative quantification method, calibration standard choose reference gene actin, measure the table of reference gene and target gene Gmsyt4-X16
Up to amount, the relative expression quantity each plant is analyzed according to formula 2- △ △ CT.
In tobacco root, the Gmsyt4-X16 gene expression amount of recipient plant NC89 under suitable condition is set as 1, other
Expression under transgenic positive plant, other treatment conditions is all in contrast to this setting value, as shown in figure 14, preference temperature
Under the conditions of, the opposite table of 7 plants of plant Gmsyt4-X16 genes for turning plant over-express vector pCAMBIA3301-Gmsyt4-X16
All it is higher than control NC89 up to amount, wherein the relative expression quantity highest of OEA10, is 2.863, is secondly OEA2(2.35) and OEA1
(2.543).After low-temperature treatment 24 hours, the Gmsyt4-X16 gene relative expression quantity of each plant is dramatically increased, explanation
Gmsyt4-X16 gene is expressed by induction of chilling stress.Compared with receptor NC89, turn plant over-express vector pCAMBIA3301-
The relative expression quantity of the plant Gmsyt4-X16 gene of Gmsyt4-X16 is higher, the relative expression quantity highest of OEA10, is 4.67,
Secondly it is OEA1(4.013) and OEA2(3.81).
In the positive plant for turning plant interference expression vector pCAMBIA3301-Gmsyt4-X16-RNAi, Gmsyt4-
The relative expression quantity of X16 gene all receives apparent inhibition, and wherein the inhibitory effect of IEA9 plant Gmsyt4-X16 gene is most
Obviously, 58.6667%, followed by IEA4(58% have been disturbed) and IEA3(52.667%).After low-temperature treatment 24 hours, each plant
Gmsyt4-X16 gene relative expression quantity dramatically increase, further illustrate Gmsyt4-X16 gene by induction of chilling stress table
It reaches.
Gmsyt4-X16 gene is analyzed in the integration of tobacco gene group
To the transgenic plant of PCR test positive, Southern hybridization check is carried out.With the riddled basins of purifying
Bar is that template prepares probe, is marked using random primering, hybridizes and other steps for detecting are according to DIG
The specification of High Prime DNA Labeling and Detection Starter KitII kit operates.
It is control with unconverted tobacco plant NC89, chooses and be overexpressed, interfere the apparent 6 plants of transgenic positives of expression effect
Plant carries out Southern hybridization check, the results showed that, 6 plants of transgenic positive tobaccos all have apparent hybridization signal, and select
The non-non-transgenic control lines amixia signal taken generates (Figure 16), illustrates that foreign gene has been integrated into the genome of tobacco
In, integration form is single copy form, and the position of each hybridization signal is different, illustrates the whole angle of striking of each transgenic plant
It is all different.
<110>Jilin Agriculture University
<120>soybean synaptotagmin gene Gmsyt4-X16 and its application
<160> 1
<210> 1
<211> 1056
<212> DNA
<213>artificial
<400> 1
ctaatgaagg gaattaactt atgccaatga tttctttaat tgccaattgt cataatgaca 60
caatgttgcc aagcatatac tgcaactctg caaggcaagc atacccaaag cccaggattg 120
attatactct gaaagctatt ggtggaagtt taacggctat tcctggaatc tcggatatga 180
ttgatgatac tgtgaactca attgttactc acatgctcca atggcctcat aggattgtgg 240
ttcaacttgg tggcattcct attgatacca gtgaattgga gcttaaatcc cagggaaaac 300
ttgcactgac tgtagtgaaa gcaactgctt taaagaatat ggaaatgatt gcaaagacag 360
atccttatgt agttgtgcat attcgaccat tatttaagta taaaaccaag gttattgata 420
acaacctgaa tcctatttgg aatgagaaat ttaagttgat tgcagaagac aaggagaccc 480
aattactcat tcttaggttc ttgataaaga cattgggcaa gataagcgat tgggaaaagc 540
acagttaccg catattggtc tatgaaatac aaactgaaaa ggagatcgaa ttgaggctac 600
tgccctcact tgatacactt aaggtgaaag ataagaaaga ttcaggaacc ttaacaatga 660
aggtcatgta ttatcaattt aacaaggaag gcagctggtt gcgctagaag cagaaaagaa 720
gatactagaa gaaaggaaga aactgaaaga agcgggagtg ataggatgca caatggatgt 780
actagatgga gcagtatcag tggtagggtc tggtgttgga ctggtttggt gaattattct 840
gcatagccat atttgttttt atcaaatagc ccttgatatc cttttgtttt gttttttgta 900
ccaaatataa aataattcta gaaagtattg caagaggctc tgtgatttca tttcgctatt 960
tgtttcttat ctcctatagg aatggaacat attttaatcg tgggccgtat tgagatgctc 1020
atgtttttat aaatatcata aagttgatga tgagaa 1056
Claims (3)
1. soybean synaptotagmin geneGmsyt4-X16, its base sequence is as shown in sequence SEQ ID NO.1.
2. a kind of plant vector pCAMBIA3301- Gmsyt4-X16, it is that base sequence is inserted in pCAMBIA3301
The gene as shown in sequence SEQ ID NO.1, and withBarFor selection markers, promoter CaMV35S.
3. soybean synaptotagmin gene according to claim 1Gmsyt4-X16In terms of cultivating cold-resistant tobacco bred
Application.
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Non-Patent Citations (7)
| Title |
|---|
| Accession NO:KHN04090;Lam H.-M. et al.;《EMBL》;20141218;全文 |
| Calcium-Dependent Freezing Tolerance in Arabidopsis Involves Membrane Resealing via Synaptotagmin SYT1;Tomokazu Yamazaki et al.;《The Plant Cell》;20081216;第20卷;第3389-3404页 |
| PREDICTED:Glycine max synaptotagmin-4-like(LOC100795666),transcript variant X4,misc_RNA，Accession NO:XR_001387984.1;GenBank;《GenBank》;20151125;全文 |
| The Arabidopsis Synaptotagmin1 Is Enriched in Endoplasmic Reticulum-Plasma Membrane Contact Sites and Confers Cellular Resistance to Mechanical Stresses;Jessica Perez-Sancho et al.;《Plant Physiology》;20150319;第168卷;第132-143页 |
| The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling;Kathryn P Harris et al.;《eLife》;20160525;第5卷;e13881 |
| 干旱胁迫下大豆抗旱突变体M18苗期生长和生理特性;莫金钢等;《中国油料作物学报》;20141231;第36卷(第6期);第770-776页 |
| 拟南芥synaptotagmin B基因的克隆及其功能初步研究;吴小容;《中国优秀硕士学位论文全文数据库 基础科学辑》;20111015(第10期);A006-146 |
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