CN109929923A - Transcribe application of the sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene - Google Patents
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Abstract
The invention discloses a kind of application of transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene, are related to biological gene technical field.Jujube fruit-shrink disease of the invention is caused by the dip dyeing of alternaria bacterium.The kind of jujube is that the July of the honey pot and sense fruit-shrink disease of nonshrink fruit disease is fresh.Transcribing sequencing technologies includes pretreatment, transcript profile sequencing and data processing.Pretreatment includes: to extract jujube total serum IgE, detection total serum IgE quality, synthesis cDNA library and real-time fluorescence quantitative PCR verifying.The present invention handles control material with the nonshrink fruit disease of jujube ' honey pot ', sense fruit-shrink disease ' July is fresh ' and its clear water, with alternaria bacterium (A.altrenata) for inductive condition, pass through the acknowledgement mechanism using technologies such as transcript profile sequencing technologies and qPCR from Molecular level study date fruit under alternaria bacterium (A.altrenata) induction, to screen jujube fruit-shrink disease disease-resistant gene, certain theoretical basis is provided for jujube breeding work.
Description
Technical field
It is disease-resistant in screening jujube fruit-shrink disease that the present invention relates to biological gene technical fields more particularly to a kind of transcription sequencing technologies
Application in gene.
Background technique
Jujube (Ziziphus jujuba Mill.) is Rhamnaceae (Rhamnaceae) jujube, and being that China is important does
The development of fruit tree kind, jujube industry is bringing high-level income to peasant and the good change of ecological environment is promoted to have ten
Divide positive effect (Liu Mengjun, 2009) kind.Jujube tree is rich, full of nutrition outstanding in China, the resistance to ridge of drought resisting and jujube fruit speed with its specialty
It is vitamin content is high, be used as medicine can also moistening herat and lung, QI invigorating support and the widely used advantage such as hold, promoting peasant economy development
Aspect occupies highly important status with improving the ecological environment.
The rapid development of jujube industry plays an important roll in terms of promoting peasant economy development and improving the ecological environment, but by
After the generation of jujube fruit-shrink disease, cause jujube fruit fall off and necrosis, the yield and quality of jujube fruit has been seriously affected, to jujube area, China
Production cause serious harm, it is very urgent to the prevention and treatment of the disease.In the 1970s, the country begins with jujube fruit-shrink disease
The report (Han Jinsheng 1979) of research, and at present for the identification of jujube fruit-shrink disease pathogen always without unified final conclusion.Initial stage recognizes
Disease is physiological disturbance thus, also there is disease (Chen Yijin etc. 1989) caused by being considered a kind of Erwinia bacteria.Mesh
Before until, the pathogen reported altogether has 6 kinds of fungies and 2 kinds of bacteriums (Zhang Chaohong etc. 2008).Qu Jianxu etc. (1992) report
The pathogen of jujube fruit-shrink disease is D.gregaria.The identification of Zheng Xiaolian etc. (1995) thinks that cause of disease is shield shell mould
Coniaothyrium olivaceum Bon, thin chain are every (poly-) the raw vesicle shell of spore bacterium Alternaria tenuis Nees. and group
3 kinds of weak parasites of bacterium Dothriorella gregaria Sacc and a kind of bacterium, and this 4 kinds of cause of diseases individually or combined inoculation all
It can produce typical symptom；Kang Shaolan etc. (1998) proposes jujube fruit-shrink disease by alternaria bacterium A.altrenata, destruction stem point
Tri- kinds of mould P.destructive, bobbin case spore bacterium Fusicoccum sp disease fungus are individually or mixed infection causes；Luo Jun etc.
(2009) pass through taken at regular intervals jujube tree and carry out tissue separation, specify that jujube fruit-shrink disease cause of disease includes alternaria bacterium
A.altrenata, bobbin case spore bacterium Fusicoccum sp, Alternaria tenuissima (A.altrenata) and olive colour shield shell are mould
Coniaothyrium olivaceum；Han Dangyue (2012) is true using the methods of tissue separation, morphologic observation and Molecular Identification
The more stable jujube fruit-shrink disease pathogen alternaria bacterium A.alternata of segregation ratio is determined；Wang Ye etc. (2013) specifies 6
Strain jujube fruit-shrink disease pathogen, i.e. horse chestnut shell shuttle embrace bacterium Fusicoccum aesculi Cord, head Phoma sp Phoma
Glomerata, Alternaria tenuissima Alternaria tenuissima (Kunze) Wiltshire, alternaria bacterium
A.alternata, pear rod method A.R.G.Roberts.Although each department are different to the report of jujube fruit-shrink disease cause of disease,
But it is widely believed that jujube fruit-shrink disease is to be caused by the above single cause of disease or two or more cause of disease mixed infections, and find rod method
The bacterial strain of category may be to cause the most common disease fungus of jujube fruit-shrink disease.
In order to control above-mentioned germ, correlative study is mainly from Physiology and biochemistry and morphosis Study on Level jujube to alternaria
The defense reaction of bacterium, and the research of molecular level is rarely reported.
Summary of the invention
In view of this, the embodiment of the invention provides a kind of transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene
Using.
In order to achieve the above objectives, invention broadly provides following technical solutions:
On the one hand, the answering in screening jujube fruit-shrink disease disease-resistant gene the embodiment of the invention provides a kind of transcription sequencing technologies
With.
Preferably, the jujube fruit-shrink disease is caused by the dip dyeing of alternaria bacterium.
Preferably, the kind of the jujube is fresh for the honey pot of nonshrink fruit disease and the July of sense fruit-shrink disease.
Preferably, the transcription sequencing technologies include preprocessing process, transcript profile sequencing procedure and data handling procedure.
Preferably, the preprocessing process include: extract jujube total serum IgE, detection total serum IgE quality, synthesis cDNA library and
Real-time fluorescence quantitative PCR verifying.
Preferably, the detailed process of the synthesis cDNA library includes:
End repair: with set terminal repair complex enzyme reaction system, in Thermomixer thermophilic reaction a period of time into
Row end is repaired, and is repaired product with kit and is carried out purification and recovery；
3 end ˊ cDNA adds A: under the action of polymerase systems, adding reparation product obtained in the previous step in 3 ends ˊ
Upper A base is prepared for the connection of next step connector；A product is added to carry out purification and recovery with kit；
The connection of Adapter: configuration ligase reaction system, thermophilic reaction a period of time makes in Thermomixer
Adapter with plus A product connect；Product after connection Adapter carries out purification and recovery with kit；
The glue recovery purifying of connection product: preparing Ago-Gel, carries out electrophoresis to the connection product of previous step；Completion is cut
Remaining blob of viscose is taken pictures in gel imaging system after glue and stores picture；Determine that there is no problem rear abandons remaining gel
Blob of viscose；The blob of viscose cut carries out purification and recovery with kit；
PCR reaction and product purification: configuration PCR reaction system expands the gel extraction product of previous step；Reaction
After to PCR reaction product carry out electrophoresis, cut glue, and carry out purification and recovery with kit, recovery product is dissolved in right amount
In Elution buffer, printed label is attached on centrifugation tube wall, and so far library preparation is completed；
The sequencing of upper machine: the c DNA library built tentatively quantify with Qubit2.0 and dilute by library inspection and the sequencing of upper machine
Annotations library value 1ng/ μ L detects c DNA library insert size using Agilent2100, after meeting demand, with q RT-
PCR carries out accurate quantitative analysis to library effective concentration to guarantee Library Quality；After library inspection is qualified, library will not had to according to effectively dense
Under degree and object table after the demand pooling of machine data volume, carried out using Illumina Hi Seq TM2500 microarray dataset
PE125 sequencing analysis；
Gene expression profile data analysis: resulting data are sequenced by IIIumina HiSeqTM 2000 and are known as raw reads
Or raw data, Quality Control then is carried out to raw reads, to determine whether sequencing data is suitable for subsequent analysis；After Quality Control, warp
High quality clean reads is obtained by filtration, is compared using the library Hisat2 platform construction unigene, with jujube genomic data
Afterwards, Differential expression analysis is carried out after eliminating biological variation using DESeq method；Using the method for RPKM to sequencing data mark
Standardization；It carries out gene expression, gene structure optimization, alternative splicing, the prediction of new transcript and annotation, SNV to test and analyze, and right
All differences expressing gene is compared with GO database and KEGG database, carries out Gene Ontology functional analysis respectively
It is enriched with and analyzes with KEGG Pathway.
Preferably, the detailed process of the real-time fluorescence quantitative PCR verifying are as follows:
RNA is extracted and cDNA synthesis: date fruit total serum IgE uses Ta Ka Ra Mini BEST Plant RNA
Extraction Kit kit extracts, and it is 200ng that reverse transcription, which synthesizes c DNA total amount, and used kit is Ta Ka Ra
Prime ScriptTM RT reagent Kit with gDNA Eraser；
Quantitative fluorescence analysis: q RT-PCR verifying is carried out to difference expression gene based on SYBR method, reference gene used is
ACT1 gene；10 μ L:SYBR Premix Ex Taq of PCR reaction system II 5.0 μ L, positive each 0.4 μ L of anti-primer, DNA profiling
1.0 μ L, 3.2 μ L of water.Reaction condition are as follows: 95 DEG C of 30s, 95 DEG C of 5s, 58 DEG C of 30min, 72 DEG C of 30s, totally 40 recycle；Wherein, institute
State primer gene I/D accession number be Zj.jz107428959, Zj.jz 107419914, Zj.jz107429464,
Zj.jz107420088、Zj.jz107417234、Zj.jz107413980、Zj.jz107420411、 Zj.jz107426176、
Zj.jz107432190、Zj.jz107429308、Zj.jz107433466、Zj.jz107434596、 Zj.jz107419438。
Preferably, the jujube fruit-shrink disease disease-resistant gene includes sulfate transporter, GAM synzyme, extension albumen, ammonia
Base acid permease, CCCH Zinc finger domain albumen, ill-resistant protein, protein N RT1/PTR family, calbindin, chlorophyll
A-b combination egg and gibberellin 2- β-dioxygenase.
Compared with prior art, the beneficial effects of the present invention are:
The present invention be understand date fruit alternaria bacterium (Alternaria.altrenata) induction under in transcript profile
Horizontal variation excavates the classification and function of important gene relevant to jujube fruit-shrink disease resistance, with the nonshrink fruit disease ' honey of jujube
Tank ', sense fruit-shrink disease ' July is fresh ' and its clear water handle control material, with alternaria bacterium (A.altrenata) be induction item
Part, by utilizing the technologies such as transcript profile sequencing technologies and qPCR from Molecular level study date fruit in alternaria bacterium
(A.altrenata) acknowledgement mechanism under inducing provides centainly to screen jujube fruit-shrink disease disease-resistant gene for jujube breeding work
Theoretical basis.
Detailed description of the invention
Fig. 1 is the 2100 total serum IgE detection figure of Agilent that the embodiment of the present invention 1 provides；
(R1: ' honey pot ' different vaccination time RNA mixed in equal amounts；R2: ' honey pot ' clear water control；S1: ' July is fresh ' no
With inoculation time RNA mixed in equal amounts；S2: ' July is fresh ' clear water control)
Fig. 2 is the difference expression gene statistical chart that the embodiment of the present invention 1 provides；
Fig. 3 is the significant difference gene GO statistic of classification figure that the embodiment of the present invention 1 provides；
Fig. 4 be the embodiment of the present invention 1 provide July it is fresh ' in significant difference gene participate in Plant hormone signal conduction generation
Thank to access diagram；
Fig. 5 be the embodiment of the present invention 1 provide July it is fresh ' in significant difference gene participate in glutathione metabolism approach show
It is intended to；
Fig. 6 is the flavonoids biosynthesis way that the embodiment of the present invention 1 provides that significant difference gene in ' July is fresh ' participates in
Diameter schematic diagram；
Fig. 7 is that the fatty acid synthesis pathway that significant difference gene participates in the offer of the embodiment of the present invention 1 ' July is fresh ' shows
It is intended to；
Fig. 8 is that the qReal Time-PCR analysis result for the nonshrink fruit disease related gene of jujube that the embodiment of the present invention 1 provides is bent
Line chart；
Fig. 9 is that disease-resistant related gene is resisting, feeling fruit-shrink disease date fruit under the different vaccination time that the embodiment of the present invention 1 provides
In qRT-PCR analyze histogram；(1:0h, 2:0.5d, 3:1d, 4:2d, 5:3d, 6:4d, 7:5；A:GMP, B:MYB21；D:
expansin-A1；G:Amino acid permease；H:zinc finger CCCH domain-containing
protein；K:disease resistance protein RPM1；)
Figure 10 is that Transcription factor MYB21 exists under the different vaccination time that the embodiment of the present invention 1 provides
QRT-PCR anti-, in sense fruit-shrink disease date fruit analyzes histogram；
Figure 11 is that disease-resistant related gene is resisting, feeling fruit-shrink disease jujube fruit under the different vaccination time that the embodiment of the present invention 1 provides
QRT-PCR in reality analyzes histogram；(C:Glucose-6-phosphate/phosphate translocator 2；E:
Purple acid phosphatase 17)
Figure 12 is that disease-resistant related gene is resisting, feeling fruit-shrink disease jujube fruit under the different vaccination time that the embodiment of the present invention 1 provides
QRT-PCR in reality analyzes histogram；(F:transcription factor bHLH30；M:protein NRT1/PTR
FAMILY 5.1)
Figure 13 is that disease-resistant related gene is resisting, feeling fruit-shrink disease jujube fruit under the different vaccination time that the embodiment of the present invention 1 provides
QRT-PCR in reality analyzes histogram；(G:Amino acid permease；I:zinc transporter 1；J:
sulfate transporter；L:aquaporin NIP3-1；)
Specific embodiment
For further illustrate the present invention to reach the technical means and efficacy that predetermined goal of the invention is taken, below with compared with
Good embodiment, to specific embodiment, technical solution, feature and its effect applied according to the present invention, detailed description is as follows.
The special characteristic, structure or feature in multiple embodiments in following the description can be combined by any suitable form.
Embodiment 1
Jujube kind used by this experiment is ' honey pot ', and it is red to pick up from Shaanxi Province Qingjian for ' July is fresh ' white ripe phase date fruit
Jujube experiment station, alternaria bacterium are ZS091 biological strain, derive from Henan Province's forest-science institute.
Inoculation and sampling: will dissolve pathogen spore with Tween 80 for examination strain after cultivating 7 days in PDA culture medium,
And diluted with sterile water, after blood counting chamber counts, being made into concentration is 1 × 108The spore suspension of a/mL.When jujube fruit into
When entering the white ripe phase, select the similar healthy honey pot jujube of growing way as test material.Nothing is completely used afterwards with 75% alcohol rinse
Bacterium water rinses 3 times, finally shakes up the spore suspension prepared, is equably sprayed at jujube fruit surface as experimental group
(Tester).As a control group (Driver) with healthy jujube fruit sprinkling sterile water simultaneously.Template is carefully put on after inoculation
Bag, and it is put into bag the sterile cotton moisturizing of immersion.Experimental group and control group sample are acquired respectively at 0.5d, 1d, 2d, 3d and 4d
Product, 3 repetitions of each sample (5 jujube fruits of each repetition), pick up from 3 plants of plant.The jujube fruit of acquisition is taken back rapidly with ice chest
Laboratory, belt leather are chipped, and are put into liquid nitrogen and freezed immediately after being wrapped with tinfoil, be placed in -80 DEG C of refrigerators save it is standby
With.
The instrument used in this experiment is shown in Table 1.
1 key instrument model of table and source
Main agents are shown in Table 2.
2 main agents of table and source
It extracts total serum IgE: when date fruit is ground into fine powdered, liquid nitrogen being constantly added thereto, to keep RNA living
Property, avoid RNA from degrading.Blade is extracted using Ta Ka Ra Mini BEST Plant RNA Extraction Kit kit
Sample total serum IgE pays attention to carrying out polysaccharide polyphenol and goes genomic DNA step:
(1) sample being ground into powder (50-100mg) is added to and (is asked really using preceding containing 450 μ l Buffer RL
Recognize and 50 × DTT Solution be added) 1.5ml sterile centrifugation tube in, blown and beaten repeatedly with pipettor until lysate in nothing
Obvious sediment, 12,000rpm, 4 DEG C of centrifugation 5min.
(2) supernatant is carefully drawn in new 1.5ml sterile centrifugation tube.
(3) dehydrated alcohol (at this time it is possible that precipitating) of 1/2 volume of supernatant or mixed liquor in 2 steps is added,
Solution is uniformly mixed using liquid-transfering gun.
(4) mixed liquor (containing precipitating) is all transferred in RNA centrifugal column (containing collecting pipe) immediately.If (mixed liquor
Volume is greater than 600 μ l, please be added portionwise, the volume being added every time is not larger than 600 μ l.)
12,000rpm, it is centrifuged 1min, abandons filtrate.
(5) the Buffer RWA of 500 μ l is added into RNA centrifugal column, 12,000rpm centrifugation 30s abandon filtrate.
(6) the Buffer RWB of 600 μ l is added into RNA centrifugal column, 12,000rpm centrifugation 30s abandon filtrate.Note:
100% ethyl alcohol of designated volume is had been added in PLSCONFM Buffer RWB.
(7) DNase I digests:
1. the preparation of DNase I reaction solution: taking 5 μ l 10 × DNase I Buffer, 4 μ l Recombinant DNaseI
(RNase free, 5U/ μ l), 41 μ l RNase free d H2O to new 1.5ml in sterile centrifugation tube, are uniformly mixed.
2. 50 μ l DNase I reaction solutions are added to the centrifugal column film center RNA, it is stored at room temperature 15min.
3. 350 μ l are added to the centrifugal column film center RNA Buffer RWB, 12,000rpm centrifugation 30s abandon filtrate.
(8) repetitive operation step 5, by the relocation of RNA centrifugal column on collecting pipe, 12,000rpm are centrifuged 2min.
(9) RNA centrifugal column is placed on the new sterile centrifugation tube of 1.5ml, is added in RNA centrifugal column film centre
RNase Free the d H2O or 0.1%DEPC of 50-200 μ l handles water, is stored at room temperature 12,000rpm centrifugation 2min after 5min
Eluted rna.
Detect total serum IgE: using 1% agarose gel electrophoresis analysis RNA palliating degradation degree and pollution level, Nanodrop
2000 detection RNA purity, Qubit 2.0 carry out accurate quantification, Agilent 2100 to RNA concentration and accurately detect the complete of RNA
Whole property.
CDNA synthetic material and method: detecting qualified RNA sample, with the enrichment with magnetic bead m RNA with Oligo (d T),
Obtained m RNA is synthesized into the first chain cDNA by corresponding program in PCR instrument.First chain cDNA and the second chain of reverse transcription is anti-
Answer system to mix, thermophilic reaction a period of time synthesizes the second chain, after reaction, with kit to two chain synthetic products into
Row purifying carries out end reparation plus A tail to it and connects sequence measuring joints, carries out clip size choosing with AMPure XP beads
It selects, finally carries out PCR and be enriched with to obtain final c DNA library.
(1) end is repaired: complex enzyme reaction system is repaired with set terminal, when thermophilic reacts one section in Thermomixer
Between carry out end reparation, repair product and with kit carry out purification and recovery.
(2) 3 end ˊ cDNA adds ' A ': under the action of polymerase systems, making reparation product obtained in the previous step at the end 3 ˊ
End adds ' A ' base, prepares for the connection of next step connector.' A ' product is added to carry out purification and recovery with kit.
(3) connection of Adapter: configuration ligase reaction system, thermophilic reaction a period of time makes in Thermomixer
Adapter with plus ' A ' product connect.Product after connection Adapter carries out purification and recovery with kit.
(4) the glue recovery purifying of connection product: preparing Ago-Gel, carries out electrophoresis to the connection product of previous step.It is complete
At remaining blob of viscose is taken pictures in gel imaging system after cutting glue and stores picture.Determining after there is no problem can will be remaining
Gel blob of viscose is abandoned into dustbin.The blob of viscose cut carries out purification and recovery with kit.
(5) PCR reaction and product purification: configuration PCR reaction system expands the gel extraction product of previous step.
Electrophoresis is carried out to PCR reaction product after reaction, cuts glue, and carry out purification and recovery with kit, recovery product is dissolved in right amount
In Elution buffer, printed label is attached on centrifugation tube wall, and so far library preparation is completed.
(6) machine is sequenced on: library inspection and the sequencing of upper machine by the c DNA library built with Qubit2.0 carries out it is preliminary quantitatively simultaneously
Library value 1ng/ μ L is diluted, c DNA library insert size is detected using Agilent2100, after meeting demand, with q RT-
PCR carries out accurate quantitative analysis (effective concentration > 2n M) to library effective concentration, to guarantee Library Quality.It, will not after library inspection is qualified
After demand pooling of the library according to machine data volume under effective concentration and object table, Illumina Hi Seq is utilized
TM2500 microarray dataset carries out PE125 sequencing analysis.Examining order is completed by Jin Weizhi (China) Biotechnology Co., Ltd.
(7) gene expression profile data is analyzed: resulting data are sequenced by IIIumina HiSeqTM 2000 and are known as raw
Reads or raw data then will carry out Quality Control (QC) to raw reads, to determine whether sequencing data is suitable for subsequent point
Analysis.After Quality Control, high quality clean reads is obtained by filtration, utilizes the library Hisat2 (v2.0.1) platform construction unigene
(Haas et al., 2013), after being compared with jujube genomic data (Liu et al., 2014), using DESeq (Anders et
Al., 2012) method carries out Differential expression analysis after eliminating biological variation, to the Standard General of differential gene screening are as follows: FDR
≤ 0.05, differential expression multiple | log2Ratio | >=2.Using RPKM (Reads Per Kilo bases per Million
Reads) method of (Mortazavi et al., 2008) standardizes sequencing data.It can carry out gene expression, gene structure
A series of subsequent analysis such as optimization, alternative splicing, the prediction of new transcript and annotation, SNV detection, and base is expressed to all differences
Cause and GO database (http://www.geneontology.org/) and KEGG database (http://www.kegg.jp/
Kegg/ it) compares, carries out Gene Ontology (GO) functional analysis and KEGG Pathway enrichment analysis respectively.
Real-time fluorescence quantitative PCR verifying:
(1) RNA is extracted and c DNA is synthesized: date fruit total serum IgE uses Ta Ka Ra Mini BEST Plant RNA
Extraction Kit kit extracts, and it is 200ng that reverse transcription, which synthesizes c DNA total amount, and used kit is Ta Ka Ra
Prime ScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time), concrete operations step
It is rapid to be carried out referring to instructions direct.
(2) q RT-PCR verifying, internal reference base used quantitative fluorescence analysis: are carried out to difference expression gene based on SYBR method
Because of ACT1 gene (Zhang C et al., 2016).The primer information is shown in Table 2-3, is synthesized by Shanghai Sangon Biotech Company. PCR
10 μ L:SYBR Premix Ex Taq of reaction system II 5.0 μ L, positive each 0.4 μ L of anti-primer, 1.0 μ L of DNA profiling, 3.2 μ L of water.
Reaction condition are as follows: 95 DEG C of 30s, 95 DEG C of 5s, 58 DEG C of 30min, 72 DEG C of 30s, totally 40 recycle.
3 primer information of table
4 PCR reaction system of table (25 μ l)
(3) data process&analysis: one-way analysis of variance (ANOVA) is carried out to data with SPSS20.0, using LSD
And Duncan ' s method is compared two-by-two, significance analysis p < 0.05, and is charted using 8.0 software of Origin Pro.
Interpretation of result:
RNA quality testing: after extracting RNA using centrifugal column RNA isolation kit, 1.5ul is taken to use Thermo Scientific
NanoDrop spectrophotometer detects its concentration 260/280, detects date fruit with 1% agarose gel electrophoresis after detection
The integrality of total serum IgE.Test sample mixes after melting centrifugation on ice, and l μ L sample is taken to dilute, and after 70 DEG C of denaturation 2min, carries out
Agilent 2100 is detected.Test sample concentration, clip size, RIN and 28S:18S ratio.Sample as the result is shown is analyzed from Fig. 3
This RNA integrality is preferable, and all samples RIN value is both greater than 1, RNA concentration greater than 8, rRNA Ratio (28s/18s) and is both greater than
100ng·μl-1, therefore a sample gross mass satisfaction builds library and sequencing requires, as shown in Figure 1.
The analysis of transcript profile sequencing data:
The assessment of transcript profile sequencing quality: 4 samples sequencing in this experiment obtains 4.88 × 10 respectively7、4.93×107、
4.74×107、4.80×107Raw reads, is filtered low quality data, can obtain respectively after removing depollution and joint sequence
To 4.87 × 107、4.91×107、4.71×107、4.78×107High quality sequence.Wherein have 4.12 × 10 respectively7、4.28×
107、4.06×107、 3.86×107Item is compared to jujube genome (Liu et al.2014), there is 3.71 × 107、3.49×107、
3.27×107、3.31×107Item obtains unique compare.In sequencing analysis, it is considered that the base of Q≤20 has reliable matter
Amount, Q20 range are the requirement that 97.08-97.35% meets that Q20 is greater than 80%, and G/C content is averagely about 44.79%.The skin of sample
Square (the R of your inferior related coefficient2) it is all larger than 0.9, show that the repeatability between different repeated materials is preferable, can be used for
Subsequent bio bioinformatics analysis.
5 transcript profile sequencing data of table statistics
Note: Q20 is percentage shared by the base of mass value >=20 Clean reads.
Pathogen inoculation after difference expression gene quantitative analysis: annotation of gene function the result shows that, A.altrenata inoculation
The gene number of ' honey pot ', ' July is fresh ' differential expression afterwards is respectively 47 and 2357, and wherein up-regulated expression gene number is respectively 39
With 1754, and lower expression then be respectively 8 and 603.
The enrichment analysis of GO functional annotation: it is found by the GO functional annotation analysis to difference expression gene, differential gene master
It is enriched in biological process (Biological process) and molecular function (Molecular function) two major classes
It not, at ' honey pot ' and ' July fresh in ' include respectively 14 and 16 function classifications.With Corrected P-value≤
0.05 is threshold value, defines the GO-term of difference expression gene significant enrichment after A.altrenata inoculation ' honey pot ' fruit, and
Carry out GO functional analysis；The result shows that ' honey pot ' after being inoculated with A.altrenata, the difference table of date fruit biological process
Up to gene significant enrichment in 5 GO-term, including positioning (localization), single creature process (single-
Organism process), stimulate the reaction (response to stimulus), metabolic process (metabolic
Process), cell processes (cellular process).Difference expression gene significant enrichment in cellular component is in 5 GO-
In term, including cellular machineries (cell part), membrane element (membrane part), film (membrane), organelle
(organelle), organelle component (organelle part) etc..And the difference expression gene in molecular function is significantly rich
Collection is in 4 GO-term, including connection (binding), transport activity (transporter activity), catalytic activity
(catalytic activity) structural molecule activity (structural molecule activity) etc..It is right
A.altrenata is inoculated with ' July is fresh ' fruit difference expression gene and carries out GO functional analysis, as a result such as Fig. 3 expression, biology mistake
The difference expression gene significant enrichment of journey in 12 GO-term, mainly include metabolic process (metabolic process),
Cell processes (cellular process), positioning (localization), single creature process (single-organism
Process), stimulate the reaction (response to stimulus), biological regulation (biological regulation) etc., with
It infects unlike ' honey pot ', that biological process is mainly enriched with is metabolic process (metabolic process), enrichment
133 genes；Difference expression gene significant enrichment in cellular component is in 8 GO-term, including cellular machineries (cell
Part), membrane element (membrane part), organelle (organelle), film (membrane), organelle component
(organelle part) etc..Compared with infecting ' honey pot ', cellular machineries (cell is equally mainly enriched in cellular component
Part), it is enriched 48 altogether；And the difference expression gene significant enrichment in molecular function is in 11 GO-term, including
Catalytic activity (catalytic activity), connection (binding), transport activity (transporter activity), electricity
Subcarrier activity (electron carrier), signal transduction activity (signal transducer activity) etc., and are invaded
Dye ' honey pot ' is compared, and that be mainly enriched with is catalytic activity (catalytic activity), is enriched 182 genes altogether.
The Pathway of difference expression gene is analyzed: in vivo, mutually coordinated its biology function of enforcement of different genes
Can, the main biochemical metabolism approach and signal transduction that difference expression gene participates in can determine that by the enrichment of Pathway conspicuousness
Approach.KEGG (Kyoto Encyclopedia of Genes and Genomes) is the main public number in relation to Pathway
According to library (Kanehisa, 2008).The enrichment analysis of Pathway conspicuousness is examined as unit of KEGG Pathway using hypergeometry
It tests, finds out compared with whole gene group background, the Pathway that conspicuousness is enriched in difference expression gene.Table 3-7 shows
Difference expression gene after A.altrenata inoculation ' July celestial ' has 745 to be enriched in 32 Pathway, chooses difference base
Pathway relevant to date fruit response pathogen infection has Plant hormone signal transduction (Plant hormone because in
Signal transduction), glutathione metabolism (Glutathione metabolism), cysteine and methionine generation
Thank (Cysteine and methionine metabolism), flavonoids biosynthesis (Flavonoid
Biosynthesis), herxheimer-liked reaction (Carotenoid biosynthesis), fatty acid metabolism (Fatty
Acid metabolism), fatty acid biological synthesis (Fatty acid biosynthesis), alpha-linolenic acid be metabolized (alpha-
Linolenic acid metabolism) etc..A.altrenata infects differential gene in ' honey pot ' fruit total right 13
A, the metabolic pathway of Pvalue≤0.05 has 13.Including photosynthesis (Photosynthesis-antenna
Proteins), Plant hormone signal transduction (Plant hormone signal transduction), pentose phosphate pathway
(Pentose phosphate pathway), glycolysis/gluconeogenesis (Glycolysis/Gluconeogenesis), diterpene are raw
Object synthesizes (Diterpenoid biosynthesis) etc. such as table 6.
6 metabolic pathway of table
July is fresh ' in resistance relevant metabolic pathway and genetic analysis:
Plant hormone signal Signal Transduction Pathways are as shown in Figure 4.
Resistance signal's approach that SA, JA/ET are mediated: studies have shown that ' July is celestial ' infected by A.altrenata after, have 1
It is right to show that SA signal pathway takes part in ' July is celestial ' for the expression that gene (1.6 times of up-regulation) participates in Downstream regulatory NPR1
The response of A.altrenata.
After ' July celestial ' is infected by A.altrenata, regulation Protein TIFY 10A related gene has 2 (on respectively
Adjust 2.3,2.2 times), the expression variation of key gene, shows that JA signal pathway is infected in A.altrenata in JA signal pathway
It plays an important role during ' honey pot '.
Ethylene is one of plant growth regulating substance of plant hormone, in terms of plant is resisted
It plays a significant role.Analysis is found, in the case where A.altrenata infects, multiple gene expressions of ' July is celestial ' Ethylene Signal access
It changes, including 2 regulation CTR1 (Constitutive Triple Response 1) genes (1 is raised 2.5 times, and one
It is a to lower 2.3 times), 2 EIN3 (Ethylene insensitive 3) genes (raising 2.9,2.8 times respectively), 3 ETR1
(Ethylene receptor 1) gene (raises 4.4,2.9,1.6) respectively, 2 ERF (Ethylene response
Factor) gene (raising 3.0,2.5 times respectively).Under A.altrenata induction, ethylene pathway in ' July is fresh ' date fruit
Multiple gene expressions change, and which imply ethylene signaling pathways to take part in date fruit to alternaria bacterium A.altrenata
Response process.
Resistance signal's approach that BRs is mediated: rape element sterol (BRs) has a variety of physiological effects, and plant can be promoted thin
The elongation and division of born of the same parents enhances tolerance of the plant in environment stress (Chen Quan helps 2013).A.altrenata infects jujube fruit
After reality, related gene expression changes in rape element sterol (BRs) signal pathway, and analysis is found, 1 regulation BSK1 gene
(up-regulation 1.8 times), 1 BAK1 gene (2.2 times of up-regulation), 1 TCH4 gene (2.5 times of up-regulation), CYCD3 gene (under
Adjust 4.7 times).BSK1 and BAK1 is BRI1 high receptor associated kinase family member, is interacted with BRI1, by plant BRs
Signal from extracellular to transduction intracellular, regulating growth of plants and it is disease-resistant between equilibrium process in play an important role
(Albrecht C et al.2012).Under alternaria bacterium A.altrenata induction,;July is fresh ' way BRs in date fruit
The multiple gene expressions of diameter change, and which imply BRs signal pathways to take part in date fruit to alternaria bacterium
The response process of A.altrenata.
Resistance signal's approach that PYR/PYL and Auxins is mediated: during the growth and development of plant, auxin
(auxin, IAA) plays important adjustment effect.The effect of auxin shows many aspects, most importantly molecular level
Effect, most of mechanisms are originated or are mediated by the gene expression of auxin regulation.A.altrenata infects
After date fruit, related gene expression changes in growth signals approach, and analysis is found, the gene of 2 regulation Aux/IAA (divides
Shang Tiao not be 2.6,1.6 times), the gene (raising 2.0,1.9 times respectively) of 2 regulation GH3, gene (one of 2 regulation SAUR
Up-regulation 1.7, one are lowered 2.8 times), they are auxin early stage responsive genes.In the case where A.altrenata infects, ' July
It is fresh ' multiple gene expressions of abscisic acid signal path change, including 1 regulation PYR/PYL (Pyrabatin
Riesistance/Pyrabatin Riesistance) gene (up-regulation 1.4 times), 5 regulation PP2C (Protein
Phosphatase, 2C) gene (respectively raise 2.7,2.0,2.5,1.8,2.6 times), 2 regulation SnRK2 (Serine/
Threonine-protein kinase SRK2I) gene (up-regulation 2.7 times, lower 2.4 times), 1 regulation ABF
The gene (1.6 times of up-regulation) of (ethylene response factor).In addition A.altrenata infects under date fruit,
It also found in MAPK signaling pathway signal transduction pathway 2 WRKY33 genes (raising 2.0,2.4 times respectively), 3
(1 is raised 2.3,2 to the gene of a respiratory burst oxidizing ferment (Respiratory Burst OxidaseHomolog, RBOH)
Lower 2.0 times).
Glutathione metabolism approach related gene: glutathione (glutathione, GSH) is widely present in plant,
It is to play an important role in plant against environmental stress.Meanwhile glutathione is also maintained in tissue anti-oxidation characteristics and is gone back to oxidation
Key effect is played in the adjusting of former sensitive signal transduction, most effective active oxygen is clear in glutathione or plant
One of clean dose, the peroxide generated when can effectively remove plant own metabolism and environment-stress (Edwards R et
al.2000).Correlative study thinks that GSH level content is related to the patience of environment-stress to it in plant, when plant by
When to environment-stress, the synthetic quantity of glutathione is added to increase, to improve self resistance.The study found that in A.altrenata
Infecting under, date fruit GSH-PX activity Metabolism-Related Genes Expression is changed, including regulation 10 regulation glutathione
The related gene (1.9,1.8,2.6,2.3,2.5,1.7,2.7,2.6,2.1,1.6) of s- transferase (GST) and 1 regulation are anti-
The gene (3.5 times of up-regulation) of bad hematic acid peroxidase (Ascorbate peroxidase, APX), a regulation spermidine close
At the gene (1.8 times of up-regulated expression) of enzyme (Spermidine synthase 2, SPDS), a regulation G-6-P 1-
The gene (lowering 2.2 times) of dehydrogenase (glucose-6-phosphate 1-dehydrogenase, GDH), a regulation core
Riboside diphosphonic acid reductase large subunit (ribonucleoside-diphosphate reductase large subunit,
RRM1 gene (lowering 1.6 times)).
Flavonoids biosynthesis and related gene: flavonoids is a kind of important secondary metabolite, biosynthesis way
Diameter is generally existing in plant, has and removes all kinds of active oxygen radicals that plant intracellular metabolite generates, and enhances peroxide
The functions such as the activity of mutase (SOD), in terms of phytomicroorganism signal transduction, plant are resisted from it is important
Effect, is one of three big protective plant protecting agents.The study found that under the infecting of A.altrenata, flavonoids biosynthesis in date fruit
Related gene expression is changed, including 1 regulation naringenin 3- dioxygenase (Naringenin, 2-oxoglutarate
3-dioxygenase, ODD) gene (up-regulation 2.6 times), the genes of 3 regulations CHS (Chalcone synthase) are (respectively
3.0,2.6,3.5 times of up-regulation), the gene (5.6 times of up-regulation) of 1 regulation CHI (Chalcone isomerase), 2 FLS
(Flavonol synthase) (raises 5.9,2.3 times) respectively, 2 LDOX (Leucoanthocyanidin
Dioxygenase) (1.9,1.7 times are raised respectively), 1 DFR (Dihydroflavonol reductase) (up-regulation 2.2
Times), 1 cinnamic acid -4- hydroxylase (Trans-cinnamate 4-monooxygenase, C4H) (2.1 times of up-regulation), one
A caffeoyl coenzyme A oxygen transmethylase (Caffeoyl CoA O-methyltransferase, CCoAOMT) (up-regulation 2.0
Times).
Fatty acid synthesis pathway and related gene: fatty acid is the main component of cell biological film rouge, wide in plant
General presence.Fatty acid plays a significant role when plant resists stress from outside.In addition, some fatty acid are also used as signaling molecule
The signal transduction process for participating in plant hormone ethylene, auxin etc. plays active role in plant defense signal path.It grinds
Hair is studied carefully under the infecting in A.altrenata, and fatty acid synthesis pathway related gene expression is changed in date fruit, packet
Include (the up-regulation 1.5 of 1 β -one acyl-acyl carrier protein reductase (β-ketoacyl-ACP reductase, FabG) gene
Times), 1 stearoylketene carrier protein desaturase enzyme 1 (StearoyI-ACF desaturase, Fab1) gene (lowering 1.4 times), 3
It is a (long-chain acyl CoA synzyme (Long-chain acyl-CoA synthetase, ACSL) gene (respectively lower 2.4,
2.0,1.6 times), 2 acetyl-acetyl carrier protein thioester enzyme (Acetyl-fatty acyl carrier protein
Thioesterase, FATB) gene (raising 2.7,2.4 times respectively), 3 acyl groups-acyl carrier protein desaturase
(Plastid acyl-ACP desaturase, FAB2) gene (raises 2.3,2.0,1.7 times) respectively.
Disease-resistant gene tissue expression specificity analyzes in ' honey pot ' date fruit genome: the present invention is from disease-resistant material ' bee
Honey jar ' in differential gene in screen disease-resistant related gene, including extension albumen (expansin-A1), GMP synthase (GMP
Synthase), aquaporin (Aquaporin NIP3-1), transcription factor MYB21 (Transcription factor
MYB 21), purple acid phosphatase (Purple acid phosphatase 17), ill-resistant protein RPM1 (Disease
Resistance protein RPM1), 2 (Glucose-6-phosphate/ of G-6-P/phosphate transporter
Phosphate translocator 2), transcription factor bHLH30 (Transcription factor bHLH30), amino acid
Permease (Amino acid permease), protein N RT1/PTR protein family (NRT1/PTR FAMILY), CCCH
Zinc finger domain albumen (Zinc finger CCCH domain-containing protein 18), sulfate transporter
(Sulfate transporter 3.3), Zinc transporter (Zinc transporter 1), wherein GMP synthase (GMP
Synthase), transcription factor bHLH30 (Transcription factor bHLH30), G-6-P/phosphoric acid transhipment
Albumen 2 (Glucose-6-phosphate/phosphate translocator 2), purple acid phosphatase (Purple
Acid phosphatase 17), CCCH Zinc finger domain albumen (Zinc finger CCCH domain-containing
Protein 18), Zinc transporter (Zinc transporter 1) in sample ' July is fresh ' also simultaneously up-regulated expression.It adopts
These disease-resistant verifyings for carrying out next step with real-time fluorescence quantitative RT-PCR to date fruit, excavate participate in disease resistance response when
Between and the differential expression situation in disease-resistant and susceptible material.
The verifying of real time fluorescent quantitative: being detected shown in its expression analysis by opposite real time fluorescent quantitative, as a whole, choosing
The 13 gene real time fluorescent quantitative testing results and Illumina sequencing result trend taken are generally consistent, but between the two
Differential gene expression multiple there are deviation, reason may be Illumina sequencing technologies compared with the high sensitivity that Q-PCR is analyzed
(Hoen PA et aj.2008), it is thus regarded that Illumina sequencing result is reliable.The nonshrink fruit disease related gene of jujube
It is as shown in Figure 8 that qReal Time-PCR analyzes result.
It is being inoculated with to further study to the related disease-resistant gene of differential expression in verifying ' honey pot ' date fruit
Specific situation of change between the 0-5d of A.altrenata, we pick out the gene (GMP of 13 with the expression of disease-resistant relevant difference
synthase、 Transcription factor MYB21、Glucose-6-phosphate/phosphate
translocator 2、Expansin-A1、Purple acid phosphatase 17、Transcription factor
bHLH30、Amino acid permease、Zinc finger CCCH domain-containing protein、Zinc
transporter 1、Sulfate transporter、Disease resistance protein RPM1、Aquaporin
NIP3-1, Protein NRT1/PTR FAMILY 5.1), pass through the expression mould of opposite Real time PCR in time
Formula.GMP synthase,Expansin-A1,Sulfate transporter,Disease resistance protein
RPM1, Zinc finger CCCH domain-containing protein gene expression quantity under the different vaccination time are first
Then up-regulation is being lowered, and be higher than susceptible strain in nonshrink fruit disease diseased plant system's expression quantity；
For Transcription factor MYB21 in 0-1d, the expression quantity in Resistant variants is higher than susceptible strain,
And expression quantity reaches peak value in 3d, and expression quantity reaches highest when in disease plant to 4d；
In disease plant the expression quantity of Glucose-6-phosphate/phosphate translocator 2 first on
Decline after rising, is to reach peak value in 2d；The expression quantity of Purple acid phosphatase 17 3d reach highest and
Expression quantity is above disease-resistant plant later；
Transcription factor bHLH30, Protein NRT1/PTR FAMILY 5.1 with the time extension
Expression quantity gradually rises, and Transcription factor bHLH30, Protein NRT1/PTR FAMILY 5.1 are susceptible
Then in downward trend after first rising in plant.
Experimental result:
In ' honey pot ' significant difference gene, sulfate transporter (sulfate transporter 3.3), GAM
Synzyme (GMP synthase) extends albumen (Expansin-A1), amino acid permease (Amino acid permease),
CCCH Zinc finger domain albumen (Zinc finger CCCH domain-containing protein 18), ill-resistant protein
(Disease resistance protein RPM1), protein N RT1/PTR family (Protein NRT1/PTR FAMILY
5.1) gene expression is raised, and expression is lowered in ' July is fresh ', and other genes such as calbindin (calcium-
Binding protein), chlorophyll a-b combines egg (Chlorophyll a-b binding protein) gibberellin 2- β-
The obviously expression up-regulation in ' honey pot ' of the genes such as dioxygenase (Gibberellin 2-beta-dioxygenase 8),
Do not occur in the difference expression gene of ' July is fresh ', illustrates that alternaria bacterium induces these lower genes to take part in jujube to pathogen
Defense reaction, and have outstanding performance in Resistant variants.Transcription factor MYB21 (transcription factor
MYB21), G-6-P/phosphate transporter (Glucose-6-phosphate/phosphate translocator
2), purple acid phosphatase 17 (Purple acid phosphatase 17), (the Zinc transporter of Zinc transporter 1
1), the genes such as aquaporin NIP3-1 (Aquaporin NIP3-1) express up-regulation in two kinds, illustrate these genes
Take part in the acknowledgement mechanism that alternaria bacterium induces lower date fruit.
And real time fluorescent quantitative the results show that disease-resistant variety and sense and strain screening 13 genes with inoculation time
Extend, expression way is different, and this difference may cause jujube fruit difference disease-resistant to fruit-shrink disease and susceptible, expression
The difference of amount illustrates that they are likely to take part in jujube fruit to the defense mechanism of fruit-shrink disease.
For extending albumen, when plant is by pathogen infection, cell wall structure changes to prevent or limit
Pathogen infection and propagation (Xu Xiao etc. 2010).Fruit gradually increases the sensibility of pathogen with maturity, mature
Fruit defends the ability of pathogen weaker relative to green fruit.In ripening of fruits, cell wall peptidoglycan network
Structure rearranges the induction that will affect plant to pathogen.Albumen, which is extended, as Cell wall loosening agent has also assisted in plant
To the response process of biotic, disease-resistant process is carried out by the expression of regulation extension protein gene, does not depend on traditional water
The signal transduction path of poplar acid and jasmonic and work, ability (the Cantu et of plant resistant pathogen may be improved
al.2009)。
Plant hormone is that a kind of organic substance that plant itself generates plays important tune in Plant defense responses
Control effect.Auxins (Auxins), gibberellin class (Gibberenllins, GA), cytokinin (Cytokinins,
CK), ethylene (Ethylene), abscisic acid (Abscisic acid, ABA), brassinosteroid (Brassinosteroids,
BRs the six major class endogenous hormones of plant) are acknowledged as.In addition, correlative study in recent years shows jasmonic (Jasmonic
Acid, JAS), salicylic acid (Salicylates, SA) etc. be also the major signaling molecule for inducing plant to generate defense reaction
(Vlot A C,2009)。
Salicylic acid (salicylic acid, SA) is the intracorporal endogenous phenolic compound of one kind of plant, and it is a variety of to participate in plant
Metabolic regulation process is the important participant during plant defense pathogen infection, by the plant infected, SA level liter
It is high to generate allergic reaction (Hypersensitive response, HR) and systemic acquired resistance to induce
(Systemic acquired resistance, SAR) (Durner J et al.1997).Resistance signal's transduction that SA is mediated
Approach is by the regulation of multiple genes, and NPR1 is the key gene that the downstream SA is acted in SAR signal transduction pathway, and NPR1 is logical
It crosses with transcription factor TGA interaction and activates a series of expression of SAR genes, plant is finally made to generate disease resistance (Mou Z et
al.2003).Studies have shown that NPR1 gene participates in different disease-resistant approach during disease resistance of plant obtains, not only it is situated between in SA
The SAR led, and important regulating and controlling effect is played in the ISR that JA is mediated is generated,
Jasmonic (Jasmonic acid, JA) is to be widely present the intracorporal signaling molecule of plant, is the growth for adjusting plant
The important compound of development, when plant is by pathogen infection, JAs biosynthesis related genes up-regulated expression, to mediate
The defense reaction of plant.GIFY gene is sent out in jasmonic (JA) signal transduction path, plant growth and development and pathogen response
Wave important function (Zhu D et al.2014).
ETR1 is one kind of ethylene receptor family, has histidine kinase activity.CTR1 is in ethylene signaling access
In play the factor (Zhang Cunli etc. 2012) of negative regulation.EIN3 is nucleoprotein specific to plant, in endonuclear ethylene
The downstream of signal transduction plays a crucial role, and is the positive regulating factor (Chao Q et.al.1997) of ethylene reaction.
TCH4 is a kind of Xyloglucan endotransglycosylase (xyloglucan endotransglycosylase, XET),
By being catalyzed the hydrolysis of xyloglucan molecule and the relaxation and reconstruction of reconnection mediated plant cell wall, studies have shown that XET energy
Enough respond the stimulation (Lu Wangjin etc. 2004) of a variety of external environments.
Aux/IAA is a transcription inhibitory factor, includes 4 conserved domains.It is free when auxin concentration is lower
Aux/IAA with auxin response factor (auxin response factor, ARF) formed heterodimer, inhibit auxin
The expression of responsive genes；When auxin concentration increases, auxin will transport inhibiting factor 1 with growth hormone receptor
(transport inhibitor response 1/auxin-binding F-box protein, TIR1/AFB) albumen knot
It closes, causes Aux/IAA ubiquitination and be degraded, ARF is released, to promote the expression (Okushima of auxin responsive genes
Y et al.2005；Kalluri U C et al.2007).GH3 gene family encodes a kind of acyl acid amide synthetase, can promote
Amino acid and IAA, jasmonic (jasmonic acid, JA) and salicylic acid (salicylic acid, SA) are combined, and change them
The concentration of biologically active form in the cell, to regulate and control the growth of plant, development and defense reaction (Park JE et
al.2007).SAUR gene family is that plant is distinctive, is a maximum family in auxin response factor, can be in life
The early stage of long element induction responds (Yang T et al.2000).
PYR/PYL/RCAR is distributed in cytoplasm and core, is the receptor protein of ABA.PYR/PYL/RCAR is in ABA letter
The upstream of number access has the function of identifying ABA signal and enabling signal transduction originally process (Zhang great Peng, 2011).PP2C is
A kind of important serine/threonine residue protein phosphatase, PP2C albuminoid phosphatase have unique tactic pattern:
The N-terminal of protein or the catalysis region of C-terminal have 11 conservative structure subprovinces (Bork P et ai.1996), and these are conservative
Region it is related with intracellular signal transduction.A large number of studies show that PP2C is negativity crucial in ABA signal transduction pathway
Regulatory factor (Pullen K E et al.2004).SnRK is to be widely present in the intracorporal a kind of serine/threonine of plant
(Ser/Thr) albuminoid kinases is divided into 3 subfamilies of SnRK1, SnRK2 and SnRK3, and wherein SnRK2 is that plant is distinctive
A kind of Ser/Thr albuminoid kinases, and SnRK2-AREB/ABF regulatory pathway is considered responding non-life by ABA in plant
Play the role of vital (Fujita Y et al.2009) in object stress.ABA participates in each of regulating growth of plants
Stage is one of hormone mostly important in plant.As " adverse circumstance hormone ", ABA copes with various environment-stress processes in plant
In also play key effect.Research shows that PYR/PYL/RCAR energy specific bond ABA, and positive can adjust ABA signal and pass
Approach is led, the physiological responses of ABA in plant are caused.
WRKY33 may also participate in the reaction that plant resists fungi, and WRKY33 mutant increases the sensibility and drop to fungi
Plant defense protein gene (Plant defensin, the PDF1.2) expression of low jasmonic regulation, and high expression WRKY33 is then
Improve the fungi disease resistance (Zheng Z et al.2006) of plant.
In glutathione synthesis approach, it is that GSH metabolic pathway is most important that GST, which is a kind of changeable protein families,
One of enzyme, play an important role (Edwards R et al.2000 in terms of maintaining plant metabolism, participating in；
Wagner U et al.2002), and the enhancing of GST expression has been considered to be plant to the important symbol of stress response it
One (Edwards R et al.2000).
In flavonoids route of synthesis, enzyme, namely chalcone isomerase (Chalcone isomerase, CHI) catalysis chalcone is formed
Naringenin enters downstream route of synthesis as important metabolite, forms various flavone compounds.Chalcone synthase
(Chalcone synthase, CHS) is the key enzyme of flavonoids route of synthesis early stage, the special construction and function base of CHS gene
Group, influences flavones, the synthesis of isoflavones etc., so that the physiology course of plant is influenced, in plant defense stress from outside, auxin
Transport etc. play an important role；Flavonols synzyme (Flavonol synthase, FLS) is flavonoids route of synthesis
Direct regulation and control enzyme is the key gene of flavonoids metabolic pathway of synthesizing.Flavonols reductase (Dihydroflavonol
Reductase, DFR) be flavonoids route of synthesis relatively after the stage key enzyme.
The present invention is by studying anti-sense kind ' honey pot ', ' July is celestial ' in alternaria bacterium using transcript profile sequencing technologies
The transcriptome differences infected between the processing control of rear and its clear water obtain a large amount of difference table through sequence assembling and gene annotation
Up to Unigene sequence and its biological pathways from the horizontal overall study jujube disease resistance mechanisms of transcript profile, while finding and jujube fruit-shrink disease
The relevant important gene of resistance and biological pathways, the studies above for disease-resistant related gene separation and utilize genetic engineering
Technology carries out genetic improvement to date fruit and is of great significance.Sequencing result extreme enrichment jujube transcript profile data resource, it is convenient
The disease-resistant molecule mechanism of jujube is further inquired into, is the element task of the follow-up studies such as screening and clone's disease-resistant gene.
The anti-sense fruit-shrink disease transcriptome analysis of date fruit: being sequenced using 2000 both-end of IIIumina HiSeqTM, carries out De
Novo assembling and data analysing method, obtain 50676507 in this 4 samples of R1, R2, S1, S2 respectively, 51136789,
49090715 and 49752767 initial data carry out low quality data using Cutadapt (version 1.9.1) software
Depollution and joint sequence are filtered and gone, filters out 48676507,49136789,47090715 and 47752767 height respectively
Mass-sequential.
The differential gene obtained by transcript profile sequencing:
(1) it is sequenced by transcript profile and carries out differential gene analysis using DESeq2 software, with FDR < 0.05 He ∣
The ∣ of log2FC≤2 carries out the screening of significant difference gene, finds sample R1VS R2 difference base up-regulated expression gene 39, lowers
Expressing gene 8.The up-regulated expression gene 17 of S1-VS-S2 54 lowers expressing gene 603.These differential genes are being planted
Object by pathogen infection during play crucial adjustment effect.
(2) the enrichment analysis for carrying out GO function to the transcript of significant difference expression is found, ' honey pot ' anti-jujube fruit-shrink disease
(R1vs R2) significant difference express transcript profile in transcript by significant enrichment to 9 terms, wherein in biological
That process is most enriched with is localization, and that be most enriched in Cellular component is cell par, and
Difference expression gene in molecular function is most enriched in binding.In ' July is celestial ' sense fruit-shrink disease disease (S1vs
S2) transcript that significant difference is expressed in transcript profile is enriched to 31 terms, and wherein biological process is most rich
Collection is metabolic process, and that be most enriched in Cellular component is cell par, in molecular
Significant difference expressing gene in function is most enriched in catalytic activity.
(3) analysis is found in the metabolic pathway that A.altrenata infects ' July is celestial ', can pass through glutathione generation
It thanks, fatty acid synthesis and metabolic pathway, flavonoids synthesis, cysteine and Methionine metabolism, JA signal, rape element sterol
(BRs), bigcatkin willow acid signal, ethylene, etc. in approach the expression regulation of key gene resist infecting for pathogen.
It (4) include extension albumen in the significant difference gene that A.altrenata infects in disease-resistant material ' honey pot '
(expansin-A1), GMP synthase (GMP synthase), aquaporin (Aquaporin NIP3-1), transcription factor
MYB21 (Transcription factor MYB 21), purple acid phosphatase (Purple acid phosphatase
17), ill-resistant protein RPM1 (disease resistance protein RPM1), G-6-P/phosphate transporter
2 (Glucose-6-phosphate/phosphate translocator 2), transcription factor bHLH30 (Transcription
Factor bHLH30), amino acid permease (Amino acid permease), CCCH Zinc finger domain albumen (Zinc
Finger CCCH domain-containing protein 18), sulfate transporter (Sulfate transporter
3.3), the disease-resistant genes such as Zinc transporter (Zinc transporter 1).These genes relevant to anti-jujube fruit-shrink disease carry out
Real time PCR under the different vaccination time, as a result, it has been found that in high disease-resistant plant and disease plant this 13 genes with
The extension of inoculation time, expression way is different, and it is disease-resistant to fruit-shrink disease and susceptible that this difference may cause jujube
Difference.
Place, those skilled in the art can not select from the prior art to the greatest extent in the embodiment of the present invention.
Disclosed above is only a specific embodiment of the invention, but scope of protection of the present invention is not limited thereto, is appointed
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with above-mentioned scope of protection of the claims
It is quasi-.
Claims (8)
1. transcribing application of the sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene.
2. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as described in claim 1, which is characterized in that
The jujube fruit-shrink disease is caused by the dip dyeing of alternaria bacterium.
3. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as described in claim 1, which is characterized in that
The kind of the jujube is that the July of the honey pot and sense fruit-shrink disease of nonshrink fruit disease is fresh.
4. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as described in claim 1, which is characterized in that
The transcription sequencing technologies include preprocessing process, transcript profile sequencing procedure and data handling procedure.
5. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as claimed in claim 4, which is characterized in that
The preprocessing process includes: to extract jujube total serum IgE, detection total serum IgE quality, synthesis cDNA library and real-time fluorescence quantitative PCR to test
Card.
6. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as claimed in claim 5, which is characterized in that
It is described synthesis cDNA library detailed process include:
It repairs end: repairing complex enzyme reaction system with set terminal, thermophilic reaction a period of time carries out end in Thermomixer
End is repaired, and is repaired product with kit and is carried out purification and recovery；
3 end ˊ cDNA adds A: under the action of polymerase systems, making reparation product obtained in the previous step in 3 ends ˊ plus A alkali
Base is prepared for the connection of next step connector；A product is added to carry out purification and recovery with kit；
The connection of Adapter: configuration ligase reaction system, thermophilic reaction a period of time makes Adapter in Thermomixer
With add A product to connect；Product after connection Adapter carries out purification and recovery with kit；
The glue recovery purifying of connection product: preparing Ago-Gel, carries out electrophoresis to the connection product of previous step；After glue is cut in completion
Remaining blob of viscose is taken pictures in gel imaging system and stores picture；Determine that there is no problem rear abandons remaining gel blob of viscose；
The blob of viscose cut carries out purification and recovery with kit；
PCR reaction and product purification: configuration PCR reaction system expands the gel extraction product of previous step；Reaction terminates
Electrophoresis is carried out to PCR reaction product afterwards, cuts glue, and carry out purification and recovery with kit, recovery product is dissolved in right amount
In Elutionbuffer, printed label is attached on centrifugation tube wall, and so far library preparation is completed；
Upper machine sequencing: the c DNA library built is carried out tentatively quantitative and dilutes text by library inspection and the sequencing of upper machine with Qubit2.0
Library value 1ng/ μ L detects c DNA library insert size using Agilent2100, after meeting demand, with q RT-PCR couple
Library effective concentration carries out accurate quantitative analysis to guarantee Library Quality；After library inspection is qualified, library will not had to according to effective concentration and mesh
It marks under table after the demand pooling of machine data volume, carries out PE125 sequencing using Illumina Hi Seq TM2500 microarray dataset
Analysis；
Gene expression profile data analysis: by IIIumina HiSeqTM 2000 be sequenced resulting data be known as raw reads or
Rawdata then carries out Quality Control to raw reads, to determine whether sequencing data is suitable for subsequent analysis；After Quality Control, pass through
Filter obtains high quality clean reads, after being compared using the library Hisat2 platform construction unigene and jujube genomic data, adopts
Differential expression analysis is carried out after eliminating biological variation with DESeq method；Sequencing data is standardized using the method for RPKM；Into
Row gene expression, gene structure optimization, alternative splicing, the prediction of new transcript and annotation, SNV are tested and analyzed, and to all differences
Expressing gene is compared with GO database and KEGG database, carries out Gene Ontology functional analysis and KEGG respectively
Pathway enrichment analysis.
7. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as claimed in claim 4, which is characterized in that
The detailed process of the real-time fluorescence quantitative PCR verifying are as follows:
RNA is extracted and cDNA synthesis: date fruit total serum IgE uses Ta Ka Ra Mini BEST Plant RNA Extraction
Kit kit extracts, and it is 200ng that reverse transcription, which synthesizes c DNA total amount, and used kit is Ta Ka Ra Prime ScriptTM
RT reagent Kit with gDNA Eraser；
Quantitative fluorescence analysis: q RT-PCR verifying is carried out to difference expression gene based on SYBR method, reference gene used is ACT1
Gene；10 μ L:SYBR Premix Ex Taq of PCR reaction system II 5.0 μ L, positive each 0.4 μ L of anti-primer, 1.0 μ L of DNA profiling,
3.2 μ L of water.Reaction condition are as follows: 95 DEG C of 30s, 95 DEG C of 5s, 58 DEG C of 30min, 72 DEG C of 30s, totally 40 recycle；Wherein, the primer
Gene I/D accession number be Zj.jz107428959, Zj.jz 107419914, Zj.jz107429464, Zj.jz107420088,
Zj.jz107417234、Zj.jz107413980、Zj.jz107420411、Zj.jz107426176、Zj.jz107432190、
Zj.jz107429308、Zj.jz107433466、Zj.jz107434596、Zj.jz107419438。
8. application of the transcription sequencing technologies in screening jujube fruit-shrink disease disease-resistant gene as described in claim 1, which is characterized in that
The jujube fruit-shrink disease disease-resistant gene includes sulfate transporter, GAM synzyme, extension albumen, amino acid permease, CCCH zinc
Finger domain albumen, ill-resistant protein, protein N RT1/PTR family, calbindin, chlorophyll a-b combine egg and gibberellin
2- β-dioxygenase.
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| CN112553223A (en) * | 2020-12-30 | 2021-03-26 | 浙江省农业科学院 | Gene SlBSK1 participating in regulation and control of tomato fruit tip type and lycopene synthesis and application thereof |
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| CN103981137A (en) * | 2014-05-23 | 2014-08-13 | 北京林业大学 | Bacterial strain antagonistic to pathogenic bacteria of jujube fruit shrink disease and application of bacterial strain |
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| CN103981137A (en) * | 2014-05-23 | 2014-08-13 | 北京林业大学 | Bacterial strain antagonistic to pathogenic bacteria of jujube fruit shrink disease and application of bacterial strain |
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| 焦小利: "细交链孢菌诱导下不同品种枣抗病基因的筛选", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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| CN112553223A (en) * | 2020-12-30 | 2021-03-26 | 浙江省农业科学院 | Gene SlBSK1 participating in regulation and control of tomato fruit tip type and lycopene synthesis and application thereof |
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