CN106244601A - Rice bacterial blight resistance and bacterial stripe ospc gene OsHsp18.0 CI and application thereof - Google Patents
Rice bacterial blight resistance and bacterial stripe ospc gene OsHsp18.0 CI and application thereof Download PDFInfo
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Abstract
The present invention relates to plant biotechnology field, it is specifically related to functional verification and the application of a kind of DNA fragmentation including paddy disease-resistant related gene OsHsp18.0 CI, strong promoter is specifically utilized to drive and transgenic technology based on RNA interference principle, overexpression carrier and the RNAi carrier of OsHsp18.0 CI gene are proceeded to the expression of OsHsp18.0 CI gene in rice varieties sage rice 806, excess and suppression Oryza sativa L..The resistance of bacterial stripe and bacterial leaf-blight is remarkably reinforced by the genetic transformation Oryza sativa L. that OsHsp18.0 CI gene expression amount significantly improves, it was demonstrated that OsHsp18.0 CI gene plays a significant role in Rice Resistance bacterial stripe and bacterial leaf-blight.
Description
Technical field
The present invention relates to field of plant genetic, it is provided that a rice bacterial blight resistance and bacterial stripe
Gene OsHsp18.0-CI and application thereof, the genetic transformation Oryza sativa L. that this gene expression amount significantly improves is to bacterial stripe and white
The resistance of leaf spoting bacteria is remarkably reinforced.
Background technology
Plant, during growth, is encroached on by multiple pathogen.The phytopathy original of a great variety, including disease
Poison, antibacterial, fungus and nematicide etc..Pathogen invaded plants causes two kinds of results: (1) pathogen is successfully numerous in host plant
Grow, cause associated conditions；(2) host plant produces disease resistance response, kills pathogen or stops it to grow.Resistant gene is utilized to provide
The disease resistance of source improvement plant, protects again the fundamental solution of environment while of being pre-disease prevention.
The disease resistance response of plant is the complex process that polygenes participates in regulation and control.The gene of involved in plant disease resistance response is divided into two
Class: (1) disease-resistant gene, also known as R (resistance) gene and (2) disease-resistant related gene.
According to the current people understanding to disease-resistant gene function, the product of this genoid primarily as receptor, directly or
Indirectly interact with pathogenic proteins, defense signaling path in startup plant (Jones and Dangl, 2006,
Nature444:323-329；Zipfel, 2014, Trends in immunology, 35:345-351).Disease-resistant gene mediation
Disease resistance response is strong, is good genetic resources.But due to following reason, make to utilize disease-resistant gene improvement plant resistance to environment stress to be limited
System: the resource-constrained of (1) disease-resistant gene；(2) disease-resistant gene has cause of disease kind and cause of disease biological strain specificity, disease-resistant scope
Limited；(3) because the rapid mutation of cause of disease, just lose after the most several years of the effect of a disease-resistant gene or more than ten years.
Disease-resistant related gene refers to the gene of all participation disease resistance responses in addition to disease-resistant gene, and their coded product participates in
Resistance signal's molecule, participation signal conduction or participation defense response etc. in synthesis plant.The common feature of this genoid is disease
After former induction, their expression raises or reduces, the difference of the expression of gene before and after therefore people can induce according to cause of disease
Plant identification disease-resistant related gene (Maleck etc., 2000, Nature Genet.26:403-410 on a large scale；Schenk etc.,
2000, Proc.Natl.Acad.Sci.USA 97:11655-11660；Zhou etc., 2002, Science in China 45:
449-467).At present, people are limited to the understanding of disease-resistant related gene.According to it has been reported that most disease-resistant related gene
Resistance capacity during independent role may be less than disease-resistant gene.But according to following reason, they are the genes being worth Devoting Major Efforts To Developing
Resource: (1) need not directly due to the product of most disease-resistant related genes and pathogen interacts, and this genoid is tool
There is the genetic resources of durable resistance；(2) disease resistance response that most of disease-resistant related genes participate in does not has a cause of disease specificity, therefore it
Be the genetic resources with resistance of wide spectrum；(3) aboundresources of this genoid.But, although Oryza sativa L. identifying a lot of anti-
Sick related gene (Zhou etc., 2002, Science in China Series C-Life Sciences 45:449-467；Chu
Deng, 2004, Molecular Genetics and Genomics 271:111-120), these genes are in paddy disease-resistant reacts
The mechanism of action and single disease-resistant related gene the change of paddy disease-resistant phenotype whether can be caused the most unclear.
Oryza sativa L. be the most important cereal crops, bacterial leaf-blight and bacterial stripe be in Rice Production two kinds important
Bacterial disease, drastically influence the yield and quality of Rice Production.Therefore, understand the pathogenesis of disease, contribute to profit
By the resistance of high effective way improvement rice varieties, control the generation of disease, loss that plant disease brought is reduced or avoided.Point
It is the premise to paddy disease-resistant study mechanism from clone's disease-resistant related gene.Report at present and cloned at least 37 white leaves of Oryza sativa L.
The main effect disease-resistant gene of rot, but these disease-resistant genes are in addition to Xa21, the most only the biological strain of minority had resistance, anti-spectrum
Narrower.In Oryza sativa L., be not yet cloned at present the main effect disease-resistant gene of bacterial stripe, only maize seed be cloned into one non-
Host disease-resistant gene Rxo1 (Zhao etc., 2005, Proc.Nati.Acad.Sci USA 102:15383-15388).Existing examination
Testing result to show, disease-resistant related gene can participate in the signal path of plant immune system, by activating or strengthening immunity
Signal improves anti-xanthomonas oryzae pv. oryzicola gene DEPG1, OsWRKY45-in the resistance of plant for disease, such as Oryza sativa L.
1, OsPGIP4 (Guo etc., 2012, Mol.Biol.rep.39:3491-3504；Tao etc., 2009, Plant Physiol.151:
936-948；Feng etc., 2016, Planta 243:1297-1308) etc..It addition, compared with the application of disease-resistant gene, disease-resistant phase
The application of correlation gene is provided that plant more wide spectrum and long-acting resistance, even provides resistance such as overexpression to multiple diseases
Os2H16 improve Oryza sativa L. to the resistance of bacterial leaf-blight and banded sclerotial blight (Li etc., 2013, Plant Cel l Tiss.Org.115:
429-441).Changing of rice varieties is carried out as the function of the disease-resistant related gene of the positive regulatory factor of disease resistance response by excess
Good, will further enhance the disease resistance of plant, widen the anti-spectrum of plant.These aspects are to use conventional plant breeding and improvement skill
Art institute is inaccessiable.The clone the most how utilizing paddy disease-resistant gene obtain disease-resistant plant become problem demanding prompt solution it
One.
Summary of the invention
The present inventor is for the situation of above-mentioned prior art, it is provided that one clone and isolate from Oryza sativa L. anti-
The DNA fragmentation of sick related gene OsHsp18.0-CI complete coding region section, utilizes this improvement of genes Oryza sativa L. or other plant to support
The ability of imperial disease.The sequence of its gene is as shown in sequence table SEQ ID NO.1, or is approximately equivalent to SEQ ID NO.1
Shown DNA sequence, or its function is equivalent to the subfragrnent of sequence shown in SEQ ID NO.1.This gene fragment expression amount shows
Write the genetic transformation Oryza sativa L. improved the resistance of bacterial stripe and leaf spot bacteria is remarkably reinforced, it is seen that use overexpression
Plant can be given afterwards to by Xanthomonas campestris PV.oryzicola (Xanthomonas oryzae pv.oryzicola) and bacterial leaf-blight
Disease caused by bacterium (Xanthomonas oryzae pv.oryzae) produces disease resistance response, it is thus achieved that high disease-resistant plant.
Inventor provide firstly a disease-resistant related gene OsHsp18.0-CI cloned and isolated from Oryza sativa L. and completely compiles
The DNA fragmentation of code section, this fragment derives from the holy rice 806 of Oryza sativa L. (Oryza sativa), and it is expanded by the primer of particular design
Rice sage rice 806 genomic DNA of surging obtains, and wherein said primer includes primer 1,5 '-TGAGAATTGAGATCACCCTCTT-
3 ' its sequences as shown in sequence table SEQ ID NO.3, primer 2,5 '-GGACCAGATTTGACGCTTTTAT-3 ' its sequence such as sequence
Shown in list SEQ ID NO.4, the nucleotide sequence of the final gene obtained is as shown in sequence table SEQ ID NO.1, and it encodes
Aminoacid sequence as shown in sequence table SEQ ID NO.2.
Strong promoter is utilized to drive afterwards and transgenic based on RNA interference (RNA interference, RNAi) principle
Technology, proceeds to rice varieties sage rice 806, excess and suppression by overexpression carrier and the RNAi carrier of OsHsp18.0-CI gene
The expression of OsHsp18.0-CI gene in Oryza sativa L..The genetic transformation Oryza sativa L. that OsHsp18.0-CI gene expression amount significantly improves is to carefully
The resistance of bacterium property Population of Xanthomonas Oryzae Pv and leaf spot bacteria is remarkably reinforced, and expression significantly reduced genetic transformation Oryza sativa L. is to bacillary bar
The resistance of pinta bacterium and leaf spot bacteria substantially weakens, it was demonstrated that OsHsp18.0-CI gene plays weight in rice bacterial blight resistance
Act on.It is therefore evident that plant can be given to by Xanthomonas campestris PV.oryzicola (Xanthomonas after this gene of overexpression
Oryzae pv.oryzicola) and leaf spot bacteria (Xanthomonas oryzae pv.oryzae) caused by disease produce
Raw disease resistance response, it is thus achieved that high disease-resistant plant.Using this transgenic technology to create disease-resistant plants is that traditional breeding technology is not caned
Reach, why adopt with the aforedescribed process, proceed to susceptible plant mainly due to by the disease-resistant related gene of clone, help
In producing new disease-resistant plants.Particularly can be with genetic transfoumation cumulative multiple resistant genes in plant, without producing
With the linked gene group sequence occurred in raw traditional breeding technology.And the clone of disease-resistant related gene is to overcome traditional breeding method not
The premise of disease-resistant related gene problem is shifted between energy plant species.
In addition to utilizing gene provided by the present invention, it is also possible to the nucleotide sequence using its gene is the most suitable
In the DNA sequence shown in SEQ ID NO.1, or its function is equivalent to the subfragrnent of sequence shown in SEQ ID NO.1, namely
As long as saying that the genetic fragment possessing the DNA sequence shown in SEQ ID NO.1 all can obtain similar effect.
In sum, the present inventor provide first one clone and isolate from Oryza sativa L. disease-resistant
The DNA fragmentation of related gene complete coding region section, OsHsp18.0-CI, and demonstrate the function of this gene, utilize its function
Plant can be given to by Xanthomonas campestris PV.oryzicola (Xanthomonas oryzae after finding eventually to use overexpression
Pv.oryzicola) the disease generation and caused by leaf spot bacteria (Xanthomonas oryzae pv.oryzae) is disease-resistant instead
Should, it is thus achieved that high disease-resistant plant.
Accompanying drawing explanation
Fig. 1. by quantitative RT-PCR technology detection holy rice 806 inoculated bacteria Population of Xanthomonas Oryzae Pv RS105 and bacterial leaf-blight respectively
The expression pattern result schematic diagram of OsHsp18.0-CI gene after bacterium PXO99；
The expression of gene after black bar diagram represents inoculation RS105 in figure, that the bar diagram of Lycoperdon polymorphum Vitt represents is inoculation PXO99
The expression of rear gene, it can be seen that the testing result of sampling after inoculation 0h, 6h, 12h, 24h and 72h.With 0h for comparison, it is seen that
After inoculation RS105 12h and 24h, OsHsp18.0-CI gene expression is significantly improved compared with the control；After inoculation PXO99
6h, 12h and 24h OsHsp18.0-CI gene has significant up-regulated expression；
T is expressed in Fig. 2 .OsHsp18.0-CI gene overexpression and suppression0For genetic transformation plant inoculated bacteria streak
Pathogenic bacteria RS105 is result signal gray-scale map after two weeks；
In figure, " WT " is wild type sage rice 806；T is expressed in suppression0For genetic transformation plant (OsHsp18.0-CI RNAi)
The scab that inoculated bacteria Population of Xanthomonas Oryzae Pv RS105 was formed after two weeks is substantially than the length of holy rice 806 (comparison)；This gene overexpression
T0For the scab formed after transformed plant (OsHsp18.0-CI OE) inoculated bacteria Population of Xanthomonas Oryzae Pv RS105 two weeks substantially than
Sage's rice 806 (comparison) is short；
Fig. 3 .OsHsp18.0-CI gene overexpression plant OE-7T1For material inoculated bacteria Population of Xanthomonas Oryzae Pv RS105 two
All sequela result schematic diagrams；
In figure, vertical coordinate represents that each individual plant inoculates RS105 morbidity length after two weeks, and " WT " is that genetic transformation receptor is wild
Raw type sage rice 806, " " represents that, compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length pole significantly reduces (t
Inspection, P < 0.01), " " represents that compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length significantly reduces (t inspection
Test, P < 0.05), during in figure, in agarose gel photograph, band is each individual plant, the PCR amplification of hygromycin gene-Hpt is shown
Being intended to, can amplify Hpt gene is positive transformants material；
Fig. 4 .OsHsp18.0-CI gene overexpression plant OE-11T1For material inoculated bacteria Population of Xanthomonas Oryzae Pv RS105
Two weeks sequela result schematic diagrams；
In figure, vertical coordinate represents that each individual plant inoculates RS105 morbidity length after two weeks, and " WT " is that genetic transformation receptor is wild
Raw type sage rice 806, " " represents that, compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length pole significantly reduces (t
Inspection, P < 0.01), " " represents that compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length significantly reduces (t inspection
Test, P < 0.05), during in figure, in agarose gel photograph, band is each individual plant, the PCR amplification of hygromycin gene-Hpt is shown
Being intended to, can amplify Hpt gene is positive transformants material；
Fig. 5 .OsHsp18.0-CI gene inhibition expresses plant RNAi-9T1For material inoculated bacteria Population of Xanthomonas Oryzae Pv RS105
Two weeks sequela result schematic diagrams；
In figure, vertical coordinate represents that each individual plant inoculates RS105 morbidity length after two weeks, and " WT " is that genetic transformation receptor is wild
Raw type sage rice 806, " " represents that, compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length pole dramatically increases (t
Inspection, P < 0.01), " " represents that compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length dramatically increases (t inspection
Test, P < 0.05), during in figure, in agarose gel photograph, band is each individual plant, the PCR amplification of hygromycin gene-Hpt is shown
Being intended to, can amplify Hpt gene is positive transformants material；
Fig. 6 .OsHsp18.0-CI gene inhibition expresses plant RNAi-12T1For material inoculated bacteria Population of Xanthomonas Oryzae Pv
Two weeks sequela result schematic diagrams of RS105；
In figure, vertical coordinate represents that each individual plant inoculates RS105 morbidity length after two weeks, and " WT " is that genetic transformation receptor is wild
Raw type sage rice 806, " " represents that, compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length pole dramatically increases (t
Inspection, P < 0.01), " " represents that compared with the morbidity length of rice 806 holy with comparison, converting material morbidity length dramatically increases (t inspection
Test, P < 0.05), during in figure, in agarose gel photograph, band is each individual plant, the PCR amplification of hygromycin gene-Hpt is shown
Being intended to, can amplify Hpt gene is positive transformants material；
Fig. 7 .OsHsp18.0-CI gene inhibition and the transformed plant T of overexpression2Pickup kind Xanthomonas campestris PV.oryzicola
Two weeks sequela result schematic diagrams of RS105,
In figure, WT is wild type sage rice 806, and OE-7 is OsHsp18.0-CI gene overexpression plant OE-7T1For material,
OE-11 is OsHsp18.0-CI gene overexpression plant OE-11T1For material, RNAi-9 is OsHsp18.0-CI gene inhibition
Express plant RNAi-9T1For material, RNAi-12 is that OsHsp18.0-CI gene inhibition expresses plant RNAi-12T1For material；
Fig. 8 .OsHsp18.0-CI gene inhibition and the transformed plant T of overexpression2Pickup kind rice leaf spot bacteria
Two weeks sequela result signal gray-scale maps of PXO99；
Fig. 9 .OsHsp18.0-CI gene inhibition and the transformed plant T of overexpression2Pickup kind rice leaf spot bacteria
Two weeks sequela result schematic diagrams of PXO99,
In figure, WT is wild type sage rice 806, and OE-7 is OsHsp18.0-CI gene overexpression plant OE-7T2For material,
OE-11 is OsHsp18.0-CI gene overexpression plant OE-11T2For material, RNAi-9 is OsHsp18.0-CI gene inhibition
Express plant RNAi-9T2For material, RNAi-12 is that OsHsp18.0-CI gene inhibition expresses plant RNAi-12T2For material.
Detailed description of the invention
Following example define the present invention further, according to above description and these embodiments, people in the art
Member may determine that the basic feature of the present invention, and without departing from the spirit and scope of the invention, can be to the present invention
Make various changes and modifications, so that its applicable various uses and condition.In addition to special indicating, of the present invention it is this
Field prior art；The separating clone of embodiment 1:OsHsp18.0-CI gene and structural analysis
1. separating clone OsHsp18.0-CI gene from rice varieties
The plant genome DNA using TIANGEN company extracts test kit and extracts rice varieties sage rice 806 to specifications
Genomic DNA, with this DNA as template, by round pcr, utilize primer 1 (5 '-TGAGAATTGAGATCACCCTCTT-3 ',
Its sequence is as shown in sequence table SEQ ID NO.3) and primer 2 (5 '-GGACCAGATTTGACGCTTTTAT-3 ', its sequence such as sequence
Shown in list SEQ ID NO.4) expand the DNA fragmentation obtaining OsHsp18.0-CI gene.
Its PCR response procedures is 98 DEG C of 2min of denaturation, 98 DEG C of 10s of degeneration, and anneal 55 DEG C of 25s, extends 72 DEG C of 30s, instead
Answer 30 circulations, 72 DEG C of 5min of rear extension.The unnamed gene obtained is OsHsp18.0-CI, its nucleotide sequence such as SEQ
Shown in NO.1, wherein 75-557 position is encoding base, encodes the heatshock protein of a kind of small-molecular-weight, by 161 aminoacid groups
Becoming, its aminoacid sequence is as shown in SEQ NO.2.
Embodiment 2:OsHsp18.0-CI gene expression pattern analysis in rice varieties
In order to confirm that OsHsp18.0-CI gene participates in the regulation and control of disease resistance response, we use quantitative RT-PCR technology analysis
Rice material sage rice 806 OsHsp18.0-CI gene after inoculated bacteria Population of Xanthomonas Oryzae Pv RS105 and leaf spot bacteria PXO99
Expression pattern.0h, 6h, 12h, 24h and 72h take inoculation blade, extracted total RNA the most after inoculation.Utilize iQTM5 quantitative PCRs
Instrument (Bio-Rad company), SYBR Green fluorescent intercalating dyes do different time points after quantitative RT PCR analysis inoculation
The differential expression of OsHsp18.0-CI gene.OsHsp18.0-CI gene specific PCR primer in quantitative RT-PCR technology is to draw
Thing 3, its sequence of 5 '-GGTGGAGAGCTTCGATTCGA-3 ' as shown in sequence table SEQ ID NO.5 and primer 4,5 '-
Its sequence of GGACCAGATTTGACGCTTTTATTT-3 ' is as shown in sequence table SEQ ID NO.6.RT-with rice actin
PCR primer as sample size concordance compare, actin PCR primers sequence be OsActin 1F (5 '-
Its sequence of TGCTATGTACGTCGCCATCCAG-3 ' is as shown in sequence table SEQ ID NO.7) and OsActin 1R (5 '-
AATGAGTAACCACGCTCCGTCA-3 ', its sequence is as shown in sequence table SEQ ID NO.8).
After analysis result display rice material sage rice 806 inoculated bacteria Population of Xanthomonas Oryzae Pv, 12h and 24hOsHsp18.0-CI
The expression 12.2 of gene and 15.7 times (Fig. 1), illustrate that OsHsp18.0-CI gene significantly can be induced by Xanthomonas campestris PV.oryzicola
Express；After inoculation leaf spot bacteria 6h, OsHsp18.0-CI gene upregulation 3 times, 12h and 24h raised 3.3 and 5.1 respectively
Times, illustrate that OsHsp18.0-CI also can be by leaf spot bacteria abduction delivering.
The plant expression vector construction of embodiment 3:OsHsp18.0-CI gene
Used carrier of the present invention is pU1301.With holy rice 806DNA as template, with primer 5 (5 '-
ATGGGTACCTGAGAATTGAGATCACCCTCTT-3 ', band underscore base is restricted enzyme KpnI recognition site, its
Sequence is as shown in sequence table SEQ ID NO.9) and primer 6 (5 '-CGGGATCCGGACCAGATTTGACGCTTT-3 ', leukorrhagia is drawn
Line base is restricted enzyme BamHI recognition site, and its sequence is as shown in sequence table SEQ ID NO.10) amplification gene sheet
Section, PCR response procedures is as follows: 98 DEG C of 2min of denaturation, 98 DEG C of 10s of degeneration, and anneal 56 DEG C of 25s, extends 72 DEG C of 30s, reacts 30
Individual circulation, 72 DEG C of 5min of rear extension, it is thus achieved that the fragment of SEQ ID NO.1.
PCR primer KpnI and BamHI carry out enzyme action, carry maize ubiquitin promoter with KpnI and BamHI enzyme action simultaneously
Genetic transformation carrier pU1301, enzyme action is complete, purification digestion products.With endonuclease bamhi and the carrier of OsHsp18.0-CI gene
Do coupled reaction, by enzyme action screening positive clone；Named pU1301::OsHsp18.0-CI, uses for overexpression.
Meanwhile, the present invention uses RNA to disturb (RNA interference, RNAi) technology, by suppression rice varieties sage rice
The expression of OsHsp18.0-CI gene in 806, verifies the function of this gene.The present invention with primer 7 (5 '-
AAGACTAGTGGTACCGCATCTTCCCGTCCTTCC-3 ', band underscore base is restricted enzyme SpeI and KpnI recognition site,
Its sequence is as shown in sequence table SEQ ID NO.11) and primer 8 (5 '-AAGGAGCTCGGATCCGGACCAGATTTGACGCTTT-
3 ', band underscore base is restricted enzyme SacI and BamHI recognition site, its sequence such as sequence table SEQ ID NO.12 institute
Show), OsHsp18.0-CI gene DNA fragment is template, and the DNA fragmentation that PCR amplification is to be transformed, PCR response procedures is as follows: become in advance
Property 98 DEG C of 2min, 98 DEG C of 10s of degeneration, anneal 56 DEG C of 25s, extends 72 DEG C of 30s, reacts 30 circulations, rear extends 72 DEG C of 5min.
Build the first repetition chain (forward chain) of OsHsp18.0-CI genetic fragment: digest part PCR with KpnI and BamHI
Product and carrier ds1301 (as shown in Figure 4), inactivate restricted enzyme at 75 DEG C of water-bath 10min after digestion completely, be placed in ice
On, it is attached reaction with T4DNA ligase.The cloned plasmids obtained after heat shock primer 7 and 8 expands screening positive clone.
Build the second repetition chain (reverse strand) of OsHsp18.0-CI genetic fragment: remaining with SpeI and SacI digestion
PCR primer and the above-mentioned positive colony plasmid being connected to the first repetition chain filtered out, 75 DEG C of water-bath 10min again after digestion completely
Inactivation restricted enzyme, is placed on ice, is attached reaction with T4DNA ligase.The cloned plasmids obtained after heat shock is used
SacI and SpeI double digested reaction screening positive clone, named ds1301::OsHsp18.0-CI, expresses as suppression
Carrier.
By the excess built and suppression expression vector pU1301::OsHsp18.0-CI and ds1301::OsHsp18.0-
CI electricity converts Agrobacterium competent cell EHA105, preserves bacterial strain and is used as agriculture bacillus mediated rice transformation.
The functional verification of embodiment 4:OsHsp18.0-CI gene
Use agrobcterium-mediated transformation (Lin and Zhang, 2005, Plant Cel l Rep.23:540-
547) by excess and suppression expression vector Introduced into Rice kind sage rice 806.The genetic transformation plant obtained is named as OE respectively
And RNAi.
The present invention obtains excess and independent transformation plant 19 and 25 strain of suppression expression OsHsp18.0-CI gene respectively, point
Ming Ming OE-1 to 19 and RNAi-1 to 25.
The transformed plant of overexpression is respectively with maize ubiquitin promoter special primer UbiF (5 '-TTTTAGCCCTGCCTTCATACGC-
3 ', its sequence is as shown in sequence table SEQ ID NO.13) and OsHsp18.0-CI gene special primer 2 (5 '-
CGGGATCCGGACCAGATTTGACGCTTT-3 ', its sequence is as shown in sequence table SEQ ID NO.9) and the screening mark of carrier
Note Hpt gene flanking sequence design primer HptF (5 '-GATCGTTATGTTTATCGGCACTTTG-3 ', its sequence such as sequence table
Shown in SEQ ID NO.14)/HptR (5 '-GTACTTCTACACAGCCATCGGTCCA-3 ', its sequence such as sequence table SEQ ID
Shown in NO.15) respectively acquisition transformed plant is carried out positive identification, identify that the material being the positive is as the positive using two pairs of primers
Plant.With the flanking sequence design primer S2F2 (5 '-TTCTAATCCCCAATCCAAA-of two chain cloning sites on ds1301 carrier
3 ', its sequence is as shown in sequence table SEQ ID NO.16)/S2R2 (5 '-TAGGCGTCTCGCATATCTC-3 ', its sequence such as sequence
Shown in list SEQ ID NO.17) and carrier selection markers Hpt gene primer respectively to obtain suppression express
The converting material of OsHsp18.0-CI carries out positive identification, using the two pairs of primers identify the material being the positive as positive plant,
The PCR response procedures carrying out positive identification is as follows: 94 DEG C of 3min of denaturation, 94 DEG C of 30s of degeneration, and anneal 55 DEG C of 30s, extends 72 DEG C
45s, reacts 30 circulations, 72 DEG C of 5min of rear extension.
Use living body puncture method at Seedling Stage inoculated bacteria Population of Xanthomonas Oryzae Pv RS105, find that excess plant resistance has difference
The enhancing of degree, suppression plant resistance has weakening in various degree；Compared with the receptor Oryza sativa L. sage rice 806 of transgenic, resistance increases
The scab contraction in length of strong transformed plant 0.3cm-0.8cm, the scab length of the transformed plant that resistance weakens
0.2cm-1.0cm (table 1) (as shown in Figure 2).
Table 1. part T0For the transformed plant reaction to Xanthomonas campestris PV.oryzicola RS105
Resistance against diseases for checking transformed plant further is the most relevant to the expression of OsHsp18.0-CI gene, this
Bright respectively to excess listed in table 1 and suppression transformed plant, extracted total RNA, utilize iQTM(Bio-Rad is public for 5 quantitative PCR apparatus
Department), SYBR Green fluorescent intercalating dyes do quantitative RT PCR analysis, compare OsHsp18.0-in transformed plant and control material
CI gene expression amount.Wherein the PCR reaction primer of excess plant be primer 3 (5 '-GGTGGAGAGCTTCGATTCGA-3 ', its sequence
Row are as shown in sequence table SEQ ID NO.5) and primer 4 (5 '-GGACCAGATTTGACGCTTTTATTT-3 ', its sequence such as sequence
Shown in table SEQ ID NO.6)；The PCR primer of suppression plant be primer 9 (5 '-CAACCAAAAAACAGCAAGACACA-3 ', its
Sequence is as shown in sequence table SEQ ID NO.18) and primer 10 (5 '-CCCAGAGGTCGAGGGAGAAG-3 ', its sequence such as sequence
Shown in table SEQ ID NO.19), compare using the RT-PCR product of rice actin as sample size concordance respectively.
The expression of experimental result display OsHsp18.0-CI gene and the resistance closely related (table 1) of plant.Disease resistance
In the transformed plant strengthened, the expression of OsHsp18.0-CI gene dramatically increases compared with control material sage rice 806, disease resistance
In the transformed plant weakened, the expression of OsHsp18.0-CI gene substantially reduces compared with control material sage rice 806.This result
Illustrate that the coded product of OsHsp18.0-CI gene plays the effect of positive regulatory factor in rice bacterial blight resistance reaction.Visible
Plant can be given after using overexpression and produce disease resistance response to by the disease caused by Xanthomonas campestris PV.oryzicola, it is thus achieved that be high
Disease-resistant plant.
For being further characterized by OsHsp18.0-CI gene in Oryza sativa L. to the effect in Xanthomonas campestris PV.oryzicola resistance, the present invention
From T0The excess in generation and suppression transformed plant respectively have chosen 2 strains and carry out T1Generation and T2The repeated inoculation checking in generation.Result shows
Show, 2 T1For in strain, the transformed plant of OsHsp18.0-CI gene overexpression is (containing carrier pU1301::OsHsp18.0-
CI) resistance is significantly higher than the holy rice 806 (P < 0.05) of comparison, and the plant without pU1301::OsHsp18.0-CI carrier is to R105
Resistance with compare holy rice 806 without significant difference (Fig. 3 and Fig. 4).Equally, 2 T that OsHsp18.0-CI gene inhibition is expressed1
For transformed plant (containing carrier ds1301::OsHsp18.0-CI), the resistance of RS105 is obviously reduced (P < 0.05), and does not contains
The plant of ds1301:OsHsp18.0-CI carrier to the resistance of R105 with compare holy rice 806 without significant difference (Fig. 5 and Fig. 6).T2
Inoculation test for plant also show equifinality, as shown in Figure 7.This further illustrates gene OsHsp18.0-CI at water
Rice is to the effect in bacterial stripe resistance.Can give after visible employing overexpression OsHsp18.0-CI plant to by
Disease caused by Xanthomonas campestris PV.oryzicola (Xanthomonas oryzae pv.oryzicola) produces disease resistance response, thus
The Oryza sativa L. resistance to Xanthomonas campestris PV.oryzicola can be improved.
It addition, the T that OsHsp18.0-CI gene excess and suppression are expressed2Bacterial blight of rice has been carried out for transformed plant
The qualification of resistance.To obtaining T2Inoculating leaf spot bacteria PXO99 for transformed plant in boot stage, each individual plant inoculates 5 completely
The blade stretched, inoculates latter 14 days and carries out data statistics, using holy rice 806 as control material, carries out t check analysis.Result shows
Showing, rice 806 holy with comparison falls ill compared with length 8.70 ± 0.4cm, after the plant inoculation PXO99 of OsHsp18.0-CI overexpression
The a length of 7.2-8.2cm of morbidity, all reached significant difference level (P < 0.05), shown overexpression OsHsp18.0-CI base
Because the Oryza sativa L. resistance (Fig. 8 and Fig. 9) to bacterial leaf-blight can be increased；And the transformed plant suppressing OsHsp18.0-CI to express is inoculated
The a length of 8.9-11.2cm of PXO99 sequela, dramatically increases (P < 0.05) than wild type sage rice 806 length of falling ill.The above results
Plant can also be given to by leaf spot bacteria (Xanthomonas oryzae after overexpression OsHsp18.0-CI is described
Pv.oryzae) disease caused by produces disease resistance response, such that it is able to the improvement Oryza sativa L. resistance to bacterial leaf-blight.
Claims (3)
1. a paddy disease-resistant related gene OsHsp18.0-CI, the nucleotide sequence of its gene such as sequence table SEQ ID NO.1
Shown in, the aminoacid sequence of its coding is as shown in sequence table SEQ ID NO.2.
Paddy disease-resistant related gene the most according to claim 1, it is characterised in that: the nucleotide sequence of its gene such as sequence
Shown in table SEQ ID NO.1, or it is approximately equivalent to the DNA sequence shown in SEQ ID NO.1, or its function is equivalent to
The subfragrnent of sequence shown in SEQ ID NO.1.
3. paddy disease-resistant related gene described in claim 1 is increasing Oryza sativa L. in bacterial stripe and bacterial leaf spot resistance
Application.
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