CN108728369B - Withered germ of water-melon RNAi component FonDCL1 deletion mutant body and its construction method - Google Patents

Abstract

The invention discloses a kind of withered germ of water-melon RNAi component FonDCL1 deletion mutant body and its construction methods.The present invention is according to 1 protein sequence of Dicer like having been reported, homologous comparison Fon genome sequence, obtain withered germ of water-melon FonDCL1 gene, using homogenotization principle, utilize Split-marker strategy, target gene FonDCL1 is replaced with hygromycin B (HPH) gene DNA fragment, by constructing the homologous recombination segment, the deletion mutant body of FonDCL1 is obtained with wild strain Fon1 genetic transformation, the FonDCL1 mutant has to the enhancing of watermelon seedlings pathogenicity, to Monensin sodium, hydroxycarbamide, fluorescent whitening agent CFW, the characteristics such as the abiotic stress factor such as Congo red CR and hydrogen peroxide is more sensitive, this participates in own growth and development for Fon1 and infects what watermelon generated in the process The function parsing of tiny RNA provides Research foundation.

Description

Withered germ of water-melon RNAi component FonDCL1 deletion mutant body and its building Method

Technical field

The present invention relates to a kind of withered germ of water-melon RNAi component FonDCL1 deletion mutant body and its construction method, Belong to technical field of bioengineering.

Background technique

Watermelon blight is invaded by Fusarium oxysporum f. sp. niveum (Fusarium oxysporum f.sp.niveum) Watermelon fibrovascular system disease caused by contaminating, cause of disease mainly passes through root wound or the intrusion of root hair apical cell gap, in host It after tube wall iuntercellular and Intracellular growth, into vascular bundle, decomposes and destroys cell, make to accumulate pectic substance in conduit, blocking Conduit influences Water Transportation, and then plant is caused to be wilted.

Watermelon blight can fall ill from seedling to strain, and it is most heavy to expand late onset with seat melon phase and melon.Seedling is aggrieved, Can be caused before emergence rotten kind be unearthed after fall ill, cotyledon, true leaf in dehydration shape wilt, basal part of stem browning shrink in it is broken fall shape, extraction The visible root yellowish-brown of young plant is rotted.Adult plant is aggrieved, and initial stage diseased plant lower blade dehydration is wilted, the upward chlorisis of vines base portion. Later period disease portion is in sepia, is felt like jelly, normal lobe, has gluey red material to overflow.When diseased plant base portion humidity, it is covered with white to pink Color mustiness cause of disease conidium, section view basal part of stem to root, it is seen that vascular bundle flavescence brown.

So far, oneself discovery watermelon blight has a biological strain, i.e. biological strain 0,1,2 and 3, and China is with biological strain 1 It is dominant.In agricultural production, for wilt disease type Fusarium oxysporum in addition to endangering watermelon, host range is extensive, can cause more than 100 kinds Plant vasular beam wilting disease, such as Flos Carthami, sugarcane, sesame, dwarf banana, pineapple and melon and beans various plants, especially There is serious harm effect to cucurbitaceous plant, banana, tomato and cotton grade high economic value crop.

There is generation in cultivating watermelon area to watermelon blight throughout our country, which can cause the yield of watermelon to decline, even Total crop failure.Cultivating watermelon especially protects ground area to expand rapidly in recent years, and continuous cropping continuous cropping area increases, and protecting field high temperature and humidity is made It is more and more common at watermelon blight, it is increasingly severe, huge economic losses are caused to watermelon industry.

MiRNA (microRNA) is that one kind is about 22nt, the endogenous non-coding tiny RNA with adjusting function.MiRNA ginseng With important physiology and pathologic process many in body, such as the growth and development of body, the generation and immune response of tumour, this makes Obtain its focus for becoming biological study.Dicer albumen is a member important in III family of RNase, to miRNA or siRNA Generation play a crucial role.Dicer albumen is usually by 1 DEXH box or H box, 1 DUF283 structural domain, 1 A PAZ structural domain, 2 III structural domains of RNase (III b of RNase III a and RNase) and 1 dsRNA binding structural domain composition. The molecular structure of Dicer albumen determines that it is played an important role in miRNAs synthesis.

Pre-miRNA can be degraded into specific length by Dicer, usually the short dsRNA segment of 21-25nt.Dicer is first First then the structure of " molecular ruler " is utilized to exist in conjunction with the PAZ structural domain of its own 3 ' the end dinucleotides of pre-miRNA Pre-miRNA is cut fixed position, and the 5 ' ends of miRNA are formed, and Dicer needs Mg to the mechanism of pre-miRNA²⁺, But do not combine Mg²⁺, catalytic activity shows that electrostatic substrate-enzyme interacting is to the function of Dicer to ionic strength sensitive It is very important.

In arabidopsis, when by virus infection, DCL4 is the first line of defence that Dicer resists virus in arabidopsis, DCL4 can produce tiny RNA and resist virus.When DCL4 cannot be played and be resisted activity, DCL2 can become Dicer and resist disease The second defence line of poison.In the deletion mutant of the Osdcl1 of rice, the quantity of miRNA can be reduced largely, can final shadow The development of Xiangshui County rice shows that OsDcr1 is related with the process of rice miRNA, is sent out by miRNA come the growth of adjusting and controlling rice It educates.DCL1 is related with the cutting of miRNA and the generation of miRISC compound in drosophila.

Germline stem cell and mature stem cells can be generated normally in normal drosophila, and in drosophila Dcr1 mutant Ovary stem cell is just dead early stage development, this has also indicated that the miRNA that Dcr1 is generated can regulate and control the life of drosophila Life activity.However the mouse as mammal Typical Representative, the missing of Dicer gene deposit it all cannot in embryo stage It is living.

In Neuraspora crassa, the double-mutant of DCL-1 and DCL-2 cannot be processed into dsRNA the siRNA of 25nt, but It is that dsRNA can be processed into siRNA by each single mutant and original strain, this has turned out the function between two Dicer Complementation with intersect.Dicer albumen sms-3 in Neuraspora crassa participates in the sexual reproduction of neurospora.Sms-3 mutant The shell of ascus mature can also can produce the ascospores of a large amount of maturations simultaneously, but the hair with the shell of ascus of original strain compared with It is white.Necessary element during CaAgo1 and CaDcr1 is the gene silencing of RNAi initiation in Candida albicans, simultaneously CaDCR1 can seriously affect the growth of itself.

Currently, people use prevention and treatment of various measures reinforcement to watermelon blight, such as graft seedling growth, breeding for disease resistance, change Prevention and treatment etc. is learned, but its effect all has certain limitation, also causes certain negative effect, graft seedling growth is to watermelon maturation There are important influence, the shortage of breeding for disease resistance long periodicity and its antigen in phase, quality, and chemical prevention is largely brought using pesticide The negative effect such as environmental pollution, ecological disruption and disease drug resistance.Dicer like as the Main Components in RNAi access, It plays an important role in the generation regulation process of miRNA.Specify they and growth and pathogenic relationship, it will help we are from another One angle illustrates the pathogenesis of withered germ of water-melon.

Summary of the invention

The present invention compares sharp spore sickle according to 1 protein sequence of Dicer like in Fusarium oxysporum tomato specialized form Fol Knife bacterium watermelon specialized form genomic DNA, finds homologous sequence FonDCL1, is turned by the building and heredity of the genetic recombination segment Change and obtains FonDCL1 deletion mutant body.

The technical solution adopted by the present invention is as follows:

A kind of withered germ of water-melon RNAi component gene FonDCL1, the nucleosides comprising its upstream and downstream 3Kb nucleotide sequence Acid sequence is as shown in SEQ ID NO:1.

The nucleotide sequence replaced with hygromycin gene HPH is as shown in SEQ ID NO:2.

The cDNA of withered germ of water-melon RNAi component gene FonDCL1 coding of the present invention, with shown in SEQ ID NO:3 Nucleotide sequence.

The protein of withered germ of water-melon RNAi component gene FonDCL1 expression of the present invention, with SEQ ID NO:4 institute The amino acid sequence shown.

The present invention also provides a kind of withered germ of water-melon FonDCL1 deletion mutant body, which passes through following Method obtains:

1) FonDCL1 gene delection recombinant dna fragment constructs

First round PCR amplification: using watermelon blight genomic DNA as template, primers F on1DCL1-LBCK/Fon1DCL1- HPH-LB-R expands FonDCL1 upstream region of gene FonDCL1-UP segment, primers F on1DCL1-HPH-RB-F/Fon1DCL1-RBCK Expand FonDCL1 downstream of gene FonDCL1-DOWN segment；Using carrier pCT74 Plasmid DNA as template, primer HYG-F/HYG-R Expand resistant gene HYG segment；

Second wheel PCR amplification: using FonDCL1-UP and HYG as template, primers F on1DCL1-LB-F/HYG-R1 amplification FonDCL1-UP+HY segment, using FonDCL1-DOWN and HYG as template, primer HYG-F1/Fon1DCL1-RB-R expands YG+ FonDCL1-DOWN segment；

3) protoplast and FonDCL1-UP+HY segment and YG+FonDCL1-DOWN segment equal proportion are mixed, is carried out FonDCL1 gene delection genetic transformation obtains mutant；

Described primers F on1DCL1-LBCK, Fon1DCL1-HPH-LB-R, Fon1DCL1-HPH-RB-F, Fon1DCL1- The nucleotide sequence of RBCK, HYG-F, HYG-R, Fon1DCL1-LB-F, HYG-R1, HYG-F1, Fon1DCL1-RB-R are successively such as SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, shown in SEQ ID NO:14.

The FonDCL1 deletion mutant body is identified by the following method:

Using the genomic DNA of transformant as template, designs four pairs of primers and is used to identify FonDCL1 deletion mutant body, It is upstream primer Fon1DCL1-LBCK/HPT-LBCK, downstream primer Fon1DCL1-RBCK/HPT-RBCK, for expanding respectively Primer HYG-F/HYG-R, the FonDCL1 gene internal primers F on1DCL1-2193/Fon1DCL1-3030 of HPH gene；

The nucleotide sequence of described primer HPT-LBCK, HPT-RBCK, Fon1DCL1-2193, Fon1DCL1-3030 are successively As shown in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18.

Compared with prior art, the beneficial effects of the present invention are:

The present invention provides FonDCL1 deletion mutant body and its construction method, which has to west Melon seedling pathogenicity weakens, to abiotic sides of body such as Monensin sodium, hydroxycarbamide, fluorescent whitening agent CFW, Congo red CR and hydrogen peroxide The characteristics such as the urgent factor is more sensitive, this participates in own growth and development for Fon1 and infects the function of the tiny RNA generated during watermelon It can parsing offer Research foundation.

Detailed description of the invention

Fig. 1 is building schematic diagram and the positive transformant PCR identification of FonDCL1 gene delection recombinant fragment；Wherein, Fig. 1- A is that FonDCL1 gene and upstream and downstream substrate section, resistant maker gene HPH are divided into two parts, respectively with FonDCL1 gene Upstream and downstream about 2Kb carries out fusion DNA vaccine amplification, and arrow is that four couples of PCR identify primer；Fig. 1-B is after four pairs of primers carry out PCR amplification Electrophoresis detection；

Fig. 2 is FonDCL1 deletion mutant body Pathogenic Tests；Wherein, Fig. 2-A is that watermelon seedlings carry out hurting root processing Afterwards, 14d morbidity result after inoculating spores suspension；Fig. 2-B is the disease index of FonDCL1 deletion mutant body morbidity；

Fig. 3 is that the speed of growth of FonDCL1 deletion mutant body measures；Wherein, Fig. 3-A is to cultivate 7d on PDA plate Afterwards, FonDCL1 deletion mutant body colonial morphology；Fig. 3-B is that the colony diameter of FonDCL1 deletion mutant body measures；

Fig. 4 is that the sporulation quantity of FonDCL1 deletion mutant body measures；

Fig. 5 is the abiotic stress sensitivity testing of FonDCL1 deletion mutant body.

Specific embodiment

Below by specific embodiment combination attached drawing, invention is further described in detail.

Experimental method used in the embodiment of the present invention is conventional method unless otherwise specified.

Material used in the embodiment of the present invention, reagent etc., are commercially available unless otherwise specified.

Embodiment one: the acquisition of withered germ of water-melon FonDCL1 deletion mutant body

1, FonDCL1 gene delection recombinant dna fragment constructs

Withered germ of water-melon FonDCL1 gene knockout uses the principle of homologous replacement, with resistant gene hygromycin B (HPH) The sequence of the DNA fragmentation of gene DNA fragment replacement target gene FonDCL1, FonDCL1 and upstream and downstream 3kb are SEQ ID NO:1, displaced FonDCL1 gene order are SEQ ID NO:2.

1) design of primers of FonDCL1 gene delection recombinant dna fragment

Using the HPH gene with promoter and the 1.4Kb of terminator as template, first round PCR amplification, designed for amplification The primer HYG-F/HYG-R of the HYG genetic fragment of 1.4Kb, for expanding the primer of the upstream FonDCL1 2Kb genetic fragment Fon1DCL1-LBCK/Fon1DCL1-HPH-LB-R, for expanding the primers F on1DCL1- of the downstream FonDCL1 2Kb genetic fragment HPH-RB-F/Fon1DCL1-RBCK；

Second wheel PCR amplification, design primer Fon1DCL1-LB-F/HYG-R1, for expanding the upstream portion 1.5Kb and HYG Sub-sequence HY, primer HYG-F1/Fon1DCL1-RB-R, for expanding HYG partial sequence YG and downstream 1.5Kb sequence；For The PCR of positive transformant is verified, the aligning primer of HPT-LBCK/HPT-RBCK and FonDCL1 gene replacement part Fon1DCL1-2193/Fon1DCL1-3030。

1 primer sequence of table

2) PCR amplification of FonDCL1 gene delection recombinant dna fragment

First round PCR amplification:

Using Fon1 genomic DNA as template, primers F on1DCL1-LBCK/Fon1DCL1-HPH-LB-R amplification gene upstream FonDCL1-UP segment (1808bp)；Primers F on1DCL1-HPH-RB-F/Fon1DCL1-RBCK amplification gene downstream FonDCL1-DOWN segment (1947bp), using carrier pCT74 Plasmid DNA as template, primer HYG-F/HYG-R expands 1376bp's HYG segment.

PCR response procedures: 95 DEG C, 4min, 1cycle；94 DEG C, 40s, 58 DEG C, 40s, 72 DEG C, 1min 30s, 32cycles；72 DEG C, 10min, 1cycle；16 DEG C, hold.

Second wheel PCR amplification:

Expanded using LA Taq, will first round PCR product dilute 100 times after be used as template, with FonDCL1-UP and HYG is template, and primers F on1DCL1-LB-F/HYG-R1 expands FonDCL1-UP+HY segment (2570bp), with FonDCL1- DOWN and HYG is template, and primer HYG-F1/Fon1DCL1-RB-R expands YG+FonDCL1-DOWN segment (2259bp).

PCR response procedures: 95 DEG C, 4min, 1cycle；94 DEG C, 40s, 58 DEG C, 1min20s, 72 DEG C, 2min 30s, 32cycles；72 DEG C, 10min, 1cycle；16 DEG C, hold.

2, the protoplast transformation that PEG is mediated

1) prepared by protoplast

5-10 block Fon1 mycelia block (culture 7-10d, size 5mm) is taken, is added in the PDB of 100ml, 28 DEG C, 150rpm Shake training 4-5d.3 layers of lens wiping paper filtering, collect the conidium in filtrate.5-10ml conidium liquid is taken, the PDB of 200ml is added In, 28 DEG C, 150rpm shakes training 12-18h.Cultured bacterium solution is passed through into 4 layers of filtered through gauze, appropriate 0.7M NaCl (8.18g NaCl uses H₂O is settled to 200ml) solution flushing, fresh and tender mycelia is collected, mycelia is transferred in 50ml centrifuge tube.8-10ml is added (storing liquid concentration is 20mg/ml, working concentration 10mg/ml to enzymolysis liquid, is dissolved after weighing driselase with 0.7M NaCl, fixed Hold).It is vortexed, so that thallus is scattered and come into full contact with enzymolysis liquid, 30 DEG C, 80-100rpm, warm bath 3-4h.After enzymatic hydrolysis, it is added 0.7M NaCl to 50ml centrifuge tube dilutes enzymolysis liquid, and three layers of lens wiping paper filtering, is rinsed with appropriate 0.7M NaCl after being mixed by inversion Filter paper；4 DEG C, protoplast is collected after being centrifuged 15min no more than 5000rpm.With STC (the 21.86g sorbierite of 10-20ml； The 1M Tris-HCl pH 7.5 of 1.0ml；0.735g CaCl₂·2H₂O adds H₂O is settled to 100ml) precipitating is sufficiently dissolved, 4500rpm 15min centrifugation, abandons supernatant.General 400 μ l STC is added and sufficiently dissolves precipitating, the amount of STC is determined according to the amount of precipitating It is fixed, guarantee that the concentration of protoplast can reach 10⁸A/ml, places it in the centrifuge tube of 1.5ml.

2) protoplast transformation

Protoplast is dispensed, guarantees sterile working.By protoplast (general 200 μ l) and FonDCL1-UP+HY segment (100 μ l) and YG+FonDCL1-DOWN segment (100 μ l) mix (gently revolving) in 1.5ml centrifuge tube, are put in 1- on ice 2h.It carries out on ice, the liquid in small centrifuge tube is transferred in the centrifuge tube of tip and (indicates accurate calibration 50ml's), is added dropwise 400 μ l PEG (the 1M Tris-HCl pH 7.5 of 30g PEG4000,0.5ml, 0.735g CaCl₂·2H₂O adds H₂O is settled to 50ml), it closes the lid and gently revolves, ice bath 15min.800 μ l PEG are added (to be added dropwise, this step can not carry out on ice, room Temperature), it gently revolves, mixes, after room temperature sets 20min.2ml RB is added.In 28 DEG C of incubators cultivate 12h after, tip from RA culture medium (glucose 10g is added in heart pipe；KCl 0.52g；MgSO₄·7H₂O 0.52g；KH₂PO₄0.25g；Sorbierite 218.5g(1.2M)；NaNO₃6g；1% agar), it is settled to 50ml, divides and changes into 5 culture dishes.It is cultivated in 28 DEG C of incubators After for 24 hours, one layer of PDA (containing hygromycin) is covered as screening in media surface, hygromycin content at least needs 50 μ g/ml, sets It is cultivated in incubator.

3) FonDCL1 deletion mutant body is identified

Using the genomic DNA of transformant as template, using four pairs of primers for identifying FonDCL1 deletion mutant body, It is upstream primer combination Fon1DCL1-LBCK/HPT-LBCK respectively, downstream primer combines Fon1DCL1-RBCK/HPT-RBCK, Primer HYG-F/HYG-R expands HPH gene, FonDCL1 gene internal primers F on1DCL1-2193/Fon1DCL1-3030. As shown if figure 1-b, the upstream and downstream primer combination of FonDCL1 deletion mutant body amplifies product and says PCR amplification result Bright transformant upstream and downstream sequence is replaced, and gene internal primer, which fails to amplify band, illustrates that FonDCL1 is replaced with HYG Change (Fig. 1-B).

The analysis of two: FonDCL1 deletion mutant body function of embodiment

1, FonDCL1 deletion mutant Pathogenic Tests

1) the FonDCL1 deletion mutant for taking culture 7d, is beaten with 5mm punch and takes 3 pieces of mycelia block, the PDB of 200ml is added In culture medium, 28 DEG C, 150rpm shakes training 3d, and 3 layers of lens wiping paper filtering collect conidium liquid, 5000rpm is centrifuged 10min, with nothing Bacterium water adjusts spore suspension concentration to 2 × 10⁶A/ml.

2) watermelon seed is taken, with 0.1% sodium hypochlorite in 28 DEG C of immersion 6-12h, cleans 3-5 with gauze package watermelon seed It is secondary, watermelon seed is evenly laid out on wet towel, vernalization 2d under 28 DEG C of dark conditions.The seed of bud will have been urged to be seeded in In nutritive cube (15 × 15cm) containing Nutrition Soils such as coco brans, 10 watermelon seeds of each nutritive cube, at 25 DEG C, Routine Management, After 7d, grow up to the watermelon seedlings of two leaves wholeheartedly, it is spare.

3) the watermelon seedlings dissection knife wound root processing by two leaves wholeheartedly, it is then that the spore suspension of 20ml is equal from root from hurting Even addition, at 25 DEG C, normal water and fertilizer management after 2d meets 3d, 5d, 7d and 14d after bacterium, records incidence, and calculate the state of an illness and refer to Number.

Watermelon seedlings Disease investigation is according to following diseased plant grade scale:

0 grade: without illness；

1 grade: 1 or 2 cotyledons slightly turn to be yellow, but grow normal；

There is necrotic plaque in 2 grades: 1-2 piece cotyledon yellow or 1 cotyledon, and true leaf slightly turns yellow, slightly here withers；

3 grades: cotyledon is withered, and here true leaf obviously withers, and plant strain growth is obstructed, few to downgrade；

4 grades: here complete stool seriously withers, and blade is withered and yellow；

5 grades: whole strain is withered, or even lodging.

Disease index calculation formula:

Disease index=Σ (sick grade strain number × represent numerical value)/(strain number summation × morbidity most heavy duty representative numerical value) × 100%

Pathogenic Tests result such as Fig. 2-A: negative control (water process), in the case where not being inoculated with wilt, watermelon children Seedling robust growth, does not have wilt disease.Positive control, is inoculated with wild strain Fon1, and watermelon shows apparent wilt disease Symptom is inoculated with FonDCL1 mutant strain, and watermelon seedlings morbidity is caused harm serious, and calculating its disease index is 92.3, is significantly higher than The 70.2 of wild strain Fon1 show that FonDCL1 mutant strain pathogenicity enhances (Fig. 2-B).

2, the FonDCL1 deletion mutant speed of growth measures

The FonDCL1 deletion mutant (Fig. 3-A) for taking culture 7d, is beaten with 5mm punch and takes mycelia block, and it is flat to be inoculated in PDA Culture 7d is inverted in plate (Φ=60mm), in 28 DEG C, 2d, 3d, 4d, 5d measure colony diameter after inoculation, measure its life Long speed.As a result as shown in Fig. 3-B, FonDCL1 deletion mutant covers with entire plate after being inoculated with 5d, and colony diameter reaches 5.1cm, and wild strain colony diameter is 4.6cm, the mutant speed of growth and wild strain Fon1 are without marked difference.

3, FonDCL1 deletion mutant sporulation quantity measures

The FonDCL1 deletion mutant for taking culture 7d, is beaten with 5mm punch and takes 3 pieces of mycelia block, and the PDB training of 200ml is added It supports in base, 28 DEG C, 150rpm shakes training 3d, takes 1ml spore suspension blood counting chamber to calculate spore concentration, and be converted into logarithm Value, statistical result showed, FonDCL1 deletion mutant can generate 10 with wild strain Fon1 after shaking training 3d⁷A/ml's Spore, after Analysis of variance, FonDCL1 deletion mutant sporulation quantity and Fon1 show FonDCL1's without marked difference (Fig. 4) Missing does not influence the spore output of Fon1.

4, FonDCL1 deletion mutant abiotic stress sensitivity testing

By shake training 3d FonDCL1 deletion mutant spore suspension respectively with sterile water adjust spore concentration most 2 × 10⁷、2×10⁶、2×10⁵、2×10⁴A/ml is dispensed into the sterile EP tube of 1.5ml, and 4 DEG C save backup.

Basic MM culture medium is prepared, MM culture medium is separately added into the abiotic stress factor, is adjusted to concentration are as follows: 0.1M MgCl₂、0.5M CaCl₂, 1.0M sorbierite (Sorbitol), 1.0M KCl, 1.0M glucose (Glucose), 0.7M NaCl, 0.05% (W/V) Monensin sodium (MMS), 1.0mg/ml hydroxycarbamide (Hydroxyurea), 6.0mg/ml histidine (Histidine), 400 μ g/ml fluorescent whitening agents (CFW), 200 μ g/ml Congo red (CR), 0.01% (W/V) SDS, 1.0 μM H₂O₂, it is made into MM plate (Φ=15cm), is uniformly crossed into the small lattice of 1.5 × 1.5cm in MM flat plate bottom, it is spare.

2.0 μ l are taken to be added drop-wise in the small lattice of MM plate respectively the spore suspension of gradient concentration, 2d is cultivated in 28 DEG C of inversions, is clapped According to the abiotic stress sensibility of observation FonDCL1 deletion mutant.As a result as shown in figure 5, Protein transport inhibitor can not be mould The FonDCL1 deletion mutant of plain sodium processing, growth are suppressed.Another hydroxycarbamide (hydroxyurea, HU), hydroxyl Urea can inhibit ribonucleotide reductase, prevent cytidine monophosphate from being changed into deoxycytidylic acid, to inhibit the synthesis of DNA, it can be selective Ground acts on S phase cell.After hydroxycarbamide is handled, the growth of FonDCL1 deletion mutant is significantly inhibited.By fluorescent brightening After the relevant stress factors processing of the cell wall integrities such as agent CFW and Congo red CR, FonDCL1 deletion mutant is more sensitive. After dioxygen water process, FonDCL1 deletion mutant is more sensitive.And other abiotic stress factor (metal ions, infiltration Gesture, detergent etc.) it does not make significant difference to FonDCL1 deletion mutant.

The above content is specific embodiment is combined, further detailed description of the invention, and it cannot be said that this hair Bright specific implementation is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, it is not taking off Under the premise of from present inventive concept, a number of simple deductions or replacements can also be made.

Sequence table

<110>Research Institute of Environment and Plant Protection, Chinese Academy of Tropi

<120>withered germ of water-melon RNAi component FonDCL1 deletion mutant body and its construction method

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ataacattca aaacattctg aaggaccgat gttcttagag cctcactaca atgttggtga 1920

agcttaatac tggggcaacg aatcagggct gtttatttgg ttcttatatg ctataaagtt 1980

ccctgcgtaa attttccgac ttgctaaact tttgaacagc tgaactttcg ttaaagcaga 2040

ctctatgtgc tccgcctcaa ctacatagat acaccccgag agtacatatc gacccgagca 2100

agcatatgat cccccgtgag gccagtatca gaatgcacgg tgacaggcaa ttttgataat 2160

tgtatgggat gacatttcct gcttgagagt tgaaggaaat tttggtgcga gcatattatg 2220

gaccatgaga gggtttctac gggcttcttt gacgtcatag attctcagtg aagaattgca 2280

tgcccaacat atgcgatttc aaccttaacc ttggccaaag aggcctcagg catgagcagt 2340

caccctgcct ggagaaggcg aacggtagag accaaagaac acactcagat gagggctttg 2400

tagacactta caaaaaaatg gtgtgtaaat attgagcgaa gctgagatga tttgaagtga 2460

gatatgcggg agagcctctc aggggttctc ctattatact tgtagtggat atgcgtccca 2520

acgatgaacc gtgtgtgagg ctctgcaaat gtggctccct cccaatggga catgcaggta 2580

ccaaacctta ctggaactgc tattcgatat ctgcataaaa ttgtatagaa gcatcgcgag 2640

gtgcgtgtgc tcgaatggtt tgatactgcg aaacagtgtc tgtgtgtacc ttgcttctgt 2700

ttgcctagtg tttgctccat gattgtggct agttaaagaa taaagaaaat agaaacagcg 2760

tgatacttga tggagattgt agcggactct tgctgttaca ctaatgcaag catccgcgca 2820

aacaacctcc ctcagtagcc tccacctcaa ggcaccttcc acctccgaca atccttttcc 2880

tgtctctctg gtattcactc gtcagaaata tcagtacagg gcttattcca ctcttccttt 2940

gatcgctctc tcattgcaaa tgtatcttca tcctgagcag taactaatcc acgcttcaac 3000

atgtcggatc acgagtacat cgacactgat gacgaagacg atcgatatcg cctcatcgtg 3060

aacccgtcga agcctcgaaa gatcacggaa aagaagctcc gagaccaagc tgctctacaa 3120

gcgcacatag aaaagacaca gagagatgcc gcaaggagtg ccgctccttt cagcgatcct 3180

agtaaacagt ccctcgccca gctcatgaac cagaacgaga gccacaagat catcgcctcg 3240

cctcgagaat accagattga actgttcgag cgcgctaaag agagaaacct ccttgtggtg 3300

ttgcctacag gtatgtttcg ctaccaatct ggaggctcgc taatagagta ggtactggca 3360

aaacactcat ctccgccttg ctgctacgac atcacctcga acaagagctg gaagctcgag 3420

ccatcggaaa gcccaagaaa gtcgcctttt tcctagtaga aaaggtcgcc ctctgtgttc 3480

agcaacatgc agtcctcagc tgcaacctcg gggcccaccc tgtcaccaag ttcgtaggcc 3540

atatgaaagg catgacgaag accaaagaat actgggatga gaagtttggc gaaaacatgg 3600

ttgtggtctg cactgctcag atcctcctcg actgcctcac ttgcggcttc atcaccatgt 3660

cccggataaa tcttctcata ttcgacgaag cacaccacac aaagaagaag cacccgtacg 3720

ctcgaattat ggatgggtat tattgcaaac acgaaggcga aaaacctcgc atactgggaa 3780

tgactgcttc gcctgtggac gcgcgcacca aggacgtgag agacactgcc ctggagcttg 3840

agaggtctct tgacagccag attgcaacga tttcagatga ggtgctgatg gaaaccatgc 3900

accgaaagaa gcaaaccgaa gaaactgtca actacagcag gcctgaagct cccgatgata 3960

caaagacgca tctatgggag cagatctttg ccctagtttc tcgcaacgaa cagttcaagt 4020

tgtcgctcga gttcacaaaa gaggcctcgt cagtcctcgg tacttggtgt gcggaccgat 4080

attgggagct cgcaatgaca gatctcgaga ctgaacggct cgcagccaga actagtcggg 4140

aatttattgg agacattgca gcaacaagag cagatctagc aacagaggcc gtccgacgtg 4200

tccaggaaat tgttcaggct cacaagttcg gtgcaatcga cccaaaggcc gaagggctct 4260

cggccaaggt gaaaagtctc cataagatct tgtcgcacgc tttcacaatg gatggcacca 4320

agcgttgcat tgtttttgtc gagaagcgat acacagcccg tttactttcg gatctctata 4380

gccaaccttc catgaggatc ccaagaattt ctgcttcata catggtgagt agtagccttt 4440

tcgggtcgac tttgctaata attgcaggtt ggctgtcaat caaccaattc gagcttcggc 4500

accatgtcgt ttcgagaaca agtcctggct ctccacgaat tcaagcgcgg caataccaac 4560

tgccttttca caacacctgt tgcagaagag ggcattgacg ttccagattg cgaccttgtt 4620

attcgtttcg atctctacaa ctccgtcatc caatatttgc aatccaaagg acgcgctcga 4680

caagcacgct ctcgatatat caccatgatg gaggagggaa atttgacaca tattcgaagt 4740

gcgaaacaag cattgagaga ttcacaggct ctggaaaagt tctgtctctc tctttctgct 4800

gaccgaaaac ttcatgatga tttgatagac gaggacatgg agatgatggt tgagcgcatg 4860

cagcatcaag tgtacgagat accttcgaca ggcgcacgcc ttacgtttcc cgccagcctt 4920

gaaatcctgg ccagatttgt ttctcacttg tcaactggca gctacgccaa agttgaatac 4980

caagtacaga aggttggcga tcgatatcta gctgacgtgg tcatgccatt acagtccccg 5040

gtcaactttg tgaagggtac ccaacagcgt agcaaattgc tggccaagtg ttctgcagca 5100

tttgaagcct gcaagatact gatcagagac aaacatgttg acggcaatct tcagccgaca 5160

ttcaagaaaa aggttcacgc tatgcggaac gcacggctag ctgtgagttc aaacaagaag 5220

gctgactacg acatgcgatt gcggccagag atttgggctg aacgagggga gtggactgaa 5280

tgttttgcga ctacaatcac catcaaagac gggggcaccc ttaggaaggc aatagccgga 5340

aagtcgttaa tacttctctc gcgcaagccg atcccgaaac tgccaggtgt tcctctattc 5400

tttggcaatg gacattcctc tatcgctgaa ttggcgccta atcaggatgc tcttaaactc 5460

aaccctgaag aagctgacgg attgaccaaa ttcactcatc gggtattcgc cgatgtgttc 5520

agcaaagaat acgataccac gtccgaccaa tatccatacc tcctcgcacc ttatgccgac 5580

agtccccaat cagacactcg atcacatatt gattggaatg ttgtcaacct tgtgagcaag 5640

aacgaggtgc tcgaattgga gaatgcgcca gaagacttct tcgtcaacaa gcttgtgact 5700

gatccatatg atggaggacg caagcttgtc atcaaaggta tcgacaaaac gaagaagtca 5760

tccgacccaa caccagaagg agtccccgaa tcaagaagca gagcttatag gtctgtggaa 5820

ccgacaatca tgcaatacag taacagtctg tactataaat ctcgccaaag ggtgaagtgg 5880

cgagatgatc agcctgtggt caaggcagag cttctttcct tgagacgaaa ccttctggat 5940

gagtttgaag ttgacgagaa ggtcaacatg gagtgttgca tcattctgga gcctctccat 6000

gtgtctcctg taagttgaat cccagccttg atgcttgtct aattttccca gctacctatc 6060

gatgtagtct ctatggccat ggcatttcca gcaatcattc accgaataga ttccgtcctg 6120

attgtgttgg atgcatgcaa tctccttgac cttgaaattc caccagaact cgcactggag 6180

gccatgacca aagacagcga caactctggg gagcatgaca cagaacagat caatttccaa 6240

acaggcatgg gcgacaacta tgaacggcta gagttcctcg gcgactgttt tctcaagatg 6300

gccaccacga tctcaatcta cacactgatg cctgacggaa atgagtgcaa ataccacgtt 6360

gagcgcatgc ttctcatttg caaccaaaac ctcttcaatc atgcggttga tcgcaaactc 6420

caagagttcg tccgatccaa ggcttttgac agaagaacat ggtaccccga tcttccactc 6480

aagaaaggaa aggcaccgaa aacttcgatg aagcataatc tggccgacaa gacgattgca 6540

gacgtatgtg aagctctcat tggggcagca tacctcacaa gcaaggccag caacatggat 6600

ctagctgtca aagccgtgac gcgaatgacc aagtcaaaga accacaggat gaaggctttc 6660

aaagattact ataaagccta caaggtgcct gagtggcagg aaggtaatgc gtccgcctct 6720

caacgcagcc ttgtgcaaaa ggtggcacaa gctacaggat atcgcttcaa gtctgcacag 6780

cttgtccaga gtgcattcaa acatccttcg tggccatatg agtctgtccc agattatcag 6840

cgcctcgaat ttctgggcga ttcacttctc gacatggcca tcgtcgatta tctctatcaa 6900

aacttcccca gagcagaccc tcagtggctg acggaacaca agatggccat ggtatcgaat 6960

cagttccttg gatgcctctg cgttaagctt ggtctgcacc aacacctact caccagcaca 7020

tcgagtcttc tcagccaaat ccgcgattat gtcgtcgaac ttgaagttgc tgaagaaaat 7080

gctcgcaagg aagcgaaaga agatgggact cccatgcgca aggacttctg gctgaaggtg 7140

gattcggcac ccaaggttta cgccgatgtg attgaagctc ttgttggagc catgtttgtc 7200

gattccaagt acaaattttc ggttgttgag aatttcttta ccaagttcat ccagccatat 7260

ttccaagaca tgagacttta cgataccttt gcgaacaagc acccagtcac gttcctctcc 7320

aagaagatgc atcaggaatt cggttgcaca aactggcgca tcagcgcaga ggctgtacca 7380

tgcagtgcag atcaaggcat ggcggcgctg aaagataccg aaatgtgcgc tgttttcatg 7440

gtgcatcaaa aggttatcgc ggatgacaca tctgagagtg ggaggtattc taagcagcgt 7500

gccgcgacga aagcgctcga gatacttgat tcgtttggtg aggatgttga ggcggcgaag 7560

aggtttttgg ggtgtgattg tcagcttgga ggaggggatg ttgaggttga tcatggaaca 7620

gctgtttgag ctgaaatcgg cactagaatt ggaggacatg tgtttcaggg acataaagcg 7680

cttcatagcg ttagaagcgt agacaaagat catagagcaa ttaaaatgtt gcgttgatgt 7740

gttcttggtc cactgagtgc atcgctgaga gacttgattg agctagcttc tcctccgacc 7800

aaggctccag acatgataga atatctcata tccaaagtat cataagaatc aatccattgc 7860

gacttgtata taatcccatt cacaactatt tagtcctccc aggtacaata ctagaaagcc 7920

cccgtctatc aggcatcacg tacccatccc tcgcctcaac actcccccac agctgcggcg 7980

aataatacac aaacggcaaa tcctcaaacg ccaaaaccga attcccctta ataaactcct 8040

caaatcccat gtctttccgc cccgccagtg caatttggat ctgctgtacc cagaacatag 8100

cctgtgtcaa accagtccat ttcgcaatct ccagcgccgt tgacgaagct agcgaagcat 8160

caccgcgttt tgtgctcgta gcggtagatt tggtcttttc tgtaagacgg ttgaatagga 8220

aacggatgag gctcgcgtgg ctggcgacgt tgagttcgga ctctgggatg agttttgctt 8280

cgtcgacgag gagtgctact aggaggtcta gatcctcagg gggtgggagg tctgtatccg 8340

ttgagcggtg gttcatggcg tttatcattt gtgaacgagc gaggtctttt cttgcttgtg 8400

atggtaaacc aaagatattg gggagaggtt tcttgtcggg gttgacgaat gtcattcgag 8460

cggcgatgct ctcccacgtt gattgggagt aatactccct ccactcatca cccttaatat 8520

cgaacagaga cttaaacgcc tcgtaagtgg taaattggct ttcggacgat tcaacctcca 8580

aggagcgggt ataggcgtga atgatctgaa tccagaaata cgcctgtgtc tcagagtacg 8640

gaggaaccga agaatccgaa gccctcaaac gcatagtcga agactgcagt gcttcaagag 8700

cctgtttaac aacccctccc cgtctaagct ttgacgaaag agtcttttga accaccgaaa 8760

acgcaaatcg aggcagccga tcaacatcgt tgacagcttt ttctcgcaaa ggcgcagaag 8820

tggcctgctt aatagtggga agaggctgta agtcaggaag actatagtga tcccgagcta 8880

gctgcgagaa caacaggttc ttggagtagt atgatctcca gaggcccgag ttcatgagat 8940

ggggtgctga gaagacgatg tcggcgtagt cttctcgaga ggggaatttg tcgaggtgtg 9000

attttgcttg gtattctatt gcagcgactt gtatttgatg aagccagaat atcgtcatcg 9060

ttctaaatag tcagtcatca gaaataggga gtggatgggt gacatacttg tgcgctgtgt 9120

tgcgaaacct ctcaggattt ccctcccgca gccgattcaa gtgcttcata aactcatcag 9180

cacactccaa caatcccaat cctttaccga aacaatcgtg catgacaaag aaacccgctc 9240

tgagatgagt gaaatgatcc cactcctcca aagttccatc taaaaacgat tcccagaacg 9300

tcttggcatc agcactctca gcattagccg ttctaatcct attgcgaagt tcctcttcac 9360

tcggcccttt ctccctctgt tcaatctcag ctttcgtatc cttagctacg ttcttcggtt 9420

tcaacgtctt tcgtatctca tccatgacgt tatgaacctg gcaaccccat agggcactta 9480

gaccaggatt atccaggatc tttatatcga gcttatcagc atatggcttg atctcagctt 9540

ctatcttagc ctttagttct tttacgactt ttggtcgttc ttcttccgag aggaggtctt 9600

gtttgttgaa gatgataaac atgtgctttg cctcgtattc aagaatcata ttcattccca 9660

tgcggagata ctcaagcgag aactcaacac gatcaggatc tgcgtcggtg cagctttgaa 9720

tgaatactag gaagcgatct tgggccatgt agtgtcgtat gagaggacga atggcatcac 9780

agcctagatt gttagctgag atcgtgggct acagaggata gcttacctcc aacgtcccag 9840

agggtaactt tctctccatc aggatagtct atagtctcga cattgaagcc aattgtcgga 9900

atggcctgca caacctcatt aaacttgagt ctgtatagaa atgttgtttt accggatgcg 9960

tcattgccta gaacgaggcc gacatgttcg cggcctccgc ccaagagcca acgagcgatg 10020

cctgggacat tagcattgag acgaccactg cttacaataa aacttacgca tgattattga 10080

ggattgaatt gagttgagga ttaaacagaa acagaaataa caggctcttt tatatgtgat 10140

tcaaagtcgc tgccttgaag tggggagctg agattagacg tggcttcaag aatctgcctt 10200

gctcttcagc ttctcaaaat aactacgcta agtgtccact ccagcccctc agattctagt 10260

tcggcaacga ggtaatgctt tacagtcttc gtgagaagat tgaactatac tattcatttc 10320

catgcaaatc tagtccacca tgctccagcc ttgggtatca atcctaatcc atagacccag 10380

caagcgcgac agcactaaac ctcaaatcct taccctccct ctccaaaaac ttcaacgccc 10440

ccagcgccag gccctttgga ctcagcacat cctcgttatc ctccagatcg cctcgtcgaa 10500

taggtccttt tcgccgtccg tcgagctccc acaatccacc gtctgtgccc ttggcgaagc 10560

atacgtagtg tagctccacg tcgtcttctg cagcgggcgc gggtgtgtcg ccttgtgtag 10620

cagcttcttt atgcgcagtc gcgagatcag gtgtcgtttc gagaactttt gcgcggccgt 10680

ggggatcaag agggatactc ttcttgatca atacatcaag cgtagaatcc tcc 10733

<210> 2

<211> 4933

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 2

ctaatgcaag catccgcgca aacaacctcc ctcagtagcc tccacctcaa ggcaccttcc 60

acctccgaca atccttttcc tgtctctctg gtattcactc gtcagaaata tcagtacagg 120

gcttattcca ctcttccttt gatcgctctc tcattgcaaa tgtatcttca tcctgagcag 180

taactaatcc acgcttcaac atgtcggatc acgagtacat cgacactgat gacgaagacg 240

atcgatatcg cctcatcgtg aacccgtcga agcctcgaaa gatcacggaa aagaagctcc 300

gagaccaagc tgctctacaa gcgcacatag aaaagacaca gagagatgcc gcaaggagtg 360

ccgctccttt cagcgatcct agtaaacagt ccctcgccca gctcatgaac cagaacgaga 420

gccacaagat catcgcctcg cctcgagaat accagattga actgttcgag cgcgctaaag 480

agagaaacct ccttgtggtg ttgcctacag gtatgtttcg ctaccaatct ggaggctcgc 540

taatagagta ggtactggca aaacactcat ctccgccttg ctgctacgac atcacctcga 600

acaagagctg gaagctcgag ccatcggaaa gcccaagaaa gtcgcctttt tcctagtaga 660

aaaggtcgcc ctctgtgttc agcaacatgc agtcctcagc tgcaacctcg gggcccaccc 720

tgtcaccaag ttcgtaggcc atatgaaagg catgacgaag accaaagaat actgggatga 780

gaagtttggc gaaaacatgg ttgtggtctg cactgctcag atcctcctcg actgcctcac 840

ttgcggcttc atcaccatgt cccggataaa tcttctcata ttcgacgaag cacaccacac 900

aaagaagaag cacccgtacg ctcgaattat ggatgggtat tattgcaaac acgaaggcga 960

aaaacctcgc atactgggaa tgactgcttc gcctgtggac gcgcgcacca aggacgtgag 1020

agacactgcc ctggagcttg agaggtctct tgacagccag attgcaacga tttcagatga 1080

ggtgctgatg gaaaccatgc accgaaagaa gcaaaccgaa gaaactgtca actacagcag 1140

gcctgaagct cccgatgata caaagacgca tctatgggag cagatctttg ccctagtttc 1200

tcgcaacgaa cagttcaagt tgtcgctcga gttcacaaaa gaggcctcgt cagtcctcgg 1260

tacttggtgt gcggaccgat attgggagct cgcaatgaca gatctcgaga ctgaacggct 1320

cgcagccaga actagtcggg aatttattgg agacattgca gcaacaagag cagatctagc 1380

aacagaggcc gtccgacgtg tccaggaaat tgttcaggct cacaagttcg gtgcaatcga 1440

cccaaaggcc gaagggctct cggccaaggt gaaaagtctc cataagatct tgtcgcacgc 1500

tttcacaatg gatggcacca agcgttgcat tgtttttgtc gagaagcgat acacagcccg 1560

tttactttcg gatctctata gccaaccttc catgaggatc ccaagaattt ctgcttcata 1620

catggtgagt agtagccttt tcgggtcgac tttgctaata attgcaggtt ggctgtcaat 1680

caaccaattc gagcttcggc accatgtcgt ttcgagaaca agtcctggct ctccacgaat 1740

tcaagcgcgg caataccaac tgccttttca caacacctgt tgcagaagag ggcattgacg 1800

ttccagattg cgaccttgtt attcgtttcg atctctacaa ctccgtcatc caatatttgc 1860

aatccaaagg acgcgctcga caagcacgct ctcgatatat caccatgatg gaggagggaa 1920

atttgacaca tattcgaagt gcgaaacaag cattgagaga ttcacaggct ctggaaaagt 1980

tctgtctctc tctttctgct gaccgaaaac ttcatgatga tttgatagac gaggacatgg 2040

agatgatggt tgagcgcatg cagcatcaag tgtacgagat accttcgaca ggcgcacgcc 2100

ttacgtttcc cgccagcctt gaaatcctgg ccagatttgt ttctcacttg tcaactggca 2160

gctacgccaa agttgaatac caagtacaga aggttggcga tcgatatcta gctgacgtgg 2220

tcatgccatt acagtccccg gtcaactttg tgaagggtac ccaacagcgt agcaaattgc 2280

tggccaagtg ttctgcagca tttgaagcct gcaagatact gatcagagac aaacatgttg 2340

acggcaatct tcagccgaca ttcaagaaaa aggttcacgc tatgcggaac gcacggctag 2400

ctgtgagttc aaacaagaag gctgactacg acatgcgatt gcggccagag atttgggctg 2460

aacgagggga gtggactgaa tgttttgcga ctacaatcac catcaaagac gggggcaccc 2520

ttaggaaggc aatagccgga aagtcgttaa tacttctctc gcgcaagccg atcccgaaac 2580

tgccaggtgt tcctctattc tttggcaatg gacattcctc tatcgctgaa ttggcgccta 2640

atcaggatgc tcttaaactc aaccctgaag aagctgacgg attgaccaaa ttcactcatc 2700

gggtattcgc cgatgtgttc agcaaagaat acgataccac gtccgaccaa tatccatacc 2760

tcctcgcacc ttatgccgac agtccccaat cagacactcg atcacatatt gattggaatg 2820

ttgtcaacct tgtgagcaag aacgaggtgc tcgaattgga gaatgcgcca gaagacttct 2880

tcgtcaacaa gcttgtgact gatccatatg atggaggacg caagcttgtc atcaaaggta 2940

tcgacaaaac gaagaagtca tccgacccaa caccagaagg agtccccgaa tcaagaagca 3000

gagcttatag gtctgtggaa ccgacaatca tgcaatacag taacagtctg tactataaat 3060

ctcgccaaag ggtgaagtgg cgagatgatc agcctgtggt caaggcagag cttctttcct 3120

tgagacgaaa ccttctggat gagtttgaag ttgacgagaa ggtcaacatg gagtgttgca 3180

tcattctgga gcctctccat gtgtctcctg taagttgaat cccagccttg atgcttgtct 3240

aattttccca gctacctatc gatgtagtct ctatggccat ggcatttcca gcaatcattc 3300

accgaataga ttccgtcctg attgtgttgg atgcatgcaa tctccttgac cttgaaattc 3360

caccagaact cgcactggag gccatgacca aagacagcga caactctggg gagcatgaca 3420

cagaacagat caatttccaa acaggcatgg gcgacaacta tgaacggcta gagttcctcg 3480

gcgactgttt tctcaagatg gccaccacga tctcaatcta cacactgatg cctgacggaa 3540

atgagtgcaa ataccacgtt gagcgcatgc ttctcatttg caaccaaaac ctcttcaatc 3600

atgcggttga tcgcaaactc caagagttcg tccgatccaa ggcttttgac agaagaacat 3660

ggtaccccga tcttccactc aagaaaggaa aggcaccgaa aacttcgatg aagcataatc 3720

tggccgacaa gacgattgca gacgtatgtg aagctctcat tggggcagca tacctcacaa 3780

gcaaggccag caacatggat ctagctgtca aagccgtgac gcgaatgacc aagtcaaaga 3840

accacaggat gaaggctttc aaagattact ataaagccta caaggtgcct gagtggcagg 3900

aaggtaatgc gtccgcctct caacgcagcc ttgtgcaaaa ggtggcacaa gctacaggat 3960

atcgcttcaa gtctgcacag cttgtccaga gtgcattcaa acatccttcg tggccatatg 4020

agtctgtccc agattatcag cgcctcgaat ttctgggcga ttcacttctc gacatggcca 4080

tcgtcgatta tctctatcaa aacttcccca gagcagaccc tcagtggctg acggaacaca 4140

agatggccat ggtatcgaat cagttccttg gatgcctctg cgttaagctt ggtctgcacc 4200

aacacctact caccagcaca tcgagtcttc tcagccaaat ccgcgattat gtcgtcgaac 4260

ttgaagttgc tgaagaaaat gctcgcaagg aagcgaaaga agatgggact cccatgcgca 4320

aggacttctg gctgaaggtg gattcggcac ccaaggttta cgccgatgtg attgaagctc 4380

ttgttggagc catgtttgtc gattccaagt acaaattttc ggttgttgag aatttcttta 4440

ccaagttcat ccagccatat ttccaagaca tgagacttta cgataccttt gcgaacaagc 4500

acccagtcac gttcctctcc aagaagatgc atcaggaatt cggttgcaca aactggcgca 4560

tcagcgcaga ggctgtacca tgcagtgcag atcaaggcat ggcggcgctg aaagataccg 4620

aaatgtgcgc tgttttcatg gtgcatcaaa aggttatcgc ggatgacaca tctgagagtg 4680

ggaggtattc taagcagcgt gccgcgacga aagcgctcga gatacttgat tcgtttggtg 4740

aggatgttga ggcggcgaag aggtttttgg ggtgtgattg tcagcttgga ggaggggatg 4800

ttgaggttga tcatggaaca gctgtttgag ctgaaatcgg cactagaatt ggaggacatg 4860

tgtttcaggg acataaagcg cttcatagcg ttagaagcgt agacaaagat catagagcaa 4920

ttaaaatgtt gcg 4933

<210> 3

<211> 4506

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 3

atgtcggatc acgagtacat cgacactgat gacgaagacg atcgatatcg cctcatcgtg 60

aacccgtcga agcctcgaaa gatcacggaa aagaagctcc gagaccaagc tgctctacaa 120

gcgcacatag aaaagacaca gagagatgcc gcaaggagtg ccgctccttt cagcgatcct 180

agtaaacagt ccctcgccca gctcatgaac cagaacgaga gccacaagat catcgcctcg 240

cctcgagaat accagattga actgttcgag cgcgctaaag agagaaacct ccttgtggtg 300

ttgcctacag taggtactgg caaaacactc atctccgcct tgctgctacg acatcacctc 360

gaacaagagc tggaagctcg agccatcgga aagcccaaga aagtcgcctt tttcctagta 420

gaaaaggtcg ccctctgtgt tcagcaacat gcagtcctca gctgcaacct cggggcccac 480

cctgtcacca agttcgtagg ccatatgaaa ggcatgacga agaccaaaga atactgggat 540

gagaagtttg gcgaaaacat ggttgtggtc tgcactgctc agatcctcct cgactgcctc 600

acttgcggct tcatcaccat gtcccggata aatcttctca tattcgacga agcacaccac 660

acaaagaaga agcacccgta cgctcgaatt atggatgggt attattgcaa acacgaaggc 720

gaaaaacctc gcatactggg aatgactgct tcgcctgtgg acgcgcgcac caaggacgtg 780

agagacactg ccctggagct tgagaggtct cttgacagcc agattgcaac gatttcagat 840

gaggtgctga tggaaaccat gcaccgaaag aagcaaaccg aagaaactgt caactacagc 900

aggcctgaag ctcccgatga tacaaagacg catctatggg agcagatctt tgccctagtt 960

tctcgcaacg aacagttcaa gttgtcgctc gagttcacaa aagaggcctc gtcagtcctc 1020

ggtacttggt gtgcggaccg atattgggag ctcgcaatga cagatctcga gactgaacgg 1080

ctcgcagcca gaactagtcg ggaatttatt ggagacattg cagcaacaag agcagatcta 1140

gcaacagagg ccgtccgacg tgtccaggaa attgttcagg ctcacaagtt cggtgcaatc 1200

gacccaaagg ccgaagggct ctcggccaag gtgaaaagtc tccataagat cttgtcgcac 1260

gctttcacaa tggatggcac caagcgttgc attgtttttg tcgagaagcg atacacagcc 1320

cgtttacttt cggatctcta tagccaacct tccatgagga tcccaagaat ttctgcttca 1380

tacatggttg gctgtcaatc aaccaattcg agcttcggca ccatgtcgtt tcgagaacaa 1440

gtcctggctc tccacgaatt caagcgcggc aataccaact gccttttcac aacacctgtt 1500

gcagaagagg gcattgacgt tccagattgc gaccttgtta ttcgtttcga tctctacaac 1560

tccgtcatcc aatatttgca atccaaagga cgcgctcgac aagcacgctc tcgatatatc 1620

accatgatgg aggagggaaa tttgacacat attcgaagtg cgaaacaagc attgagagat 1680

tcacaggctc tggaaaagtt ctgtctctct ctttctgctg accgaaaact tcatgatgat 1740

ttgatagacg aggacatgga gatgatggtt gagcgcatgc agcatcaagt gtacgagata 1800

ccttcgacag gcgcacgcct tacgtttccc gccagccttg aaatcctggc cagatttgtt 1860

tctcacttgt caactggcag ctacgccaaa gttgaatacc aagtacagaa ggttggcgat 1920

cgatatctag ctgacgtggt catgccatta cagtccccgg tcaactttgt gaagggtacc 1980

caacagcgta gcaaattgct ggccaagtgt tctgcagcat ttgaagcctg caagatactg 2040

atcagagaca aacatgttga cggcaatctt cagccgacat tcaagaaaaa ggttcacgct 2100

atgcggaacg cacggctagc tgtgagttca aacaagaagg ctgactacga catgcgattg 2160

cggccagaga tttgggctga acgaggggag tggactgaat gttttgcgac tacaatcacc 2220

atcaaagacg ggggcaccct taggaaggca atagccggaa agtcgttaat acttctctcg 2280

cgcaagccga tcccgaaact gccaggtgtt cctctattct ttggcaatgg acattcctct 2340

atcgctgaat tggcgcctaa tcaggatgct cttaaactca accctgaaga agctgacgga 2400

ttgaccaaat tcactcatcg ggtattcgcc gatgtgttca gcaaagaata cgataccacg 2460

tccgaccaat atccatacct cctcgcacct tatgccgaca gtccccaatc agacactcga 2520

tcacatattg attggaatgt tgtcaacctt gtgagcaaga acgaggtgct cgaattggag 2580

aatgcgccag aagacttctt cgtcaacaag cttgtgactg atccatatga tggaggacgc 2640

aagcttgtca tcaaaggtat cgacaaaacg aagaagtcat ccgacccaac accagaagga 2700

gtccccgaat caagaagcag agcttatagg tctgtggaac cgacaatcat gcaatacagt 2760

aacagtctgt actataaatc tcgccaaagg gtgaagtggc gagatgatca gcctgtggtc 2820

aaggcagagc ttctttcctt gagacgaaac cttctggatg agtttgaagt tgacgagaag 2880

gtcaacatgg agtgttgcat cattctggag cctctccatg tgtctcctct acctatcgat 2940

gtagtctcta tggccatggc atttccagca atcattcacc gaatagattc cgtcctgatt 3000

gtgttggatg catgcaatct ccttgacctt gaaattccac cagaactcgc actggaggcc 3060

atgaccaaag acagcgacaa ctctggggag catgacacag aacagatcaa tttccaaaca 3120

ggcatgggcg acaactatga acggctagag ttcctcggcg actgttttct caagatggcc 3180

accacgatct caatctacac actgatgcct gacggaaatg agtgcaaata ccacgttgag 3240

cgcatgcttc tcatttgcaa ccaaaacctc ttcaatcatg cggttgatcg caaactccaa 3300

gagttcgtcc gatccaaggc ttttgacaga agaacatggt accccgatct tccactcaag 3360

aaaggaaagg caccgaaaac ttcgatgaag cataatctgg ccgacaagac gattgcagac 3420

gtatgtgaag ctctcattgg ggcagcatac ctcacaagca aggccagcaa catggatcta 3480

gctgtcaaag ccgtgacgcg aatgaccaag tcaaagaacc acaggatgaa ggctttcaaa 3540

gattactata aagcctacaa ggtgcctgag tggcaggaag gtaatgcgtc cgcctctcaa 3600

cgcagccttg tgcaaaaggt ggcacaagct acaggatatc gcttcaagtc tgcacagctt 3660

gtccagagtg cattcaaaca tccttcgtgg ccatatgagt ctgtcccaga ttatcagcgc 3720

ctcgaatttc tgggcgattc acttctcgac atggccatcg tcgattatct ctatcaaaac 3780

ttccccagag cagaccctca gtggctgacg gaacacaaga tggccatggt atcgaatcag 3840

ttccttggat gcctctgcgt taagcttggt ctgcaccaac acctactcac cagcacatcg 3900

agtcttctca gccaaatccg cgattatgtc gtcgaacttg aagttgctga agaaaatgct 3960

cgcaaggaag cgaaagaaga tgggactccc atgcgcaagg acttctggct gaaggtggat 4020

tcggcaccca aggtttacgc cgatgtgatt gaagctcttg ttggagccat gtttgtcgat 4080

tccaagtaca aattttcggt tgttgagaat ttctttacca agttcatcca gccatatttc 4140

caagacatga gactttacga tacctttgcg aacaagcacc cagtcacgtt cctctccaag 4200

aagatgcatc aggaattcgg ttgcacaaac tggcgcatca gcgcagaggc tgtaccatgc 4260

agtgcagatc aaggcatggc ggcgctgaaa gataccgaaa tgtgcgctgt tttcatggtg 4320

catcaaaagg ttatcgcgga tgacacatct gagagtggga ggtattctaa gcagcgtgcc 4380

gcgacgaaag cgctcgagat acttgattcg tttggtgagg atgttgaggc ggcgaagagg 4440

tttttggggt gtgattgtca gcttggagga ggggatgttg aggttgatca tggaacagct 4500

gtttga 4506

<210> 4

<211> 1501

<212> PRT

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 4

Met Ser Asp His Glu Tyr Ile Asp Thr Asp Asp Glu Asp Asp Arg Tyr

1 5 10 15

Arg Leu Ile Val Asn Pro Ser Lys Pro Arg Lys Ile Thr Glu Lys Lys

20 25 30

Leu Arg Asp Gln Ala Ala Leu Gln Ala His Ile Glu Lys Thr Gln Arg

35 40 45

Asp Ala Ala Arg Ser Ala Ala Pro Phe Ser Asp Pro Ser Lys Gln Ser

50 55 60

Leu Ala Gln Leu Met Asn Gln Asn Glu Ser His Lys Ile Ile Ala Ser

65 70 75 80

Pro Arg Glu Tyr Gln Ile Glu Leu Phe Glu Arg Ala Lys Glu Arg Asn

85 90 95

Leu Leu Val Val Leu Pro Thr Val Gly Thr Gly Lys Thr Leu Ile Ser

100 105 110

Ala Leu Leu Leu Arg His His Leu Glu Gln Glu Leu Glu Ala Arg Ala

115 120 125

Ile Gly Lys Pro Lys Lys Val Ala Phe Phe Leu Val Glu Lys Val Ala

130 135 140

Leu Cys Val Gln Gln His Ala Val Leu Ser Cys Asn Leu Gly Ala His

145 150 155 160

Pro Val Thr Lys Phe Val Gly His Met Lys Gly Met Thr Lys Thr Lys

165 170 175

Glu Tyr Trp Asp Glu Lys Phe Gly Glu Asn Met Val Val Val Cys Thr

180 185 190

Ala Gln Ile Leu Leu Asp Cys Leu Thr Cys Gly Phe Ile Thr Met Ser

195 200 205

Arg Ile Asn Leu Leu Ile Phe Asp Glu Ala His His Thr Lys Lys Lys

210 215 220

His Pro Tyr Ala Arg Ile Met Asp Gly Tyr Tyr Cys Lys His Glu Gly

225 230 235 240

Glu Lys Pro Arg Ile Leu Gly Met Thr Ala Ser Pro Val Asp Ala Arg

245 250 255

Thr Lys Asp Val Arg Asp Thr Ala Leu Glu Leu Glu Arg Ser Leu Asp

260 265 270

Ser Gln Ile Ala Thr Ile Ser Asp Glu Val Leu Met Glu Thr Met His

275 280 285

Arg Lys Lys Gln Thr Glu Glu Thr Val Asn Tyr Ser Arg Pro Glu Ala

290 295 300

Pro Asp Asp Thr Lys Thr His Leu Trp Glu Gln Ile Phe Ala Leu Val

305 310 315 320

Ser Arg Asn Glu Gln Phe Lys Leu Ser Leu Glu Phe Thr Lys Glu Ala

325 330 335

Ser Ser Val Leu Gly Thr Trp Cys Ala Asp Arg Tyr Trp Glu Leu Ala

340 345 350

Met Thr Asp Leu Glu Thr Glu Arg Leu Ala Ala Arg Thr Ser Arg Glu

355 360 365

Phe Ile Gly Asp Ile Ala Ala Thr Arg Ala Asp Leu Ala Thr Glu Ala

370 375 380

Val Arg Arg Val Gln Glu Ile Val Gln Ala His Lys Phe Gly Ala Ile

385 390 395 400

Asp Pro Lys Ala Glu Gly Leu Ser Ala Lys Val Lys Ser Leu His Lys

405 410 415

Ile Leu Ser His Ala Phe Thr Met Asp Gly Thr Lys Arg Cys Ile Val

420 425 430

Phe Val Glu Lys Arg Tyr Thr Ala Arg Leu Leu Ser Asp Leu Tyr Ser

435 440 445

Gln Pro Ser Met Arg Ile Pro Arg Ile Ser Ala Ser Tyr Met Val Gly

450 455 460

Cys Gln Ser Thr Asn Ser Ser Phe Gly Thr Met Ser Phe Arg Glu Gln

465 470 475 480

Val Leu Ala Leu His Glu Phe Lys Arg Gly Asn Thr Asn Cys Leu Phe

485 490 495

Thr Thr Pro Val Ala Glu Glu Gly Ile Asp Val Pro Asp Cys Asp Leu

500 505 510

Val Ile Arg Phe Asp Leu Tyr Asn Ser Val Ile Gln Tyr Leu Gln Ser

515 520 525

Lys Gly Arg Ala Arg Gln Ala Arg Ser Arg Tyr Ile Thr Met Met Glu

530 535 540

Glu Gly Asn Leu Thr His Ile Arg Ser Ala Lys Gln Ala Leu Arg Asp

545 550 555 560

Ser Gln Ala Leu Glu Lys Phe Cys Leu Ser Leu Ser Ala Asp Arg Lys

565 570 575

Leu His Asp Asp Leu Ile Asp Glu Asp Met Glu Met Met Val Glu Arg

580 585 590

Met Gln His Gln Val Tyr Glu Ile Pro Ser Thr Gly Ala Arg Leu Thr

595 600 605

Phe Pro Ala Ser Leu Glu Ile Leu Ala Arg Phe Val Ser His Leu Ser

610 615 620

Thr Gly Ser Tyr Ala Lys Val Glu Tyr Gln Val Gln Lys Val Gly Asp

625 630 635 640

Arg Tyr Leu Ala Asp Val Val Met Pro Leu Gln Ser Pro Val Asn Phe

645 650 655

Val Lys Gly Thr Gln Gln Arg Ser Lys Leu Leu Ala Lys Cys Ser Ala

660 665 670

Ala Phe Glu Ala Cys Lys Ile Leu Ile Arg Asp Lys His Val Asp Gly

675 680 685

Asn Leu Gln Pro Thr Phe Lys Lys Lys Val His Ala Met Arg Asn Ala

690 695 700

Arg Leu Ala Val Ser Ser Asn Lys Lys Ala Asp Tyr Asp Met Arg Leu

705 710 715 720

Arg Pro Glu Ile Trp Ala Glu Arg Gly Glu Trp Thr Glu Cys Phe Ala

725 730 735

Thr Thr Ile Thr Ile Lys Asp Gly Gly Thr Leu Arg Lys Ala Ile Ala

740 745 750

Gly Lys Ser Leu Ile Leu Leu Ser Arg Lys Pro Ile Pro Lys Leu Pro

755 760 765

Gly Val Pro Leu Phe Phe Gly Asn Gly His Ser Ser Ile Ala Glu Leu

770 775 780

Ala Pro Asn Gln Asp Ala Leu Lys Leu Asn Pro Glu Glu Ala Asp Gly

785 790 795 800

Leu Thr Lys Phe Thr His Arg Val Phe Ala Asp Val Phe Ser Lys Glu

805 810 815

Tyr Asp Thr Thr Ser Asp Gln Tyr Pro Tyr Leu Leu Ala Pro Tyr Ala

820 825 830

Asp Ser Pro Gln Ser Asp Thr Arg Ser His Ile Asp Trp Asn Val Val

835 840 845

Asn Leu Val Ser Lys Asn Glu Val Leu Glu Leu Glu Asn Ala Pro Glu

850 855 860

Asp Phe Phe Val Asn Lys Leu Val Thr Asp Pro Tyr Asp Gly Gly Arg

865 870 875 880

Lys Leu Val Ile Lys Gly Ile Asp Lys Thr Lys Lys Ser Ser Asp Pro

885 890 895

Thr Pro Glu Gly Val Pro Glu Ser Arg Ser Arg Ala Tyr Arg Ser Val

900 905 910

Glu Pro Thr Ile Met Gln Tyr Ser Asn Ser Leu Tyr Tyr Lys Ser Arg

915 920 925

Gln Arg Val Lys Trp Arg Asp Asp Gln Pro Val Val Lys Ala Glu Leu

930 935 940

Leu Ser Leu Arg Arg Asn Leu Leu Asp Glu Phe Glu Val Asp Glu Lys

945 950 955 960

Val Asn Met Glu Cys Cys Ile Ile Leu Glu Pro Leu His Val Ser Pro

965 970 975

Leu Pro Ile Asp Val Val Ser Met Ala Met Ala Phe Pro Ala Ile Ile

980 985 990

His Arg Ile Asp Ser Val Leu Ile Val Leu Asp Ala Cys Asn Leu Leu

995 1000 1005

Asp Leu Glu Ile Pro Pro Glu Leu Ala Leu Glu Ala Met Thr Lys Asp

1010 1015 1020

Ser Asp Asn Ser Gly Glu His Asp Thr Glu Gln Ile Asn Phe Gln Thr

1025 1030 1035 1040

Gly Met Gly Asp Asn Tyr Glu Arg Leu Glu Phe Leu Gly Asp Cys Phe

1045 1050 1055

Leu Lys Met Ala Thr Thr Ile Ser Ile Tyr Thr Leu Met Pro Asp Gly

1060 1065 1070

Asn Glu Cys Lys Tyr His Val Glu Arg Met Leu Leu Ile Cys Asn Gln

1075 1080 1085

Asn Leu Phe Asn His Ala Val Asp Arg Lys Leu Gln Glu Phe Val Arg

1090 1095 1100

Ser Lys Ala Phe Asp Arg Arg Thr Trp Tyr Pro Asp Leu Pro Leu Lys

1105 1110 1115 1120

Lys Gly Lys Ala Pro Lys Thr Ser Met Lys His Asn Leu Ala Asp Lys

1125 1130 1135

Thr Ile Ala Asp Val Cys Glu Ala Leu Ile Gly Ala Ala Tyr Leu Thr

1140 1145 1150

Ser Lys Ala Ser Asn Met Asp Leu Ala Val Lys Ala Val Thr Arg Met

1155 1160 1165

Thr Lys Ser Lys Asn His Arg Met Lys Ala Phe Lys Asp Tyr Tyr Lys

1170 1175 1180

Ala Tyr Lys Val Pro Glu Trp Gln Glu Gly Asn Ala Ser Ala Ser Gln

1185 1190 1195 1200

Arg Ser Leu Val Gln Lys Val Ala Gln Ala Thr Gly Tyr Arg Phe Lys

1205 1210 1215

Ser Ala Gln Leu Val Gln Ser Ala Phe Lys His Pro Ser Trp Pro Tyr

1220 1225 1230

Glu Ser Val Pro Asp Tyr Gln Arg Leu Glu Phe Leu Gly Asp Ser Leu

1235 1240 1245

Leu Asp Met Ala Ile Val Asp Tyr Leu Tyr Gln Asn Phe Pro Arg Ala

1250 1255 1260

Asp Pro Gln Trp Leu Thr Glu His Lys Met Ala Met Val Ser Asn Gln

1265 1270 1275 1280

Phe Leu Gly Cys Leu Cys Val Lys Leu Gly Leu His Gln His Leu Leu

1285 1290 1295

Thr Ser Thr Ser Ser Leu Leu Ser Gln Ile Arg Asp Tyr Val Val Glu

1300 1305 1310

Leu Glu Val Ala Glu Glu Asn Ala Arg Lys Glu Ala Lys Glu Asp Gly

1315 1320 1325

Thr Pro Met Arg Lys Asp Phe Trp Leu Lys Val Asp Ser Ala Pro Lys

1330 1335 1340

Val Tyr Ala Asp Val Ile Glu Ala Leu Val Gly Ala Met Phe Val Asp

1345 1350 1355 1360

Ser Lys Tyr Lys Phe Ser Val Val Glu Asn Phe Phe Thr Lys Phe Ile

1365 1370 1375

Gln Pro Tyr Phe Gln Asp Met Arg Leu Tyr Asp Thr Phe Ala Asn Lys

1380 1385 1390

His Pro Val Thr Phe Leu Ser Lys Lys Met His Gln Glu Phe Gly Cys

1395 1400 1405

Thr Asn Trp Arg Ile Ser Ala Glu Ala Val Pro Cys Ser Ala Asp Gln

1410 1415 1420

Gly Met Ala Ala Leu Lys Asp Thr Glu Met Cys Ala Val Phe Met Val

1425 1430 1435 1440

His Gln Lys Val Ile Ala Asp Asp Thr Ser Glu Ser Gly Arg Tyr Ser

1445 1450 1455

Lys Gln Arg Ala Ala Thr Lys Ala Leu Glu Ile Leu Asp Ser Phe Gly

1460 1465 1470

Glu Asp Val Glu Ala Ala Lys Arg Phe Leu Gly Cys Asp Cys Gln Leu

1475 1480 1485

Gly Gly Gly Asp Val Glu Val Asp His Gly Thr Ala Val

1490 1495 1500

<210> 5

<211> 23

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 5

ggctatggtc ccaagaatca tca 23

<210> 6

<211> 46

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 6

acctccacta gctccagcca agcgagtgaa taccagagag acagga 46

<210> 7

<211> 46

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 7

gaatagagta gatgccgacc gggtgatgtg ttcttggtcc actgag 46

<210> 8

<211> 21

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 8

tctcgcttga gtatctccgc a 21

<210> 9

<211> 22

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 9

cttggctgga gctagtggag gt 22

<210> 10

<211> 23

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 10

cccggtcggc atctactcta ttc 23

<210> 11

<211> 23

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 11

ctgagccctt gtcttgagcc ata 23

<210> 12

<211> 20

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 12

ggatgcctcc gctcgaagta 20

<210> 13

<211> 20

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 13

cgttgcaaga cctgcctgaa 20

<210> 14

<211> 22

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 14

tctcagagcg ggtttctttg tc 22

<210> 15

<211> 20

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 15

gacagacgtc gcggtgagtt 20

<210> 16

<211> 21

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 16

tctggaccga tggctgtgta g 21

<210> 17

<211> 21

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 17

acaatcacca tcaaagacgg g 21

<210> 18

<211> 21

<212> DNA

<213>withered germ of water-melon (Fusarium oxysporum f. sp. niveum)

<400> 18

ccaacacaat caggacggaa t 21

Claims (4)

1. a kind of withered germ of water-melon RNAi component gene FonDCL1, which is characterized in that comprising thereon, downstream nucleotide sequence Nucleotide sequence as shown in SEQ ID NO:1.

2. a kind of withered germ of water-melon RNAi component gene FonDCL1 according to claim 1, which is characterized in that described The length of upstream and downstream nucleotide sequence is 3Kb.

3. a kind of construction method of withered germ of water-melon FonDCL1 deletion mutant body, which is characterized in that pass through following steps It obtains:

1) FonDCL1 gene delection recombinant dna fragment constructs

First round PCR amplification: using watermelon blight genomic DNA as template, primers F on1DCL1-LBCK/Fon1DCL1-HPH- LB-R expands FonDCL1 upstream region of gene FonDCL1-UP segment, primers F on1DCL1-HPH-RB-F/Fon1DCL1-RBCK amplification FonDCL1 downstream of gene FonDCL1-DOWN segment；Using carrier pCT74 Plasmid DNA as template, primer HYG-F/HYG-R amplification Resistant gene HYG segment；

Second wheel PCR amplification: using FonDCL1-UP and HYG as template, primers F on1DCL1-LB-F/HYG-R1 amplification FonDCL1-UP+HY segment, using FonDCL1-DOWN and HYG as template, primer HYG-F1/Fon1DCL1-RB-R expands YG+ FonDCL1-DOWN segment；

2) protoplast and FonDCL1-UP+HY segment and YG+FonDCL1-DOWN segment are mixed, carries out FonDCL1 gene Deleting genetic conversion, obtains mutant；

The primers F on1DCL1-LBCK, Fon1DCL1-HPH-LB-R, Fon1DCL1-HPH-RB-F, Fon1DCL1-RBCK, The nucleotide sequence of HYG-F, HYG-R, Fon1DCL1-LB-F, HYG-R1, HYG-F1, Fon1DCL1-RB-R are successively such as SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10、SEQ ID NO: 11, SEQ ID NO:12, SEQ ID NO:13, shown in SEQ ID NO:14；

The nucleotide sequence of the watermelon blight genomic DNA is as shown in SEQ ID NO:1.

4. a kind of construction method of withered germ of water-melon FonDCL1 deletion mutant body according to claim 3, special Sign is that the FonDCL1 deletion mutant body is identified by the following method:

Using the genomic DNA of transformant as template, four pairs of primers are designed for identifying FonDCL1 deletion mutant body, respectively It is upstream primer Fon1DCL1-LBCK/HPT-LBCK, downstream primer Fon1DCL1-RBCK/HPT-RBCK, for expanding HYG base Primer HYG-F/HYG-R, the FonDCL1 gene internal primers F on1DCL1-2193/Fon1DCL1-3030 of cause；

The nucleotide sequence of described primer HPT-LBCK, HPT-RBCK, Fon1DCL1-2193, Fon1DCL1-3030 are successively such as SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, shown in SEQ ID NO:18.