CN111334492A

CN111334492A - Watermelon chitinase and coding gene and application thereof

Info

Publication number: CN111334492A
Application number: CN202010120487.2A
Authority: CN
Inventors: 许勇; 李茂营; 宫国义; 张海英; 郭绍贵; 张洁; 任毅
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2020-02-26
Filing date: 2020-02-26
Publication date: 2020-06-26

Abstract

The invention belongs to the field of genetic engineering, and relates to watermelon chitinase, a coding gene and application thereof. The watermelon chitinase protein has a sequence composition shown in a sequence 1 in a sequence table, and a coding gene of the watermelon chitinase protein has a sequence composition shown in a sequence 2 in the sequence table. The invention also relates to the application of the watermelon chitinase protein and the coding nucleic acid sequence of the watermelon chitinase in the resistance of watermelon fusarium wilt. The invention also provides a method for obtaining the watermelon fusarium wilt-resistant strain. The watermelon chitinase and the coding gene thereof provided by the invention are closely related to the resistance of the blight, and the higher the expression level is, the stronger the resistance to the blight is; therefore, by over-expressing the watermelon chitinase in watermelon plants, stable watermelon strains resistant to the fusarium wilt can be obtained, and a quick and efficient way is provided for breeding the fusarium wilt-resistant watermelons.

Description

Watermelon chitinase and coding gene and application thereof

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to watermelon chitinase, and a coding gene and application thereof.

Background

Chitin, a straight-chain polymeric compound of N-acetyl-D-glucosamine linked by β -1, 4-glycosidic bonds, is the main component constituting the fungal cell wall skeleton, while the cell wall of plant cells does not contain chitin.

Watermelon (watermelon land (Thunb.) Matsum & Nadai) is an important horticultural crop in the world, and the watermelon cultivation area, the total yield and the per-capita consumption of China all reside in the first place of the world. Due to continuous cropping obstacles caused by long-term continuous cropping in greenhouse planting, the physical and chemical properties of soil are changed, pathogenic bacteria are increased, diseases are easily caused, and particularly large-scale outbreak of blight becomes an important factor for restricting watermelon production.

Watermelon Fusarium wilt is a soil-borne fungal disease caused by the obligate parasitism of Fusarium Oxysporum (FON) of deuteromycete subgenus, wherein the physiological race of pathogenic bacteria is 4, and the physiological race 1(FON1) has the strongest pathogenicity and the widest existing range in China. Early genetic studies show that the watermelon fusarium wilt resistant gene is controlled by a single dominant gene Fon-1.

For years, people adopt the traditional conventional breeding method and other methods to solve the problem of blight resistance of watermelon varieties, but the traditional breeding method is high in cost, high in workload and low in selection efficiency. Therefore, how to solve these problems in conventional breeding and rapidly obtain stable watermelon fusarium wilt resistant strains is an important technical problem which is urgently needed in practice.

Disclosure of Invention

Through a large number of comprehensive experiments of the inventor, the gene ClCHI1 of watermelon chitinase (also called ClCHI1 protein) plays an important role in resisting blight of watermelon. On the basis, the inventor clones the watermelon chitinase gene ClCHI1, constructs a transgenic knockout and overexpression vector of ClCHI1 to transform watermelon to obtain transgenic watermelon materials, and defines the function of ClCHI1 in wilt resistance. Based on the gene, the invention provides watermelon chitinase, a coding gene ClCHI1 and application thereof.

The invention provides a watermelon chitinase protein in a first aspect, which is any one of the following:

1) protein shown as a sequence 1 in a sequence table;

2) a fusion protein obtained by connecting a label to the N end and/or the C end of the protein in the step 1);

3) 1) protein with watermelon chitinase activity obtained by substitution and/or deletion and/or addition of one or more amino acid residues;

4) a protein having an identity of 98% or more to the protein of 1) and having a watermelon chitinase activity.

In a second aspect, the invention provides a coding nucleic acid sequence of chitinase of watermelon, which is any one of the following:

1) the coding region is a DNA molecule shown in a sequence 2 in a sequence table, namely a watermelon chitinase gene ClCHI 1;

2) a DNA molecule which has more than 90% of identity with 1) and codes watermelon chitinase;

3) a DNA molecule which is hybridized with any one of the limited nucleotide sequences 1) or 2) under strict conditions and codes for the watermelon chitinase.

In a third aspect, the invention provides the use of a watermelon chitinase protein provided according to the first aspect of the invention or a nucleic acid sequence encoding a watermelon chitinase provided according to the second aspect of the invention for resistance to watermelon wilt disease.

According to the third aspect of the present invention, preferably, said use comprises the operation of overexpressing a watermelon chitinase-encoding nucleic acid sequence in a watermelon plant.

According to the third aspect of the present invention, preferably, the operation of overexpressing a watermelon chitinase-encoding nucleic acid sequence in a watermelon plant comprises the operations of constructing a recombinant vector comprising a watermelon chitinase-encoding nucleic acid sequence and transforming a watermelon plant with the recombinant vector.

According to the third aspect of the present invention, preferably, the recombinant vector is a Super1300-GFP-ClCHI1 recombinant plasmid.

According to the third aspect of the present invention, preferably, the operation of transforming the watermelon plant with the recombinant vector comprises introducing the recombinant vector into agrobacterium strain GV3101, and then introducing the coding nucleic acid sequence of watermelon chitinase into the watermelon plant by using the agrobacterium strain GV3101 into which the recombinant vector is introduced, so that the coding nucleic acid sequence of watermelon chitinase is over-expressed in the watermelon plant.

In a fourth aspect, the invention provides a method for obtaining a watermelon anti-fusarium wilt disease strain, which comprises the steps of performing overexpression on a watermelon chitinase encoding nucleic acid sequence according to the second aspect of the invention in a watermelon plant, then screening a strain stably overexpressing watermelon chitinase in the watermelon plant, and continuously selfing the strain stably overexpressing watermelon chitinase to obtain a stable anti-fusarium wilt watermelon strain.

According to the fourth aspect of the present invention, preferably, said stable overexpression watermelon chitinase line is selfed continuously for at least three generations, thereby obtaining a stable blight-resistant watermelon line.

The watermelon chitinase and the coding gene thereof provided by the invention are closely related to the resistance of the blight, and the higher the expression level is, the stronger the resistance of the watermelon chitinase to the blight is. Therefore, by over-expressing the watermelon chitinase in watermelon plants, stable watermelon strains resistant to the fusarium wilt can be obtained, and a quick and efficient way is provided for breeding the fusarium wilt-resistant watermelons.

Drawings

FIG. 1 is a schematic diagram showing the relative expression levels of ClCHI1 gene in each tissue site of watermelon in example 1.

FIG. 2 is a diagram showing the comparison of the relative expression amounts of the wild-type YX in example 3 and the ClCHI1 genes in transgenic homozygous lines OE1 and OE2 in which ClCHI1 is overexpressed.

FIG. 3 is a schematic diagram showing the identification of field disease resistance to blight by ClCHI1 gene knock-out watermelon lines (CRISPR-1 and CRISPR-2), ClCHI1 gene overexpression transgenic watermelon lines (OE1 and OE2), anti-blight JX2 and blight-susceptible YX in example 4.

Detailed Description

In order to make the technical solution, objects and advantages of the present invention clearer, the present invention is further described in detail by the following specific embodiments. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The methods in the following examples are conventional methods unless otherwise specified. The materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical vendors. The quantitative tests in the following examples, all set up three replicates and the results averaged. From the following description and these examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions.

The pBSE401 plasmid is described in the following documents: CRISPR/Cas9 toolkit for μ ltiplex genome editing in plants.xing HL, Dong L, Wang ZP, Zhang HY, Han CY, Liu B, Wang xc, Chen qj.bmc Plant biol.2014nov 29; 327.10.1186/s12870-014-0327-y (14) (1).

BM culture medium: 0.44g of MS medium, 3g of sucrose and 0.8g of agar were dissolved in 100mL of deionized water, the pH was adjusted to 5.8, and the mixture was sterilized by autoclaving for 15 min. MS medium is a product of PhytoTech company.

Co-culture medium: BM medium containing 1.5 mg/L6-BA.

Selection medium 1: co-cultivation medium containing 100mg/L Timentin and 1.5mg/L Basta.

Selection medium 2: co-cultivation medium containing 100mg/L Timentin and 2.0mg/L Basta.

Shoot elongation medium: BM medium containing 0.1 mg/L6-BA, 0.01mg/L NAA, 100mg/L Timentin and 1.5mg/L LBAsta.

Rooting culture medium: BM Medium containing 1mg/L IBA.

Example 1

This example illustrates the discovery, cloning and localization of watermelon chitinase protein and its coding gene.

Discovery of watermelon chitinase protein and coding gene thereof

Through the previous fine positioning and function research of the FON1 gene, a watermelon database is used for searching and comparing related gene sequences of other species, a new protein is obtained from a watermelon variety 97103 and is named as watermelon chitinase, and the sequence of the new protein is shown as a sequence 1 (consisting of 291 amino acid residues) in a sequence table. The gene coding watermelon chitinase is named ClCHI1 gene, and the open reading frame of the cDNA is shown as a sequence 2 in a sequence table (consisting of 876 nucleotides).

To analyze the differences between the ClCHI1 gene and homologous gene sequences of other species, M μ Ltalinwebsite (M.mu.L.) (http://mμLtalin.toμLouse.inra.fr/mμLtalin/) The a hierachincalstering proparaach homologous alignment of the amino acid sequence of the watermelon chitinase and the amino acid sequences of homologous genes of other species. The multiple sequence alignment results are as follows: the N-terminal amino acid sequence of these homologous genes contains a Cys-rich signal peptide sequence (mainly functioning to bind chitin) of about 40 amino acids in length, which is then linked via a hinge region to a highly conserved enzyme-active catalytic domain.

Cloning of ClCHI1 Gene

Watermelon leaf RNA was extracted using a Huayuyo kit (the kit was purchased from Huayuyo Biotech Co., Ltd., and the extraction was performed according to the provided instructions), and first-strand cDNA was synthesized using an M-MLV reverse transcription kit (purchased from Promega Co., Ltd., and was performed according to the kit instructions), and the obtained first-strand cDNA was used for amplification of the full length of the ClCHI1 gene.

According to the ClCHI1 gene sequence obtained from the watermelon genome (http:// cucurbitangenomics. org/, V1) database, two specific primers are designed for PCR amplification of the ClCHI1 gene sequence, and the two primers are as follows:

an upstream primer: 5'-ATGGCTTCCCACAAAATAAC-3', respectively;

a downstream primer: 5'-TCAGATGCTGCCTTTAATGG-3' are provided.

20 μ L of PCR reaction system is 10 × Buffer 2 μ L, 10mmol/L dNTPs 2 μ L, MgSO₄1.4. mu.L, template cDNA 1.2. mu. L, KOD-Plus enzyme 0.4. mu.L, upstream primer 0.3. mu.L, downstream primer 0.3. mu. L, ddH₂O 12.4μL。

The PCR amplification procedure was: stage 1: 94 ℃ for 2 min; and (2) stage: 94 ℃, 15s, 56 ℃, 30s, 68 ℃ and 3min for 30 cycles; and (3) stage: extension at 68 ℃ for 10 min.

The PCR product was electrophoresed on a 1% agarose gel. After the electrophoresis, the band of interest (i.e., 876bp of SEQ ID NO: 2, located near 900bp on agarose gel) was excised under an ultraviolet lamp, and purified using an agarose gel DNA recovery kit (purchased from Tiangen Biotechnology Ltd.), according to the protocol of the kit.

The end of the recovered fragment needs to be added with A, the 10 muL reaction system for adding A is 10 × Buffer 1 mu L, dATP 1 mu L, Taq enzyme 0.5 muL and the recovered fragment 7.5 muL, the reaction is carried out at 72 ℃ for half an hour, the recovered fragment after adding A is connected with PMD18-T carrier (purchased from TaKaRa company), the operation is carried out according to the instruction of the TaKaRa company, the reaction system for connecting is that the fragment after adding A is 4.5 mu L, PMD 18-T0.5 muL and Solution I5 muL, the total volume is 10 muL, and the connection is carried out at 16 ℃ overnight.

Taking 5 mu L of the ligation product, transforming Escherichia coli DH5 α by heat shock method (refer to J. SammBruk, et al, Huangpetang, et al, molecular cloning instruction (third edition), scientific Press, 2002 edition), screening positive clones in LB solid plate containing 50mg/L ampicillin, selecting 5 clones for sequencing verification (sequencing work is completed by Beijing Opticalk New technology Co., Ltd.), the sequencing result shows that the sequence has a total length of 876bp and is just the complete ORF reading frame of 291 amino acids of the coding sequence 1.

Tissue-specific expression of ClCHI1 Gene

The tissue-specific expression of the ClCHI1 gene in watermelon variety 97103 was analyzed by fluorescent real-time quantitative PCR.

The PCR template for detecting tissue specific expression is cDNA obtained by reverse transcription of total RNA of each part of the watermelon. The cDNA was obtained as described above.

The primer pairs used for the fluorescent real-time quantitative PCR were as follows:

an upstream primer: 5'-GGAGTCAGTGGACGACGTTT-3', respectively;

a downstream primer: 5'-GTCGCTGTAGCCACTGTCAA-3' are provided.

Watermelon Actin gene was used as control. The primer pair for identifying the watermelon Actin gene is as follows:

an upstream primer: 5' -CCTACAACTCAATTATGAAGTGTG-3;

a downstream primer: 5'-GAAATCCACATCTGCTGGAAGGTG-3' are provided.

The PCR procedure was: denaturation at 94 ℃ for 30 s; denaturation at 94 ℃ for 5s, annealing at 60 ℃ for 35s, and 40 cycles.

The fluorescence real-time quantitative PCR was performed using ABI 7500fast (applied biosystem) and 2 relative expression levels of ClCHI1 gene^-ΔΔCtAnd (4) calculating by using the method. The relative expression level of ClCHI1 gene in each tissue site of watermelon is shown in FIG. 1. As can be seen from FIG. 1, ClCHI1 gene has a certain expression level in different tissues of watermelon, while its expression level in root is obviously higher.

The expression level of the ClCHI1 gene in the watermelon root is obviously higher, which indicates that the gene may have important relation with the watermelon wilt resistance.

Example 2

This example illustrates the acquisition of CRISPR knockout ClCHI1 plants.

Construction of CRISPR-ClCHI1 knockout vector

1. Four-primer PCR amplification was performed using pCBC-DT1T2 at a final concentration of 10 ng/. mu.L as a template.

The sequence composition of the four primers is as follows.

The sequence composition of DT1-BsF-ClCHI 1:

5’-ATATATGGTCTCGATTG ATTGGACACTGCGGCGCGGGTT-3’；

the sequence composition of DT1-F0-ClCHI 1:

5’-TG ATTGGACACTGCGGCGCGGGTTTTAGAGCTAGAAATAGC-3’；

DT2-R0-ClCHI1 sequence composition:

5’-AACCGGCTTTGGCAAGATTTGACAATCTCTTAGTCGACTCTAC-3’；

the sequence composition of DT2-Bsr-ClCHI 1:

5’-ATTATTGGTCTCGAAAC CGGCTTTGGCAAGATTTGA CAA-3’。

20 μ L of PCR reaction system comprises pCBC-DT1T2 template, 10 × Buffer 2 μ L, 10mmol/L dNTPs 2 μ L, MgSO₄1.4. mu. L, cDNA 1.2.2. mu. L, KOD-Plus enzyme 0.4. mu.L, four primers 0.3. mu. L, ddH, respectively₂O 11.8μL。

In the four primers, the concentrations of DT1-BsF-ClCHI1 and DT1-F0-ClCHI1 are both 100 mu mol/L; the concentrations of DT2-R0-ClCHI1 and DT2-Bsr-ClCHI1 were 5. mu. mol/L.

The PCR amplification procedure was:

stage 1: 94 ℃ for 2 min; and (2) stage: 15s at 94 ℃; 56 ℃ for 30 s; at 68 ℃ for 3 min; a total of 30 cycles; and (3) stage: extension at 68 ℃ for 10 min.

The PCR product was electrophoresed on a 1% agarose gel. After completion of the electrophoresis, the band of interest (626bp) was excised under an ultraviolet lamp and purified using an agarose gel DNA recovery kit (purchased from Tiangen Biochemical technology Co., Ltd.), according to the instructions for use of the kit.

2. And (3) establishing the following enzyme digestion-connection system by using the PCR product purified and recovered in the step (1):

3. and (3) taking 5 mu L of the enzyme digestion-connection product obtained in the step (2) to transform the escherichia coli competent cells. Transformed E.coli competent cells were then screened using Kan plates and the resulting positive E.coli clones were PCR amplified using the primer pair U626-IDF and U629-IDR of the PBSE401 vector. Wherein, the primer sequences of U626-IDF and U629-IDR are as follows:

U626-IDF:5’-TGTCCCAGGATTAGAATGATTAGGC-3’；

U629-IDR:5’-AGCCCTCTTCTTTCGATCCATCAAC-3’。

the reaction system for PCR amplification (13. mu.L) was:

1.3 μ L of MgCl containing 15mM₂10 × EasyTaq PCR Buffer, 0.8. mu.L of dNTPs with a concentration of 2.5mM, 0.4U PCR Taq DNA polymerase, 0.5. mu.L of 10mM U626-IDF, 0.5. mu.L of 10mM U629-IDR, template DNA, ddH₂O make up to 13. mu.L.

Wherein, 1.0 μ L of template DNA is positive Escherichia coli liquid with OD value of 0.8(OD value is at least 0.8, generally 0.8-1.0); taq DNA polymerase, reaction buffer and dNTPs were purchased from Beijing Quanji Biotech Ltd.

The reaction procedure for PCR amplification was:

stage 1: pre-denaturation at 94 ℃ for 5 min; and (2) stage: 34 cycles of 94 ℃ for 15s, 55 ℃ for 20s, and 72 ℃ for 20 s; and (3) stage: extending for 4min at 72 ℃; and (4) stage: storing at 4 ℃.

The PCR instrument was a Veriti 96well Thermal Cycler from Applied Biosystems.

The PCR product is sent to Beijing Optimalaceae biotechnology limited to be sequenced by using primers U626-IDF and U629-IDF, and subsequent operation can be carried out if the sequencing is correct.

The primer sequence of U626-IDF is as described above, and the primer sequence of U629-IDF is as follows:

U629-IDF:5’-TTAATCCAAACTACTGCAGCCTGAC-3’。

4. extracting the positive escherichia coli with correct sequencing obtained in the step 3 into a plasmid (the plasmid is a CRISPR-ClCHI1 knockout vector), and transforming agrobacterium GV3101 by using the plasmid. Then screening by using a Kan plate, carrying out PCR identification (the positive agrobacterium GV3101 bacterial liquid with 1.0 mu L of template DNA in the step and an OD value of 0.8 (the OD value is at least 0.8, generally 0.8-1.0) according to the method in the step 3), identifying the clone capable of amplifying the target fragment as a positive agrobacterium GV3101 clone, and using the positive agrobacterium GV3101 clone for subsequent watermelon transformation.

Genetic transformation process of CRISPR-ClCHI1 knockout transgenic watermelon

1. Taking full JX2 watermelon seeds, carefully peeling off the seed coats (avoiding damaging the seed kernels as much as possible), sterilizing for 15min by using a 10% (m/v, mass percent concentration) sodium hypochlorite aqueous solution, washing for 3 times by using sterile water, gently placing the watermelon seeds in a culture dish containing a BM culture medium (sterilized by high pressure), and performing dark culture at 28 ℃ for 3 days to ensure that the watermelon seeds germinate.

2. Taking the healthy germinated kernels obtained in the step 1, cutting the kernels from the near-axial end of the cotyledon into explants of 1.5mm × 1.5.5 mm, putting the explants into a culture dish (the specification is 9cm) containing 10mL of MS liquid culture medium, then adding 50 mu L of bacterial liquid of Agrobacterium GV3101 clone with OD600 of 0.8-1.0 into the culture dish, uniformly mixing, soaking for 10min, draining the bacterial liquid, transferring the explants into a co-culture medium, and co-culturing for 4 days at 28 ℃ in the dark to obtain the co-cultured explants.

3. The co-cultured explants obtained in step 2 were subcultured on selection medium 1 alternately in light and dark at 25 ℃ (14h light/10 h dark; light intensity of about 2000lx) for 2-4 weeks, 1 time per week (i.e., once selection medium 1 was changed). Then the explants are transferred to a selective medium 2 and cultured alternately in light and dark (14h light/10 h dark; the light intensity is about 2000lx) at 25 ℃ for 2-4 weeks, and subculture is carried out for 1 time every week, thus obtaining the plant tissues containing green buds.

4. And (3) transferring the green buds on the plant tissues containing the green buds obtained in the step (3) to a bud elongation culture medium, alternately culturing in light and dark at 25 ℃ (14h light/10 h dark; light intensity is about 2000lx) for 4 weeks, and subculturing for 1 time per week to obtain resistant seedlings.

5. And (4) transferring the resistant seedlings obtained in the step (4) to a rooting culture medium, and carrying out light-dark alternate culture (14h light/10 h dark; the light intensity is about 2000lx) at the temperature of 25 ℃ for 7 days to obtain regenerated plants, namely T0 transgenic plants.

Thirdly, identifying transgenic plants of T0 generation:

1. and identifying T0 generation transgenic plants by using a Bar immunoassay test strip. Specifically, 0.1g of leaf of the T0 generation transgenic plant obtained in the step two is taken and put into a 2mL centrifuge tube, and distilled water is added for grinding to obtain a sample solution.

2. And (2) vertically inserting Bar immune detection test paper (a product of Beijing Ogaojin Biotech limited) into the sample liquid of the 2mL centrifuge tube in the step (1), submerging the end of the test paper into the sample liquid to a depth of about 0.5cm, taking out the test paper after 1min, and flatly reading the detection result.

3. The detection line and the control line can appear within 1-2min generally, and the detection standard is as follows: only one mauve quality control line appears on the test strip, and the result is negative; when two purple red strips (one of which is a purple red detection line and the other is a purple red quality control line) appear on the detection strip, a positive result is obtained, namely, the plant corresponding to the sample is a T0 generation positive transgenic plant.

And through identification, 54 positive transgenic plants of T0 generation are obtained.

Fourthly, molecular detection of mutation type and acquisition of homozygous knockout ClCHI1 plant

1. And respectively carrying out PCR amplification by using genome DNA of the T0 positive transgenic plant leaves as a template and ClCHI1-IDF and ClCHI1-IDR as primers.

The primer sequences for ClCHI1-IDF and ClCHI1-IDR consisted of:

ClCHI1-IDF：5’-ATGGCTTCCCACAAAATAAC-3’；

ClCHI1-IDR：5’-GATGCTGCCTTTAATGGCG-3’。

20 mu L of PCR reaction system comprises a template (genome DNA of a positive transgenic plant leaf of the T0 generation), 10 × Buffer 2 mu L and 10mmol/L dNTPs 2 mu L, MgSO₄1.4 μ L, cDNA 1.2 μ L, KOD-Plus enzyme 0.4 μ L, ClCHI1-IDF primer 0.3 μ L, ClCHI1-IDR primer 0.3 μ L, ddH₂O12.4μL。

The procedure for PCR amplification was: stage 1: 94 ℃ for 2 min; and (2) stage: 94 ℃, 15s, 56 ℃, 30s, 68 ℃ and 3min for 30 cycles; and (3) stage: extension at 68 ℃ for 10 min.

Sequencing the obtained PCR amplification product. Sequencing results show that in 54 positive transgenic plants of T0 generation, the genotype of 37 plants is completely consistent with that of a wild type (namely, the plants of JX2 of a non-transgenic watermelon), the target region is not edited, all or part of + G in the target region of 17 plants are heterozygous mutations, and the mutant form of the ClCHI1 gene in the heterozygous mutant is that a G is inserted after the 530 th nucleotide residue from the 5' end of a sequence 2 (namely, the ClCHI1 gene) in a sequence table.

2. Selfing 17 transgenic plants containing ClCHI1 gene heterozygous knockout in the step 1, harvesting seeds to obtain T1 generation seeds, and identifying whether the T1 generation plants are transgenic and contain ClCHI1 mutation by adopting the method in the step 1. The identification result shows that the homozygous plants containing the ClCHI1 mutation account for about 25% of the T1 generation plants.

3. T1 homozygous plants containing the ClCHI1 mutation from different strains are respectively selfed and bred to obtain T2 transgenic seeds from different strains with ClCHI1 gene knockout. Sowing T2 transgenic seeds from two different strains to obtain two homozygous strains CRISPR-1 and CRISPR-2 with ClCHI1 gene knockout for subsequent disease-resistant identification experiments.

Example 3

This example illustrates the acquisition of transgenic over-expressing ClCHI1 plants.

First, construction of recombinant plasmid Super1300-GFP-ClCHI1

1. Total RNA of the leaf of the blight-resistant watermelon variety JX2 is extracted and reverse transcribed into cDNA.

2. According to the multiple cloning site of a plant binary transformation vector super1300 (the vector is provided by the university of Chinese agriculture biotechnology institute, which is well-entrenched in the laboratory of the college of encyclopedia of loyalty and professor), and the coding region sequence of the ClCHI1 gene, forward primers and reverse primers for amplifying the whole coding region of the ClCHI1 gene are designed, corresponding enzyme cutting sites (the sequences of the enzyme cutting sites are underlined and are used as Xba1 and Kpn1) are respectively added at the 5' ends of the forward primers and the reverse primers, and a protective base is added outside the recognition sequence of the enzyme cutting sites so as to facilitate the smooth proceeding of enzyme cutting reaction. The DNA sequences of the corresponding primer pairs are as follows:

forward primer F1: 5' -CTCTAGAATGGCTTCCCACAAAATAAC-3’。

Wherein the underlined partTCTAGAThe sequence of the restriction site of Xba 1.

Reverse primer R1: 5' -GGGTACCGATGCTGCCTTTAATGGCG-3’。

Wherein the underlined partGGTACCThe sequence of the enzyme cutting site is Kpn 1.

Using the cDNA obtained in step 1 as a template, and performing PCR amplification by using a primer pair consisting of the forward primer F1 and the reverse primer R1 to obtain a PCR amplification product (namely, ClCHI1 gene).

20 μ L of PCR reaction system is 10 × Buffer 2 μ L, 10mmol/L dNTPs 2 μ L, MgSO₄1.4. mu.L, template cDNA 1.2. mu. L, KOD-Plus enzyme 0.4. mu.L, forward primer F1 and reverse primer R1 were 0.3. mu. L, ddH₂O 12.4μL。

3. The PCR amplification product obtained in step 2 was double-digested with restriction enzymes XbaI and Kpn1, and the digested product was recovered. The Super1300-GFP vector was digested with restriction enzymes XbaI and Kpn1, and the vector backbone was recovered. Then the enzyme digestion product is connected with the vector framework to obtain the recombinant plasmid Super1300-GFP-ClCHI 1.

Second, obtaining transgenic plants

1. The recombinant plasmid Super1300-GFP-ClCHI1 was introduced into Agrobacterium strain GV3101 to obtain recombinant Agrobacterium.

2. ClCHI1 was overexpressed in multiple lines of susceptible watermelon variety YX following the watermelon genetic transformation procedure of example 2, repeated selfing and harvesting multiple lines of T3 transgenic overexpressing seeds. Transgenic seeds with over-expressed ClCHI1 genes from T3 generations of two different strains are sown to obtain two homozygous strains OE1 and OE2 with over-expressed ClCHI1 genes, and the two homozygous strains are used for subsequent disease resistance identification tests.

3. The expression level of ClCHI1 gene in ClCHI1 overexpression transgenic homozygous lines OE1 and OE2 was analyzed (i.e., molecular characterization) by fluorescent real-time quantitative PCR (the same method as "tissue-specific expression of ClCHI1 gene" in example 1).

The PCR template for detecting the expression quantity of the ClCHI1 gene is cDNA obtained by reverse transcription of total RNA of root systems in overexpression transgenic homozygous strains OE1 and OE 2. The cDNA was obtained as described in example 1.

an upstream primer: 5'-GGAGTCAGTGGACGACGTTT-3', respectively;

a downstream primer: 5'-GTCGCTGTAGCCACTGTCAA-3' are provided.

an upstream primer: 5' -CCTACAACTCAATTATGAAGTGTG-3;

a downstream primer: 5'-GAAATCCACATCTGCTGGAAGGTG-3' are provided.

The fluorescence real-time quantitative PCR was performed using ABI 7500fast (applied biosystem) and 2 relative expression levels of ClCHI1 gene^-ΔΔCtAnd (4) calculating by using the method.

As can be seen from the comparative schematic diagram of the relative expression amounts of the ClCHI1 genes in the wild-type YX and ClCHI1 overexpressed transgenic homozygous lines OE1 and OE2 shown in FIG. 2, ClCHI1 genes with high expression amounts are respectively present in the ClCHI1 overexpressed transgenic homozygous lines OE1 and OE2, and the expression amounts of the ClCHI1 genes are respectively 4 times and 5 times of that of the wild type.

Example 4:

this example illustrates the disease-resistant response of different transgenic lines to blight.

1. Disease resistance analysis of watermelon overexpression transgenic homozygous lines

Transgenic homozygous lines OE1, OE2 overexpressing ClCHI1 obtained in example 3, and blast-sensitive receptor material YX were germinated separately. Taking vermiculite after high-temperature sterilization as a substrate, covering the substrate with the vermiculite after sowing, covering the substrate with a heat-insulating and moisture-preserving film, and controlling the seedling raising temperature at 24-28 ℃ in the daytime and about 18 ℃ at night. Referring to the artificial inoculation identification method for watermelon wilt disease reported by Gunn Lihua and the like, when the leaves of watermelon materials are tested to be unfolded, the seedlings in the seedling tray are gently taken out, and the roots are cleaned by clear water. Soaking the root system of the inoculated seedling in FON1 Fusarium oxysporum spore suspension for 15min by adopting a root soaking method to prevent cotyledon from being stained with bacterial liquid, and shaking the spore suspension for 3-5 times during the period to prevent spore settlement. While the blank control plants were rooted in sterile water for 15 min. After inoculation, seedlings are planted in a matrix irrigated thoroughly in advance, and the mixture ratio of the grass peat to the soil is 2:1 through high-pressure damp-heat sterilization. Shading and seedling revival are carried out 3d after inoculation by using a shading net, wilted seedlings are removed, and the host starts to develop diseases after 7d, and the disease condition is investigated. During the artificial inoculation identification period, the temperature is required to be controlled at 28-30 ℃ in the daytime and is not lower than 21 ℃ at night.

The watermelon wilt seedling disease identification refers to the classification standard of Martyn and the like. Level 0: no symptoms; level 1: mild wilting of cotyledon margins at noon; and 2, stage: 2 cotyledons or true leaves wither and cannot recover; and 3, level: leaf and more than 60% of true leaves will wither, hindering growth; 4, level: the whole plant wilts and heart leaves survive; and 5, stage: the stem shrinks and turns brown, and the whole plant withers and dies. Wherein, the disease-resistant type is 0-2 grade, and the disease-susceptible type is 3-5 grade.

The results show that both over-expressed transgenic lines OE1, OE2 have stronger disease resistance than the control YX, with very significant differences (see fig. 3).

2. Disease resistance analysis of watermelon CRISPR knockout transgenic homozygous lines

The ClCHI1 knockout transgenic homozygous lines CRISPR-1, CRISPR-2 obtained in example 2, and the disease-resistant receptor JX2 were separately pregerminated. FON1 fusarium oxysporum spores were inoculated according to the method of step 1 in this example, and the results showed that the disease resistance of both CRISPR-1 and CRISPR-2 CRISPR knockout transgenic lines was significantly lower than that of the control JX2 (see FIG. 3).

As described in examples 1 to 4 above, it was found that ClCHI1 gene was significantly reduced in resistance to blight by knocking out ClCHI 3578 gene in JX2 resistant to blight; after the watermelon ClCHI1 gene is transferred into YX which is infected with the blight, the resistance of the watermelon to the blight is obviously improved. This indicates that the watermelon ClCHI1 gene is closely related to the resistance to blight, the higher the expression level, the stronger the resistance to blight, and since the main infection site of blight is the root, the expression level of ClCHI1 gene in the root is significantly higher than that of other sites.

Prospect of application of the invention

With the continuous development of molecular biology, the cultivation of watermelon varieties resistant to blight is becoming possible by using biotechnology means. Although the current gene cloning work of the watermelon has achieved certain achievements, the gene cloning work of the watermelon is far behind that of grain crops such as rice, corn, wheat and the like, and particularly, the research on the blight resistance of the watermelon, especially the research on the molecular level, is not many. The CRISPR is an effective reverse genetics technology and is widely used for identifying the plant genome function, endogenous genes can be successfully knocked out at different parts of a plant through CRISPR gene knockout, and a feasible means is provided for researching the gene functions in different growth periods. The invention clones the ClCHI1 gene of watermelon on the basis of early genetic location and map-based cloning, verifies the knockout efficiency of the CRISPR technology on the watermelon, and explores the biological function of the ClCHI1 gene.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

<110> agriculture and forestry academy of sciences of Beijing City

<120> watermelon chitinase and coding gene and application thereof

<160>2

<170>SIPOSequenceListing 1.0

<210>1

<211>291

<212>PRT

<213> watermelon (Citrullus lanatus)

<400>1

Met Ala Ser His Lys Ile Thr Thr Thr Leu Ser Ile Ile Phe Leu Leu

1 5 10 15

Ser Ser Ile Phe Arg Ser Ser Asp Ala Ala Gly Ile Ala Ile Tyr Trp

20 25 30

Gly Gln Asn Gly Asn Glu Gly Ser Leu Ala Ser Thr Cys Ala Thr Gly

35 40 45

Asn Tyr Lys Phe Val Asn Ile Ala Phe Leu Ser Ser Phe Gly Asn Gly

50 55 60

Gln Thr Pro Val Leu Asn Leu Ala Gly His Cys Asn Pro Asp Asn Asn

65 70 75 80

Gly Cys AlaPhe Leu Ser Asp Glu Ile Asn Ser Cys Lys Ser Leu Gly

85 90 95

Ile Lys Val Leu Leu Ser Ile Gly Gly Gly Ala Gly Ser Tyr Ser Leu

100 105 110

Ser Ser Ala Glu Asp Ala Arg Asp Val Ala Asn Phe Leu Trp Asn Asn

115 120 125

Phe Leu Gly Gly Gln Ser Ser Ser Arg Pro Leu Gly Asp Ala Val Leu

130 135 140

Asp Gly Ile Asp Phe Asp Ile Glu Ser Gly Ser Gly Gln Trp Trp Asp

145 150 155 160

Glu Leu Ala Arg Gln Leu Lys Gly Phe Gly Gln Val Leu Leu Ser Ala

165 170 175

Ala Pro Gln Cys Pro Ile Pro Asp Ala His Leu Asp Ala Ala Ile Lys

180 185 190

Thr Gly Leu Phe Asp Phe Val Trp Val Gln Phe Tyr Asn Asn Pro Pro

195 200 205

Cys Met Phe Ala Asp Asn Ala Asp Asn Leu Leu Asn Ser Trp Ser Gln

210 215 220

Trp Thr Thr Phe Pro Ala Ala Ser Leu Phe Met Gly Leu Pro Ala Ala

225 230 235 240

Pro Glu Ala Ala Pro Ser Gly Gly Phe Ile Pro Ala Asp Val Leu Ile

245 250 255

Ser Gln Val Leu Pro Thr Ile Lys Thr Ser Ser Asn Tyr Gly Gly Val

260 265 270

Met Leu Trp Ser Lys Ala Phe Asp Ser Gly Tyr Ser Asp Ala Ile Lys

275 280 285

Gly Ser Ile

290

<210>2

<211>876

<212>DNA

<213> watermelon (Citrullus lanatus)

<400>2

atggcttccc acaaaataac tacaactctt tctatcatct ttctcctctc ctccatattt 60

agatcttcgg atgcggccgg aatcgccatc tactggggcc aaaacggcaa cgaaggctct 120

cttgcctcca cctgtgccac cggaaactac aagttcgtca acatagcatt tctctcctcc 180

ttcggcaatg gtcaaacccc ggtcctcaac cttgccggtc actgcaaccc tgacaacaac 240

ggttgcgctt ttttgagcga cgaaataaat tcttgcaaaa gtctaggcat caaagtcctc 300

ctctctatcg gcggcggcgc agggagctat tcactctcct ccgccgaaga tgcaagagac 360

gtcgcaaact tcctttggaa caacttcctc ggcgggcagt cgagttcgag gccactcggc 420

gacgccgttt tggacggcat tgatttcgat atcgaatctg gctcagggca gtggtgggac 480

gaactagctc ggcagctgaa gggcttcggt caagtccttc tctccgccgc gccgcagtgt 540

ccaatccccg acgcacacct agacgcggcc attaaaacgg gtttgtttga tttcgtttgg 600

gttcaattct acaacaaccc gccatgcatg tttgcagata acgccgacaa tctcctgaat 660

tcttggagtc agtggacgac gtttccggct gccagtctct tcatggggct gccggcggcc 720

cctgaggccg cgccgagcgg cggctttatt ccggcggatg tgcttatttc tcaagttctt 780

ccgaccatta aaacttcttc caactatgga ggagttatgt tatggagcaa ggcgtttgac 840

agtggctaca gcgacgccat taaaggcagc atctga 876

Claims

1. The watermelon chitinase protein is any one of the following proteins:

1) protein shown as a sequence 1 in a sequence table;

2. The coding nucleic acid sequence of the watermelon chitinase is any one of the following nucleic acid sequences:

3. Use of the watermelon chitinase protein of claim 1 or the nucleic acid sequence encoding watermelon chitinase of claim 2 for resistance to watermelon wilt disease.

4. The use of claim 3, wherein said use comprises overexpressing a watermelon chitinase-encoding nucleic acid sequence in a watermelon plant.

5. The use of claim 4, wherein said act of overexpressing a watermelon chitinase-encoding nucleic acid sequence in a watermelon plant comprises the act of constructing a recombinant vector comprising a watermelon chitinase-encoding nucleic acid sequence and transforming a watermelon plant with said recombinant vector.

6. The use according to claim 5, wherein the recombinant vector is a Super1300-GFP-ClCHI1 recombinant plasmid.

7. The use of claim 5 or 6, wherein transforming a watermelon plant with said recombinant vector comprises introducing said recombinant vector into Agrobacterium strain GV3101, and introducing said nucleic acid sequence encoding watermelon chitinase into a watermelon plant using Agrobacterium strain GV3101 into which the recombinant vector has been introduced, thereby allowing overexpression of said nucleic acid sequence encoding watermelon chitinase in the watermelon plant.

8. A method for obtaining a watermelon anti-fusarium wilt disease strain is characterized by comprising the steps of performing overexpression on a coding nucleic acid sequence of watermelon chitinase in a watermelon plant, then screening a strain stably overexpressing the watermelon chitinase in the watermelon plant, and continuously selfing the strain stably overexpressing the watermelon chitinase to obtain the stable anti-fusarium wilt watermelon strain.

9. The method of claim 8, wherein said stable overexpression watermelon chitinase is selfed for at least three consecutive generations to obtain a stable blight resistant watermelon line.