CN112195186A

CN112195186A - Application of SlBBX20 gene in regulation and control of tomato gray mold resistance

Info

Publication number: CN112195186A
Application number: CN202011067333.8A
Authority: CN
Inventors: 王涛涛; 罗丹; 熊程; 孙文慧; 张小燕; 叶志彪; 张俊红; 杨长宪; 李汉霞
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-10-06
Filing date: 2020-10-06
Publication date: 2021-01-08
Anticipated expiration: 2040-10-06
Also published as: CN112195186B

Abstract

The invention relates to an application of SlBBX20 gene in regulation of tomato gray mold resistance, which reduces tomato gray mold resistance by improving expression of SlBBX20 in tomato and improves tomato gray mold resistance by reducing or eliminating expression of SlBBX20 in tomato. Compared with the prior art, the invention has the beneficial effects that: (1) the gene editing technology is utilized to obtain plants with strong gray mold resistance, is more economical and effective than the traditional breeding method, is a good method for creating disease-resistant materials, and greatly shortens the breeding period; (2) compared with the traditional control method, the resistance of the SlBBX20 knockout strain to gray mold is obviously enhanced, the usage amount of pesticides can be reduced, and the environment is protected; (3) provides a plant and a method for reducing the gray mold resistance, and provides a material for the subsequent research and control of gray mold.

Description

Application of SlBBX20 gene in regulation and control of tomato gray mold resistance

Technical Field

The invention belongs to the field of genetic engineering, and particularly relates to an application of a SlBBX20 gene in regulation and control of tomato gray mold resistance.

Background

Botrytis cinerea is a broad-spectrum pathogenic fungus, the host of the fungus can be more than two hundred, and the fungus can invade organs such as flowers, stems, leaves and fruits of plants, thereby causing great threat to the yield and quality of fruits and vegetables in the world every year. Botrytis cinerea is a saprophytic pathogenic bacterium, and can secrete toxic substances after invading plants, and utilize a self-defense mechanism to destroy host cells, obtain nutrition and propagate by self.

Gray mold, the second fungal disease in the world, causes great losses to agricultural production. The majority of domestic control of botrytis cinerea still stays in the aspect of researching biological characteristics of botrytis cinerea, and agricultural control, biological control and chemical control methods are adopted in production. For example, the prevention and control methods of seedbed seedling raising, high-temperature greenhouse closing, reasonable fertilization, cultivation management strengthening, crop rotation, stubble changing, ecological prevention and the like have certain effects, but are influenced by various factors, the effects are not good, and the chemical prevention and control are mainly used in production. But chemical prevention and control not only affects the food quality of people, but also causes harm to the environment, so that the cultivation of disease-resistant varieties is particularly important.

Disclosure of Invention

In order to solve the technical problems, the invention provides an application of a SlBBX20 gene in regulation and control of tomato gray mold resistance.

The specific technical scheme is as follows:

application of SlBBX20 gene in regulation and control of tomato gray mold resistance.

Compared with the prior art, the SlBBX20 gene is adopted to regulate the resistance of the botrytis cinerea of the tomato, and plants with different resistance of the botrytis cinerea can be prepared.

Further, the botrytis resistance of tomatoes is improved by reducing or eliminating the expression of SlBBX20 in tomatoes; the sequence of the SlBBX20 gene is shown in SEQ ID NO. 1.

Further, the expression level of the SlBBX20 gene is improved by transferring the segment selected from the segment containing the SlBBX20 gene ORF in tomato, and the sequence of the segment containing the SlBBX20 gene ORF is shown in SEQ ID No. 2.

Furthermore, the SlBBX20 gene is mutated through gene editing, and the SlBBX20 gene in tomato is knocked out.

The tomato gray mold resistance gene is characterized in that the tomato gray mold resistance gene is prepared by mutation of the SlBBX20 gene, and the SlBBX20 gene is shown as SEQ ID No. 1.

Further, the mutant segment of the SlBBX20 gene comprises a first target segment of a SlBBX20 gene and/or a second target segment of a SlBBX20 gene, the sequence of the first target segment of the SlBBX20 gene is shown as SEQ ID No.3, and the sequence of the second target segment of the SlBBX20 gene is shown as SEQ ID No. 4.

Compared with the prior art, the SlBBX20 mutant gene capable of improving the resistance of tomato gray mold is provided.

The SlBBX20 gene knockout vector is characterized in that the SlBBX20 gene knockout vector comprises a first target segment of a SlBBX20 gene and a second target segment of a SlBBX20 gene, wherein the sequence of the first target segment of the SlBBX20 gene is shown as SEQ ID No.3, and the sequence of the second target segment of the SlBBX20 gene is shown as SEQ ID No. 4.

Further, a gene segment to be transferred containing the first target segment of the SlBBX20 gene and the second target segment of the SlBBX20 gene is amplified by using a target primer, the gene segment to be transferred is inserted into a linearized transfer vector, and after connection, the gene segment to be transferred is transferred into escherichia coli, and the recombinant vector is obtained after extraction.

Further, the primer sequence of the first target fragment of the SlBBX20 gene is shown as SEQ ID No.5, and the primer sequence of the second target fragment of the SlBBX20 gene is shown as SEQ ID No. 6.

Further, the transfer vector is a PTX041 plasmid, and the PTX041 plasmid is cut by BsaI for linearization treatment.

Compared with the prior art, the knockout vector capable of knocking out the SlBBX20 gene is provided, and the SlBBX20 gene can be knocked out by using the knockout vector, so that the gray mold resistance of a plant is improved.

A method of increasing the resistance of a tomato plant to gray mold, the difference being that the method of increasing the resistance of a tomato plant to gray mold comprises:

step S1, transferring the SlBBX20 gene knockout vector into agrobacterium;

and S2, infecting tomato plants with the agrobacterium transformed into the SlBBX20 gene knockout vector.

Compared with the prior art, the SlBBX20 gene is knocked out by using the SlBBX20 gene knockout vector to obtain a plant with strong gray mold resistance, so that the method is more economical and effective compared with the traditional breeding method, is a good method for creating a disease-resistant material, and greatly shortens the breeding period; the resistance of the SlBBX20 knockout strain to gray mold is obviously enhanced, the usage amount of pesticides can be reduced, and the environment is protected.

A method of reducing gray mold resistance in a tomato plant, said method comprising:

step A1: transferring a vector with an ORF comprising a SlBBX20 gene into agrobacterium;

step A2: carrying out dip-dyeing on the tomato plant by the agrobacterium transformed with the SlBBX20 gene knockout vector; the sequence of the ORF containing the SlBBX20 gene is shown in SEQ ID NO. 2.

Further, the preparation method of the vector containing SlBBX20 gene ORF and SlBBX20 gene ORF comprises the following steps:

amplifying the ORF containing the SlBBX20 gene by using a primer containing the SlBBX20 gene ORF and a tomato plant gene as a template; the front primer containing the SlBBX20 gene ORF is shown as SEQ ID NO.7, and the rear primer containing the SlBBX20 gene ORF is shown as SEQ ID NO. 8; and transferring the ORF containing the SlBBX20 gene into a linearized pHellsgate8 vector, transferring the vector into escherichia coli after connection, and extracting to obtain the vector containing the SlBBX20 gene ORF and the SlBBX20 gene ORF.

Further, the preparation method of the tomato plant gene comprises the following steps: extracting total RNA in the tomato, and then synthesizing a tomato gene by reverse transcription, wherein the sequence of the tomato gene is shown as SEQ ID NO. 9.

Further, the pHellsgate8 vector was linearized by digestion with XhoI and XbaI.

The invention provides a plant and a method for reducing gray mold resistance by transferring the ORF fragment of the SlBBX20 gene, and provides a material for the subsequent research and prevention and treatment of gray mold.

Drawings

FIG. 1 is the analysis of the overexpression level of SlBBX20 gene of T0 generation line SlBBX 20-OE;

FIG. 2 is a drawing of an alignment analysis of the editing status of SlBBX20 gene in SlBBX20-CR homozygous lines;

FIG. 3 shows the disease condition of 72h of Botrytis cinerea inoculated on the excess of SlBBX20 and the knocked-out tomato plant leaves;

FIG. 4 is a statistics of excess of SlBBX20 and disease spots of knockout tomato plants inoculated with Botrytis cinerea for 72 h;

FIG. 5 shows the disease-resistant material TS179 inoculated with Botrytis cinerea for 72 h;

FIG. 6 shows the onset of infection of disease-sensitive material TS100 with Botrytis cinerea for 72 h;

FIG. 7 Botrytis cinerea induced expression profile of SlBBX20 in disease-resistant material TS 179;

FIG. 8 Botrytis cinerea-induced expression profile of SlBBX20 in susceptible material TS 100.

Detailed Description

The principles and features of this invention are described below in conjunction with the following drawings, which are set forth by way of illustration only and are not intended to limit the scope of the invention.

Example 1

Botrytis cinerea induced expression profile of SlBBX20 gene in anti-botrytis cinerea-infected material

The botrytis resistant material TS179 (variety selected from TGRC, American tomato genetic resource center) and the botrytis sensitive material TS100 (variety selected from TGRC, American tomato genetic resource center) are sowed, and the seedling with the size of 4 weeks is subjected to a botrytis inoculation experiment. The disease-resistant material TS179 (see figure 5) and the disease-susceptible material TS100 (see figure 6) are photographed respectively and inoculated with the botrytis cinerea for 72 h. The scab is smaller in the disease-resistant material TS179, and the scab is greatly spread after the disease-sensitive material TS100 is inoculated with the botrytis cinerea for 72 h. Sampling at different time points (0h, 24h, 48h and 72h) of inoculating the botrytis cinerea, extracting RNA, performing reverse transcription to form cDNA, and performing a real-time fluorescence quantitative PCR experiment to detect the botrytis cinerea-induced expression level of SlBBX20 in a disease-resistant material TS179 (shown in figure 7) and a disease-sensitive material TS100 (shown in figure 8). The SlBBX20 gene can be greatly induced to express after the botrytis cinerea is inoculated in the susceptible material TS100 for 72h, and the induced expression level is lower in the disease-resistant material TS 179.

The reverse transcription system is as follows:

(1)

RNase free ddH₂O to 16μL

4*gDNAwiperMix 4μL

template RNA 1. mu.g

Gently pipetting and beating with a pipette, and mixing uniformly at 42 ℃ for 2 min.

(2)

Directly adding 5 HiScript II Qrt Supermix II 4. mu.L into the reaction tube in the first step, and gently blowing and mixing by a pipette, wherein the mixture is at 50 ℃ for 15min and at 85 ℃ for 5 s. After the procedure is finished, cDNA can be obtained, and the product can be directly used for qPCR reaction.

Example 2

Preparation of SlBBX20 gene knockout vector

The DNA sequence of SlBBX20 gene is searched on SGN (https:// www.sgn.cornell.edu/search/locus) website, the DNA sequence of the gene is input into CRISPR design website (http:// CRISPR. hzau. edu. cn/cgi-bin/CRISPR2/CRISPR), and two suitable targets are selected according to the off-target rate and the like: the gene sequence of the first target fragment of the SlBBX20 gene is shown as SEQ ID No.3, and the sequence of the second target fragment of the SlBBX20 gene is shown as SEQ ID No. 4.

And (2) gene synthesis of a primer containing two targets, wherein the primer sequence of the first target fragment of the SlBBX20 gene is shown as SEQ ID No.5, and the primer sequence of the second target fragment of the SlBBX20 gene is shown as SEQ ID No. 6.

PCR fragments containing Two targets were amplified using primers containing sequences of the targets artificially synthesized, using pCBC _ DT1T2_ SlU6p plasmid as template (from the trexia topic group of vegetable and flower institute, farm J, Zhang S, Wang X, et al. variations in Both FTL1 and SP5G, Two Tomato FT Paralogs, Control Day-Neutral Flouring [ J ]. Molecular Plant,2020,13(7) of Chinese academy of agriculture).

The amplification system was as follows:

the amplification conditions were as follows:

and (3) performing amplification procedures of 2-4, wherein 34 cycles of amplification are performed, and the rest are 1 cycle.

BsaI single enzyme digestion pTX041 vector (provided by the topic group of Li-biogenesis institute of genetics and developmental biology, Deng, L., Wang, H., Sun, C., Li, Q., Jiang, H., Du, M., Li, C. -B.and Li, C. (2018) efficiency generation of pin-free protocols using CRISPR/Cas9 system. journal of genetics and Genomics,45,51-54) was used, 3, 1.0% agarose gel digestion was performed at 37 ℃ to recover the amplified gene fragment 608bp and the vector large fragment of pTX 041. Mixing the recovered amplification product with the pTX041 vector subjected to enzyme digestion according to the volume ratio of 1:5, adding 5 XCE II Buffer 2 mu L and Exnase II 1 mu L, supplementing 10 mu L of sterile water, reacting at 37 ℃ for 30min, quickly transferring into ice water, placing for 5min, carrying out 42-degree heat shock conversion on the homologous recombination product to enter an Escherichia coli DH5 alpha strain, screening positive clones by a Kan resistance plate, carrying out PCR detection on a bacterial liquid, then sending the bacterial liquid to a sequencing company for sequencing, comparing the sequencing result with a known sequence, determining a bacterial liquid containing two target points, and then shaking the bacterial liquid to extract plasmids.

Example 3

Preparation of vector carrying ORF gene fragment of SlBBX20

Extracting total RNA in tomato by using Trizol (Vazyme, China) reagent, then utilizing a reverse transcription kit (Vazyme, China) to carry out reverse transcription to synthesize cDNA (sequence is shown as SEQ ID NO. 9) of tomato genes, utilizing designed primers, wherein the sequence of a front primer is shown as SEQ ID NO.7, the sequence of a rear primer is shown as SEQ ID NO.8, obtaining ORF (open reading frame) of SlBBX20 genes through PCR amplification, the sequence is shown as SEQ ID NO.2, detecting by 1.0% agarose gel, and recovering target fragments by using a recovery kit (OMEGA, USA).

The reverse transcription system is as follows:

(1)

RNase free ddH₂O to 16μL

4*gDNAwiperMix 4μL

template RNA 1. mu.g

(2)

Directly adding 5 HiScript II Qrt Supermix II 4. mu.L into the reaction tube in the first step, and gently blowing and mixing by a pipette, wherein the mixture is at 50 ℃ for 15min and at 85 ℃ for 5 s. After the procedure is finished, cDNA can be obtained, and the product can be directly used for qPCR reaction. Simultaneously, performing double enzyme digestion on pHellsgate8 vector by Xho I and XbaI, performing enzyme digestion at 37 ℃ for 3 hours, detecting 1.0% agarose gel, mixing an amplified fragment and pHellsgate8 vector after enzyme digestion according to an optimal volume ratio required by a kit (Vazyme, China), adding 2 muL of 5 XCE II Buffer and 1 muL of Exnase II, supplementing the volume to 10 muL of sterile water, reacting at 37 ℃ for 30min, quickly transferring to ice water, standing for 5min, performing 42-degree heat shock transformation on the homologous recombination product to enter an Escherichia coli DH5 alpha strain, screening positive clones by a Spec resistance plate, performing PCR detection on bacterial liquid, sending to a company for sequencing, comparing a sequencing result with a known sequence, and determining a correct Escherichia coli liquid and then shaking the bacterial liquid to extract plasmids.

Example 4

Preparation of Gene-edited plants

4.1

The recombinant vectors prepared in example 2 and example 3 were respectively transformed into Agrobacterium by the following method:

transforming the recombinant plasmid with correct detection into agrobacterium tumefaciens C58 by an electric converter, screening by an LB solid plate containing Rif50 mg/L recombinant vector, selecting positive clones, carrying out shaking culture at the temperature of 28 ℃ and at the speed of 200r/min overnight, taking 1 mu L of supernatant as a template, and carrying out PCR detection by using gene specific primers.

4.2

The agrobacterium transformed into the SlBBX20 gene knockout vector in the example 2 and the agrobacterium transformed into the ORF gene fragment vector with SlBBX20 in the example 3 are respectively dip-dyed with tomato plants, and the operation method is as follows:

tomato seeds, Alisa Craig (variety selected from TGRC, American tomato genetic resource center), were washed with 75% ethanol for 1min, sterilized with sodium hypochlorite for 15min, sown on 1/2MS medium (pH 5.8), grown at 25 + -2 deg.C until two cotyledons were completely spread. Selecting cotyledons, cutting the cotyledons into 2-3 explants, putting the explants on a KCMS culture medium, and performing dark culture for 1 d. Putting the pre-cultured explant into a suspension, dip-dyeing agrobacterium liquid which is resuspended to OD600 & lt 0.5 & gt for 3-5min, sucking excess bacteria liquid by using sterilized filter paper, putting back to a KCMS culture medium again, co-culturing for 2d under a dark condition, transferring to a screening culture medium 2Z for resistance screening, carrying out subculture once every two weeks, and transferring the explant to a 0.2Z culture medium after resistant buds appear. Cutting off the resistant buds after 20 days, inserting the cut resistant buds into a rooting culture medium to induce rooting, transplanting the plants with developed root systems into a nutrition pot, wherein the nutrition systems of the culture media are as follows:

KCMS culture medium:

PH＝5.8

2Z and 0.2Z culture media, namely adding different antibiotics into MS culture media, which is as follows:

the agrobacterium transferred into the SlBBX20 gene knockout vector in the embodiment 2 is a SlBBX20 knockout plant, the subsequent marker is a SlBBX20-CR-n or CR-n (n is a number and represents different serial numbers in the same series) plant, the agrobacterium transferred into the ORF gene fragment vector with SlBBX20 in the embodiment 3 is a SlBBX20 excessive plant, and the subsequent marker is a SlBBX20-OE-n or OE-n (n is a number and represents different serial numbers in the same series) plant.

Example 5

Detecting the expression quantity of the SlBBX20 hyperplants, wherein the detection method comprises the following steps:

and (3) obtaining a T0 generation of a SlBBX20 overexpression plant through genetic transformation, and extracting genome DNA of a leaf by using a CTAB method. Then, the CaMV35S pre-primer and the gene specific post-primer are used, the gDNA of a T0 generation plant is used as a template, the over-expression plasmid is used as a positive control, and the wild background material is used as a negative control for carrying out PCR detection.

The primer sequence is as follows:

CaMV35S:ACGCACAATCCCACTATCCTTC；

SlBBX20-OE-RV:GGAAAAAACTACACATCTGGTCG。

designing primers for positive T0 generation plants detected by PCR, and carrying out real-time fluorescence quantitative experiment to detect the level of overexpression of SlBBX20 gene (see figure 1), wherein the primer sequence is as follows:

SlBBX20-Qpcr-FW:AAATTCAATATCCCCAGCTGGA；

SlBBX20-Opcr-RV:TGTGTCGATTCTGTAGTACTCG；

wherein the expression level of SlBBX20 genes of 14 SlBBX20-OE lines is 100 times greater than that of the genes in a wild type, WT is a wild plant without gene editing, and the same is as below.

Example 6

Sequencing SlBBX20 knockout plants

The T0 generation of a plant is knocked out by SlBBX20, and the genomic DNA of the leaf is extracted by a CTAB method. When in test, forward and reverse primers of the vector are firstly used for carrying out PCR reaction to detect whether the vector pTX041 is inserted into a T0 generation plant or not, and the primer sequence is as follows:

pTX041-Fw：AGCGGATAACAATTTCACACAGGA；

pTX041-Rv：GCAGGCATGCAAGCTTATTGG。

PCR amplification is carried out on the SlBBX20 gene by taking gDNA of a T0 generation of a SlBBX20 plant edited by the inserted CRISPR/Cas9 gene with the carrier pTX041 as a template, the sequencing result is counted after sequencing (see table 1), the sequencing result is compared (see figure 2), and whether the gene is edited or not is detected.

35 strains are shared in the T0 generation by SlBBX20-CR, vectors are detected, the vectors are inserted, 23 strains are sequenced, 6 strains are unedited, four strains are homozygous, 13 strains are bimodal, and three knockout lines of CR13, CR18 and CR19 are selected in a later stage botrytis cinerea resistance experiment.

TABLE 1 sequencing results of SlBBX20-CR plants

Example 7

And detecting gray mold resistance of the SlBBX20 hyperplants and homozygous SlBBX20 knockout plants by the following detection method:

sowing 3 homozygous knockout strains obtained by screening, 3 SlBBX20-OE strains and a background material AC, and taking the third and fourth leaves growing for 4 weeks from top to bottom to perform a botrytis inoculation experiment. The B05.10 strain was selected to inoculate 1 drop of spore suspension per leaf at a spore suspension concentration of 105, each drop having a volume of 10. mu.L. After the inoculation of the botrytis cinerea, the mixture is sealed by a preservative film for moisture preservation treatment, and meanwhile, the mixture is placed in an incubator at the temperature of 22 +/-2 ℃ for observing the morbidity of the mixture. Three days after inoculation with Botrytis cinerea, the disease of the leaf was photographed (see FIG. 3), and the lesion area was counted (see FIG. 4). The lesion area of SlBBX20-OE-23, -29, -40 is extremely larger than that of wild-type leaves, and the lesion area of SlBBX20-CR-13, -18, -19 three homozygous lines is extremely smaller than that of wild-type background materials.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Sequence listing

<110> university of agriculture in Huazhong

Application of <120> SlBBX20 gene in regulation and control of tomato gray mold resistance

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Claims

Application of SlBBX20 gene in regulation and control of tomato gray mold resistance.
2. The use of the SlBBX20 gene according to claim 1 to modulate resistance to tomato gray mold by reducing or eliminating expression of SlBBX20 in tomato to increase resistance to tomato gray mold; the sequence of the SlBBX20 gene is shown in SEQ ID NO. 1.
3. The tomato gray mold resistance gene is characterized by being prepared by mutating the SlBBX20 gene of claim 1, wherein the SlBBX20 gene is shown as SEQ ID No. 1.
4. The tomato gray mold resistance gene as claimed in claim 3, wherein the mutant segment of the SlBBX20 gene comprises a first target segment of SlBBX20 gene and/or a second target segment of SlBBX20 gene, the sequence of the first target segment of the SlBBX20 gene is shown as SEQ ID No.3, and the sequence of the second target segment of the SlBBX20 gene is shown as SEQ ID No. 4.
5. The SlBBX20 gene knockout vector is characterized by comprising a first target fragment of a SlBBX20 gene and a second target fragment of a SlBBX20 gene, wherein the sequence of the first target fragment of the SlBBX20 gene is shown as SEQ ID No.3, and the sequence of the second target fragment of the SlBBX20 gene is shown as SEQ ID No. 4.
6. The method for preparing the SlBBX20 gene knockout vector of claim 5 is characterized in that a gene segment to be transferred containing the first target segment of the SlBBX20 gene and the second target segment of the SlBBX20 gene is amplified by using a target primer, the gene segment to be transferred is inserted into a linearized transfer vector, and after connection, the gene segment to be transferred is transferred into escherichia coli, and the recombinant vector is obtained after plasmid extraction.
7. The method for preparing the SlBBX20 gene knockout vector according to claim 5, wherein a primer sequence of the first target fragment of the SlBBX20 gene is shown as SEQ ID No.5, and a primer sequence of the second target fragment of the SlBBX20 gene is shown as SEQ ID No. 6.
8. The method for preparing the SlBBX20 gene knockout vector as claimed in claim 5, wherein the transfer vector is a PTX041 plasmid, and the PTX041 plasmid is digested with BsaI and linearized.
9. A method of increasing the resistance of a tomato plant to gray mold, said method comprising:

step S1, transferring the SlBBX20 gene knockout vector of claim 5 into agrobacterium;

and S2, infecting tomato plants with the agrobacterium transformed into the SlBBX20 gene knockout vector.
10. A method of reducing gray mold resistance in a tomato plant, said method comprising:

step A1: transferring a vector with an ORF-containing SlBBX20 gene into agrobacterium;

step A2: carrying out dip-dyeing on the tomato plant by the agrobacterium transformed with the SlBBX20 gene knockout vector; the sequence of the SlBBX20 gene containing ORF is shown in SEQ ID NO. 2.