CN114292943B - Application of tomato SlC H39 gene as negative regulatory factor in improving low temperature resistance of tomato - Google Patents

Abstract

The invention discloses an application of a tomato SlC H39 gene as a negative regulatory factor in improving low temperature resistance of tomatoes, wherein the nucleotide sequence of a protein coding region of the SlC H39 gene is shown as SEQ ID NO:1, the approach of the application is to improve the low temperature resistance of tomatoes by knocking out SlC H39 genes. The invention provides gene resources for cultivating new varieties of low-temperature-resistant tomatoes, proves the application of SlC H39 genes as negative regulation factors in improving low-temperature resistance of tomatoes, and lays a theoretical foundation for researching the mechanism of stress signal response of tomato plants and the molecular mechanism of adverse environment tolerance.

Description

Application of tomato SlC H39 gene as negative regulatory factor in improving low temperature resistance of tomato

Technical Field

The invention relates to the fields of genetic engineering, molecular biology, physiology and the like, in particular to application of tomato SlC H39 serving as a negative regulatory factor in improving low temperature resistance of plants.

Background

The tomato origin is tropical areas in south america, is one of the most widely cultivated vegetable crops in the world, and is also the facility vegetable crop with the largest cultivation area in China. Tomatoes are warm-loving plants, whose growth and development are extremely sensitive to environmental changes, especially temperature changes. Along with the frequent occurrence of global extreme climate phenomenon, and the problems of low facility equipment level, poor temperature light environment controllability and the like commonly existing in tomato facility production in China, the low-temperature stress has become a main factor for influencing the growth and development and yield of tomato crops. Therefore, the research on the physiological and biochemical changes and molecular mechanisms of tomatoes in response to low-temperature stress has important theoretical significance and practical significance, not only can effectively excavate low-temperature resistant mechanisms and key genes of plants, but also can provide important theoretical support for improving low-temperature resistance of tomatoes, improving yield and reducing economic loss caused by low temperature.

Among the many types of transcription factors in plants, zinc finger proteins are one of the most family members of the transcription factor family in plants and have a rich diversity. Zinc finger proteins are involved in transcriptional regulation, RNA binding, protein interactions, and the like, and play an important role in plant growth and development and stress response. CCCH-type zinc finger proteins are a broad class of zinc finger proteins, members of which contain one or more zinc ion binding motifs consisting of three cysteines and one histidine. CCCH-type zinc finger proteins in plants are widely present in various species with very limited research. In recent years, CCCH zinc finger proteins AtSZF1 and AtSZF2 in arabidopsis have been shown to play a positive regulatory role in the pathway of salt stress. In cotton, salt stress, osmotic stress and salicylic acid treatment all induced the expression of GhZFP 1. Meanwhile, the over-expression of GhZFP1 in tobacco enhances the salt resistance and disease resistance.

However, the role of tomato CCCH zinc finger protein in low temperature stress and its regulatory mechanism are not reported. In recent years, CRISPR/Ca9 gene editing technology has been rapidly developed, and compared with traditional breeding, the technology can precisely knock out genes in genome, thereby precisely changing the characters of crops and greatly shortening breeding time. By utilizing the technology, slC H39 genes in tomatoes are knocked out, so that the resistance of the tomatoes to low-temperature stress is improved, and adversity stress gene resources are excavated.

Disclosure of Invention

In view of this, the embodiment of the invention provides an application of the tomato SlC H39 gene as a negative regulatory factor in improving the low temperature resistance of tomatoes.

In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:

application of tomato SlC H39 gene as negative regulatory factor in improving low temperature resistance of tomato, wherein the nucleotide sequence of protein coding region of SlC H39 gene is shown in SEQ ID NO:1, the approach of the application is to improve the low temperature resistance of tomatoes by knocking out SlC H39 genes.

Further, the gene knockout technology is specifically as follows:

selecting a target fragment containing a motif PAM structure adjacent to a prosomal sequence from a protein coding region of a tomato SlC H39 gene, designing corresponding primers based on the first 20 bases of the target fragment, and constructing a CRISPR/Cas9 vector;

and (3) introducing the CRISPR/Cas9 vector into a host cell, and then infecting a target plant by using the CRISPR/Cas9 vector, and screening a positive transgenic plant to obtain a low-temperature-resistant transgenic plant.

Further, the host cell is an E.coli cell or an Agrobacterium cell.

Further, the host cell is preferably an EHA105 agrobacterium cell.

Further, the nucleotide sequence of the first 20 bases of the PAM structure containing the motif of the proscenium sequence is shown in SEQ ID NO: 3.

The beneficial effects of the invention are as follows: construction of tomato SlC H39 gene knockout plants by gene knockout technology, and research on the regulation and control mechanism of low-temperature resistance of tomatoes by regulating the protein level of the gene SlC H39, and as a result, it is found that SlC H39 gene knockout plants show a low-temperature resistant phenotype at low temperature. The invention provides gene resources for cultivating new low-temperature-resistant tomato varieties, has better potential application value, and lays a theoretical foundation for researching the mechanism of the tomato plants for responding to stress signals and the molecular mechanism of the adverse environment. The invention constructs a tomato SlC H39 gene knockout plant for the first time and performs functional research. Through low-temperature treatment experiments, the SlC3H39 gene is found to play a negative regulation role in low-temperature stress resistance of tomatoes.

Drawings

In order to make the objects, technical solutions and advantages of the present invention more apparent, the following detailed description of the embodiments of the present invention will be given with reference to the accompanying drawings.

FIG. 1 shows the DNA sequencing result of tomato SlC H39 knockout line in example 2 of the present invention;

FIG. 2 shows the relative expression levels of SlC H39 gene of wild-type tomato of example 3 of the present invention after treatment at 25℃and 4 ℃;

FIG. 3 shows the protein accumulation of SlC H39 of wild type tomato of example 3 of the present invention after treatment at 25℃and 4 ℃;

FIG. 4 shows the phenotype of tomato SlC H39 knockout line after low temperature treatment in example 3 of the present invention;

FIG. 5 shows the relative electrolyte permeability of tomato SlC H39 knockout line after low temperature treatment in example 3 of the present invention;

FIG. 6 shows the maximum photochemical efficiency of the tomato SlC H39 knockout line of example 3 of the present invention after low temperature treatment.

Detailed Description

The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. It is intended that the present invention cover other modifications and variations within the scope and spirit of the present disclosure.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botanicals, tissue culture, molecular biology, biophysical biochemistry, DNA recombination, and bioinformatics, which will be apparent to one of skill in the art. These techniques are well explained in the literature.

Example 1: construction of SlC H39 CRISPR/Cas9 Gene knockout vector

Construction of SlC H39 Gene mutation vector

The CRISPR-P website is utilized to design a SlC H39 gene target sequence, and the specific sequence is shown in SEQ ID No:3, ttatccatgt ttaaatcaga. The synthesized target sequence was annealed and ligated to the Bbs I site of AtU6-sgRNA-AtUBQ-Cas9 vector, and then the newly obtained AtU-sgRNA-AtUBQ-Cas 9 fragment was ligated to the HindIII/Kpn I site of pCAMBIA1301 vector. The ligation product was transformed into E.coli DH 5. Alpha. Competent cells, single colonies were picked up and cultured overnight in liquid LB medium containing 50mg/L kanamycin (Kan) at 37℃with shaking at 200 rpm. And then sent to the sequencing confirmation of the department of the Optimum.

Example 2: construction and detection of tomato SlC H39 transgenic material

The gene editing vector pCAMBIA1301: atU6-sgRNA (SlC H39) -AtUBQ-Cas9 was used. Agrobacterium EHA105 is transformed, tomato cotyledon infection is carried out, tissue culture seedlings are obtained through inducing callus, resistance inducing differentiation and rooting culture, positive SlC H39 mutant plants are verified by utilizing a PCR and sequencing technology, slc3h39#1 is found to be deleted for two bases, slc3h39#2 is increased by one base, mutation is carried out at the 4 th base of an original adjacent motif (PAM), and stop codon translation is carried out in advance (fig. 1).

Example 3: low temperature resistance detection of SlC H39 gene transgenic material

First five-leaf and one-heart wild tomato seedlings were treated at 25℃and 4℃and the results showed that the expression of SlC H39 gene was induced at 6 hours at low temperature (FIG. 2) while the accumulation of SlC H39 protein was promoted at 9 hours at low temperature (FIG. 3), which indicates that tomato SlC3H39 gene was responsive to low temperature stress.

Five-leaf, heart, wild-type tomato seedlings and SlC H39 mutant strains obtained in example 2 were subjected to treatment at 25 ℃ and 4 ℃ in an artificial incubator, after 7 days of low temperature treatment, the low temperature stress treatment group was compared with a control group which was not subjected to low temperature treatment under the same conditions, and the changes in phenotype (fig. 4), conductivity (fig. 5) and PSII maximum photochemical quantum yield (Fv/Fm, fig. 6) of the tomato plants of the wild-type and mutant strains were observed, and as a result, it was shown that the mutant tomato plants were capable of significantly improving the low temperature resistance of tomatoes (fig. 4) and the relative electrolyte permeability was significantly lower than that of the wild-type (WT) plants (fig. 5). In addition, fv/Fm (FIG. 6) was also higher in the mutant plants than in the wild-type tomato (WT). From this, it can be seen that tomato SlC3H39 negatively regulates the low temperature tolerance of plants.

Based on the above examples, SEQ ID NO:1 and the nucleotide sequence with the function of not changing the original nucleotide sequence is mutated by utilizing a gene editing technology, agrobacterium is transformed, then agrobacterium is used for invading tomato cotyledons, plant tissue culture is carried out, positive mutant plants are screened, and low-temperature resistant mutant tomatoes can be obtained.

Based on the above examples, the sequence will be identical to SEQ ID NO:1, mutating the nucleotide sequence with the same function obtained by hybridization of the sequence shown in the formula 1 by utilizing a gene editing technology, then transforming agrobacterium, then invading tomato cotyledon by using the transformed agrobacterium, performing plant tissue culture, screening positive mutant plants, and obtaining the low-temperature-resistant mutant tomatoes. The stringent conditions are hybridization in 0.1 XSSPE containing 0.1% SDS or 0.1 XSSC containing 0.1% SDS at 65℃and washing the membrane with the solution;

based on the above examples, a nucleotide sequence having a homology of 90% or more with the nucleotide sequence of 1), 2) or 3) and encoding the same functional protein was mutated in tomato using a gene editing technique, and then agrobacterium was transformed, and then the tomato cotyledon was infiltrated with the transformed agrobacterium and plant tissue culture was performed, and positive mutant plants were screened, and also low temperature resistant mutant tomatoes could be obtained.

While the invention has been described in detail in the foregoing general description and specific embodiments, the invention is not limited to the above examples, but is capable of numerous modifications and improvements as will be apparent to those skilled in the art. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.

Sequence listing

<110> university of Zhejiang

Application of <120> tomato SlC H39 gene as negative regulatory factor in improving low temperature resistance of tomato

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2088

<212> DNA

<213> tomato (Solanum lycopersicum)

<400> 1

atgtgtactg gttcaaggag taaagtttgt ccttctgatt taaacatgga taagaaagat 60

gggcatggta gaaattgttc aaaattgctt gaattgtcag cttcagatga tcttgctagc 120

ttcatatgtg aagttgagaa aggttgtgct gttgatgagt tcagcttttg gtatgggaga 180

agatttggtt caaaaaagat ggggtttgaa gagagaactc ctctgatgat tgcttccatg 240

tatggaagta ttgagatttt gaagtatatt gttggaactg gtaaggttga tgtcaacagg 300

gcttgtggct ctgatggtgc aactgctctt cactgtgctg cagctggtgg gtcggaatca 360

tcggtcgagg ttttaaagat cttgattgat gcttctgctg atgttaatgt ctgtgattct 420

agtggaaaca ggccttgtga tgtgattgct tcttatccta agtctttgag gaattcgaaa 480

aggaaatcgc tggagttgtt gttgaatggc tgcttggctg agcttgctga gttggaggag 540

gaggaagaag gaaaaacagc aattcagatg acgaaggaag gtagcgagaa gaaagagtat 600

ccggtcgata cttccttgcc tgacataaat gatgggattt atgggagtga tgatttcagg 660

atgtattgtt ttaaggttaa gccatgttca agggcttatt ctcatgactg gactgaatgc 720

ccttttgttc atccagggga gaacgcgagg agacgcgatc caaggaagta taactatact 780

tgtgttccat gtcccgagtt caagaaaggc gtttgtgcaa agaatgattc ttgtgagtat 840

gctcatggtg tatttgagtc atggcttcac cctgctcaat atagaacccg tctctgcaag 900

gatgagaccg ggtgctcaag gaaagtttgc ttctttgctc acaagcccga agagctgcgc 960

cccttgtatg cgtccactgg ttcagctatc ccttctccaa aggctagtcc agttgggtcg 1020

atggacatgt caacattgag ccctctggct cttggttctt catccatgtt gttgcctggt 1080

acttcaacgc caccgatgtc tcctgctgtt acatgttcgt ctccaatggg tggaaacatg 1140

tggcagaaca aagcgaacat caccccacct gcattacagc tacctggcag taggctaaag 1200

acatctctaa acgctcggga cttggacttg gacatggaca tgcttggctt ggaaagcatc 1260

cgcacgcagc agcaactaag acagcaattg atagatgaga tggctggtct ctcttcaccg 1320

tcttattgga acaaggacaa tcggatggca gatttgaagc ctacgaatct tgatgatgtt 1380

ttcggatcca tggattctca attgctgtcc caatttcaag gcctttctcc gagagttaca 1440

tccaccacca gctctcagtt gtactctcca tcggtttctc atttgcaagg tctctcacct 1500

aaggtgtcaa gtaacggctc ccagatgcaa tctccaaccg ggctacagat gcgacaaaac 1560

atgaaccaat ttcaatcaag ctatactaac attcctcaat catcgtctcc gatgaggaag 1620

ccctcaacct atggattcga ctcctcagca gcagtggctc aagctgtcat gaactcgagg 1680

tctgctgctt ttgcaaagcg tagccagagt tttattgacc gtagtggaat gggccatcgt 1740

gctgttccca atggtgttgc taactcacca cctttgatgt catcagactg gggctcccct 1800

gatgggaaac tggaatgggg cttcaacagt gatgacacaa acaagctcaa gagatctcaa 1860

tcttttggtt ttcgcggtgg aaatggtgct ccaacaagat caacaatcac accctctcca 1920

ctcaacgagc cagacgtgtc gtgggttcat tccttggtga aagatgtgtc ctccactggt 1980

actggactat acagttcaga gcagcagaag cacggtggtg gtgttcgcga ctccatccca 2040

ccttggctgg aacaaatgta catagaccag gagcgtatcg tggcttaa 2088

<210> 2

<211> 695

<212> PRT

<213> tomato (Solanum lycopersicum)

<400> 2

Met Cys Thr Gly Ser Arg Ser Lys Val Cys Pro Ser Asp Leu Asn Met

1 5 10 15

Asp Lys Lys Asp Gly His Gly Arg Asn Cys Ser Lys Leu Leu Glu Leu

20 25 30

Ser Ala Ser Asp Asp Leu Ala Ser Phe Ile Cys Glu Val Glu Lys Gly

35 40 45

Cys Ala Val Asp Glu Phe Ser Phe Trp Tyr Gly Arg Arg Phe Gly Ser

50 55 60

Lys Lys Met Gly Phe Glu Glu Arg Thr Pro Leu Met Ile Ala Ser Met

65 70 75 80

Tyr Gly Ser Ile Glu Ile Leu Lys Tyr Ile Val Gly Thr Gly Lys Val

85 90 95

Asp Val Asn Arg Ala Cys Gly Ser Asp Gly Ala Thr Ala Leu His Cys

100 105 110

Ala Ala Ala Gly Gly Ser Glu Ser Ser Val Glu Val Leu Lys Ile Leu

115 120 125

Ile Asp Ala Ser Ala Asp Val Asn Val Cys Asp Ser Ser Gly Asn Arg

130 135 140

Pro Cys Asp Val Ile Ala Ser Tyr Pro Lys Ser Leu Arg Asn Ser Lys

145 150 155 160

Arg Lys Ser Leu Glu Leu Leu Leu Asn Gly Cys Leu Ala Glu Leu Ala

165 170 175

Glu Leu Glu Glu Glu Glu Glu Gly Lys Thr Ala Ile Gln Met Thr Lys

180 185 190

Glu Gly Ser Glu Lys Lys Glu Tyr Pro Val Asp Thr Ser Leu Pro Asp

195 200 205

Ile Asn Asp Gly Ile Tyr Gly Ser Asp Asp Phe Arg Met Tyr Cys Phe

210 215 220

Lys Val Lys Pro Cys Ser Arg Ala Tyr Ser His Asp Trp Thr Glu Cys

225 230 235 240

Pro Phe Val His Pro Gly Glu Asn Ala Arg Arg Arg Asp Pro Arg Lys

245 250 255

Tyr Asn Tyr Thr Cys Val Pro Cys Pro Glu Phe Lys Lys Gly Val Cys

260 265 270

Ala Lys Asn Asp Ser Cys Glu Tyr Ala His Gly Val Phe Glu Ser Trp

275 280 285

Leu His Pro Ala Gln Tyr Arg Thr Arg Leu Cys Lys Asp Glu Thr Gly

290 295 300

Cys Ser Arg Lys Val Cys Phe Phe Ala His Lys Pro Glu Glu Leu Arg

305 310 315 320

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

325 330 335

Pro Val Gly Ser Met Asp Met Ser Thr Leu Ser Pro Leu Ala Leu Gly

340 345 350

Ser Ser Ser Met Leu Leu Pro Gly Thr Ser Thr Pro Pro Met Ser Pro

355 360 365

Ala Val Thr Cys Ser Ser Pro Met Gly Gly Asn Met Trp Gln Asn Lys

370 375 380

Ala Asn Ile Thr Pro Pro Ala Leu Gln Leu Pro Gly Ser Arg Leu Lys

385 390 395 400

Thr Ser Leu Asn Ala Arg Asp Leu Asp Leu Asp Met Asp Met Leu Gly

405 410 415

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

420 425 430

Glu Met Ala Gly Leu Ser Ser Pro Ser Tyr Trp Asn Lys Asp Asn Arg

435 440 445

Met Ala Asp Leu Lys Pro Thr Asn Leu Asp Asp Val Phe Gly Ser Met

450 455 460

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

465 470 475 480

Ser Thr Thr Ser Ser Gln Leu Tyr Ser Pro Ser Val Ser His Leu Gln

485 490 495

Gly Leu Ser Pro Lys Val Ser Ser Asn Gly Ser Gln Met Gln Ser Pro

500 505 510

Thr Gly Leu Gln Met Arg Gln Asn Met Asn Gln Phe Gln Ser Ser Tyr

515 520 525

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

530 535 540

Gly Phe Asp Ser Ser Ala Ala Val Ala Gln Ala Val Met Asn Ser Arg

545 550 555 560

Ser Ala Ala Phe Ala Lys Arg Ser Gln Ser Phe Ile Asp Arg Ser Gly

565 570 575

Met Gly His Arg Ala Val Pro Asn Gly Val Ala Asn Ser Pro Pro Leu

580 585 590

Met Ser Ser Asp Trp Gly Ser Pro Asp Gly Lys Leu Glu Trp Gly Phe

595 600 605

Asn Ser Asp Asp Thr Asn Lys Leu Lys Arg Ser Gln Ser Phe Gly Phe

610 615 620

Arg Gly Gly Asn Gly Ala Pro Thr Arg Ser Thr Ile Thr Pro Ser Pro

625 630 635 640

Leu Asn Glu Pro Asp Val Ser Trp Val His Ser Leu Val Lys Asp Val

645 650 655

Ser Ser Thr Gly Thr Gly Leu Tyr Ser Ser Glu Gln Gln Lys His Gly

660 665 670

Gly Gly Val Arg Asp Ser Ile Pro Pro Trp Leu Glu Gln Met Tyr Ile

675 680 685

Asp Gln Glu Arg Ile Val Ala

690 695

<210> 3

<211> 20

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

ttatccatgt ttaaatcaga 20

Claims (5)

1. Tomato (tomato)SlC3H39The application of the gene as a negative regulatory factor in improving the low temperature resistance of tomatoes is characterized in that the nucleotide sequence of a protein coding region of the SlC H39 gene is shown as SEQ ID NO:1, the approach of the application is to increase the low temperature resistance of tomato by knocking out SlC H39 gene, wherein the low temperature is 4 ℃.

2. The use according to claim 1, wherein the gene knockout technique is specifically as follows:

in tomatoSlC3H39Selecting a target fragment containing a PAM structure of a sequence adjacent motif of a pre-region from a protein coding region of a gene, designing corresponding primers based on the first 20 bases of the target fragment, and constructing a CRISPR/Cas9 vector;

and (3) introducing the CRISPR/Cas9 vector into a host cell, and then infecting a target plant by using the CRISPR/Cas9 vector, and screening a positive transgenic plant to obtain a low-temperature-resistant transgenic plant.

3. The use according to claim 2, wherein the host cell is an e.

4. The use according to claim 3, wherein the host cell is an EHA105 agrobacterium cell.

5. The use according to claim 2, wherein the nucleotide sequence of the first 20 bases comprising the motif PAM structure is set forth in SEQ ID NO: 3.

Images