CN109777811A - Tomato SlMYB0 gene and its application in raising plant frigostabile - Google Patents

Abstract

It is 756bp that the invention discloses tomato SlMYB0 genes improving the application in plant frigostabile ability, the nucleotide sequence length of the gene SlMYB0, and sequence is SEQ ID NO:1, and corresponding amino acid sequence is as shown in SEQ ID NO:2.Tomato SlMYB0 is constructed by gene means and is overexpressed plant and gene knockout plant, regulates and controls the expression of the gene SlMYB0 to study its regulatory mechanism to tomato low-temperature resistance.As a result, it has been found that SlMYB0 is overexpressed the expression that can induce tomato low-temperature resistance gene under low temperature, the low-temperature resistance of tomato is improved.The present invention provides genetic resources to cultivate low temperature resistant New Tomato Variety, has preferable potential using value, to study the mechanism of tomato plants response adverse circumstance signal and the molecule mechanism based theoretical of tolerance adverse environment.

Description

Tomato SlMYB0 gene and its application in raising plant frigostabile

Technical field

The present invention relates to the fields such as genetic engineering, molecular biology and physiology, specifically, being related to tomato SlMYB0 base Clone, transgene carrier building and the conversion of tomato of cause more particularly to SlMYB0 gene are in improving plant frigostabile ability Application.

Background technique

Tomato is the annual or herbaceos perennial in Solanaceae Solanum, and alias tomato, tomato are world wides It is interior to cultivate most wide, the maximum vegetable crop of consumption figure.Tomato variety type is abundant, can not only eat raw and make vegetables but also can make fruit, kind Lycopene ingredient in eggplant can prevent various cancers, be one of most popular healthy food in the world.Tomato is in facilities vegetable In occupy an important position, therefore improve tomato reply poor environment ability, cultivate resistant variety have important actual production Meaning.

In recent years, with the extreme variation of global climate, low temperature, which increasingly becomes a kind of, influences extensive abiotic stress, Normal growth and agricultural production to plant cause great destruction.Therefore, the physiology for studying plant responding Chilling stress is raw Change variation and molecule mechanism, find the key gene of regulation plant low temperature response, provides weight to obtain the acquisition of low temperature resistant plant The theories integration wanted, to improve vegetables yield, quality and economic benefit, and ensure vegetables stable market supply all have it is very heavy The science and realistic meaning wanted.

Myb transcription factor takes part in as one kind transcription factor maximum in plant including cell cycle, physiological metabolism Many vital movements including balance and environment response etc..With finding that the quantity of myb gene increases sharply in plant, MYB turns The information such as the determination of regulatory mechanism, gene expression profile and target gene of the factor are recorded also to enrich constantly.Have now been found that multiple MYB The response of transcription factor participation plants against abiotic stress.In drought stress, most of myb transcription factor is related to ABA, Drought stress response is participated in as there are 43 genes to be induced in 156 myb transcription factors of soybean by ABA.It is some in addition to drought stress Myb transcription factor also assists in the Resistant process of plant.The TaPIMP1 of wheat, AtMYB46, AtMYB72 of arabidopsis, AtMYB73 respectively can the invasion to pinta bacterium, ash arrhizus bacteria, root-rot from compacted spore bacterium make response adjusting；Arabidopsis AtMYB102/ AtM4 and AtMYB41 can be by causing dehydration to resist the invasion of extraneous pest after by insect bite, and arabidopsis AtMYB96 passes through Regulate and control salicylic acid route of synthesis and improves disease resistance.In addition, arabidopsis AtMYB30 passes through very-long-chain fatty acid route of synthesis, The infringement of plants by phytopathogenic bacterium, which generates in hypersensitivity reaction process, plays positive control.These illustrate that myb transcription factor participates in planting Object is to the answering of environment stress, however effect and its regulatory mechanism of the tomato myb transcription factor in low temperature stress are fresh It appears in the newspapers.Therefore, the tomato material of variable expression SlMYB0 is cultivated by the clone to SlMYB0 gene, transgenic technology, In terms of improving tomato to the resistance of low temperature stress and excavation environment stress genetic resources, have a good application prospect.

Summary of the invention

The technical problem to be solved in the present invention is to provide gene SlMYB0 to improve the application in tomato low temperature tolerance ability.

In order to achieve the object of the present invention, gene SlMYB0 provided by the invention is improving answering in tomato low temperature tolerance ability With the nucleotide sequence of the gene SlMYB0cDNA are as follows:

1) nucleotide sequence shown in SEQ ID NO:1；Or

2) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and tool There is the nucleotide sequence of identical function；Or

3) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function；Or

4) there is 90% or more homology and nucleotides sequence with the same function with nucleotide sequence 1), 2) or 3) Column.

Invention also provides a kind of protein of said gene coding, which has shown in SEQ ID NO:2 Amino acid sequence.It should be appreciated that those skilled in the art can disclosed amino acid sequence according to the present invention, do not influencing its work Under the premise of property, replaces, lacks and/or increase one or several amino acid, obtain the mutant nucleotide sequence of the albumen.It should be understood that In view of the degeneracy of codon and the preferences of different plant species codon, those skilled in the art be can according to need using suitable Close the codon of particular species expression.

The present invention also provides cloning vectors or all kinds of tables containing the low temperature resistant SlMYB0 gene order of tomato or its segment Up to carrier, the host cell containing the carrier, the conversion plant cell containing the gene order or its specific fragment and turn Gene plant.

Plant of the present invention is tomato.The low temperature resistant SlMYB0 gene of tomato is overexpressed in tomato and is compiled using gene The technology of collecting is mutated, and foreign gene can be guided to be overexpressed in plant and gene editing knockout technology including the use of any Carrier carry out.

The present invention also provides a kind of method for constructing low temperature resistant transgene tomato, including gene overexpression, the methods Specifically: tomato total serum IgE is extracted, reverse transcription obtains cDNA, and using cDNA as template, F and R are primer, expand SlMYB0 gene, will Amplified production is building up on plant over-express vector；Utilize CRISPR-P website design SlMYB0 gene target sequence, the target of synthesis The Bbs I site of AtU6-sgRNA-AtUBQ-Cas9 carrier, the AtU6- that then will newly obtain are connected to after sequence anneals SgRNA-AtUBQ-Cas9 segment is connected to the Hind III/Kpn I site of pCAMBIA1301 carrier, constructs tomato SlMYB0 gene C RISPR expression vector.Then Agrobacterium is converted with the recombinant expression carrier obtained, then with the agriculture bar of conversion Bacterium invades tomato cotyledon, and carries out Plant Tissue Breeding, screens positive transgenic tomato plant, obtains low temperature resistant transgenosis kind Eggplant.Wherein, the nucleotide sequence of the primers F and R are as shown in SEQ ID NO:3 and 4.The nucleotide sequence of the sgRNA is such as Shown in SEQ IN NO:5.

The plant expression vector that the present invention uses is the expression vector with 35S promoter, such as carrier pFGC1008-HA Or pCAMBIA1301.

The present invention goes back while providing a kind of host cell, which is Bacillus coli cells, agrobatcerium cell.Its In, the preferably described Agrobacterium is GV3101.

The present invention goes back while providing the purposes of said gene: for constructing transgene tomato, the transgene tomato energy Enough resistances improved to low temperature stress.It, can be to used load for the ease of being identified Transgenic Tomato Plants and being screened Body is processed, such as plant alternative label or resistant antibiotic marker is added.Overexpression noted earlier carries It joined 3HA label protein in body pFGC1008.

The present invention constructs the transgenic plant of tomato SlMYB0 gene overexpression and gene knockout for the first time, and carries out function Research.It is tested by low-temperature treatment, discovery SlMYB0 gene plays the role of positive regulation in the low temperature resistant stress of tomato.

SlMYB0 gene provided by the invention provides genetic resources to cultivate low temperature resistant New Tomato Variety, has preferable Potential using value establishes theory for the mechanism for studying tomato plants response adverse circumstance signal and the molecule mechanism for being resistant to adverse environment Basis.

Detailed description of the invention

To make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawing to of the invention specific Embodiment is described in further detail.

Fig. 1 is the vegetable protein Western Blot inspection of SlMYB0 gene overexpression tomato strain in the embodiment of the present invention 3 Survey result.

Fig. 2 is the sequencing result of the sgRNA sequence of SlMYB0 gene knockout tomato strain in the embodiment of the present invention 3.

Fig. 3 be in the embodiment of the present invention 4 under room temperature and cryogenic conditions and its result of protein content；Wherein, A is The expression of SlMYB0 gene, B are the level of SlMYB0 protein content.

Fig. 4 is transgene tomato OE#16 and OE#18, myb0#13 in the embodiment of the present invention 4 under room temperature and cryogenic conditions Low temperature resistant phenotype is showed with myb0#14.

Fig. 5 is transgene tomato OE#16 and OE#18, myb0#13 in the embodiment of the present invention 4 under room temperature and cryogenic conditions With the conductivity variations of myb0#14.

Fig. 6 is transgene tomato OE#16 and OE#18, myb0#13 in the embodiment of the present invention 4 under room temperature and cryogenic conditions Change with the PSII maximum Photochemical quantum yield (Fv/Fm) of myb0#14.

Fig. 7 is transgene tomato OE#16 and OE#18, myb0#13 in the embodiment of the present invention 4 under room temperature and cryogenic conditions With the variation for resisting cold gene expression dose in myb0#14；Wherein, A is the expression of tomato CBF1 gene, and B is tomato CBF2 The expression of gene, C are the expression of tomato CBF3 gene.

Fig. 8 is in the embodiment of the present invention 4 under room temperature and cryogenic conditions, and the different MYB screened in wild-type tomatoes are transcribed The variation of factor expression level, including SlMYB0 (Solyc03g005570), SlMYB10 (Solyc05g055030), SlMYB15 (Solyc09g090130), SlMYB17 (Solyc05g048830), SlMYB30 (Solyc06g069850), SlMYB31 (Solyc03g116100), SlMYB46 (Solyc11g065840), SlMYB50 (Solyc06g071690), SlMYB61 (Solyc01g102340), SlMYB96 (Solyc02g067340), the base of SlMYB106 (Solyc01g010910) Because of the comparison between expression.

Specific embodiment

The present invention is further explained in the light of specific embodiments, but the present invention is not limited to following embodiments.This Invent other changes and modification being intended to cover in scope and spirit disclosed by the invention.

Unless otherwise indicated, implementation of the invention will use the obvious botany routine skill of those skilled in the art Art, tissue cultures, molecular biology, biological physiology are biochemical, DNA is recombinated and bioinformatics technique.These technologies are in the literature Abundant explanation is carried out.

The building of embodiment 1:SlMYB0 gene overexpression carrier

For the molecular mechanism for understanding plant responding low temperature, SlMYB0 gene has been cloned from tomato dna group.According to coding Region sequence analysis, designs specific primer SlMYB0-F and SlMYB0-R, and adds restriction enzyme site respectively on primer (Asc I and Kpn I), sequence is as shown in SEQ ID NO:3 and 4.With PrimerSTAR high fidelity enzyme PCR amplification SlMYB0 piece Then section carries out digestion to pcr amplified fragment and carrier, SlMYB0 segment is connected on pFGC1008-HA, is overexpressed Carrier OE.Above-mentioned recombinant plasmid is sent to the sequencing confirmation of Qing Ke company, the nucleotide sequence such as SEQ of resulting gene SlMYB0 Shown in ID No:1；The result shows that the sequence (Solyc03g005570) one announced in the sequence and Solgenomics cloned It causes.

The amino acid sequence of the protein of gene coding is as shown in SEQ ID No:2.

The building of embodiment 2:SlMYB0 gene mutation carrier

Using CRISPR-P website design SlMYB0 gene target sequence, particular sequence is as shown in SEQ ID No:5 CGAGCTTGAAGAAAGGGTCA.The Bbs I of AtU6-sgRNA-AtUBQ-Cas9 carrier is connected to after the target sequence annealing of synthesis Then the AtU6-sgRNA-AtUBQ-Cas9 segment newly obtained is connected to the Hind III/ of pCAMBIA1301 carrier by site Kpn I site.Above-mentioned recombinant plasmid is sent to the sequencing confirmation of Qing Ke company.

Embodiment 3: the building and detection of tomato SlMYB0 transgenic line

The over-express vector pFGC1008::SlMYB0-HA that embodiment 1 obtains and the gene editing that embodiment 2 obtains are carried Body pCAMBIA1301::AtU6-sgRNA (SlMYB0)-AtUBQ-Cas9 converts Agrobacterium GV3101 respectively, and carries out tomato Cotyledon infects, and by callus induction, induction of resistance differentiation and culture of rootage obtain tissue-cultured seedling, by T2 for mutant seeds and It is overexpressed seed and carries out the test of kalamycin resistance and chlorampenicol resistant respectively, select that 3/4 is resistant and remaining 1/4 does not have The strain of resistance illustrates that the over-express vector that target gene is connected in the strain is inserted into the form of single copy.By these plant It removes, then carries out single plant sowing.It is overexpressed positive transgenic plant using Western Blot verifying SlMYB0, it is wild as the result is shown Raw type does not have protein band, and is overexpressed the band (Fig. 1) that strain has SlMYB0-HA, is verified using PCR and sequencing technologies positive SlMYB0 mutation transgene plant, discovery myb0#13 lack 7 bases, and myb0#14 lacks 14 bases, respectively in original phase It is mutated at the 7th of adjacent motif (PAM) and the 17th base, and stops translation (Fig. 2) immediately.

The low temperature tolerance ability of embodiment 4:SlMYB0 gene transgenic material detects

The wild-type tomatoes seedling of five leaves wholeheartedly is subjected to 25 DEG C and 4 DEG C processing first, it is as the result is shown (Fig. 3, A and B), low Temperature can induce the expression (Fig. 3 A) of SlMYB0 gene for 6 hours, while promote within low temperature 12 hours a large amount of accumulation of SlMYB0 albumen (Fig. 3 B), this illustrates that tomato SlMYB0 responds low temperature stress.

By SlMYB0 gene overexpression strain and mutation obtained in five leaves wild-type tomatoes seedling wholeheartedly and embodiment 3 Body strain carries out 25 DEG C and 4 DEG C processing in artificial incubator, after low-temperature treatment 7 days, by low temperature stress processing group and identical item The control group for not carrying out low-temperature treatment under part is compared, and observation wild type is overexpressed strain and mutant strain tomato plant Phenotype (Fig. 4), conductivity (Fig. 5), PSII maximum Photochemical quantum yield (Fv/Fm, Fig. 6) and tomato resist cold gene C BF1 The variation of (Fig. 7 A), CBF2 (Fig. 7 B) and CBF3 (Fig. 7 C) expression.The results show that being overexpressed tomato plant can significantly mention The lower temperature resistance (Fig. 4) of high tomato, conductivity are substantially less than wild type (WT) and myb0 mutating strain series (Fig. 5).In addition, being overexpressed Fv/Fm (Fig. 6) the He Kangleng gene (CBF1, CBF2 and CBF3, Fig. 7) of plant is above wild-type tomatoes (WT), and mutant Strain is minimum.It can be seen that tomato SlMYB0 is just regulating and controlling the low temperature tolerance ability of plant.

In addition, the common knowledge as this field, the nucleotide sequence shown in SEQ ID NO:1 has above-mentioned effect In the case of, carrying out certain change to it by conventional means will not influence its function, such as be substituted, lack and/ Or increases one or more nucleotide and have and do not change protokaryon nucleotide sequence function, nucleotides sequence with the same function Column；Or hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function, it is described stringent Condition be in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS, hybridize at 65 DEG C, and use this Solution washes film；Or the nucleotide sequence with 90% or more homology and coding identical function protein.

Sequence table

<110>Zhejiang University

<120>tomato SlMYB0 gene and its application in raising plant frigostabile

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 756

<212> DNA/RNA

<213> Solanum lycopersicum

<400> 1

atggacagag caccttcttg tgagaaaacg agcttgaaga aagggtcatg ggatcacgaa 60

gaagaccaaa ttctcatctc ttacattaac aaaaatggcc atggcaattg gcgcgcactc 120

cctagacaag ctggattatt gagatgtgga aaaagttgca gattgaggtg gatgaattat 180

ttgagtccac atattaagag gggaaatttc accaaagaag aagaagataa tatcatcaat 240

ttacatgaaa ttcttggaaa tagatggtca gcaatagcag caagattacc gggaagaacg 300

gataatgaaa taaaaaatat ttggcacacc cacttgaaga aaaggctaaa aaattgtcag 360

tctcctcgaa gcttcaaaag acacaaaaaa attaatcaaa taaataattg catgcatgct 420

cctaactcac cagaaatatc gtctagcgag acgtcaatca tgacaaccaa ttcatcatca 480

agtggtactt tcgatacgac gatcgacgat caaatagtga ttaaacaaga agaaatggac 540

tcatcggagt atttcgccaa gatcgatgag agcttctgga cggatgaatt aacgatagag 600

aacaatttcg actttgttac taatatggat ggtgaagaat ttgaaagctt agacatgttt 660

aatagtacaa caataatgga ggatgacatt gacatggact ttttgttcaa ctttttcata 720

aagactgagg aattttcaga attaccagaa ctttga 756

<210> 2

<211> 251

<212> PRT

<213> Solanum lycopersicum

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Met Asp Arg Ala Pro Ser Cys Glu Lys Thr Ser Leu Lys Lys Gly Ser

1 5 10 15

Trp Asp His Glu Glu Asp Gln Ile Leu Ile Ser Tyr Ile Asn Lys Asn

20 25 30

Gly His Gly Asn Trp Arg Ala Leu Pro Arg Gln Ala Gly Leu Leu Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Met Asn Tyr Leu Ser Pro His

50 55 60

Ile Lys Arg Gly Asn Phe Thr Lys Glu Glu Glu Asp Asn Ile Ile Asn

65 70 75 80

Leu His Glu Ile Leu Gly Asn Arg Trp Ser Ala Ile Ala Ala Arg Leu

85 90 95

Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Ile Trp His Thr His Leu

100 105 110

Lys Lys Arg Leu Lys Asn Cys Gln Ser Pro Arg Ser Phe Lys Arg His

115 120 125

Lys Lys Ile Asn Gln Ile Asn Asn Cys Met His Ala Pro Asn Ser Pro

130 135 140

Glu Ile Ser Ser Ser Glu Thr Ser Ile Met Thr Thr Asn Ser Ser Ser

145 150 155 160

Ser Gly Thr Phe Asp Thr Thr Ile Asp Asp Gln Ile Val Ile Lys Gln

165 170 175

Glu Glu Met Asp Ser Ser Glu Tyr Phe Ala Lys Ile Asp Glu Ser Phe

180 185 190

Trp Thr Asp Glu Leu Thr Ile Glu Asn Asn Phe Asp Phe Val Thr Asn

195 200 205

Met Asp Gly Glu Glu Phe Glu Ser Leu Asp Met Phe Asn Ser Thr Thr

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Lys Thr Glu Glu Phe Ser Glu Leu Pro Glu Leu

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<212> DNA

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cgagcttgaa gaaagggtca 20

Claims (7)

1. tomato SlMYB0 gene, which is characterized in that the gene is following 1) -4) in any one nucleotide sequence:

1) nucleotide sequence shown in SEQ ID NO:1；

2) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and has not Change protokaryon nucleotide sequence function, nucleotide sequence with the same function；

3) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function, it is described tight Glazing bar part is to hybridize at 65 DEG C, be used in combination in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS The solution washes film；

4) there is 90% or more homology with nucleotide sequence 1), 2) or 3) and encodes the nucleotides sequence of identical function protein Column.

2. by tomato SlMYB0 gene described in claim 1 coding obtain protein, which is characterized in that its with it is following it One amino acid sequence:

1) amino acid sequence shown in SEQ ID No:2；

2) amino acid sequence shown in SEQ ID No:2 is substituted, lacks and/or increases one or more amino acid and has same The protein as derived from 1) of isoreactivity.

3. tomato SlMYB0 gene improve plant frigostabile ability in application, which is characterized in that the gene be it is following 1)- 4) any one nucleotide sequence in:

1) nucleotide sequence shown in SEQ ID NO:1；

2) nucleotide sequence shown in SEQ ID NO:1 is substituted, lacks and/or increases one or more nucleotide and has not Change protokaryon nucleotide sequence function, nucleotide sequence with the same function；

3) hybridize under strict conditions with sequence shown in SEQ ID NO:1 and nucleotide sequence with the same function, it is described tight Glazing bar part is to hybridize at 65 DEG C, be used in combination in 0.1 × SSPE containing 0.1%SDS or 0.1 × SSC solution containing 0.1%SDS The solution washes film；

4) there is 90% or more homology with nucleotide sequence 1), 2) or 3) and encodes the nucleotides sequence of identical function protein Column.

4. application according to claim 3, which is characterized in that make the tomato SlMYB0 by gene overexpression technology The expression of gene rises.

5. application according to claim 4, which is characterized in that the gene overexpression technology is specific as follows:

Tomato total serum IgE is extracted, reverse transcription obtains cDNA, and using cDNA as template, F and R are primer, expands SlMYB0 gene, will expand Volume increase object is building up on plant over-express vector；The nucleotide sequence of the primers F and R are as shown in SEQ ID NO:3 and 4；

Plant over-express vector is imported in host cell, it is recycled to infect purpose plant, positive transgenic plant is screened, obtains Obtain low temperature resistant transgenic plant.

6. application according to claim 5, which is characterized in that over-express vector plasmid is pFGC1008::SlMYB0-HA, Gene editing vector plasmid pCAMBIA1301::AtU6-sgRNA (SlMYB0)-AtUBQ-Cas9.

7. application according to claim 5, which is characterized in that the host cell is that Bacillus coli cells or Agrobacterium are thin Born of the same parents.