CN103739685A - Transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof - Google Patents
Transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof Download PDFInfo
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Abstract
The invention provides a transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof. The transcription factor is one of two bHLH transcription factors (Csa5G157230 and Csa5G156220) which are found in cucumber genome firstly and used for controlling synthesis of bitter taste; the two bHLH transcription factors are used for controlling the forming of the bitter taste in fruits and leaves respectively; simple crossing of yeast, a gel tissue system and instantaneous expression of tobacco prove that the two transcription factors can be combined to a bitter taste synthesis promoter region and used for activating transcription of a synthesis gene; meanwhile, the bitter taste characteristics of cucumber plants can be restored by expressing the corresponding transcription factors in the leaves or fruits of the cucumber free of bitter taste abundantly. The invention further discloses a molecular mechanism for forming bitter taste of cucumber and provides theoretical basis for breeding cucumber free of bitter taste and molecular assisted breeding goals.
Description
Technical field
The present invention relates to genetically engineered and biology field, specifically, relate to and participate in synthetic transcription factor Csa5G157230 and the application thereof of regulation and control cucumber cucurbitacin C.
Background technology
The bitter taste of cucumber is that the triterpenoid that is called cucurbitacin C by a class causes.In cucumber fruits, accumulate mouthfeel and quality that cucurbitacin C can have a strong impact on cucumber.The bitter taste that cucumber is found in early stage classical genetics test is by Bi and two Gene Handling of Bt.Research recently finds that a gene cluster by 8 genomic constitutions participates in the synthetic of cucumber bitter taste.Wherein Bi genes encoding squalene oxide cyclase, its catalysis generates cucurbit 2 enols, is the synthetic the first step of cucurbitacin C, is also a step of synthetic middle most critical.
Although substantially determined the synthetic molecular mechanism of bitter taste in cucumber, the molecular mechanism that regulation and control bitter taste forms is not clear at present, and relevant synthetic gene is not cloned yet.
Summary of the invention
The object of this invention is to provide and participate in synthetic transcription factor Csa5G157230 and the application thereof of regulation and control cucumber cucurbitacin C.
In order to realize the object of the invention, a kind of synthetic transcription factor Csa5G157230 of cucumber cucurbitacin C that participates in of the present invention, its aminoacid sequence is as shown in SEQ ID No.4, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
The present invention also provides the gene of the described transcription factor Csa5G157230 of coding, and its nucleotide sequence is as shown in SEQ ID No.3.
The present invention also provides the plant of the carrier, engineering bacteria, host cell and the conversion that contain the described transcription factor Csa5G157230 gene of encoding.
The application of the gene that the present invention also provides the described transcription factor Csa5G157230 of coding in regulation and control cucumber bitter taste is synthetic.
The present invention also provides the gene of the described transcription factor Csa5G157230 of coding in the application without in bitter taste cucumber molecular breeding.
The present invention also provide a kind of in plant the method for transient expression gene, adopt agriculture bacillus mediated method, the carrier that contains the described transcription factor Csa5G157230 gene of encoding is proceeded in plant materials (as tobacco), obtain goal gene great expression.
The present invention further provides for the encode primer pair of described transcription factor Csa5G157230 gene of pcr amplification, comprise upstream primer 5 '-TGTATACAATCTCCATTCCCTTTTGA-3 ' and downstream primer 5 '-TATTCCAAGTTAGTCAATTTATTCTGT-3 '.
Cucumber mutant (XY-3) from a blade from Hunan without bitter taste, it is by bitter taste cucumber (XY-2) spontaneous mutation.In mutant XY-3, the expression of the bitter taste synthetic gene of having found before having detected, finds very low of the expression amount of bitter taste synthetic gene bunch in mutant.Infer that thus sudden change may occur in this mutant, to regulate and control the synthetic gene of bitter taste.The genomic dna of XY-2 and XY-3 is checked order.By icp gene group sequence, emphasis is found the inside sudden change that occurs in transcription factor.By analyzing, find a SNP, be positioned at the inside of this transcription factor of Csa5G156220.In conjunction with transcribing group data, find only great expression in blade of this gene.Further detect the expression of this gene in mutant XY-3, find that the expression of this transcription factor in mutant also significantly reduces.In addition, also find that the adverse circumstance processing such as arid and ABA can make this transcription factor and bitter taste synthetic gene expression raise, and have very high consistence.Therefore, infer that this sudden change may cause this transcription factor expression amount to reduce, thereby synthetic gene expression is significantly reduced, finally caused blade to become from hardship or not.
For further checking, carried out yeast one-hybrid and gel retardation assasy, prove that this transcription factor can be attached to the promoter region of bitter taste synthetic gene really.In addition, also the promotor of bitter taste synthetic gene is connected to reporter gene (LUC), in tobacco, detect the activation of transcription factor to promoter region, find that it can activate the expression of reporter gene.Because stable cucumber transformation system is also immature, therefore, the present invention has set up cucumber cotyledons transient expression system, utilize Agrobacterium by transcription factor import cucumber XY-3 without without in bitter taste cotyledon, after one week, detect cotyledon, find transcription factor and synthetic gene great expression, thereby cause cotyledon by the not bitter hardship that becomes.Comprehensive above-mentioned result of study, the present invention has found to regulate and control the transcription factor of bitter taste synthetic gene expression first in blade.By this unnamed gene, be Bf, thereby it further regulate and control the formation of blade bitter taste by the expression of regulation and control synthetic gene.
Genetic analysis before finds that cucumber fruits gene is by Bt Gene Handling.Due to wild cucumber fruits hardship very, therefore, in cucumber domestication process, must there is sudden change in Bt gene, causes cucumber fruits not bitter, and this sudden change is subject to artificial selection and remains.In research before, 114 parts of cucumber Core Germplasms materials are weighed to sequencing analysis, the region occurring by the domestication of bioinformatic analysis cucumber, then in conjunction with map based cloning, in the interval of the Bt assignment of genes gene mapping to 5 karyomit(e) 442kb.There are 68 candidate genes in this interval.That further analyzes these genes transcribes group data, finds that Csa5G157230 wherein only expresses in the fruit of raw cucumber out of office, does not express in cultivated cucumber fruit.And the synthetic gene of Csa5G157230 gene and the regulation and control blade bitter taste of simultaneously finding is homologous gene, is all bHLH class transcription factor.By detecting the expression conditions of the synthetic Bi gene of Csa5G157230 gene and bitter taste in different cucumber varieties and in Bt gene isolation colony individuality.Find they expression amount and fruit in the content of bitter principle be proportionate.The expression amount that is Csa5G157230 gene is higher, and Bi gene expression amount is higher, thereby cucumber fruits is more bitter.According to these information, infer that this gene is exactly probably Bt gene, it is synthetic by the bitter taste in the synthetic mechanism regulating cucumber fruits of similar Bf regulation and control bitter taste.
Based on the research system of having reported before, as yeast one-hybrid, gel blocking prove the expression that Csa5G157230 gene really can transcriptional activation bitter taste synthetic gene.Because stable cucumber transformation system is also immature, therefore the present invention has set up cucumber fruits transient expression system, utilize Agrobacterium that Csa5G157230 gene is imported in the close thorn fruit in Xintai City, after three weeks, detect the fruit of injected area, find Csa5G157230 gene and synthetic gene great expression, thereby cause fruit by the not bitter hardship that becomes.Comprehensive above-mentioned result of study, thinks that Csa5G157230 gene is Bt gene.Thereby it is by the further formation of regulating fruit bitter taste of expression of regulation and control synthetic gene.
Accompanying drawing explanation
Fig. 1 is that the XY-3 that relates in the embodiment of the present invention 1 is without bitter taste mutant relevant information; Wherein, A is the relative concentration of cucurbitacin C in mutant, and XY-2 is bitter taste cucumber; B is the expression of bitter taste synthetic gene in mutant; C attaches most importance to checking order and finds that the position of sudden change is positioned at the inside of Bf transcription factor.
Fig. 2 is the expression of processing rear Csa5G156220 and bitter taste synthetic gene in the embodiment of the present invention 1 with arid and ABA.
Fig. 3 be in the embodiment of the present invention 1 with bioinformatic analysis in conjunction with map based cloning, by the Bt assignment of genes gene mapping in No. 5 chromosomal situations; Wherein, have 67 candidate genes, their expression in wild and cultivated cucumber fruit and blade are distinguished with gradient color, more deeply represent that content is higher.
Fig. 4 is bitter taste synthetic gene Bi in the individuality detecting in the embodiment of the present invention 1 in different types of cucumber and Bt segregating population, the expression of bitter taste regulatory gene Bt and the content of cucurbitacin C.
Fig. 5 proves that with yeast-one-hybrid system and tobacco transient expression system Bf can be incorporated in the promotor of bitter taste synthetic gene in the embodiment of the present invention 1, and activates downstream reporter gene and transcribe.
Fig. 6 proves that by gel blocking system Bf can be incorporated in the promotor of bitter taste synthetic gene in the embodiment of the present invention 1.
Fig. 7 proves that with yeast-one-hybrid system and tobacco transient expression system Bt can be incorporated in the promotor of bitter taste synthetic gene in the embodiment of the present invention 1, and activates downstream reporter gene and transcribe.
Fig. 8 proves that by gel blocking system Bf can be incorporated in the promotor of bitter taste synthetic gene in the embodiment of the present invention 1.
Fig. 9 is the biological function that proves respectively Bf and Bt in the embodiment of the present invention 2 with blade and fruit transient expression system, when causing bitter taste synthetic gene Bt to express after Bf or Bt expression, raises, and finally makes the content of bitter substance cucurbitacin C raise.
Embodiment
Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.If do not specialize, embodiment is all according to normal experiment condition, as Sambrook equimolecular cloning experimentation handbook (Sambrook J & Russell DW, Molecular cloning:a laboratory manual, 2001), or according to the condition of manufacturer specification sheets suggestion.
1, the excavation of candidate gene
In Hunan, found the cucumber mutant (XY-3) of a blade without bitter taste, it is by bitter taste cucumber (XY-2) spontaneous mutation (Fig. 1, A).In mutant XY-3, the expression amount of bitter taste synthetic gene bunch in mutant low (Fig. 1, B) very, infers that sudden change may occur in this mutant, to regulate and control the synthetic gene of bitter taste.By the genomic dna of XY-2 and the XY-3 order of resurveying.Utilize bioinformatic analysis, find the inside sudden change that occurs in transcription factor.A SNP is found in this analysis, is positioned at the inside (Fig. 1, C) of this transcription factor of Csa5G156220.Transcribe only great expression in blade of this gene of group data presentation, and the expression of this transcription factor in mutant also significantly reduces (Fig. 1, B).In addition, the processing of the adverse circumstance such as arid and ABA can make this transcription factor and bitter taste synthetic gene expression rising (Fig. 2).Therefore, classify this transcription factor as candidate gene, called after Bf.
Genetic analysis before finds that cucumber fruits bitter taste is by Bt Gene Handling.In cucumber domestication process, must there is sudden change in Bt gene, causes cucumber fruits not bitter.114 parts of cucumber Core Germplasms materials are weighed to sequencing analysis, the region occurring by the domestication of bioinformatic analysis cucumber, then in conjunction with map based cloning, in the interval of the Bt assignment of genes gene mapping to 5 karyomit(e) 442kb.There are 68 candidate genes (Fig. 3) in this interval.That further analyzes these genes transcribes group data, finds that Csa5G157230 wherein only expresses in the fruit of raw cucumber out of office, does not express (Fig. 3) in cultivated cucumber fruit.Detect the expression conditions of the synthetic Bi gene of Csa5G157230 gene and bitter taste in different cucumber varieties and in Bt gene isolation colony individuality.Find they expression amount and fruit in the content of bitter principle be proportionate (Fig. 4).The expression amount that is Csa5G157230 gene is higher, and Bi gene expression amount is higher, thereby cucumber fruits is more bitter.And the synthetic gene of Csa5G157230 gene and the regulation and control blade bitter taste that we find is homologous gene, is all bHLH class transcription factor.According to these information, infer that this gene is exactly probably Bt gene, it is synthetic by the bitter taste in the synthetic mechanism regulating cucumber fruits of similar Bf regulation and control bitter taste.
2, cucumber Csa5G157230 gene function checking
First prepare the cDNA library of cucumber leaves and fruit, then utilize forward primer and reverse primer to carry out pcr amplification (primer sequence is in Table 1).
Table 1 primer sequence (5 '-3 ')
| Csa5G156220-TVector-F | TCTCTTTCCTCTCTCATTACGGGTGA |
| Csa5G156220-TVector-R | CGCACATCAAGGCAATCAACTCG |
| Csa5G157230-TVector-R | TGTATACAATCTCCATTCCCTTTTGA |
| Csa5G157230-TVector-R | TATTCCAAGTTAGTCAATTTATTCTGT |
PCR reaction system is counted with 20 μ L: 10-20ng/ μ L template 1 μ L, 10pmol/ μ L forward, the each 1 μ L of reverse primer, 10mmol/L dNTP mix0.4 μ L, 0.5U/ μ L high-fidelity Taq archaeal dna polymerase 1 μ L, 10 × PCR reaction buffer, 2 μ L, surplus is water.
PCR reaction conditions is: 94 ℃ 5 minutes; 94 ℃ 20 seconds, 55 ℃ 20 seconds, 72 ℃ 1 point, 35 circulations; 72 ℃ 10 minutes.
The fragment that amplification is obtained is connected with T-carrier (TAKARA), and not sudden change is confirmed in order-checking.Shown in the nucleotide sequence SEQ ID No.3 of cucumber Csa5G157230 gene, the aminoacid sequence of its proteins encoded is as shown in SEQ ID No.4.
Adopt yeast one-hybrid to prove that Bf can be attached to the promoter region (Fig. 5, A) of bitter taste synthetic gene.In addition, also the promotor of bitter taste synthetic gene is connected to reporter gene (LUC), in tobacco, detect the activation of transcription factor to promoter region, find that it can activate the expression (Fig. 5, B) of reporter gene.Subsequently, by gel retardation assasy, also proved the mutual work (Fig. 6) of Bf and promotor.Utilize identical experimental system, proved the mutual work (Fig. 7 and Fig. 8) of Bt and bitter taste synthetic gene promotor.
Because stable cucumber transformation system is also immature, therefore set up in the present embodiment cucumber cotyledons and fruit transient expression system, further verify the function of Bf and Bt.
By Csa5G156220(SEQ ID No.2), Csa5G157230 gene (SEQ ID No.3) is building up on binary vector pBIN-Plus.Utilize chemical conversion to import respectively in Agrobacterium EA105.The Agrobacterium that contains Csa5G156220 gene is cultured to OD
600approximately 1.0, then with injection damping fluid, be diluted to OD
600approximately 0.3 left and right, uses the not syringe with syringe needle Agrobacterium to be injected to the cucumber seedlings of about 10 days.After one week, collect blade, with the compound in methanol extraction blade, then carry out UPLC-Q-TOF(Agilent) detect and correlated expression component analysis.Result as shown in Figure 9 A.The expression of Bf gene in cotyledon causes the expression of the gene of Bi, thereby impels synthesizing of cucurbitacin C.
Adopt similar method, the Agrobacterium that contains Csa5G157230 gene is cultured to OD
600approximately 1.0, then with injection damping fluid, be diluted to OD
600approximately 0.8 left and right, uses the syringe with syringe needle Agrobacterium to be injected to the cucumber fruits of about 2 each months.After three weeks, collect fruit injection site, do correlation detection.Result as shown in Figure 9 B.The expression of Bt gene in fruit causes the expression of the gene of Bi, thereby impels synthesizing of cucurbitacin C.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. participate in the synthetic transcription factor Csa5G157230 of regulation and control cucumber cucurbitacin C, it is characterized in that, its aminoacid sequence is as shown in SEQ ID No.4, or this sequence is through replacing, lack or adding one or several amino acids formed aminoacid sequence with same function.
2. the gene of transcription factor Csa5G157230 described in coding claim 1.
3. gene as claimed in claim 2, is characterized in that, its nucleotide sequence is as shown in SEQ ID No.3.
4. contain the carrier of gene described in claim 2 or 3.
5. contain the engineering bacteria of gene described in claim 2 or 3.
6. the application of gene in regulation and control cucumber bitter taste is synthetic described in claim 2 or 3.
Described in claim 2 or 3 gene in the application without in bitter taste cucumber molecular breeding.
8. a method for transient expression gene in plant, is characterized in that, adopts agriculture bacillus mediated method, and carrier claimed in claim 4 is proceeded in plant materials, obtains goal gene great expression.
9. method according to claim 8, is characterized in that, described plant includes but not limited to cucumber, tobacco.
10. for the primer pair of gene described in pcr amplification claim 2 or 3, it is characterized in that, comprise upstream primer 5 '-TGTATACAATCTCCATTCCCTTTTGA-3 ' and downstream primer 5 '-TATTCCAAGTTAGTCAATTTATTCTGT-3 '.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106518994A (en) * | 2016-12-08 | 2017-03-22 | 中国农业科学院蔬菜花卉研究所 | Transcription factor participating in regulating watermelon bitter principle and application thereof |
| CN106749572A (en) * | 2016-12-08 | 2017-05-31 | 中国农业科学院蔬菜花卉研究所 | Participate in transcription factor and its application of regulation and control muskmelon bitter principle synthesis |
| CN104357441B (en) * | 2014-10-10 | 2017-08-01 | 中国农业科学院蔬菜花卉研究所 | The SNP marker related to cucumber fruits bitter taste character and its application |
| CN109183158A (en) * | 2018-08-31 | 2019-01-11 | 中国烟草总公司郑州烟草研究院 | A kind of construction method in overall length transcription factor yeast one-hybrid library |
| CN114805511A (en) * | 2022-03-21 | 2022-07-29 | 云南师范大学 | Transport protein of cucumber bitter substance cucurbitacin C and application thereof |
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2013
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104357441B (en) * | 2014-10-10 | 2017-08-01 | 中国农业科学院蔬菜花卉研究所 | The SNP marker related to cucumber fruits bitter taste character and its application |
| CN106518994A (en) * | 2016-12-08 | 2017-03-22 | 中国农业科学院蔬菜花卉研究所 | Transcription factor participating in regulating watermelon bitter principle and application thereof |
| CN106749572A (en) * | 2016-12-08 | 2017-05-31 | 中国农业科学院蔬菜花卉研究所 | Participate in transcription factor and its application of regulation and control muskmelon bitter principle synthesis |
| CN106518994B (en) * | 2016-12-08 | 2019-05-24 | 中国农业科学院蔬菜花卉研究所 | Participate in the transcription factor and its application of regulation watermelon bitter principle synthesis |
| CN110317829A (en) * | 2016-12-08 | 2019-10-11 | 中国农业科学院蔬菜花卉研究所 | Participate in the transcription factor and its application of regulation muskmelon bitter principle synthesis |
| CN106749572B (en) * | 2016-12-08 | 2019-11-12 | 中国农业科学院蔬菜花卉研究所 | Participate in the transcription factor and its application of regulation muskmelon bitter principle synthesis |
| CN110317829B (en) * | 2016-12-08 | 2021-11-05 | 中国农业科学院蔬菜花卉研究所 | Transcription factor participating in regulation and control of synthesis of muskmelon bitter principle and application thereof |
| CN109183158A (en) * | 2018-08-31 | 2019-01-11 | 中国烟草总公司郑州烟草研究院 | A kind of construction method in overall length transcription factor yeast one-hybrid library |
| CN109183158B (en) * | 2018-08-31 | 2021-11-23 | 中国烟草总公司郑州烟草研究院 | Construction method of full-length transcription factor yeast single hybrid library |
| CN114805511A (en) * | 2022-03-21 | 2022-07-29 | 云南师范大学 | Transport protein of cucumber bitter substance cucurbitacin C and application thereof |
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