CN103483435B - Gene cluster participating in synthesis of cucumber cucurbitacine C and application thereof - Google Patents

Gene cluster participating in synthesis of cucumber cucurbitacine C and application thereof Download PDF

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CN103483435B
CN103483435B CN201310376574.4A CN201310376574A CN103483435B CN 103483435 B CN103483435 B CN 103483435B CN 201310376574 A CN201310376574 A CN 201310376574A CN 103483435 B CN103483435 B CN 103483435B
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黄三文
尚轶
张忠华
马永硕
张慧敏
李颖
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Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
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Abstract

The invention discovers a gene cluster participating in synthesis of cucurbitacine C in a cucumber genome for the first time. The gene cluster totally consists of 8 genes, wherein 5 genes are located in a 35kb range of No. 6 chromosome. The 1st to 4th steps of reaction for synthesizing cucurbitacine C are analyzed in a yeast system, and the 8 genes are co-expressed in a tobacco system, so that synthesis of cucurbitacine C can be realized. The invention further discloses a molecular mechanism of cucumber bitter formation, which provides a theoretical basis and a molecular aided breeding goal for breeding bitter-free cucumber; meanwhile, precious experience is provided for in-vitro artificial synthesis of cucurbitacine.

Description

Participate in gene cluster and the application thereof of cucumber cucurbitacine C synthesis
Technical field
The present invention relates to genetically engineered and biology field, specifically, relate to the gene cluster and application thereof that participate in cucumber cucurbitacine C synthesis.
Background technology
The bitter taste of cucumber is called that the triterpenoid of cucurbitacin C causes by a class.Mouthfeel and quality that cucurbitacin C can have a strong impact on cucumber is accumulated in cucumber fruits.Early stage classical genetics test finds that the bitter taste of cucumber is by Bi and Bt two Gene Handling.Find Bi genes encoding squalene oxide cyclase in CN103013966, its catalysis generates cucurbit 2 enol, is the first step that cucurbitacin C synthesizes, and is also a step of most critical in synthesis.The synthesis of cucurbitacin C generates cucurbit 2 enol except the first step, also need to modify through a series of catalysis, but how cucurbit 2 enol is modified, and final synthesis cucurbitacin C, it be not immediately clear.
Major part cucurbitacin section plant all has cucurbitacin, and such as, cucurbitacin C derives from cucumber, and Cucurbitacin E derives from muskmelon, and Cucurbitacin B derives from watermelon.But their synthesis path it be unclear that, relevant synthetic gene is not also cloned acquisition.Therefore, cucurbitacin mainly extracts and obtains from plant, can not external direct synthesis.
Summary of the invention
The object of this invention is to provide the gene cluster and application thereof that participate in cucumber cucurbitacine C synthesis.
In order to realize the object of the invention, the invention provides the albumen participating in cucumber cucurbitacine C synthesis, comprise Csa6G088690, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700, their aminoacid sequence is respectively as shown in SEQ ID No.1-8.
The present invention also provides the gene cluster participating in cucumber cucurbitacine C synthesis, described gene cluster is altogether by 8 genomic constitutions, Csa6G088690, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700, their nucleotide sequence is respectively as shown in SEQ ID No.12-19.
The present invention also provides the plant of the carrier of the gene cluster of synthesizing containing described participation cucumber cucurbitacine C, engineering bacteria, host cell and conversion.
The application of the gene cluster that the present invention also provides described participation cucumber cucurbitacine C to synthesize in the synthesis of regulation and control cucumber bitter taste.
The application of the gene cluster that the present invention also provides described participation cucumber cucurbitacine C to synthesize in the external synthesis of regulation and control cucurbitacin.
The gene cluster that the present invention also provides described participation cucumber cucurbitacine C to synthesize is without the application in bitter taste cucumber molecular breeding.
The present invention also provides the construction process of a kind of transgenic plant, adopts agriculture bacillus mediated method, proceeds in plant materials (such as tobacco etc.) by the carrier of the gene cluster of synthesizing containing described participation cucumber cucurbitacine C, and screening obtains transgenic plant.
In research before, weight sequencing analysis is carried out to 114 parts of cucumber Core Germplasms materials, bitter taste phenotypic evaluation is carried out to these 114 parts of materials simultaneously.Adopt method and colony's mapping of whole-genome association (GWAS), have found the Bi candidate gene controlling blade bitter taste fast.Bi encodes a cucurbit 2 enol cyclase gene, is the key enzyme that cucurbitacin C synthesizes.Cucurbit 2 enol also needs oxidized and acetylize further could generate cucurbitacin C.In order to resolve other steps that cucurbitacin C synthesizes, cucumber transcript profile data (RNA-Seq) are analyzed, find and the P450 gene of Bi gene co-expressing and acetyl transferase gene, in Cucumber germplasm, have found altogether 6 P450 genes (Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550 and Csa1G044890) and 1 acetyl transferase gene (Csa6G088700).This 7 genes and Bi(Csa6G088690) expression status of gene in each tissue of cucumber be closely similar, only great expression in blade.In addition, except 3 P450 genes (Csa3G903540, Csa3G903550 and Csa1G044890), other genes and Bi gene are all positioned at the scope of No. 6 karyomit(e) 35kb, form the structure of a similar gene cluster.At present, participate in the synthesis of important secondary metabolite with the form of gene cluster extensively to be found in higher plant, therefore infer that these 7 candidate genes that the present invention finds probably form cucurbitacin C synthetic gene bunch together with Bi gene, synthesize bitter substance in cucumber leaves.
Utilize yeast expression system further, in yeast, express these candidate genes, detect yeast extract with GC-MS or UPLC-Q-TOF instrument, resolve bitter taste synthetic mesophase product.First, in the yeast of expressing Bi, coenzyme CPR and Csa3G903540, a special product has been found with GC-MS.The MS detected result of this product, compared with cucurbit 2 enol, detects that large fragment ion has lacked 2 hydrogen atoms, this means that cucurbit 2 enol is probably oxidized by Csa3G903540.By this product, from yeast, purifies and separates is out further, nucleus magnetic resonance (UMR) is utilized to resolve its structure, finding No. 19 carbon atom generation hydroxylations of cucurbit 2 enol, and then prove the oxidation of Csa3G903540 catalysis cucurbit 2 enol, is the second step of bitter taste synthesis.Continue to utilize yeast expression system, express more candidate gene, by the accurate molecular weight of UPLC-Q-TOF assay products, infer contingent oxidation reaction product.Pass through the method, only in expression Bi, CPR, Csa3G903540 and Csa6G088160 yeast extract, find that molecular weight is the product of 459.3833 [M+H], compared with the molecular weight of the product (C30H50O2) generated with Csa3G903540 catalysis, the just quality of a much better Sauerstoffatom, and occur without any specific product in the yeast of expressing other candidate P450 genes, infer that Csa6G088160 participates in the 3rd step of cucurbitacin C synthesis thus.Adopt and use the same method, find to find that molecular weight is the product of 497.3601 [M+Na] in the yeast extract of expressing Bi, CPR, Csa3G903540, Csa6G088160 and Csa3G903550, compared with the molecular weight of the product (C30H50O3) generated with Csa6G088160 catalysis, just many again Sauerstoffatoms, infer that Csa3G903550 participates in the 4th step of cucurbitacin C synthesis.
Except resolving the synthesis path of cucurbitacin C, the present invention finds that Bi gene and HMGR and the FPS gene reported are expressed in tobacco simultaneously before also being added by find 7 candidate genes.Itself be there is not cucurbitacin C in tobacco leaf, but after successful gene expression cluster gene, in tobacco leaf, detect the synthesis of cucurbitacin C, demonstrate the function of this gene cluster.In addition, utilize same experimental system, also find to lack any one gene in 7 candidate genes, tobacco leaf all can not generate cucurbitacin C, thus the gene cluster coordinate expression in cucumber leaves found Cucumber germplasm is demonstrated from another aspect, the common synthesis participating in bitter substance.
The present invention finds the gene cluster participating in cucurbitacin C synthesis first in Cucumber germplasm, and altogether by 8 genomic constitutions, wherein 5 genes are all positioned at the scope of No. 6 karyomit(e) 35kb.The the 1st to the 4th step reaction that the cucurbitacin C that utilized yeast system to resolve synthesizes, and in tobacco system these 8 genes of coexpression, the synthesis of cucurbitacin C can be realized.Invention further discloses the molecular mechanism that bitter taste is formed, for providing theoretical foundation and marker assisted selection target without bitter taste breed cucumber, can be used for Large-scale Screening breeding material, greatly accelerating the breeding process of high nutrition cucumber; Simultaneously also for the external synthetic of cucurbitacin provides invaluable experience, the alternative traditional method extracting bitter principle (cucurbitacin) from vegetable material of the present invention, realize external synthesis bitter principle.
Accompanying drawing explanation
Fig. 1 is that the present invention passes through to analyze RNA-Seq data, finds to synthesize relevant gene cluster to bitter principle (cucurbitacin C) from Cucumber germplasm.
Fig. 2 is that the present invention utilizes GC-MS to detect the result of participation bitter principle synthesis second step reaction; Wherein, A is the specific product of the Csa5G903540 that GC-MS detects, B is the MS detected result of this specific product, and the little frame of dotted line is the MS detected result of cucurbitacin 2 enol.
Fig. 3 is that the present invention utilizes UPLC-Q-TOF detection participation bitter principle to synthesize the result of three-step reaction.
Fig. 4 is that the present invention UPLC-Q-TOF detects the result participating in bitter principle and synthesize four-step reaction.
Fig. 5 is after the present invention utilizes tobacco transient expression system coexpression and bitter principle to synthesize relevant gene cluster and HMGR and FPS gene, detects the result of tobacco leaf extract with UPLC-Q-TOF.
Embodiment
Following examples for illustration of the present invention, but are not used for limiting the scope of the invention.If do not specialize, the conventional means that technique means used in embodiment is well known to those skilled in the art, is raw materials usedly commercial goods.
Embodiment 1 participates in excavation and the acquisition of the gene cluster of cucumber cucurbitacine C synthesis
The excavation of 1 gene cluster
Bi gene great expression in cucumber leaves, as clue, analyze cucumber transcript profile data (RNA-Seq) to find and the P450 gene of Bi coexpression and acetyl transferase gene, in Cucumber germplasm, have found altogether 6 P450 genes (Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550 and Csa1G044890) and 1 acetyl transferase gene (Csa6G088700).This 7 genes and Bi(Csa6G088690) expression status of gene in each tissue of cucumber be closely similar, only great expression (Fig. 1) in blade.In addition, except 3 P450 genes (Csa3G903540, Csa3G903550 and Csa1G044890), other genes and Bi gene are all positioned at the scope of No. 6 karyomit(e) 35kb, define the structure (Fig. 1) of a similar gene cluster.Cucurbitacin C distributes very similar to the expression pattern of this gene cluster (Fig. 1) in each tissue of cucumber, is all the highest in blade, therefore thinks that this gene cluster participates in the synthesis of bitter substance.
The acquisition of 2 cucumber Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550 and Csa1G044890 genes
First prepare cucumber leaves cDNA library, then utilize forward primer and reverse primer to carry out pcr amplification (primer sequence is in table 1).
Table 1 primer sequence (5 '-3 ')
Csa6G088690-TVector-F AGATTAAAAGTGGGAAAAG
Csa6G088690-TVector-R CAGTTTTGAGCTACCC
Csa6G088700-TVector-F ATGGCTCTAAAAGTAGATATC
Csa6G088700-TVector-R GACTTCTTGTAAACCTAAT
Csa6G088160-TVector-F GGAAGACTCTATCTTACAA
Csa6G088160-TVector-R CGTGATACATTTCGTTC
Csa6G088170-TVector-F AGGGCAATGAGAGCTATTACCAAACCA
Csa6G088170-TVector-R TGCTTGCTTCTTTGTCGCCAAGA
Csa6G088710-TVector-F TCACCGGCGTCGACATTACTGA
Csa6G088710-TVector-R TCATGTCAGCTTCTCAGCAAAAGCT
Csa3G903540-TVector-F TGCAACACACAAAGCTATCAAAGAATGG
Csa3G903540-TVector-R AGGCCCATTGGCTTGGCACT
Csa3G903550-TVector-F ATTGGAGTTGTTTTAGGGGTTTGTTTGTTG
Csa3G903550-TVector-R AATAATTTTGCAAAGGCAATTTTCTGGCCG
Csa1G044890-TVector-F GCCTCGTCATTCCGAGCTCGTCT
Csa5G044890-TVector-R CCATTCAATCATTCCTTGGGAGTGAAGT
PCR reaction system is counted with 20 μ l: 10-20ng/ μ l template 1 μ l, 10pmol/ μ l forward, reverse primer each 1 μ l, 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 DEG C 5 minutes; 94 DEG C 20 seconds, 55 DEG C 20 seconds, 72 DEG C 2 points 30 seconds, 35 circulations; 72 DEG C 10 minutes.
By increasing, the fragment obtained is connected with T-carrier (TAKARA), and order-checking confirms not sudden change.The nucleotide sequence of cucumber Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550 and Csa1G044890 gene is respectively as shown in SEQ ID No.13-18.
Embodiment 2 utilizes yeast expression system to verify the function of bitter taste synthesis candidate gene
Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 gene and coenzyme CPR gene are building up to expression vector pYES2(Invitrogen respectively) on, and transformed yeast bacterium.Induction yeast expression albumen, collects yeast.Normal hexane extract compounds is used after cracking.Sample preparation well uses GC-MS(Agilent afterwards) or UPLC-Q-TOF detect.Result respectively as shown in figs 2-4.
CPR gene (SEQ ID No.20) is the coenzyme gene of P450 gene in oxidation substrates, adds CPR and is conducive to improving efficiency.
Embodiment 3 utilize agriculture bacillus mediated tobacco instant expression method checking cucumber bitter taste gene cluster function
By gene constructed to Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700 on binary vector pBIN-Plus.Chemical conversion is utilized to import respectively in Agrobacterium EA105.Will respectively containing Bi(Csa6G088690), the Agrobacterium of HMGR, FPS, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700 gene is cultured to OD 600about 1.0, then balanced mix is to one piece, with syringe, Agrobacterium is injected the tobacco of about 6 weeks.Collect tobacco leaf after one week, with the compound in methanol extraction blade, then carry out UPLC-Q-TOF(Agilent) detect.Result as shown in Figure 5.
Because HMGR gene (SEQ ID No.21) and FPS gene (SEQ ID No.22) participate in the substrate oxidation squalene of synthesis Bi gene, by these two gene co-injection tobaccos, improve the content of oxidosqualene in tobacco, be conducive to the output improving end product.
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.
Sequence illustrates:
SEQ ID No. 1-11 is respectively the aminoacid sequence of Csa6G088690, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890, Csa6G088700, CPR, HMGR and FPS albumen.
SEQ ID No. 12-22 is respectively the base sequence of gene C sa6G088690, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890, Csa6G088700, CPR, HMGR and FPS.
SEQ ID No. 23-38 is the primer sequence related in the present invention.
Reference:
1.Seki?H,et?al.(2008)LicoriceCE≤-amyrin11-oxidase,a?cytochrome?P450with?a?key?role?in?the?biosynthesis?of?the?triterpene?sweetener?glycyrrhizin.Proceedings?of?the?National?Academy?of?Sciences105(37):14204.
2.van?Herpen?TWJM,et?al.(2010)Nicotiana?benthamiana?as?a?Production?Platform?for?Artemisinin?Precursors.PLoS?One5(12):e14222.

Claims (9)

1. participate in the albumen of cucumber cucurbitacine C synthesis, comprise Csa6G088690, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700, their aminoacid sequence is respectively as shown in SEQ ID No.1-8.
2. participate in the gene cluster of cucumber cucurbitacine C synthesis, it is characterized in that, described gene cluster is by following 8 genomic constitutions, Csa6G088690, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700, their nucleotide sequence is respectively as shown in SEQ ID No.12-19.
3. the carrier containing gene cluster described in claim 2.
4. the engineering bacteria containing gene cluster described in claim 2.
5. the application of gene cluster described in claim 2 in the synthesis of regulation and control cucumber bitter taste.
6. the application of gene cluster described in claim 2 in the external synthesis of regulation and control cucurbitacin C.
7. gene cluster described in claim 2 is without the application in bitter taste cucumber molecular breeding.
8. a construction process for transgenic plant, is characterized in that, adopts agriculture bacillus mediated method, proceeds in plant materials by carrier according to claim 3, and screening obtains transgenic plant.
9. method according to claim 8, is characterized in that, described plant is tobacco.
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CN105039274A (en) * 2015-07-13 2015-11-11 中国农业科学院蔬菜花卉研究所 Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster
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
CN111411099B (en) * 2020-05-14 2022-09-27 云南农业大学 Hemsleya amabilis acetyl transferase, coding gene thereof and application of hemsleya amabilis acetyl transferase in preparation of cucurbitacin
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CN116334106A (en) * 2022-09-15 2023-06-27 云南农业大学 Application of hemsleyadin cytochrome oxidase gene and transgenic engineering bacteria thereof in preparation of cucurbitacin intermediate

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