CN109439676A - The application of Siraitia grosvenorii epoxide hydrolase Sgeph5 gene - Google Patents

Abstract

The invention discloses a kind of Momordica-Glycosides synthesis regulation genesSgeph5And its application, belong to field of biotechnology.The present invention is analyzed by Siraitia grosvenorii genome sequencing and different tissues RNA-seq sequence, and utilizes tissue specificity and the associated secondary metabolism approach of character, obtains the specifically expressed Siraitia grosvenorii epoxide hydrolase of Lo Han Guo fruitSgeph5Gene.Sweet tea salidroside content in the controllable Lo Han Guo fruit of expression by adjusting the gene, to obtain the Siraitia grosvenorii new varieties or strain of high sweet tea salidroside content, research cucurbitaceous plant fruit mouthfeel mechanism and Curcurbitaceae fruits/plant mouthfeel breeding work are had great theoretical and practical significance.

Description

The application of Siraitia grosvenorii epoxide hydrolase Sgeph5 gene

Technical field

The invention belongs to plant biotechnology fields, and in particular to cucurbitaceous plant breeding method, including improve sweet tea glucoside and contain Amount and improvement mouthfeel, relate more specifically to the nucleic acid molecules and its molecular breeding material of a kind of sweet tea salidroside content controlling gene Sgeph5 And its application in Curcurbitaceae improvement of fruit breeding work.

Technical background

Siraitia grosvenorii (Siraitia grosvenorii (Swingle) C.Jeffrey) is used as Chinese medicine has several generation The history of discipline.Traditional Chinese medicine treats hypertension, pulmonary tuberculosis, gastritis, asthma, pertussis, acute and chronic tracheitis and urgency with it Chronic tonsillitis etc..Research in recent years also found that Siraitia grosvenorii contains anticancer component, cause the great attention of domestic and international medical field. Siraitia grosvenorii is rich in sweet tea glucoside, flavones, vitamin, protein, amino acid, mannitol, scavenger substance, various trace elements etc. Effective component, nutritive value is high, natural flavour mountaineous strong, and mellowness is sweet, can also be widely used in food, health care product.Sweet tea in Siraitia grosvenorii Glycosides V is one of most strong sweet substance in the world, and sugariness is 2 times of stevioside, 6 times of glycyrrhizin, 300 times of sweetness of cane sugar, most Big advantage is free from heat, and physicochemical property is stablized, and good water solubility is without any side effects, is suitble to all groups' long-term consumption. Siraitia grosvenorii mainly originates in the mountain area of lingui county,gui lin and Yongfu county, is the rare local and special products in Guilin, Southern Hunan, Guizhou, The provinces and regions such as Guangdong and Jiangxi are also distributed.

The Study on tissue culture of Siraitia grosvenorii starts from 1980, and Lin Rong etc., for explant, is carried out with Siraitia grosvenorii plumular axis, stem apex etc. The Primary Study of culture of leaving one's post.So far rapid propagation in vitro and virus-free training are concentrated mainly on about the Study on tissue culture of Siraitia grosvenorii It supports.Siraitia grosvenorii transgenic research is almost blank.Siraitia grosvenorii is pest-resistant and metabolic gene engineering research progress includes:

1. Insect resistant gene engineer

Root knot nematode disease is a kind of important disease of Siraitia grosvenorii, is threatened greatly production, and flowering of plant result is caused to be postponed, As a result few, fruit is small, or even does not bloom, and main root rots when serious, and plant is integrally withered, seriously restricts the fast of Siraitia grosvenorii industry Speed develops in a healthy way.Some effects and income can be obtained using chemicals is disease-resistant, but long-time service is also easy to produce drug resistance and agriculture is residual Problem cannot fundamentally solve disease, will be for the hair of Siraitia grosvenorii using new and high technologies such as modern molecular breeding and genetic engineerings New world is opened in exhibition.

2. metabolic gene engineering

The main active of Siraitia grosvenorii includes triterpene and its glycoside, also contains flavones, polysaccharide, amino acid, mannitol, dimension Raw element etc..Mogroside category is cucurbitane triterpenes, early in the 1970s and 1980s in last century Japanese scholars just to the triterpene of Siraitia grosvenorii Constituents have carried out more systematic research, isolated siamenoside Ⅰ (sianenoside I), Momordia grosvenori aglycone from dry fruit II E, (II E of mogroside), Momordia grosvenori aglycone (mogroside III), mogroside Ⅲ E (III E of mogroside), mogroside IV The cucurbitanes type triterpene compounds such as (mogroside IV), Momordia grosvenori aglycone V (mogroside V).Momordia grosvenori aglycone V therein It is the main component of Siraitia grosvenorii dry fruit, accounts for about the 0.5% of dry fruit.Mogroside IV, Momordia grosvenori aglycone V, siamenoside Ⅰ ten thousand/ The aqueous solution of one concentration is 392 times, 425 times, 563 times of 5% aqueous sucrose solution sugariness respectively.Hereafter exerting by multidigit scholar Power, but separation identifies the ingredients such as a large amount of triterpenoid saponin, triterpene ester more than 30 from Siraitia grosvenorii.Squalene is all triterpene soaps Glycosides, Betulinic Acid, plant hormone class compound common precursor, be to be urged by squalene synthetase (Squalence synthase, SS) Change made of pyrophosphoric acid Acacia ester (Farnesyl diphosphate, FPP) the tail tail condensation of two molecules.Therefore, Ke Longdai Key enzyme is the effective way for improving sweet tea glucoside in Siraitia grosvenorii during thanking.

It is concentrated mainly in terms of intellectual property more in terms of product drink formula, external major company is main with drink formula Research direction, studies in China is mainly in terms of cultivating and growing and some Siraitia grosvenorii tea-drinkings.

The main active of Siraitia grosvenorii includes triterpene and its glycoside, also contains flavones, polysaccharide, amino acid, mannitol, dimension Raw element etc..Mogroside category is cucurbitane triterpenes, early in the 1970s and 1980s in last century Japanese scholars just to the triterpene of Siraitia grosvenorii Constituents have carried out more systematic research, isolated siamenoside Ⅰ (sianenoside I), Momordia grosvenori aglycone from dry fruit II E (II E of mogroside), Momordia grosvenori aglycone (mogroside III), mogroside Ⅲ E (III E of mogroside), mogroside IV The cucurbitanes type triterpene compounds such as (mogroside IV), Momordia grosvenori aglycone V (mogroside V).Momordia grosvenori aglycone V therein It is the main component of Siraitia grosvenorii dry fruit, accounts for about the 0.5% of dry fruit.Mogroside IV, Momordia grosvenori aglycone V, siamenoside Ⅰ ten thousand/ The aqueous solution of one concentration is 392 times, 425 times, 563 times of 5% aqueous sucrose solution sugariness respectively.The life of Momordia grosvenori aglycone in Siraitia grosvenorii Object route of synthesis is not fully understood.Based on to Triterpene saponins in other plant (Isoprenoid pathway) route of synthesis Solution, and about the limited report of Momordia grosvenori aglycone synthesis, by the effort of multidigit scholar, and from Siraitia grosvenorii, separation is identified greatly The ingredients such as triterpenoid saponin, the triterpene ester of amount more than 30.Previous studies propose a kind of Momordica-Glycosides biosynthesis pathway (see Fig. 1), pass through mevalonic acid (MVA) and plastid 2-C methyl D-carob alcohol -4- phosphate (MEP) synthesis three in upstream pathway Terpenoid and be related to farnesyl diphosphate (FPP) and be converted into squalene, then to 2,3- oxidosqualene, then according to It is secondary to be cyclized, the modification such as oxidation.Squalene synthetase (SQS) is catalyzed two kinds of FPP and is converted into squalene, this is sterol, triterpene With first step of Brassinosteroids (BRs) biosynthesis.The steroid on the film of endoplasmic reticulum (ER) occurs for this enzymatic reaction Pure and mild BRs plays an important role in the mobility and permeability of film, also can be used as the molecule in signal transduction plant growth and development. Epoxide hydrolase (Epoxide hydrolases, EPH) is prevalent in the plant, animal and microorganism of nature, Epoxides can prepare chiral alcohol compound with optical activation and selection by selective opening under the action of it Property retain have important value epoxides.

In order to carry out the work of Siraitia grosvenorii genetic engineering and illustrate Momordica-Glycosides metabolic mechanism, Siraitia grosvenorii gene cloning It is extremely important with the work of Function Identification, it is research and the application foundation of Siraitia grosvenorii molecular breeding.EPH is in many species Identification, overexpression may will increase phytosterol and triterpene compound.

The present invention passes through research and utilization Pacbio RSII platform (Pacfic Biosciences；USA) to arhat fruit gene Group carries out fine single-molecule sequencing, and carries out genome assembling by SMRT technology.In addition, utilizing Illumina HiSeq X-Ten platform (Illumina；CA, USA) RNA-Seq sequencing is carried out to Momordica grosvenori root, leaf and fruit, and gene family is combined to lose Evolutionary analysis is passed, sweet tea glucoside synthesis related gene is screened, successfully positions and cloned a new epoxide hydrolase (Epoxide hydrolases, EPH) gene Sgeph5.The function that EPH gene is undertaken during evolution is extremely important, The expression product of EPH gene may adjust the conjunction of sweet tea glucoside by the synthesis of control intermediate product in Momordica-Glycosides synthesis process At.EPH product has the function of various aspects, has very high specificity to the catalysis of substrate, in different development stage and different groups High special in cell is knitted to express.EPH in Momordica-Glycosides synthesis by cucurbit enol to Momordica-Glycosides during play Important function, can determine the quantity of variety classes sweet tea glucoside, influence last sugariness.Expression quantity of the gene in fruit is aobvious Be higher than its hetero-organization, this shows in the synthesis of sweet tea glucoside in fruit in the competition of precursor, can be by adjusting the expressing promoting of the gene Into the improvement of sweet tea salidroside content in fruit.The present invention also provides a kind of methods for obtaining high sweet tea salidroside content Siraitia grosvenorii kind, to sieve Chinese fruit breeding work is of great significance and value.

Summary of the invention

All bibliography being mentioned herein all are incorporated herein by reference.

Unless there are indicating on the contrary, all technical and scientific terms used herein all have common with fields of the present invention The identical meaning that technical staff is generally understood.Unless there are indicating on the contrary, technology that is used herein or mentioning is ability Standard technique well known to the those of ordinary skill of domain.Material, method and example are only used as to illustrate, rather than limit.

The present invention provides the adjusting gene Sgeph5 that one improves sweet tea salidroside content in fruit, the gene crosses scale Up to that can influence its content to Lo Han Guo fruit Momordica-Glycosides, nucleotide sequence is selected from following group of one of sequence:

(a) nucleotide sequence as shown in SEQ ID NO:1 or 2；

(b) its encoding amino acid sequence nucleotide sequence as shown in SEQ ID NO:3.

Those skilled in the art should know, the adjusting gene Sgeph5 of the present invention for improving sweet tea salidroside content in fruit Further include with the nucleotide sequence of Sgeph5 gene or protein sequence very high homology, and have same by adjusting the gene Expression promotes the valence body sequence of the very high homologies such as improvement function of sweet tea salidroside content in fruit.The function equivalence body of the very high homology Sequence include under high stringency conditions can with the DNA sequence dna of the DNA hybridization with sequence shown in SEQ ID NO:1 or 2 or its Protein amino acid sequence shown in the amino acid sequence and SEQ ID NO:3 of coding has the nucleotides sequence of 85% or more similitude Column.

Function equivalence body sequence further include have at least 80% with sequence shown in Sgeph5 gene disclosed in this invention, 85%, 90%, 95%, 98% or 99% sequence similarity, and there is the DNA sequence dna of regulation sweet Momordica grosvenori salidroside content function, it can It is obtained with being separated from any plant.Wherein, the percentage of sequence similarity can by well known bioinformatics come It obtains, including Myers and Miller algorithm, Needleman-Wunsch overall comparison method, Smith-Waterman Local Alignment Method, Pearson and Lipman similarity-searching, Karlin and Altschul algorithm, this for those skilled in the art come Say it is well known.

Gene order of the present invention can be separated from any plant and be obtained, including but not limited to Btassica, corn, Wheat, sorghum, two section shepherd's purse categories, sinapsis alba, castor bean, sesame, cottonseed, linseed, soybean, Arabidopsis, Phaseolus, peanut, lucerne Mu, oat, rapeseed, barley, oat, rye (Rye), grain, chinese sorghum, triticale, einkorn, Si Peierte wheat (Spelt), emmer, flax, gramagrass (Gramma grass), friction standing grain, false chinese sorghum, fescue grass, perennial ryegrass, sweet Sugarcane, crowberry, papaya, banana, safflower, oil palm, muskmelon, apple, cucumber, dendrobium nobile, gladiolus, chrysanthemum, Liliaceae, cotton, Eucalyptus, sunflower, rape, beet, coffee, ornamental plant and conifer etc..Preferably, plant include corn and soybean, safflower, leaf mustard, Wheat, barley, rye, rice, cotton and sorghum.

The present invention also provides a kind of expression cassette, the expression cassette contains fertility disclosed in this invention and adjusts gene The DNA sequence dna of Sgeph5, the nucleotide sequence that the sweet tea salidroside content adjusts gene are selected from following group of one of sequence:

(a) nucleotide sequence as shown in SEQ ID NO:1 or 2；

(b) its encoding amino acid sequence nucleotide sequence as shown in SEQ ID NO:3.

Specifically, the Siraitia grosvenorii squalene synthetase gene also operability in above-mentioned expression cassette is connected with one and can drive it The promoter of expression, the promoter include but is not limited to composition type expression promoter, inducible promoter, fruit specific expression Promoter, space-time specific expression promoter etc..The gene expression of constitutive promoter of the present invention do not have tissue and when Between specificity, the exogenous gene expression that extraneous factor starts constitutive promoter has little effect.The composing type starting Son including but not limited to CaMV35S, FMV35S, rice actin (Actin1) promoter, maize ubiquitin (Ubiquitin) open Mover etc..Tissue-specific promoter of the present invention also has enhancer in addition to comprising due general promoter element And the characteristic of silencer, the advantages of such promoter be can promotor gene in the expression at specific plant tissues position, avoid The unnecessary expression of foreign gene, to save the overall power consumption of plant.Inducible promoter of the present invention is Refer under certain specific physically or chemically stimulations of signal, the promoter of the transcriptional level of gene can be significantly increased, Current separated inducible promoter includes but is not limited to adverse circumstance inducing expression promoter, photoinduction expression promoter, heat Inducing expression promoter, wound-inducible expression promoter, fungal induction expression promoter and symbiotic bacteria inducing expression promoter Deng.The present invention uses the own promoter of Siraitia grosvenorii epoxide hydrolase gene, nucleotide sequence such as SEQ ID NO:4 institute Show.

The above-mentioned expression cassette of the present invention, also further may include a screening-gene, the screening-gene can be used for Plant containing the expression cassette, plant tissue cell or vector selection are come out.The screening-gene includes but is not limited to antibiosis Plain resistant gene anti-herbicide gene or fluorescence protein gene etc..Specifically, the screening-gene includes but unlimited In: chloramphenicol resistance gene, hygromycin gene, streptomycin resistance gene, miramycin resistant gene, sulfamido resistance base Cause, glyphosate gene, glufosinate-resistant gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan are glimmering Aequorin, yellow fluorescent protein gene, luciferase gene, green fluorescence protein gene etc..

The present invention also provides a kind of methods that sweet Momordica grosvenori salidroside content is turned up, by the nucleotide for influencing Sgeph5 gene Sequence regulates and controls the transcriptional expression of Sgeph5 gene to regulate and control the fertility of plant.The influence sweet Momordica grosvenori salidroside content refers to logical The expression of regulation Sgeph5 gene is crossed, so that the secondary metabolism of the plant be made to change, such as leads to plant sweet tea glucoside synthesis way The amount of substrate in diameter.Specifically, concrete application demand is depended on, Sgeph5 gene can be influenced by a variety of methods in plant Intracorporal expression, to achieve the effect that regulate and control sweet tea salidroside content.More specifically, the expression of regulation Sgeph5 gene can be used perhaps Tool obtained by more those of ordinary skill in the art carries out, for example, passing through mutation, mutagenesis, being transferred to of antisense gene, co-suppression Or introducing, complementation of gene of hairpin structure etc..

The present invention also provides a kind of method for obtaining Sgeph5 fruit mouthfeel mutant material, the method is planted by mutation The endogenous Secondary Metabolic Regulation of Callus gene Sgeph5 of object, or the nucleotide sequence of mutation and the gene of its very high homology, make the plant Body secondary metabolite is at the process for being grouped as change.The nucleotide sequence such as SEQ of the Secondary Metabolic Regulation of Callus gene Sgeph5 Shown in ID NO:1 or 2, the amino acid sequence of the sterility changing gene Sgeph5 is as shown in SEQ ID NO:3." mutation " Including but not limited to following methods, such as the gene mutation caused by method physically or chemically, chemical method includes with EMS etc. Mutagenesis caused by mutagens processing, the mutation can also be point mutation, be also possible to DNA missing or insertion mutation, can also To be by the gene silencings such as RNAi means or by the method for site-directed point mutation, the method packet of the site-directed point mutation Include but be not limited to the gene editings methods such as ZFN directed mutagenesis method, TALEN directed mutagenesis method, and/or CRISPR/Cas9.

The application of the above-mentioned mutant material of the present invention further includes above-mentioned DNA sequence dna or mutant material following (a) extremely Any one of (b) application in:

(a) Siraitia grosvenorii kind or strain are cultivated；

(b) the other plant varieties of Curcurbitaceae or strain are cultivated.

The present invention also provides the promoter of a Sgeph5 gene, the sequence of corresponding promoter be Sgeph5 gene from The sequence that ATG is formed to upstream about 1500bp nucleotide, more specifically, in rice, the Sgeph5 gene promoter Nucleotide sequence is as shown in SEQ ID NO:4.Containing nucleotide sequence shown in SEQ ID NO:4, or comprising with SEQ ID Listed nucleotide sequence has the nucleotide sequence of 90% or more similitude in NO:4, or comprising deriving from SEQ ID NO:4 sequence 500 and 500 or more continuous nucleotide fragments on column, and the nucleotide being operatively connected with the promoter can be driven Expression of the sequence in plant different tissues.Expression vector, transgenic cell line and host strain containing above-mentioned sequence etc. are equal It belongs to the scope of protection of the present invention.Expand drawing for any nucleotide fragments of SEQ ID NO:4 promoter disclosed in this invention Object is to also within protection scope of the present invention.

Promoter nucleotide sequence provided by the present invention can also be used to separate phase from other plants other than Siraitia grosvenorii Sequence is answered, homologous clone is especially carried out from other cucurbitaceous plants.According to promoter listed by these corresponding sequences and this paper Sequence homology between sequence, or the homology with this promoter gene are identified using the technologies such as such as PCR, hybridization and separate this A little corresponding sequences.Therefore, the sequence between the SEQ ID NO:4 promoter sequence (or its segment) according to listed by them and the present invention Similitude and isolated respective segments, are also included in embodiment.

" promoter " of the present invention refers to a kind of DNA regulatory region, generally comprises energy guide RNA polymerase II and exists The TATA box of the suitable transcription initiation site starting RNA synthesis of specific coding sequence.Promoter also may include other identification sequences, These identification sequences are usually located at the upstream or 5 ' ends of TATA box, commonly known as upstream promoter element, play regulatory transcription effect The effect of rate.Those skilled in the art should know, although having identified the core for promoter region disclosed by the invention Nucleotide sequence, but separate and identify other tune of the TATA box upstream region for the specific promoter region identified in the present invention It is also within the scope of the invention to control element.Therefore, promoter region disclosed herein is usually further defined as comprising upstream Controlling element, such as those of tissue expression for regulating and controlling coded sequence and temporal expressions function element, enhancer etc..With Identical mode can be identified, isolate the promoter element for making it possible to be expressed in purpose plant, by itself and other cores Heart promoter is used together, to verify it in the expression of purpose plant.

The function of the gene promoter can be analyzed by the following method: can by promoter sequence and reporter gene It is operatively connected, forms transformable carrier, then the carrier is transferred in plant, in obtaining transgenic progeny, pass through observation Expression of the reporter gene in each histoorgan of plant confirms its expression characterization；Or above-mentioned carrier is subcloned into For the expression vector of transient expression experiment, promoter or the function of its control region are detected by transient expression experiment.

Host will be depended on and by the table for the selection of test starting or the appropriate expression vector of regulatory region function The method for introducing host up to carrier, such methods are well known to those of ordinary skill in the art.For eucaryote, in carrier In region include control transcription initiation and control processing region.These regions are operably connected to reporter gene, institute Stating reporter gene includes YFP, UidA, gus gene or luciferase.Table comprising the presumption control region being located in genomic fragment It can be introduced into complete tissue, such as interim pollen up to carrier, or introduce callus, to carry out functional verification.

In addition, promoter of the invention can also be connected with the nucleotide sequence of not Sgeph5 gene, it is other different to express Exogenous nucleotide sequence.Promoter nucleotide sequence of the invention and its segment and variant can assemble together with heterologous nucleotide sequence In an expression cassette, for being expressed in purpose plant.The expression cassette has suitable restriction enzyme site, for being inserted into The promoter and heterologous nucleotide sequence.These expression cassettes can be used for carrying out genetic manipulation to any plant, to be wanted Corresponding phenotype.

It is of the present invention that nucleotide sequence, carrier or expression cassette are transferred to plant or introduces plant or plant is turned Change, refers both to that nucleotide sequence, carrier or expression cassette are transferred to recipient cell or recipient plant by conventional transgenic method In.Any transgenic method known to plant biotechnology field technical staff can be used to for recombinant expression carrier being transformed into In plant cell, to generate genetically modified plants of the invention.Method for transformation may include method for transformation directly or indirectly.Suitably Direct method include polyethylene glycol induction DNA intake, liposome-mediated conversion, using particle gun importing, electroporation and Microinjection.The method for transformation also includes the methods for plant transformation etc. of mediated by agriculture bacillus.

The present invention also provides a kind of production methods of high sweet tea glucoside Siraitia grosvenorii comprising:

(a) expression cassette provided by the present invention is constructed；

(b) expression cassette for obtaining step (a) imports plant cell；

(c) genetically modified plants are regenerated；With

(d) genetically modified plants are selected；And

(e) optionally, the plant that amplification step (d) obtains is to obtain offspring.

Genetically modified plants of the invention are prepared using method for transformation known to plant biotechnology field technical staff.It is any Method can be used for for recombinant expression carrier being transformed into plant cell, to generate genetically modified plants of the invention.Method for transformation It may include method for transformation directly or indirectly.Suitable direct method includes that the DNA of polyethylene glycol induction takes in, is liposome-mediated Conversion, use particle gun to import, electroporation and microinjection etc..In a specific embodiment of the invention, the present invention makes With the transformation technology based on agrobacterium (reference can be made to Horsch RB etc. (1985) Science 225:1229；White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.15-38；The .Techniques such as Jenes B For Gene Transfer, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Agrobacterium bacterial strain (such as Agrobacterium tumdfaciens or hair root soil bar Bacterium) it include plasmid (Ti or Ri plasmid) and T-DNA element, the plasmid and element are transferred to plant after with Agrobacterium transfection Object, and T-DNA is integrated into the genome of plant cell.T-DNA can be located on Ri- plasmid or Ti- plasmid, or independently wrap It is contained in so-called binary vector.Agrobacterium-mediated method for transformation is described in for example.Agrobacterium-mediated conversion is most It is suitble to dicotyledon, but is also suitble to monocotyledon.Agrobacterium is described in for example the conversion of plant.Conversion can be led Cause instantaneous or stable conversion and expression.Although nucleotide sequence of the invention, which can be inserted into, falls into appointing in these broad varieties In what plant and plant cell, but it is particularly suitable for crop plants cell.

Compared with prior art, the present invention is with following the utility model has the advantages that the present invention provides a kind of Siraitia grosvenorii epoxy materials Hydrolase gene and its promoter, the regulating and expressing system of the gene, which is used to be turned up sweet Momordica grosvenori salidroside content or changes Curcurbitaceae, plants The breeding of object fruit mouthfeel produces, significant for breaking through and improveing Siraitia grosvenorii and other cucurbitaceous plants.

Detailed description of the invention

Fig. 1 is the biosynthesis pathway schematic diagram of Momordica-Glycosides.

Fig. 2 is that the experimental material of Siraitia grosvenorii is grown on Hunan Province Huaihua City Zhongfang County, is picked on time according to requirement of experiment.Figure In include: the staminiferous plant to bloom；The female plant bloomed；Lo Han Guo fruit growth；The growing environment of Siraitia grosvenorii.

Fig. 3 is that Siraitia grosvenorii full-length genome is sequenced by single-molecule sequencing technology, obtains a new high-quality Siraitia grosvenorii genome.Assembling has used 31Gb (~73.8x) long unimolecule that (SMRT) reading is sequenced in real time.Last genome About 467.1Mb is assembled, contig N50 length is 556347bp.

Fig. 4 is the assembling assessment of Siraitia grosvenorii genome, i.e., the est sequence pair obtained using the RNA-seq of different tissues organ The assessment that genome carries out.

Fig. 5 is the expression thermal map in the significantly high expression transcript of fruit.By the enrichment of fruit differential gene, discovery and sweet tea Glucoside synthesizes relevant secondary metabolism approach gene.

Fig. 6 is candidate function transcript KEGG access enrichment analysis.

Fig. 7 is Siraitia grosvenorii epoxide hydrolase compared with the evolution of cucurbitaceous plant cucumber, cucurbita pepo and mountain cucurbit, Green is cucumber；Red is cucurbita pepo；Yellow is mountain cucurbit；Blue is Siraitia grosvenorii.

Specific embodiment

It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation Example.

Embodiment 1, Siraitia grosvenorii full-length genome three generations sequencing

The seedling of Siraitia grosvenorii sample is purchased from Guangxi province Guilin City, area, Yongfu, for the green peel fruit kind planted the most extensively. Seedling is planted in Hunan Province Huaihua City Zhongfang County (as shown in Figure 2).Momordica grosvenori root, bud, leaf, male flower are acquired during the 8-10 month With the fruit of different times after female flower, pollination.It is stored in after sample liquid nitrogen frozen in -80 degrees Celsius of refrigerator.Siraitia grosvenorii stem apex Due to extracting genomic DNA, tested for gene order-checking.

This research is extracted the DNA of high quality, constructs the library PacBio RS II that 2 Insert Fragments are 20kb, benefit Non- amplification long segment is carried out to DNA with third generation microarray dataset PacBio RS II to be sequenced, in total 44 SMRT cells, obtain Siraitia grosvenorii genome raw sequencing data.

In this research, the Raw data data volume of about 31Gb is obtained in 44 SMRT (unimolecule is real-time) cells, is covered Lid depth about reaches 73.8 times, and the average length of raw reads is 11401bp, and N50 length is 15754bp.Original reads After removal low quality reads, connector pollution, subreads average length is up to 7781bp, and subreads N50 long Degree is 11898bp.After removing low quality base and sequence measuring joints, 82% raw data data volume is finally remained, altogether About 25.42Gb, average overburden depth reach 60.52 times.Siraitia grosvenorii kind green peel fruit gene group has been carried out more than 100x's Sequence is resurveyed, has then carried out k-mer analysis using the read that the both-end of about 50G is sequenced.By KmerGene software to reading progress The estimation of row optimum k value, obtaining optimum k value is 112, and carries out distributional analysis to the sequence of 112-mer.

Siraitia grosvenorii full-length genome is sequenced by single-molecule sequencing technology, obtains the arhat of a new high-quality Fruit gene group (as shown in Figure 3).Assembling has used 31Gb (~73.8x) long unimolecule that (SMRT) reading is sequenced in real time.Last Genome assembles about 467.1Mb, and contig N50 length is 556347bp.

The est sequence obtained using the RNA-seq of different tissues organ is assessed (as shown in Figure 4) to genome. The homologous coverage rate of reads is more than 80% in most samples, and ensemble average reaches 84%, and error rate is below one thousandth. Est sequence coverage more than 80% shows that most of code area is embodied in well in the result of genome assembling.Siraitia grosvenorii Genome integrality with higher and accuracy.There are 1440 orthologs in plant in albumen database, have 1167 (81%) can find in the genome of splicing, including 877 single copy genes and 290 and multicopy base Cause.

Embodiment 2, the sequencing of Siraitia grosvenorii transcript profile, assembling and analysis

The kind of Siraitia grosvenorii transcript profile sequencing is consistent with gene order-checking kind (as shown in Figure 5), is green peel fruit, from wide The Yongfu county Xi Sheng is introduced, and is planted in Hunan Province Huaihua City Zhongfang County.In different growth and development stages, female plants are taken respectively Blade, male plants blade, the blade of neighbouring fruit, fresh young root, the fruit (3DAA) after fertilization three days, Yi Jishou Essence 20 days fruit (20DAA) totally 6 samples, each sample, at least there are two biology to repeat.After all samples acquisition, Use ddH₂O cleaning wrap rapidly three times, after then being dried with blotting paper be put in liquid nitrogen store it is spare.

RNA is extracted to carry out according to step shown in kit, and after sample detection is qualified, each sample takes 3 μ g total serum IgEs to be used as Beginning raw material constructs transcript profile sequencing library.According toUltra^TMRNA Library Prep Kit forThe operating instruction of (#E7530L, NEB) chooses different index labels respectively and builds library, and uses HiSeq X-Ten Sequenator carries out transcript profile sequencing.In originally grinding and making internal disorder or usurp, we are using Trimmomatic (v0.33) software to original sequencing number According to progress quality control.After the expression for obtaining gene, we carry out point of difference expression gene using DESeq software Analysis.The input data of analysis of gene differential expression is Readcount data obtained in gene expression dose analysis, right first (Normalization) is normalized in Read count, and the meter of hypothesis testing probability (P value) is then carried out according to model It calculates, finally carries out multiple hypothesis test correction, obtain FDR value (false discovery rate).In KEGG enrichment analysis, cucumber gene is used Group annotation has carried out preliminary analysis as relationship of the background to this period blade and fruit gene expression and metabolic pathway.Fruit Early development is mainly the growth course of fruit, and various plants hormone signal is synthesized in fruit, conducted after pollinating, and is started The building process of fruit and seed, a large amount of new metabolic processes are activated or metabolic pathway is enhanced.There are 16 class metabolic pathway bases Because of obvious up-regulation.Wherein the gene expression of Plant hormone signal transduction approach is obviously raised, while with Momordica-Glycosides synthesizes relevant metabolic pathway, including Phenylpropanoid biosynthesis, Phenylalanine metabolism、Glycolysis/Gluconeogenesis、Biosynthesis of secondary metabolites Deng, it is also obvious to raise, illustrate that related gene expression enhances, secondary metabolite starts to increase synthesis.Have in this period A little metabolic pathways start not yet or no longer need thus gene expression lowered, as the accumulation of reserve substance in seed does not have also There are beginning, Fatty acid metabolism, Fatty acid biosynthesis, Cysteine and methionine The metabolic pathway gene expression relevant to Zhi Fang, protein accumulation such as metabolism is also in the stage of downward.Generation in plant The development of the expression regulation and cell tissue of thanking to related gene is closely related, and the up-regulation and downward of gene expression are sent out in plant difference There is its particularity in the stage of educating.

Candidate function transcript KEGG access enrichment analysis display (as shown in Figure 6), there is 12 classes in the fruit of Siraitia grosvenorii Metabolic pathway gene obviously raises.Wherein the gene expression of Plant hormone signal transduction approach is obvious Up-regulation, while metabolic pathway relevant to Momordica-Glycosides synthesis, including Phenylpropanoid biosynthesis, Phenylalanine metabolism、Glycolysis/Gluconeogenesis、Biosynthesis of secondary Metabolites etc., also obvious up-regulation, illustrates that related gene expression enhances, and secondary metabolite starts to increase synthesis.By Sweet tea glucoside in Siraitia grosvenorii is cucurbitane triterpene compounds, and a series of and sesquiterpenoid is related in its route of synthesis Gene relevant with triterpenoid, so this result is consistent with KEGG access enrichment analysis result.

The clone of embodiment 3, Siraitia grosvenorii epoxide hydrolase gene (Sgeph5)

Genome sequencing and transcript profile sequencing, respectively obtain Siraitia grosvenorii epoxide hydrolase gene (Sgeph5) sequence Information.The genomic DNA and RNA for extracting Siraitia grosvenorii ripening fruits, are respectively synthesized primer by round pcr, have cloned this Its unnamed gene is Sgeph5 by segment, cDNA segment and the promoter of gene.Including promoter dna fragment (SEQ ID NO:4)；CDNA sequence (SEQ ID NO:2), genome sequence (SEQ ID NO:1) and amino acid sequence (SEQ ID NO:3).

Fig. 7 is Siraitia grosvenorii epoxide hydrolase compared with the evolution of cucurbitaceous plant cucumber, cucurbita pepo and mountain cucurbit, Green is cucumber；Red is cucurbita pepo；Yellow is mountain cucurbit；Blue is Siraitia grosvenorii.The EPH gene of Siraitia grosvenorii and cucumber, west The EPH gene affiliation of the cucurbitaceous plants such as cucurbit and mountain cucurbit is more complicated.Siraitia grosvenorii EPH gene often has on chromosome The close copy of affiliation, while also having special small cluster；4 kinds of plants cucurbitaceous are all in most small cluster There is respective copy, but there are also do not have the copy of Siraitia grosvenorii in small cluster.This illustrates EPH gene height during evolution It repeats and disproportionation, the function of being undertaken in various plants is more specifically changed.

The expression analysis of embodiment 4, Sgeph5 gene in Lo Han Guo fruit

According to the cDNA sequence design primer of Sgeph5 gene, while using Siraitia grosvenorii Ubiquitin gene as internal reference pair According to design primer.The total serum IgE of Siraitia grosvenorii material different tissues is extracted respectively and synthesizes cDNA template, recycles real time fluorescent quantitative PCR method, analysis Sgeph5 gene respectively in Siraitia grosvenorii different organ and tissue or the expression in period, including: root, Stem, leaf, different times fruit, gynoecium and male flower.Sgeph5 gene respectively develops rank in the root at florescence, stem, leaf, gynoecium and male flower Duan Zhongjun is not detected, but relatively high in Lo Han Guo fruit, which shows expression and the Siraitia grosvenorii fruit of Sgeph5 gene Real secondary metabolism is closely connected.

Embodiment 5, Siraitia grosvenorii epoxide hydrolase gene (Sgeph5) with sweet tea glucoside synthesize TIME CORRELATION EXPERIMENTS

The Lo Han Guo fruit of different developmental phases, the expression of quantitative PCR detection Sgeph5 gene are taken, while being extracted corresponding Momordica grosvenori mogroside V in stage Lo Han Guo fruit, and detection level.Experiment showed in fruit development early stage (after pollination 1-50 days) The expression of Sgeph5 gene is positively correlated with sweet tea salidroside content.

Sequence table

<110>the emerging Kechuang Bioisystech Co., Ltd of Huaihua

<120>application of Siraitia grosvenorii epoxide hydrolase Sgeph5 gene

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2875

<212> DNA

<213>Siraitia grosvenorii (Siraitia grosvenorii (Swingle) C.Jeffrey)

<400> 1

atggcgaaag gcgggttttt tgtcgataag attcggagat gtctaaggac gttgttcttt 60

atggtggcga tggtggcgtc actgctcgtg tcgtcgttgc cagtgcttgt ggccatcgga 120

gacatgttgg tgccctgtat tttgatctcc agctttacgt gcgttacgtg ctacggcttc 180

aaagagcatt tgcatcgata cgctttcaaa agttctttga ctgatatccc tttcgtttcg 240

atgatcagat ctctcattat tatctgtacg attccgattt ccgatcatcc tgctatttcg 300

tctatttaaa cctgcatctc tcaatcttta acaaacagat tccctcgagc atttcttcct 360

tcaattttgt atgctgggtc gttgagaaat gagagatatt agatgttaat tcgattatca 420

tcatcagggt ttcagttttt cgcttgttgg tgatgtttct ggttttgatc gcaggtgttt 480

attcaatgtg tgatggccct gctctttcaa atggtcctta cctgggaaca gtgactttat 540

gttccttcat ctccattctt gttctttcta ttaaggtttg tgtctttaca gtaaactccc 600

aaatcgaggc tgaagcttcg tcttccccct caaggcagaa gcttcactta aaaaaatcat 660

gggggatgcc agttttgttt ctatcttcag tagcctttgc ccttggccat acggtggtgg 720

cttacagaac aagctgcaga gcacgtagaa agcttctatt gcatcgagtt gacccagaag 780

ctgtaagttt taaagcttta actacaaaaa agaagaaaat ggcttgttcc tgtttttaaa 840

ttttgtagtt gagcctgctc taataaacct ccaatttgac ttggcaggcc ctttcttgca 900

aaaatgtatt ctctggctac cagaagatcc cacgatcccc cactccttct ggatccaaaa 960

ccccaaagag tgacagtgaa atcaagtgga aggcttcagg caatgctcgc gacgagagtg 1020

aactgccggt tagattgctc gctgacattg atagtttatt catcacatgt caaggtctta 1080

ccatccatta caagatcagc ttgcccgggt caccaccacg gtcgttgtcc tccgccgctt 1140

ttctcgaacc cggttatagt tgcaactccc caaagaaggc cataggaagg ccggtagttg 1200

acaggcatcc atttagtgtt ttatcaaaaa accaacacaa catccacagg agctatagca 1260

accagtttca cagttcatcc ctctatgatc cactattgga tggttctgca acaacttctc 1320

cagttctttg tgatgaaatc ccagtgataa gtctagatga agttgaagaa gaggaattga 1380

gcaaagatag catagatgga aactcagaga gcaacgggca attcggtatc gtgttggtgc 1440

atggtttcgg tggaggagtg ttctcgtgga gacatgtgat gggggtgctt gcaaggcaga 1500

ctggttgcag agttgctgct tacgaccggc ctggttgggg attaacttca aggctgcgtg 1560

cagaagactg ggaagaaaaa gaattgccta acccctacaa gcttgaaagt caggtaacta 1620

aagtcgtgca tttttctctc ttttttcttt tttggtagag ccactccttg tgtttgggta 1680

ggcactatgc atttttcttc tgacctttta ttgtcttttg cagtgaaagg gaaggggtct 1740

atgatttgaa atctagatca ttttgcctgt tacattgttt ttcctatgtt ttccatccct 1800

tgtatagttc ctaaaattcc aaattcttgt ttagtcccta aaccttgcat taccgtagat 1860

ggcatagtga tatgagagta tatggattat tggttgtttg gtgttttttc aggtggagct 1920

gctgctttca ttttgctcgg agatggggtt ttcttcagtg gtgctggtgg gtcatgatga 1980

cggaggtctg ctagctctca aggcagcgca aaggcttcaa ggatcaatga actcattcaa 2040

tgtaagtttg tttttgtgtt taattgaaca aagtagtaat gaatttgttg ggtttgatgt 2100

caatggtgat ggtgatggtg atggtgatgg tgatgggggg caggtttcga tcagaggagt 2160

ggtgttgctg agtgtaagct tatcaagaga ggtggttcct gggtttgcga ggattctgct 2220

acgaacatcg ctggggaaga agcacttggt tcgtcctctg ctgcgaaccg aaataactca 2280

ggtggtgaac cggcgtgcct ggtacgatgc caccaagtta acaaccgagg tcctaaatct 2340

ctataaggta acgccatatt acattcacga aacctcgtcc ttgtactcaa gatatgccct 2400

ctgcctggct gctgtttgaa tttattatca attatttgtt tgtttgtgaa tgaagcagag 2460

ggcgttgtgt gtggaagggt gggatgaagc gttgcatgag atagcgagat tatcgtacga 2520

gaccctgctt tctccaacaa atgcagaatc gttgctgaag gctgttgaag agatgccggt 2580

gttggtggtg ggtggtgttg aggatgccct cgtctccctc aaatcttctc aagcaatggc 2640

ctccaaactc ccaaattctg taaggcttca ttctctcctc atcctatttt tcttataata 2700

atgatgatta caataacaat aataaataat aatgcaaatg cagagactga ttacgatatc 2760

tggatgcgga catctcccac acgaggaatg ccccagcgcc ttgcttgctg cagtatcccc 2820

cttcatcacc agaattttgc aaaacccacc acaccacttc ctccaaaccc aatag 2875

<210> 2

<211> 1965

<212> DNA

<213>Siraitia grosvenorii (Siraitia grosvenorii (Swingle) C.Jeffrey)

<400> 2

atggcgaaag gcgggttttt tgtcgataag attcggagat gtctaaggac gttgttcttt 60

atggtggcga tggtggcgtc actgctcgtg tcgtcgttgc cagtgcttgt ggccatcgga 120

gacatgttgg tgccctgtat tttgatctcc agctttacgt gcgttacgtg ctacggcttc 180

aaagagcatt tgcatcgata cgctttcaaa agttctttga ctgatatccc tttcgtttcg 240

atgatcagat ctctcattat tatctgtgtt tattcaatgt gtgatggccc tgctctttca 300

aatggtcctt acctgggaac agtgacttta tgttccttca tctccattct tgttctttct 360

attaaggttt gtgtctttac agtaaactcc caaatcgagg ctgaagcttc gtcttccccc 420

tcaaggcaga agcttcactt aaaaaaatca tgggggatgc cagttttgtt tctatcttca 480

gtagcctttg cccttggcca tacggtggtg gcttacagaa caagctgcag agcacgtaga 540

aagcttctat tgcatcgagt tgacccagaa gctgcccttt cttgcaaaaa tgtattctct 600

ggctaccaga agatcccacg atcccccact ccttctggat ccaaaacccc aaagagtgac 660

agtgaaatca agtggaaggc ttcaggcaat gctcgcgacg agagtgaact gccggttaga 720

ttgctcgctg acattgatag tttattcatc acatgtcaag gtcttaccat ccattacaag 780

atcagcttgc ccgggtcacc accacggtcg ttgtcctccg ccgcttttct cgaacccggt 840

tatagttgca actccccaaa gaaggccata ggaaggccgg tagttgacag gcatccattt 900

agtgttttat caaaaaacca acacaacatc cacaggagct atagcaacca gtttcacagt 960

tcatccctct atgatccact attggatggt tctgcaacaa cttctccagt tctttgtgat 1020

gaaatcccag tgataagtct agatgaagtt gaagaagagg aattgagcaa agatagcata 1080

gatggaaact cagagagcaa cgggcaattc ggtatcgtgt tggtgcatgg tttcggtgga 1140

ggagtgttct cgtggagaca tgtgatgggg gtgcttgcaa ggcagactgg ttgcagagtt 1200

gctgcttacg accggcctgg ttggggatta acttcaaggc tgcgtgcaga agactgggaa 1260

gaaaaagaat tgcctaaccc ctacaagctt gaaagtcagg tggagctgct gctttcattt 1320

tgctcggaga tggggttttc ttcagtggtg ctggtgggtc atgatgacgg aggtctgcta 1380

gctctcaagg cagcgcaaag gcttcaagga tcaatgaact cattcaatgt ttcgatcaga 1440

ggagtggtgt tgctgagtgt aagcttatca agagaggtgg ttcctgggtt tgcgaggatt 1500

ctgctacgaa catcgctggg gaagaagcac ttggttcgtc ctctgctgcg aaccgaaata 1560

actcaggtgg tgaaccggcg tgcctggtac gatgccacca agttaacaac cgaggtccta 1620

aatctctata agagggcgtt gtgtgtggaa gggtgggatg aagcgttgca tgagatagcg 1680

agattatcgt acgagaccct gctttctcca acaaatgcag aatcgttgct gaaggctgtt 1740

gaagagatgc cggtgttggt ggtgggtggt gttgaggatg ccctcgtctc cctcaaatct 1800

tctcaagcaa tggcctccaa actcccaaat tctagactga ttacgatatc tggatgcgga 1860

catctcccac acgaggaatg ccccagcgcc ttgcttgctg cagtatcccc cttcatcacc 1920

agaattttgc aaaacccacc acaccacttc ctccaaaccc aatag 1965

<210> 3

<211> 654

<212> PRT

<213>Siraitia grosvenorii (Siraitia grosvenorii (Swingle) C.Jeffrey)

<400> 3

Met Ala Lys Gly Gly Phe Phe Val Asp Lys Ile Arg Arg Cys Leu Arg

1 5 10 15

Thr Leu Phe Phe Met Val Ala Met Val Ala Ser Leu Leu Val Ser Ser

20 25 30

Leu Pro Val Leu Val Ala Ile Gly Asp Met Leu Val Pro Cys Ile Leu

35 40 45

Ile Ser Ser Phe Thr Cys Val Thr Cys Tyr Gly Phe Lys Glu His Leu

50 55 60

His Arg Tyr Ala Phe Lys Ser Ser Leu Thr Asp Ile Pro Phe Val Ser

65 70 75 80

Met Ile Arg Ser Leu Ile Ile Ile Cys Val Tyr Ser Met Cys Asp Gly

85 90 95

Pro Ala Leu Ser Asn Gly Pro Tyr Leu Gly Thr Val Thr Leu Cys Ser

100 105 110

Phe Ile Ser Ile Leu Val Leu Ser Ile Lys Val Cys Val Phe Thr Val

115 120 125

Asn Ser Gln Ile Glu Ala Glu Ala Ser Ser Ser Pro Ser Arg Gln Lys

130 135 140

Leu His Leu Lys Lys Ser Trp Gly Met Pro Val Leu Phe Leu Ser Ser

145 150 155 160

Val Ala Phe Ala Leu Gly His Thr Val Val Ala Tyr Arg Thr Ser Cys

165 170 175

Arg Ala Arg Arg Lys Leu Leu Leu His Arg Val Asp Pro Glu Ala Ala

180 185 190

Leu Ser Cys Lys Asn Val Phe Ser Gly Tyr Gln Lys Ile Pro Arg Ser

195 200 205

Pro Thr Pro Ser Gly Ser Lys Thr Pro Lys Ser Asp Ser Glu Ile Lys

210 215 220

Trp Lys Ala Ser Gly Asn Ala Arg Asp Glu Ser Glu Leu Pro Val Arg

225 230 235 240

Leu Leu Ala Asp Ile Asp Ser Leu Phe Ile Thr Cys Gln Gly Leu Thr

245 250 255

Ile His Tyr Lys Ile Ser Leu Pro Gly Ser Pro Pro Arg Ser Leu Ser

260 265 270

Ser Ala Ala Phe Leu Glu Pro Gly Tyr Ser Cys Asn Ser Pro Lys Lys

275 280 285

Ala Ile Gly Arg Pro Val Val Asp Arg His Pro Phe Ser Val Leu Ser

290 295 300

Lys Asn Gln His Asn Ile His Arg Ser Tyr Ser Asn Gln Phe His Ser

305 310 315 320

Ser Ser Leu Tyr Asp Pro Leu Leu Asp Gly Ser Ala Thr Thr Ser Pro

325 330 335

Val Leu Cys Asp Glu Ile Pro Val Ile Ser Leu Asp Glu Val Glu Glu

340 345 350

Glu Glu Leu Ser Lys Asp Ser Ile Asp Gly Asn Ser Glu Ser Asn Gly

355 360 365

Gln Phe Gly Ile Val Leu Val His Gly Phe Gly Gly Gly Val Phe Ser

370 375 380

Trp Arg His Val Met Gly Val Leu Ala Arg Gln Thr Gly Cys Arg Val

385 390 395 400

Ala Ala Tyr Asp Arg Pro Gly Trp Gly Leu Thr Ser Arg Leu Arg Ala

405 410 415

Glu Asp Trp Glu Glu Lys Glu Leu Pro Asn Pro Tyr Lys Leu Glu Ser

420 425 430

Gln Val Glu Leu Leu Leu Ser Phe Cys Ser Glu Met Gly Phe Ser Ser

435 440 445

Val Val Leu Val Gly His Asp Asp Gly Gly Leu Leu Ala Leu Lys Ala

450 455 460

Ala Gln Arg Leu Gln Gly Ser Met Asn Ser Phe Asn Val Ser Ile Arg

465 470 475 480

Gly Val Val Leu Leu Ser Val Ser Leu Ser Arg Glu Val Val Pro Gly

485 490 495

Phe Ala Arg Ile Leu Leu Arg Thr Ser Leu Gly Lys Lys His Leu Val

500 505 510

Arg Pro Leu Leu Arg Thr Glu Ile Thr Gln Val Val Asn Arg Arg Ala

515 520 525

Trp Tyr Asp Ala Thr Lys Leu Thr Thr Glu Val Leu Asn Leu Tyr Lys

530 535 540

Arg Ala Leu Cys Val Glu Gly Trp Asp Glu Ala Leu His Glu Ile Ala

545 550 555 560

Arg Leu Ser Tyr Glu Thr Leu Leu Ser Pro Thr Asn Ala Glu Ser Leu

565 570 575

Leu Lys Ala Val Glu Glu Met Pro Val Leu Val Val Gly Gly Val Glu

580 585 590

Asp Ala Leu Val Ser Leu Lys Ser Ser Gln Ala Met Ala Ser Lys Leu

595 600 605

Pro Asn Ser Arg Leu Ile Thr Ile Ser Gly Cys Gly His Leu Pro His

610 615 620

Glu Glu Cys Pro Ser Ala Leu Leu Ala Ala Val Ser Pro Phe Ile Thr

625 630 635 640

Arg Ile Leu Gln Asn Pro Pro His His Phe Leu Gln Thr Gln

645 650

<210> 4

<211> 1500

<212> DNA

<213>Siraitia grosvenorii (Siraitia grosvenorii (Swingle) C.Jeffrey)

<400> 4

ctcctccccc cttttgcttc cacttctctt tctccgccgc acctattatt tttacttttt 60

ttttttctct tacactttta ttcagatcat ttcaaatata tatgtaaaac taaaagatta 120

aattacatgt taaatatcta aatttttata attatattta atatattcat aaatttttat 180

ttttattttt aataaattta gaaattttaa aaaatttcta atatattaag aatcaattca 240

atgacatatt aaacacaaaa tttaaagttt aaaaatctat tagacacttt ttaaagttag 300

aaaacttatt aaacacaatt ttaaaagttc atagatcaaa tttgtagttt aacctaacta 360

aaaatattaa tacaaaaata taccttaatt ggttaagaaa tccagtaatc tccttaaaga 420

ttgaaatctg cctactctct tgttgaaata tatataaatt gaatgaaaat aaagaactac 480

gaaatcaagg gttgtcaaat tatatttatc tacttaaaca aaatttcttt gttttgtatt 540

ctactatttt cataagtaaa ttatacatct tgaaaaccaa tataataatt tatcaaaaat 600

atactttttt ttttctttga tttcagttaa gattttaaat gtgtcttcaa agtaaaagat 660

atttttttaa aaaaaaatgt atgactgttt aatatatcaa atttctattt ttacactaaa 720

tatttagtaa taattttgtt gttatcaatt attatttcct aacaaaagga ccatttattg 780

agcgagaaat cttaccttag agattgaatt acattattaa taattaaggg ccacttcgaa 840

tttagagaga gagagcgctg gatgtaaaaa gggtattttg gtaaaatgga aaatggtgcc 900

aggtttgcaa cgggttgggc actggattaa ttaatatttt taatataaaa gatttttctt 960

ttttgcgaat accgtcctat gatcctcacg atgcttacga agaatcccta attttcaaat 1020

attattttaa ttaaaaaaga gagagagaga gagagaggga gacagaggga gagaattcaa 1080

aatccaacat caacatcttt caggaaacgc acacaaggaa aagggaaggt tttttatttc 1140

aaagctttca atacctattg atctcctctc cgctcatccc tctctgtaag agaaggaccc 1200

cttttgaagc cctagccatt tcccgtccat ggcgttccta ttggcttcca cccacctttt 1260

ccggcttctt taagatcgtc gtcttgcttt tttaagtcgc cgccgggaaa tggccgataa 1320

gccgcctgca gatgggttct tcttaacctc gccgccaccg gctagggttt cactgtgctc 1380

tcggaatccc acataacttt ttttgttggg attgccttct gacgatttgt ccgatcaatc 1440

ggaggcattt ggagcattaa agggccgtgg gttggttggt tggttggttt cgtcgttttc 1500

Claims (10)

1. a kind of method for improving sweet tea salidroside content in fruit, by changing epoxide hydrolase geneSgeph5Expression quantity come It influences it to regulate and control the content of Lo Han Guo fruit Momordica-Glycosides, it is characterised in that the nucleosides of the epoxide hydrolase gene Acid sequence is selected from following group of one of sequence:

(a) nucleotide sequence as shown in SEQ ID NO:1 or 2；

(b) be substituted, lack or add in the nucleotide sequence shown in SEQ ID NO:1 or 2 one or several nucleotide and The nucleotide sequence as derived from 1) with same function.

2. application as described in claim 1, which is characterized in that the epoxide hydrolase geneSgeph5Amino acid sequence It is classified as:

(a) albumen that the amino acid sequence shown in SEQ ID NO:3 forms；Or

(b) by the amino acid sequence of SEQ ID NO:3 by one or several amino acid residues substitution and/or missing and/or Add as derived from SEQ ID NO:3 and keep the albumen of protein function shown in SEQ ID NO:3.

3. a kind of expression cassette, which is characterized in that the ring described in claim 1 under the regulating and controlling sequence regulation effectively connected Oxide hydrolase gene.

4. expression cassette according to claim 3, which is characterized in that the regulating and controlling sequence contains specific expressing promoter, more Body, promoter isSgeph5Promoter, nucleotide sequence is as shown in SEQ ID NO:4.

5. a kind of DNA construct comprising adjusting any one of gene or claim the 2-4 expression cassette described in claim 1.

6. a kind of recombinant vector comprising DNA construct described in claim 5.

7. a kind of preparation method of mutant material, it is characterised in that the mutant material is by epoxide hydrolase geneSgeph5Mutation caused by, wherein the nucleotide sequence of the fertile gene is selected from following group of one of sequence:

(a) nucleotide sequence as shown in SEQ ID NO:1 or 2；

(b) be substituted, lack or add in the nucleotide sequence shown in SEQ ID NO:1 or 2 one or several nucleotide and The nucleotide sequence as derived from 1) with same function.

8. method according to any one of claims 8, wherein the mutation is point mutation or DNA missing or insertion mutation, or logical Cross the generation of the gene silencings means such as RNAi, rite-directed mutagenesis.

9. any method of claim 1-2 and 7-8 and its mutant material obtained are appointed in following (a) to (b) Application in one:

(a) Siraitia grosvenorii kind or strain are cultivated；

(b) the other plant varieties of Curcurbitaceae or strain are cultivated.

10. a kind of production method of high sweet tea glucoside Siraitia grosvenorii comprising:

(a) expression cassette described in claim 3-4 is constructed；

(b) expression cassette for obtaining step (a) imports plant cell；

(c) genetically modified plants are regenerated；With

(d) genetically modified plants are selected；And

(e) optionally, the plant that amplification step (d) obtains is to obtain offspring.