CN111500592A

CN111500592A - Identification method for GAPC1 gene mRNA transportation between rootstocks and ears of Cucurbitaceae

Info

Publication number: CN111500592A
Application number: CN202010200731.6A
Authority: CN
Inventors: 张文娜; 刘梓溪
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2020-03-20
Filing date: 2020-03-20
Publication date: 2020-08-07

Abstract

The invention provides a molecular identification method for GAPC1 gene mRNA transferred between rootstock and scion of Cucurbitaceae, comprising the following steps: 1) the cucumber is used as a scion, and the pumpkin is used as a stock for grafting; 2) designing specific primers of GAPC1 genes of the cucumbers and the pumpkins respectively; 3) carrying out RT-PCR on the cucumber and the pumpkin as well as the grafted cucumber and the grafted pumpkin by using the primer, and comparing the conditions of RT-PCR products before and after grafting; 4) the cloning vector for GAPC1 was constructed. The method provided by the invention is rapid, sensitive, high in accuracy, simple and convenient, and can be applied to other cucurbitaceae plants.

Description

Identification method for GAPC1 gene mRNA transportation between rootstocks and ears of Cucurbitaceae

Technical Field

The invention relates to the field of plant molecular biology, in particular to a molecular identification method for long-distance transmission of GAPC1 gene mRNA molecules between rootstocks and scions of cucurbitaceae plants.

Background

Grafting is a technique of grafting a bud or a branch of a plant to an appropriate part of another plant and joining the two to form a new plant. In the field of gardening, grafting is commonly used for obtaining the characters of resistance, dwarfing, high quality and the like and improving the product quality. A large number of researches show that the root absorption capacity of the vegetable grafted seedling can be enhanced and the stress resistance of the grafted plant can be improved by utilizing the resistant rootstock for grafting.

A great deal of research shows that RNA as an information molecule is mediated by intercellular and long-distance information networks to be transported between stock spikes of grafted plants, so that the relative capacities of plant growth and development and stress resistance are regulated. Therefore, the transport condition of mRNA obtained by transcription of specific genes among self-heterografting plants of cucumber is identified, and the long-distance mRNA molecular regulation mechanism among rootstock ears of the grafted cucumber is explored. Therefore, the cucumber growth regulator is used for regulating and controlling the growth and quality characters of the cucumber and improving the yield of the cucumber in winter and spring, and has important significance for the efficient overwintering cultivation and production of the cucumber.

Recent studies show that a large amount of signal molecules such as mRNA, small-molecule RNA and protein exist between rootstocks and scions of grafted horticultural crops, locally move between adjacent cells through plasmodesmata, enter phloem for transportation, participate in rootstock and scion interaction, regulate transcription, participate in gene expression and protein translation, and regulate organ growth and development and adaptability to adversity stress (L ucas et al, 2001, Turgeon et al, 2009).

To date, many gene mRNAs and their encoded proteins have been discovered that can be transported over long distances in the phloem of cucurbitaceae. As the results of immunoblot analysis showed, proteins PP1 and PP2 of squash were transferred from squash rootstocks, accumulated in cucumber scions, localized to sieve molecules and parasporal cells in the outer phloem, but mRNA signals of squash PP1 and PP2 were not detected in cucumber, indicating that long distance transport of PP proteins occurred (Golecki et al, 1999). Xenografting experiments demonstrated that some of the mRNA could be transferred remotely from the melon rootstock to the pumpkin scion (Omid et al, 2007).

Currently, there are many methods for detecting long-distance transportation of plant phloem mRNA, such as RNA-protein co-immunoprecipitation (Ham et al, 2009), nested RT-PCR (Harada et al, 2009), Northern hybridization, and the like. Although these methods can detect the transmissibility of genes in phloem relatively accurately, the procedures are complicated, many requirements are required, the operation is not easy, and many difficulties are added to the experimental procedures of the method.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a molecular identification method for transporting GAPC1 gene mRNA between cucurbitaceae scions.

In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:

a pumpkin GAPC1 gene has a nucleotide sequence shown in SEQ ID NO 1.

The amino acid sequence of the protein coded by the pumpkin GAPC1 gene is shown as SEQ ID NO 2.

A cucumber GAPC1 gene has a nucleotide sequence shown in SEQ ID NO 3.

The amino acid sequence of the protein coded by the cucumber GAPC1 gene is shown as SEQ ID NO 4.

A molecular identification method for GAPC1 gene mRNA transportation between rootstocks and ears of Cucurbitaceae family comprises the following steps:

(1) self-grafting the cucumber; carrying out self-grafting on the pumpkin; cucumber is used as scion, and pumpkin is used as stock to perform hypocotyl grafting; pumpkin is used as scion, and cucumber is used as stock to perform hypocotyl grafting;

(2) analyzing the GAPC1 gene sequences of cucumber and pumpkin, and numbering as follows: CmoCh18G001950 and CsGy1G 009330; searching for different fragments of the two, and respectively designing specific primers of a cucumber GAPC1 gene and a pumpkin GAPC1 gene to ensure that the specific primer of one cannot generate a band in the cDNA of the other;

(3) respectively extracting total RNA of the rootstock and the scion after the cucumber self-grafting, the pumpkin self-grafting, the cucumber as the scion, the pumpkin as the rootstock, the pumpkin as the scion and the cucumber as the rootstock are subjected to cross-grafting in two modes, carrying out reverse transcription to obtain cDNA, respectively designing primers of a cucumber GAPC1 gene fragment and a pumpkin GAPC1 gene fragment, and respectively amplifying a cucumber GAPC1 gene fragment and a pumpkin GAPC1 gene fragment by using PCR;

(4) gel electrophoresis to detect whether there is an electrophoresis band: if the RNA transmission exists in the grafting system, a specific strip of the scion appears in an electrophoretogram of an amplification product of the rootstock, and a specific strip of the rootstock also appears in an electrophoretogram of an amplification product of the scion; if no transmission occurs, the electrophoretogram of the amplified product shows the bands of the corresponding samples without other miscellaneous bands, namely, the products amplified by PCR of cucumber specific primers in the cross-grafted pumpkin part are transported if the bands exist, and are not transported if the bands do not exist.

Further, the sample of total RNA is selected from grafted and heterografted leaves and roots.

Further, the specific primers of the cucumber GAPC1 gene in the step (2) are as follows:

upstream primer 5'-CTGCTTGATGGTGTGGTCGT-3'

A downstream primer 5'-TTTGGTAGGGACAGGGGAGA-3';

specific primers of pumpkin GAPC1 gene

Upstream primer 5'-CCATATTCAGCTCTCGCCCAA-3'

The downstream primer 5'-CAGCAGCTTTCTCTTTGTCGG-3'.

Further, in the step (3), the primers for amplifying the cucumber GAPC1 gene fragment are as follows:

upstream primer 5'-CAATGGGATTTGGAAGAATTGGCA-3'

A downstream primer 5'-AGACAGAGGCAATGTGGACAATC-3';

the primers for amplifying the pumpkin GAPC1 gene fragment are as follows:

upstream primer 5'-ATGGCCAAGATCAAGATCGGA-3'

The downstream primer 5'-TTAATTGACAGATGCAACGTGAGA-3'.

Cloning genes, constructing an expression vector: extracting total RNA of cucumber and pumpkin, and carrying out reverse transcription to obtain cDNA; designing a specific primer, obtaining a target gene by PCR, constructing a cloning vector, transferring into escherichia coli, detecting a construction result by PCR, and screening out a successfully constructed plasmid;

homology analysis was performed on the target fragment on the plasmid.

Drawings

The invention has the following drawings:

FIG. 1 is a first diagram of the results of a transport assay.

FIG. 2 is a second diagram showing the results of the detection of the transportability assay.

FIG. 3 is a diagram showing the results of PCR products of the GAPC1 gene of cloned pumpkin and cucumber.

FIG. 4 shows a homology comparison of the amino acid sequence of GAPC 1. Cucurbita moschata in the figure: pumpkin; cucurbita pepo: zucchini; cucurbita maxima: winter squash; cucumis sativus: cucumber; cucumismelo: and (5) melon.

FIG. 5 shows a phylogenetic tree analysis of the amino acid sequence of GAPC 1.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1: cucumber and pumpkin grafting

(1) Cultivating scions and rootstocks: and respectively growing seedlings of the rootstock and the scion seeds, wherein when the scion grows to one leaf and one core, cotyledons of the rootstock are completely unfolded, and the thicknesses of hypocotyls of the rootstock and the scion are kept consistent.

(2) Grafting hypocotyls: cutting a 30-degree inclined plane obliquely downwards at a position 1cm below a scion cotyledon by using a blade, removing all cotyledons of the stock, obliquely cutting 1 knife at a position 1cm below the stock cotyledon by using the blade, wherein the length of a tangent plane is consistent with that of the scion, aligning and tightly adhering the scion and the tangent plane of the stock, and fixing the scion and the tangent plane of the stock by using a grafting clip; then the cotyledon part of the rootstock is grafted to the section of the original scion, and the grafting opening is fixed by a grafting clip.

(3) And (3) management after grafting: placing the grafted seedlings in a seedling raising set, fully wetting the transparent plastic top cover by using a watering can, and thoroughly watering the seedling raising matrix to keep the humidity of the small environment in the seedling raising set; sealing the seedling growing suit by using a preservative film, placing the seedling growing suit into an incubator at the temperature of 28 +/-2 ℃ for dark culture for 2-3d, then transferring to low-light culture for 3-4d, and then carrying out normal light culture.

Example 2: total RNA extraction

Total RNA was extracted using CTAB method, which comprises the following steps:

(1) 2g of leaf material was taken and ground into a fine powder in liquid nitrogen.

(2) 10ml of 65 ℃ preheated CTAB extract and 500. mu.l of β -mercaptoethanol were added to each tube, vortexed vigorously and homogenized, incubated in a 65 ℃ water bath for 30 min.

(3) 10ml of chloroform was added to each tube: isoamyl alcohol (24: 1), vortex mixing, ice bath 10 min. Centrifuge at 12000rpm for 10min at 4 ℃.

(4) The supernatant was taken and precipitated overnight at-20 ℃ by adding 1/3 volumes of 8M L iCl and 500. mu.l of β -mercaptoethanol.

(5) Centrifuge at 12000rpm for 20min at 4 ℃.

(6) The supernatant was discarded, the precipitate was dissolved in 4ml of guanidine isothiocyanate denatured solution, 120. mu.l of β -mercaptoethanol and 880. mu.l of 2M NaAc (pH4.0) were added thereto, and after mixing, 5ml of phenol, chloroform, isoamyl alcohol (25: 24: 1) was added thereto, followed by vortex mixing, and centrifugation at 12000rpm for 10min under ice-cooling at 5 min.4 ℃.

(7) Taking the supernatant, adding equal volume of phenol: chloroform: isopentanol (25: 24: 1), vortex and mix well, ice bath 5 min. Centrifuge at 12000rpm for 10min at 4 ℃.

(8) The supernatant was taken and an equal volume of chloroform was added: isoamyl alcohol (24: 1), vortex and mix well, ice bath 5 min. Centrifuge at 12000rpm for 10min at 4 ℃.

(9) The supernatant was taken, and 1/10 volumes of 3M NaAc (pH5.2) and 2.5 volumes of absolute ethanol were added, and the mixture was left overnight at-20 ℃.

(10) Centrifuge at 14000rpm for 20min at 4 ℃. The supernatant was discarded, the pellet was rinsed 2 times with pre-cooled 75% ethanol, and after each wash, centrifuged at 14000rpm for 10min, and the supernatant discarded. The RNA pellet was dried on ice.

(11) Add 100. mu.l RNase-free-ddH₂And dissolving the precipitate by using O. The concentration and purity of the RNA sample were determined by diluting 10. mu.l 200-fold. Taking 5 mu g for electrophoresis detection, detecting the integrity of rRNA, adding 3 times of volume of absolute ethyl alcohol into the rest samples, and storing at-70 ℃.

Example 3: reverse transcription of RNA into cDNA

Using TaKaRa Biochemical reagent, PrimeScript^TMRT reagent Kit with gDNA Eraser (Perfect Real Time), method according to its company instructions:

(1) reactions for removing genomic DNA

The reaction mixture is prepared on ice according to the following components, in order to ensure the accuracy of the preparation of the reaction solution, Master Mix is prepared according to the reaction number +2, then the Master Mix is subpackaged into each reaction tube, and finally the RNA sample is added.

42 ℃ for 2min (or room temperature for 5min)

4℃

(2) Reverse transcription reaction

The reaction was prepared on ice. In order to ensure the accuracy of the preparation of the reaction solution, the Master Mix should be prepared in the amount of the reaction number +2, and then 10. mu.l of the Master Mix should be dispensed into each reaction tube. After gentle mixing, reverse transcription reaction is carried out immediately.

< TB Green qPCR method >

37℃15min

85℃5sec

4℃

Example 4: transport assay

Specific primers are designed according to different fragments of the GAPC1 genes of cucumbers and pumpkins, and the specific primer of one of the cucumbers and the pumpkins cannot generate a band in the cDNA of the other pumpkin.

The designed differential primer sequences were as follows:

pumpkin: upstream primer 5'-CCATATTCAGCTCTCGCCCAA-3'

Downstream primer 5'-CAGCAGCTTTCTCTTTGTCGG-3'

Cucumber: upstream primer 5'-CTGCTTGATGGTGTGGTCGT-3'

Downstream primer 5'-TTTGGTAGGGACAGGGGAGA-3'

The primers were synthesized by Biotechnology Ltd of New Engineers of Okins, Beijing.

The PCR reaction system is as follows:

the PCR reaction procedure was as follows

Pre-denaturation at 95 ℃ for 3 min;

95℃30s；

60℃30s；

72℃1min；

39 cycles.

Final extension at 72 ℃ for 5 min.

And (3) detecting a PCR product: preparing 2% agarose gel according to the size of the target fragment, adding nucleic acid dye (ten-thousandth), 0.1% TAE electrophoresis buffer, performing 110-120v electrophoresis for about 25min, and detecting the size of the PCR product fragment under an ultraviolet lamp to obtain a band (FIG. 1, FIG. 2). Wherein S is the scion receiving portion and R is the anvil portion. Observing the bands, if the PCR product using the cucumber cDNA as a template and the cucumber specific primer PCR product has bands, and the PCR product using the cDNA of the heterograft pumpkin part as a template also has bands, the mRNA generated by the GAPC1 is proved to be transported in the phloem of the graft plant, and the opposite is also true.

Example 5: full-Length cloning of the GAPC1 Gene

Cloning primers are designed according to the GAPC1 gene segments of the cucumbers and the pumpkins.

The designed primer sequences are as follows:

pumpkin: upstream primer 5'-ATGGCCAAGATCAAGATCGGA-3'

Downstream primer 5'-TTAATTGACAGATGCAACGTGAGA-3'

Cucumber: upstream primer 5'-CAATGGGATTTGGAAGAATTGGCA-3'

Downstream primer 5'-AGACAGAGGCAATGTGGACAATC-3'

And (3) PCR reaction system:

max DNA Polymerase (TaKaRa Co.)

The PCR reaction program using pumpkin cDNA as template is as follows

Pre-denaturation at 95 ℃ for 3 min;

95℃30s；

60℃30s；

72℃2min；

39 cycles.

Final extension at 72 ℃ for 5 min.

The PCR reaction procedure with cucumber cDNA as template was as follows:

the PCR reaction procedure was as follows

Pre-denaturation at 95 ℃ for 3 min;

95℃30s；

60℃30s；

72℃1min；

39 cycles.

Final extension at 72 ℃ for 5 min.

And (3) detecting a PCR product: preparing 1% agarose gel according to the size of the target fragment, adding nucleic acid dye (ten-thousandth), 0.1% TAE electrophoresis buffer, performing 110-120v electrophoresis for about 25min, and detecting the size of the PCR product fragment under an ultraviolet lamp to obtain a band (FIG. 3).

Example 6: agarose gel recovery of the fragment of interest

The recovery kit of Bomader Biotechnology Ltd was used, and the method was as follows:

(1) under a long-wave ultraviolet lamp, a clean blade is used for cutting off a DNA band required to be recovered, and the gel without DNA is cut off as much as possible, so that the smaller the volume of the obtained gel is, the better the gel is.

(2) The excised gel containing the DNA band was placed into a 1.5ml or 2ml centrifuge tube.

(3) Add 500. mu.l volume of sol/binding solution DB.

(4) The mixture was left in a water bath at 50 ℃ for 10min until the gel was completely dissolved. The centrifuge tube was turned gently up and down during the period to ensure that the gel block was fully dissolved.

(5) Adding the solution obtained in the previous step into a trace DNA adsorption column (the adsorption column is placed into a collection tube), standing at room temperature for 1min, centrifuging at 12000rpm for 1min, and pouring off the waste liquid in the collection tube.

(6) Adding 500 μ l of rinsing liquid WB (added with anhydrous ethanol), centrifuging at 12000rpm for 1min, and discarding the waste liquid.

(7) Operation 6 is repeated.

(8) The micro DNA adsorption column was returned to the empty collection tube and centrifuged at 12000rpm for 1 min.

(9) The DNA adsorbing column was removed and placed in a clean 1.5ml centrifuge tube and left at room temperature for several minutes.

(10) Mu.l of elution buffer EB10 heated to 65 ℃ was dropped on the middle part of the adsorption membrane, and centrifuged at 12000rpm for 1 min. If a larger amount of DNA is required, the resulting solution can be re-loaded into the adsorption column and centrifuged for 1 min.

(11) The concentration of 1 μ l of the recovered product was measured and placed at-20 ℃ for further use.

Example 7: t-vector ligation and transformation of fragments of interest

The purified PCR product was added to pClone007 vector and buffer (available from Oncorhynchus).

A connection system:

mixing, and standing at room temperature for 10 min.

And (3) transforming escherichia coli:

the competent cells (DH5 α, Ongjingke Co.) were thawed in ice-water bath, the desired DNA was added to the cells and the tube bottom was pricked with a finger and gently mixed.

The mixture was left in an ice-water bath for 30 minutes without shaking.

The mixture was heated at 42 ℃ for 60 seconds without shaking.

The mixture was left in an ice-water bath for 2 minutes without shaking.

Add 500. mu.l sterile L B medium.

Placing the mixture in a shaker at 37 ℃, and shaking and reviving the mixture at 200rpm for 60 minutes.

Spreading appropriate amount of the bacterial solution on L B plate containing AMP resistance, after the liquid is blotted dry, inverting the plate, and culturing at 37 deg.C for 12-16 hr.

Selecting single colony, inoculating into 1ml L B + Amp liquid culture medium, mixing, shaking for several hours at 37 deg.C, PCR identification with the mixture as template, agarose gel electrophoresis to detect the size of inserted fragment, selecting positive clone bacterial liquid, sequencing with Beijing Strongco Biotechnology Limited company, and sequencing to show that the sizes of the two target fragments are 1014bp and 987 bp.

Example 8: plasmid extraction containing cloning vector

The fast plasmid miniprep kit (DP105) from tiangen biochemistry science and technology limited was used, the method being described in the company's instructions:

1. 1-4ml of overnight-cultured broth was taken, added to a centrifuge tube, centrifuged at 12000rpm (. about.13,400 13,400 × g) for 1min using a conventional tabletop centrifuge, and the supernatant was aspirated.

2. To the centrifuge tube where the pellet of bacteria was left, 150. mu.l of solution P1 (RNaseA and TIANRed had been added) was added, and the pellet of bacteria was thoroughly suspended using a pipette or vortex shaker.

3. Adding 150 μ l of solution P2 into the centrifuge tube, and gently turning the tube up and down for 6-8 times to fully lyse the thallus, so that the solution is clear purple.

4. Adding 350 mu l of solution P5 into a centrifuge tube, immediately and quickly turning upside down and mixing for 12-20 times, fully mixing, wherein flocculent precipitates appear at the moment, centrifuging at 12000rpm (13-400 13,400 × g) for 2min to enable the solution to be clear yellow, 5, transferring the supernatant collected in the previous step into an adsorption column CP3 by using a pipette (the adsorption column is placed into a collection tube), centrifuging at 12000rpm (13-400 13,400 × g) for 30sec, pouring waste liquid in the collection tube, and placing the adsorption column CP3 into the collection tube.

6. To the adsorption column CP3, 300. mu.l of a rinsing solution PWT (to which absolute ethanol had been added) was added, centrifuged at 12000rpm (. about.13,400 13,400 × g) for 30sec, the waste liquid in the collection tube was discarded, and the adsorption column CP3 was put into the collection tube.

7. The adsorption column CP3 was placed in a collection tube and centrifuged at 12000rpm (. about.13,400 13,400 × g) for 1 min.

8. The adsorption column CP3 was placed in a clean centrifuge tube, 50-100. mu.l of elution buffer TB was added dropwise to the middle of the adsorption membrane, and the plasmid solution was collected by centrifugation at 12000rpm (. about.13,400 13,400 × g) for 30sec at-20 ℃.

Example 9: bioinformatics analysis of pumpkin and cucumber GAPC1 genes

Comparing the obtained sequence with homology and predicting the molecular weight in software provided on websites such as http:// www.ncbi.nlm.nih.gov and the like; software such as MEDA (Medo-electric-mechanical data acquisition) is used for carrying out multiple sequence alignment analysis and evolutionary tree drawing analysis.

Blast alignment on NCBI showed: the amino acid sequences of the pumpkin and cucumber GAPC1 genes have higher similarity with other plants, wherein the similarity with pumpkin (Cucurbita pepo) is highest (98%), and then the pumpkin (Cucurbita maxima) and the melon (Cucumis melo) are sequentially adopted. The amino acid sequence encoded by the cucumber and squash GAPC1 gene contains 1 domain (fig. 4), identical to domain DH _ N of the GAPC1 gene known from other plants.

A phylogenetic tree of 13 GAPC gene amino acid sequences of plants such as cucumber, pumpkin and the like is constructed through MEDA. The evolutionary tree shows that the obtained amino acid sequence has a closest relationship with Cucurbita maxima, Cucurbita pepo and melon (Cucumis melo) and a further relationship with Nicotiana benthamiana, tomato and common tobacco (Nicotiana tabacum). But most distant from Arabidopsis (Arabidopsis thaliana), barley (Hordeum vulgare), maize (Zea mays), japonica rice (Oryza sativa Japonica Group) and Indica rice (Oryza sativa Indica Group) (FIG. 5). The phylogenetic tree obtained by the research is consistent with the classification result of botany to a certain extent. The sequence evolution of the amino acid is consistent with the evolution of the plant, the homologous plants are in the same branch on the evolutionary tree, the relatives are far away and are in different branches, and the homology percentage of the amino acid is reduced along with the distance of the species relatives.

Those not described in detail in this specification are within the skill of the art.

<110> university of agriculture in China

<120> identification method for transporting GAPC1 gene mRNA between rootstocks and ears of Cucurbitaceae

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

1 5 10 15

Arg Asp Asp Val Glu Leu Val Ala Val Asn Asp Pro Phe Ile Thr Thr

20 25 30

Asp Tyr Met Thr Tyr Met Phe Lys Tyr Asp Ser Val His Gly His Trp

35 40 45

Lys His Ser Asp Val Lys Val Lys Asp Ser Lys Ser Leu Leu Phe Gly

50 55 60

Glu Lys Ala Val Thr Val Phe Gly Phe Arg Asn Pro Asp Glu Ile Pro

65 70 75 80

Trp Gly Glu Thr Gly Ala Glu Tyr Ile Val Glu Ser Thr Gly Val Phe

85 90 95

Thr Asp Lys Asp Lys Ala Ala Ala His Leu Lys Gly Gly Ala Lys Lys

100 105 110

Val Ile Ile Ser Ala Pro Ser Lys Asp Ala Pro Met Phe Val Val Gly

115 120 125

Val Asn Glu Lys Glu Tyr Lys Pro Glu Leu Asp Val Ile Ser Asn Ala

130 135 140

Ser Cys Thr Thr Asn Cys Leu Ala Pro Leu Ala Lys Val Ile Asn Asp

145 150 155 160

Arg Phe Gly Ile Val Glu Gly Leu Met Thr Thr Val His Ala Met Thr

165 170 175

Ala Thr Gln Lys Thr Val Asp Gly Pro Ser Ser Lys Asp Trp Arg Gly

180 185 190

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

195 200 205

Lys Ala Val Gly Lys Val Leu Pro Ala Leu Asn Gly Lys Leu Thr Gly

210215 220

Met Ser Phe Arg Val Pro Thr Val Asp Val Ser Val Val Asp Leu Thr

225 230 235 240

Val Arg Leu Glu Lys Lys Ala Ser Tyr Glu Asp Ile Lys Ala Ala Ile

245 250 255

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

260 265 270

Asp Asp Val Val Ser Ser Asp Phe Val Gly Asp Ser Arg Ser Ser Ile

275 280 285

Phe Asp Ala Lys Ala Gly Ile Ala Leu Asn Asp Asn Phe Val Lys Ile

290 295 300

Val Ser Trp Tyr Asp Asn Glu Val Gly Tyr Ser Thr Arg Val Val Asp

305 310 315 320

Leu Ile Val His Ile Ala Ser Val

325

Claims

1. A pumpkin GAPC1 gene has a nucleotide sequence shown in SEQ ID NO 1.

2. The protein encoded by the pumpkin GAPC1 gene of claim 1, wherein the amino acid sequence of the protein is shown as SEQ ID NO 2.

3. A cucumber GAPC1 gene has a nucleotide sequence shown in SEQ ID NO 3.

4. A protein encoded by the cucumber GAPC1 gene of claim 3, having an amino acid sequence as set forth in SEQ ID NO 4.

5. A molecular identification method for GAPC1 mRNA molecules transmitted between cucurbitaceae scions is characterized by comprising the following steps:

(2) analyzing the nucleotide sequence of the gene of claim 1 and claim 3, searching for different fragments, designing specific primers of cucumber GAPC1 gene and pumpkin GAPC1 gene respectively, and ensuring that the specific primer of one cannot generate a band in the cDNA of the other;

(4) gel electrophoresis to detect whether there is an electrophoresis band: if the RNA transmission exists in the grafting system, a specific strip of the scion appears in an electrophoretogram of an amplification product of the rootstock, and a specific strip of the rootstock also appears in an electrophoretogram of an amplification product of the scion; if no transfer occurs, the electrophorogram of the amplified product shows the bands of the respective samples without other bands.

6. The molecular characterization method of claim 5 wherein the sample of total RNA is selected from the group consisting of grafted and heterografted leaves and roots.

7. The molecular identification method of claim 5, wherein the specific primers of the cucumber GAPC1 gene in step (2) are:

upstream primer 5'-CTGCTTGATGGTGTGGTCGT-3'

A downstream primer 5'-TTTGGTAGGGACAGGGGAGA-3';

specific primers of pumpkin GAPC1 gene

Upstream primer 5'-CCATATTCAGCTCTCGCCCAA-3'

The downstream primer 5'-CAGCAGCTTTCTCTTTGTCGG-3'.

8. The molecular characterization method of claim 5, wherein in step (3), the primers for amplifying the cucumber GAPC1 gene fragment are:

upstream primer 5'-CAATGGGATTTGGAAGAATTGGCA-3'

A downstream primer 5'-AGACAGAGGCAATGTGGACAATC-3';

the primers for amplifying the pumpkin GAPC1 gene fragment are as follows:

upstream primer 5'-ATGGCCAAGATCAAGATCGGA-3'

The downstream primer 5'-TTAATTGACAGATGCAACGTGAGA-3'.

9. Use of the molecular characterization method according to any one of claims 5 to 8 for characterization of inter-tassel GAPC1 mRNA molecular delivery in other cucurbits.