CN108330182B - Fluorescence identification method for transmitting mRNA molecules between plant rootstock and scion - Google Patents

Abstract

The invention discloses a molecular identification method for transmitting mRNA molecules between rootstock and scion of a plant, and relates to the field of plant molecular biology. The invention constructs a carrier with a gene carrying a fluorescent protein label for transgenosis, carries out micro-grafting on a transgenic rootstock and a wild type scion in a sterile glass bottle, and judges whether the gene is subjected to long-distance transmission between the rootstock and the scion or not by detecting the existence of fluorescence emitted by the adventitious root of the scion through a laser confocal microscope due to the characteristic that the scion is easy to grow the adventitious root in a humid environment; or whether the specific strips appear on the rootstock and the scion is verified by nested PCR (polymerase chain reaction) to judge whether the genes are remotely transferred between the rootstock and the scion or not, and finally the purpose of visually observing the genes to be remotely transferred between the rootstock and the scion is realized.

Description

Fluorescence identification method for transmitting mRNA molecules between plant rootstock and scion

Technical Field

The invention relates to the field of plant molecular biology, in particular to a fluorescence identification method for transmitting mRNA molecules between rootstock and scion of plants.

Background

In the practical process of horticultural crop production, the application of grafting technology can improve the plant resistance, increase the yield and improve the fruit quality, and has very important significance for the improvement of varieties and the improvement of economic value, wherein the grafting is widely applied to the production of fruit trees. In the aspect of fruit tree production, different rootstocks can generate different influences on the physiological and biochemical characteristics, flowering and fructification, environmental adaptability, tree growth and development and the like of scions, and the influences finally play a key role in the formation of fruits and the later economic value.

To date, many endogenous mRNA molecules have been discovered that can be delivered remotely from the phloem of plants, such as Arabidopsis Aux/IAA18 and Aux/IAA28(Notaguchi et al, 2012), pumpkin CmNAP (Ruiz-Mederano et al, 1999), CmPP16 (Xoconstle-Cazares et al, 1999), CmGAIP (Haywood et al, 2005), tomato the PFP-LeT6fusion gene (Kim et al, 2001), potato StBEL5 (Banerjeet al, 2006; Hannapel,2010), POTH1(a KNOTTED 1-Liquidization factor) (Mahahan jan et al, 2012), and the Like herbs. Currently, examples of endogenous mRNA molecules identified on the fruit trees of woody plants that can be transmitted remotely are GAI endogenous to apples (Xu et al, 2010), KN1 endogenous to pears (Zhang na, 2012), GAI (Zhang et al, 2012), NACP (Zhang et al, 2013), and WoxT1 (dual et al, 2015). These mRNA molecules can be transmitted from the rootstock to the scion, affecting the physiological and developmental processes of the scion. Therefore, by identifying and researching the mechanism of mRNA molecules transmitted between the rootstock and the scion in a long distance manner, not only can a theoretical basis be provided for the research of the mechanism of mRNA transmitted between the plants in a long distance manner and the mechanism of interaction between the rootstock and the scion, but also a novel rootstock breeding method capable of directionally improving the scion character can be developed by utilizing the found transmittable mRNA, and then the scion character is regulated and controlled by a grafting means, so that the transgenic product is prevented from being harvested, the product with specific excellent character can be obtained, the purpose of improving the economic benefit is achieved, and guidance can be provided for the breeding and production of horticultural crops in future.

3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) is the first important rate-limiting enzyme in the mevalonate pathway for synthesizing terpenoids, is an important regulatory site in the metabolism of cytoplasmic terpenoids (Bach, 1986; Choi et al, 1992), and plays a very important role in the growth and development of plants. The HMGR polypeptide chain parts are all composed of four parts, an N-terminal region, a joining region, a transmembrane region, and a C-terminal region in plants, where the C-terminal catalytic region is formed by the near 3' end coding in genes and is highly conserved in most plants (Roberts, 2007). Currently, pyrus PbHMGR1 has been cloned (huangjing et al, 2015), and its mRNA transitivity between tassels has also been demonstrated.

However, as for the method for identifying whether the plant gene mRNA can be transmitted in a long distance, only the technologies such as RT-PCR detection after transgenic grafting (Haywood et al, 2005), nested RT-PCR (Kanehira et al, 2010), RT-PCR-CAPS (Xu et al, 2010) and the like are carried out by constructing a vector of a target gene carrying a specific label, although the technologies detect the gene transmission more accurately, the defects exist in the methods are that the PCR amplification reaction is easy to be polluted to generate false positive, the transmitted signal cannot be detected visually, the transmission position and direction of the signal cannot be determined, and the current method for detecting intuitively is to mark the mRNA with a specific molecular probe, the probe of which has fluorescence, and detect the fluorescence diffusion condition after microinjection (Xoconsonsile-Cazare et al, 1999), however, the method still has the defects that the signal for observing the mRNA transmission exists only between one cell and two cells, belongs to the identification method of intercellular short-distance transmission, and the required instrument is very expensive, the operation technical requirement is high, the cost is high, and the common laboratory is difficult to achieve, so the harsh requirement for researching the process of carrying out the mRNA long-distance transmission between the scions greatly limits the quantity of the identified mRNA, the difficulty is increased for researching the plant endogenous mRNA grafting transmission, the research of the plant on the aspect of the mRNA long-distance transmission is difficult to advance, and the new endogenous mRNA in the long-distance transmission is difficult to discover.

Disclosure of Invention

The invention aims to overcome the problem that mRNA can not be visually observed to be transmitted after transgenic grafting is carried out by constructing a vector with a gene carrying a specific label in the prior art, and provides a fluorescence identification method for transmitting mRNA molecules between plant stock ears.

The invention provides a fluorescence identification method for transmitting mRNA molecules between plant rootstock and scion, which is to prepare a transgenic plant by constructing a vector of which the gene carries a fluorescent protein label; the transgenic plant is taken as a stock, the transgenic plant and the wild type scion are subjected to micro grafting under the aseptic condition, the fluorescence occurrence conditions of the stock and the scion are observed, and if fluorescence is observed on the adventitious roots of the stock and the scion, the mRNA molecules of the genes are transmitted between the stock and the scion of the plant.

On the other hand, the invention provides a fluorescence identification method for transmitting mRNA molecules between plant rootstock and scion, which prepares a transgenic plant by constructing a vector of which the gene carries a fluorescent protein label; the transgenic plant is taken as a rootstock, the transgenic plant and the wild scion are subjected to micro-grafting under the aseptic condition, nested PCR detection is adopted, and if the nested PCR products of the rootstock and the scion all have specific bands, the mRNA molecules of the genes are transmitted between the rootstock and the scion of the plant.

The fluorescent protein is green fluorescent protein, red fluorescent protein, yellow fluorescent protein, blue fluorescent protein, orange fluorescent protein or blue-green fluorescent protein.

In an embodiment of the invention, the gene is pyrus betulaefolia HMGR 1.

The vector is an overexpression GFP-PbHMGR1 vector, and is prepared by the following method: extracting the RNA of the birch pear genome, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification, recovering a target segment, purifying, connecting a T vector, transforming a competent cell, and selecting a positive clone with correct sequencing to obtain the final product, wherein the sequence of a primer pair used for PCR amplification is shown as SEQ ID NO. 2-3.

When the nested PCR is carried out to detect whether the scion has a specific strip, the sequence of a primer pair adopted by the first round of PCR reaction is shown as SEQ ID NO.4-5, and the sequence of a primer pair adopted by the second round of PCR reaction is shown as SEQ ID NO. 6-7.

Further, the invention provides a fluorescence identification method for HMGR1mRNA molecule transmission between rootstocks and ears of plants, which comprises the following steps:

(1) respectively extracting the total RNA of the pyrus betulaefolia, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification on the full length of the HMGR1 gene;

(2) constructing a carrier of a birchleaf pear HMGR1 gene carrying a fluorescent protein label, and preparing a transgenic plant;

(3) the transgenic plant is a stock, the transgenic plant and a wild type scion are subjected to micro grafting under the aseptic condition, the fluorescence occurrence conditions of the stock and the scion are observed, and if the stock and the scion have adventitious roots and corresponding fluorescence is observed, the transfer of mRNA molecules of the birch HMGR1 gene between the stock and the scion of the plant is indicated.

The sequence of the primer pair adopted by the PCR amplification in the step (1) is shown as SEQ ID NO. 2-3.

The fluorescent protein selected in the embodiment of the invention is GFP, after micro-grafting, the adventitious root and the stock root which grow out from the scion are taken and placed under a laser confocal microscope to observe GFP fluorescence, the GFP fluorescence conditions of the scion and the stock are compared, if mRNA transmission exists in a grafting system, GFP fluorescence expressed by GFP-PbHMGR1 appears on the stock root, and GFP fluorescence expressed by GFP-PbHMGR1 due to the long-distance transmission of the mRNA between the stocks and the scion also appears on the adventitious root of the scion tobacco; if no transmission occurs, GFP fluorescence expressed by GFP-PbHMGR1 appears only in the root of the stock tree, and GFP fluorescence does not appear in the adventitious root of the scion tobacco.

Wherein, the sample for amplifying the fragments with enzyme cutting sites of the avocado HMGR1 gene and extracting the total RNA selects the phloem of the avocado tissue culture seedling; for identifying the over-expression GFP-PbHMGR1 gene, the sample for extracting the total RNA selects the leaves of the resistant bud regenerated by the leaf disc method callus; the method is used for identifying the GFP-PbHMGR1 gene after micro-grafting, and selecting the stem segment of the micro-grafted 14d scion tobacco and the rootstock tobacco stem segment from a sample for extracting total RNA.

In another aspect, the present invention provides a method for identifying HMGR1mRNA molecules by fluorescence transmitted between rootstocks and ears of plants, comprising the following steps:

(1) respectively extracting the total RNA of the pyrus betulaefolia, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification on the full length of the HMGR1 gene;

(2) constructing a carrier of a birchleaf pear HMGR1 gene carrying a fluorescent protein label, and preparing a transgenic plant;

(3) the transgenic plant is a rootstock, the transgenic plant and the wild scion are subjected to micro-grafting under the aseptic condition, nested PCR detection is adopted, and if specific strips with the length of 1084bp appear in the nest PCR product of the rootstock and the scion, the transmission of HMGR1 gene mRNA molecules among the rootstock and the scion of the plant is indicated.

In the step (3), the sequences of the primer pairs adopted by the first round of nested PCR reaction are shown as SEQ ID NO.4-5, and the sequences of the primer pairs adopted by the second round of nested PCR reaction are shown as SEQ ID NO. 6-7.

The invention provides application of the fluorescence identification method in improvement of plant germplasm resources.

The invention provides an application of the fluorescence identification method in identifying genes capable of remotely transmitting mRNA molecules between rootstocks and ears of plants.

The method provided by the invention solves the problem that the transfer of mRNA can not be observed visually after the transgenic grafting is carried out by constructing a vector with a gene carrying a specific label, and also solves the problems that the microinjection requirement and the cost for marking a specific molecular probe on the mRNA are high, the general laboratory is difficult to finish, and the RT-PCR identification of the gene transfer property is easy to be polluted. The invention utilizes the vector of the constructed gene carrying Green Fluorescent Protein (GFP) label to carry out transgenosis, carries out micro-grafting on the transgenic rootstock and the wild type scion in a sterile glass bottle, and judges whether the gene has long-distance transmission between the rootstock and the scion by detecting the existence of GFP fluorescence emitted by the adventitious root of the scion through a laser confocal microscope due to the characteristic that the scion is easy to grow the adventitious root in a humid environment, thereby finally solving the problem that whether the gene can be intuitively observed to carry out the long-distance transmission between the rootstock and the scion.

The method provided by the invention can realize rapid, sensitive and high-accuracy visual identification of whether any gene can be remotely transferred between the rootstock and the scion, and the tobacco is used as a model plant, has wide universality and wide market prospect. The method has the advantages of simple process, low requirement, high transgenic efficiency and low cost, provides a method for identifying gene transfer for researchers on the one hand, can directly observe the gene transfer on the other hand, provides direct evidence for experiments, saves the energy spent on carrying out a large number of experiments to supplement and explain problems, indirectly shortens the experiment time, saves the time cost and improves the working efficiency. The method can be widely applied to various plants, the problem that the long-distance transmission of the gene between the rootstock and the scion cannot be observed visually is solved, the problem that the requirement and the cost are high due to the microinjection of the specific molecular probe on the mRNA mark, the completion of a common laboratory is difficult, and the problem that the RT-PCR identification of the gene transmissibility is easy to pollute is solved.

Drawings

FIG. 1 shows the PCR amplification electrophoresis picture of the enzyme cutting site of the amplified Du pear HMGR1 gene. In the figure, M: DNA molecular weight standard; 1: and (4) birchleaf pear.

FIG. 2 shows a schematic diagram of the construction of the vector overexpressing GFP-PbHMGR 1.

FIG. 3 shows a: tobacco leaf disk method transgenosis; b: RT-PCR identification of a tobacco positive line of the transgenic GFP-PbHMGR1, wherein M: DNA molecular weight standards, lanes 1, 2, 3, 4, 5, 6, 7 are different tobacco lines, respectively; c: rooting the transgenic GFP-PbHMGR1 tobacco; d: transgenic GFP-PbHMGR1 tobacco flowers and fruits; e: transgenic GFP-PbHMGR1 tobacco seeds were harvested.

FIG. 4 shows a: one month after the sowing of the transgenic GFP-PbHMGR1 tobacco seeds; b: transgenic GFP-PbHMGR1 tobacco is used as a rootstock, and wild tobacco is used as scion for micro-grafting; c: the transgenic GFP-PbHMGR1 tobacco micro-grafted 14d scions grew adventitious roots.

FIG. 5 shows the observation of GFP fluorescence of adventitious roots and rootstock roots of tobacco scions, at a bar of 50 μm.

FIG. 6 shows nested PCR results of scion stem and rootstock stem, with the positive control being transgenic tobacco rootstock stem and the negative control being wild tobacco stem.

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 extraction of Gene RNA

The extraction of total RNA in the phloem of the pyrus betulaefolia adopts a CTAB method (Zhangiang et al, 2005), is dissolved in 30 mu L DEPC water, and is stored in a refrigerator at-80 ℃ for standby after electrophoresis detection.

(1) Removing DNA in RNA:

(2) treating at 37 deg.C for 30 min; adding 550 μ L RNase-free water (0.1% DEPC treated), adding 600 μ L CI with the same volume, and mixing;

(3) centrifuging at 4 deg.C at 10000rpm for 10min, sucking supernatant, adding equal volume CI, and mixing; centrifuging at 10000rpm for 10min at 4 deg.C;

(4) sucking supernatant, adding 2 times of anhydrous ethanol into the tube, and precipitating at-20 deg.C for 1 h;

(5) centrifuging at 12000rpm for 20min at 4 deg.C; discarding the supernatant, adding 1mL of 75% ethanol for rinsing and precipitating, centrifuging at 12000rpm for 5min, rinsing for 2 times, and sucking out the excessive ethanol by using a gun head;

(6) blow-drying in a clean bench, dissolving in 30-50 μ L DEPC water, and storing in a refrigerator at-80 deg.C;

(7) the integrity of the extracted nucleic acids was checked by 1% agarose gel electrophoresis and the concentration of the extracted RNA was calculated by measuring the absorbance at 260nm using an ultraviolet spectrophotometer.

The extraction of the total RNA of the plant tobacco material adopts a Trizol method to extract the total RNA from the scion stem segment and the rootstock stem segment, 30 mu L DEPC water is used for dissolving, 1 percent agarose gel electrophoresis is used for detecting the integrity of the extracted nucleic acid, an ultraviolet spectrophotometer is used for calculating the concentration of the extracted RNA by measuring the absorbance at 260nm, and the extracted RNA is placed in a refrigerator at-80 ℃ for storage after the electrophoresis detection.

Example 2 overexpression of GFP-PbHMGR1 vector construction reverse transcription of RNA into cDNA

The RNA obtained in example 1 was reverse-transcribed into cDNA according to a conventional method, and the obtained cDNA was used as a template for PCR amplification described below.

According to the sequence information of the Pyrus betulaefolia Bunge (Pyrus betulaefolia Bunge) plant HMGR1 gene, referring to the enzyme cutting site on pCAMBIA1305 vector, Bgl II and Spe I are selected as the gene insertion site by PbHMGR1CDS, the enzyme cutting site is underlined, and the primers are designed as follows:

an upstream primer: 5' -AGATCTATGGACGTCCGAAGGC-3’，(SEQ ID NO.2)

A downstream primer: 5' -ACTAGTTTAAGCGGACGCAACAG-3’(SEQ ID NO.3)。

The above primers were synthesized by Zhongmeitai and Biotechnology Ltd.

And (3) PCR reaction system: 2 XEs Taq MasterMix (Dye) was purchased from (Beijing Kang is a century Biotechnology Co., Ltd., CW0682S)

The PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 57 ℃, 2min at 72 ℃ and 34 cycles; final extension at 72 ℃ for 10 min.

And (3) detecting a PCR product: 1% agarose gel is prepared according to the size of the target fragment, 0.1% TAE electrophoresis buffer solution, electrophoresis is carried out for about 15min at a voltage of 70-110V, ethidium bromide is used for staining, and the size of the PCR product fragment is detected under an ultraviolet lamp (see figure 1).

After the target fragment is recovered and purified, the T-simple vector is connected, the escherichia coli competence DH 5 α is transformed, the colony PCR detection is carried out, the positive clone bacterial liquid is selected, sequencing is carried out by Mimetai and biotechnology limited company, and the obtained sequencing correct vector and pCAMBIA1305 plasmid are respectively subjected to double enzyme digestion by Bgl II and Spe I.

The enzyme digestion system is as follows: 10 XBuffer 2. mu.L, BglII 1. mu.L, speI 1. mu.L, plasmid DNA 16. mu.L in a total volume of 20. mu.L.

And (4) carrying out agarose gel electrophoresis on the enzyme digestion product, and recovering and connecting.

Placing in a metal bath at 16 ℃ for connection for 9-10h, transforming escherichia coli competence DH 5 α, performing colony PCR detection, selecting positive clone bacterial liquid, sequencing by Mimetai and biotechnology limited company, extracting plasmids from the obtained correctly sequenced bacterial liquid, and obtaining recombinant plasmids (shown in figure 2). Du pear GFP-Bgl II-PbHMGR 1-SpeI sequence is shown as SEQ ID NO. 1.

Example 3 preparation and transformation of Agrobacterium tumefaciens competence

1. Preparation of Agrobacterium tumefaciens competent cells

(1) Individual colonies were picked and added to 20mL YEP liquid medium containing 20mg/L Rif, shake-cultured (28 ℃,200 rpm, dark conditions) for 24-48h, and Agrobacterium was shake-cultured to an appropriate OD 600 value of 0.5.

(2) Performing ice bath for 30 min;

(3) centrifuging at 4 deg.C and 5000rpm for 10 min;

(4) operating in a super clean bench, discarding the supernatant, adding 0.15M NaCl, and resuspending;

(5) performing ice bath for 20min, and centrifuging at 6000rpm for 5 min;

(6) the supernatant was discarded and 20mM CaCl was added₂Resuspending, subpackaging, adding 200 μ L per tube, adding glycerol with the same volume of 50%, and preserving at-80 deg.C.

2. Transformation of Agrobacterium tumefaciens competent cells

(1) 100 μ L of competent cells were taken, placed on ice, and after complete lysis, the cells were gently suspended.

(2) Adding 5 μ L of the constructed plant expression vector plasmid, mixing, and ice-cooling for 30 min.

(3) And performing cold quenching in liquid nitrogen for 60 s.

(4) Heat shock at 37 deg.C for 5min, and standing on ice for 2 min.

(5) 500. mu.L of YEP medium was added and the mixture was cultured at 28 ℃ for 4 hours with shaking at 180 rpm.

(6) The cells were collected at 6000rpm at room temperature, 400. mu.L of supernatant was discarded, and the cells were suspended in the remaining medium.

(7) Bacteria were spread evenly on solid YEP medium supplemented with antibiotics 50mg/L Kan and 20mg/L Rif.

(8) The plates were incubated at 28 ℃ overnight (24-48h) in an inverted position.

Example 4 preparation of tobacco preculture and bacterial liquid

1. Tobacco pre-culture

Tobacco W38(Nicotiana tabacum var. winsconsin 38) was transformed using the leaf disc method (Murashigeet al, 1962). Cutting two sides of main vein of tobacco leaf into 0.5cm × 0.5cm slices, spreading on MS +2.0 mg/L6-BA +0.5mg/L NAA culture medium, culturing at 25 deg.C under light for 72 hr.

2. Preparation of bacterial liquid

20mL of YEP liquid culture medium containing 20mg/L Rif and 50mg/L Kan was shake-cultured for 6-9h, the Agrobacterium was grown to logarithmic plateau, and shake-cultured to an appropriate OD 600 value of 0.8-1.0.

Example 5 tobacco transformation into shoots

The infection method was described in Hotsch et al (1985) and modified. Placing the leaves in the bacterial liquid, gently shaking for 3min, taking out, removing the excess bacterial liquid with sterile filter paper, placing back to the original pre-culture solid culture medium, and performing dark culture at 25 deg.C for 48 h. Transferring into a solid culture medium (pre-culture medium + cefamycin 250mg/L) without bacteria, and continuing culturing under light. After 7-10 days, transferring to a screening solid medium (sterile solid medium + hygromycin 20mg/L) after the leaf has callus, and inducing the formation of resistant buds (see a picture of figure 3).

Cutting resistant seedlings, inoculating the cut resistant seedlings to a medium containing MS (Murashige and Skoog), IBA (1 mg/L) and cefamycin (250 mg/L) for inducing rooting, obtaining a positively regenerated transgenic plant about 20 days, extracting DNA and RNA of the transgenic plant for RT-PCR (shown in a picture b of a picture 3), carrying out amplification propagation and rooting culture on the obtained positive plant (shown in a picture c of the picture 3), after inducing rooting in a rooting medium for 2-3 weeks, keeping the transgenic plant at room temperature for one week, and transplanting the transgenic plant to culture soil (peat: vermiculite ═ 1: 1) for waiting for flowering and seed harvesting (shown in a picture d and a picture e of the picture 3).

EXAMPLE 6 sterile sowing of tobacco seeds and sterile glass bottle tobacco seedling micro-grafting

(1) Placing approximately 100 harvested seeds into a 2mL EP tube;

(2) adding 12.5% sodium hypochlorite for disinfection for 10min, and sucking the solution;

(3) washing the seeds with sterile distilled water for 5 times;

(4) the seeds are evenly sown on the MS culture medium.

Selecting rootstock transgenic tobacco and scion tobacco W38(Nicotiana tabacumvar. Winsconnin 38) seed seedlings (shown in a picture a of figure 4) after sowing for 30d, wherein the culture environment is a constant light source, the light intensity is 1500lx, the illumination is 14h/10h, the day and night cycle is carried out, the room temperature is 23-25 ℃, and the relative humidity is 85%.

Under the aseptic condition, the transgenic tobacco seedling is cut flatly and the head is removed, and a long stem section of about 1cm is reserved as a stock; taking tobacco seedlings with the height of 3-5 cm, reserving 2-3 leaves at the top ends, horizontally cutting the base parts, aligning the cuts of the stock scions, fixing the cuts by using a silica gel sleeve, and placing the cut in a culture medium MS for growth (see a picture b of figure 4).

Example 7 GFP fluorescence identification of PbHMGR1mRNA after Micrografting, transferred between transgenic tobacco rootstock and wild-type tobacco scion

At 14d after the micro-grafting, the expression of GFP fluorescent protein at 1-3mm of the root was observed with an objective lens of 20X and at 488nm excitation wavelength of excitation light using a confocal laser microscope Olympus FluoView FV1000 focal laser scanning microscopical (Olympus, Tokyo, Japan), and the adventitious roots (see the c diagram in FIG. 4) and the root segments of the rootstock transgenic tobacco grown from the wild type tobacco of the scion, respectively (see FIG. 5).

The results show that: GFP fluorescent protein is strongly expressed in the roots of transgenic tobacco plants, after grafting, GFP fluorescent protein also appears in adventitious roots grown by the scions, and the GFP fluorescent protein is not expressed in the adventitious roots grown by the wild tobacco scions grafted on wild tobacco stocks in the control group.

Example 8 nested PCR identification of PbHMGR1mRNA delivery between transgenic tobacco rootstock and wild-type tobacco scion after micrografting

Total RNA was extracted as in example 1, and nested PCR was performed after reverse transcription (see FIG. 6). The cDNA of the wild tobacco scion grafted on the wild tobacco rootstock is used as a negative control, and the cDNA of the stem segment of the transgenic tobacco rootstock is used as a positive control.

First round PCR reaction System: 2 XEs Taq MasterMix (Dye) 10. mu.L, 10. mu.L each of the primers shown in SEQ ID NO.4 and 5, 1. mu.L of cDNA, 7. mu.L of double distilled water, and 20. mu.L in total.

The PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 55 ℃, 2min at 72 ℃ and 26 cycles; final extension at 72 ℃ for 10 min.

Second round PCR reaction System: 2 XEs Taq MasterMix (Dye) 10. mu.L, 10. mu.L each of the primers shown in SEQ ID NO.6 and 7, 0.5. mu.L of the first PCR reaction product, 7.5. mu.L of double distilled water, and 20. mu.L in total.

The PCR reaction procedure was as follows: pre-denaturation at 94 ℃ for 5 min; 30s at 94 ℃, 30s at 60 ℃, 1min at 72 ℃ and 34 cycles; final extension at 72 ℃ for 10 min.

The results show that: the rootstock has 1084bp specific bands, the GFP-PbHMGR1 fusion gene is transcribed in a large quantity in a tobacco plant of the transgenic rootstock, the scion also has the specific bands of the rootstock after grafting, and the scion in a control group has no specific bands.

Although the invention has been described in detail hereinabove by way of general description, specific embodiments and experiments, it will be apparent to those skilled in the art that many modifications and improvements can be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

SEQUENCE LISTING

<110> university of agriculture in China

<120> fluorescent identification method for transmitting mRNA molecules between rootstock and scion of plant

<130>KHP161119381.3

<160>7

<170>PatentIn version 3.5

<210>1

<211>2556

<212>DNA

<213> Du pear GFP-Bgl II-PbHMGR 1-Spe I

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cccgctgcgg tgaactggat cgaaggtcgt ggaaaatcgg tggtctgtga ggctgtgatc 2040

aagggtgatg tggtgcagaa ggtgttgaaa accaatgtgg cgtccctgtg cgagcttaac 2100

atgctcaaga accttactgg gtctgcaatg gctggagccc tcggtggatt caacgcacat 2160

gccagcaaca tcgtctctgc catctacatc gctaccggcc aagacccagc tcagaatgtg 2220

gagagttctc actgcattac catgatggaa cccatcaatg atgggcagga ccttcacgtg 2280

tctgtcacca tgccttcaat tgaggttggt actgttggag gtgggaccca acttgcatct 2340

caatcagctt gtctgaacct tcttggagtg aagggtgcta acagggaggc accaggatct 2400

aatgcaagat tgttggccac tgttgtggct ggttctgttc ttgctggaga gctttctctc 2460

atgtctgcta tctcagctgg acagcttgtg aatagtcaca tgaaatacaa cagatcaagc 2520

aaagatgtct cagctgttgc gtccgcttaa actagt 2556

<210>2

<211>22

<212>DNA

<213> Artificial sequence

<400>2

agatctatgg acgtccgaag gc 22

<210>3

<211>23

<212>DNA

<213> Artificial sequence

<400>3

actagtttaa gcggacgcaa cag 23

<210>4

<211>20

<212>DNA

<213> Artificial sequence

<400>4

agaagaacgg catcaaggtg 20

<210>5

<211>23

<212>DNA

<213> Artificial sequence

<400>5

actagtttaa gcggacgcaa cag 23

<210>6

<211>20

<212>DNA

<213> Artificial sequence

<400>6

ggtggtcaag tccgtcgtgg 20

<210>7

<211>21

<212>DNA

<213> Artificial sequence

<400>7

tcctggtgcc tccctgttag c 21

Claims (10)

1. A fluorescence identification method for transmitting mRNA molecules between plant rootstock and scion is characterized in that a vector of which the gene carries a fluorescent protein label is constructed to prepare a transgenic plant; the transgenic plant is taken as a stock, the transgenic plant and the wild type scion are subjected to micro grafting under the aseptic condition, the fluorescence occurrence conditions of the stock and the scion are observed, and if fluorescence is observed on the adventitious roots of the stock and the scion, the mRNA molecules of the genes are transmitted between the stock and the scion of the plant.

2. An identification method for transmitting mRNA molecules between plant rootstock and scion is characterized in that a vector of which the gene carries a fluorescent protein label is constructed to prepare a transgenic plant; carrying out micro-grafting on a transgenic plant serving as a stock and a wild type scion under an aseptic condition, and adopting nested PCR detection to indicate that mRNA molecules of the gene are transferred between plant stocks and scions if nested PCR products of the stocks and the scions have specific bands;

the gene is birch pear HMGR 1.

3. The method of claim 1 or 2, wherein the fluorescent protein is a green fluorescent protein, a red fluorescent protein, a yellow fluorescent protein, a blue fluorescent protein, an orange fluorescent protein, or a blue-green fluorescent protein.

4. The method of claim 1, wherein the gene is pyrus betulaefolia HMGR 1.

5. The method of claim 2 or 4, wherein the vector is an over-expression GFP-PbHMGR1 vector, which is prepared by: extracting the RNA of the birch pear genome, carrying out reverse transcription to obtain cDNA, carrying out PCR amplification, recovering a target segment, purifying, connecting a T vector, transforming a competent cell, and selecting a positive clone with correct sequencing to obtain the final product, wherein the sequence of a primer pair used for PCR amplification is shown as SEQ ID NO. 2-3.

6. The method of claim 2, wherein the nested PCR reaction uses primer pairs of sequences shown in SEQ ID NO.4-5 for the first round of PCR reaction and SEQ ID NO.6-7 for the second round of PCR reaction.

7. A fluorescence identification method for HMGR1mRNA molecule transmission between rootstocks and ears of plants comprises the following steps:

(1) respectively extracting the total RNA of the pyrus betulaefolia, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification on the full length of the HMGR1 gene;

(2) constructing a carrier of a birchleaf pear HMGR1 gene carrying a fluorescent protein label, and preparing a transgenic plant;

(3) the transgenic plant is a stock, the transgenic plant and a wild type scion are subjected to micro grafting under the aseptic condition, the fluorescence occurrence conditions of the stock and the scion are observed, and if fluorescence is observed on the stock and the scion with adventitious roots, the mRNA molecules of the birch HMGR1 gene are transmitted between the stock and the scion of the plant.

8. A method for identifying the transmission of HMGR1mRNA molecules between rootstocks and ears of plants, which comprises the following steps:

(1) respectively extracting the total RNA of the pyrus betulaefolia, carrying out reverse transcription to obtain cDNA, and carrying out PCR amplification on the full length of the HMGR1 gene;

(2) constructing a carrier of a birchleaf pear HMGR1 gene carrying a fluorescent protein label, and preparing a transgenic plant;

(3) the transgenic plant is a rootstock, the transgenic plant and the wild scion are subjected to micro-grafting under the aseptic condition, nested PCR detection is adopted, and if specific strips with the length of 1084bp are generated on nested PCR products of the rootstock and the scion, the transmission of HMGR1 gene mRNA molecules among the rootstock and the scion of the plant is indicated.

9. Use of the identification method of any one of claims 1 to 8 for the improvement of germplasm resources of a plant.

10. Use of the method of any one of claims 1 to 8 for identifying a gene capable of remotely transmitting an mRNA molecule between tassels of a plant.

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