CN111593056A - Molecular identification method for transporting NAC2 gene mRNA between cucurbitaceae rootstock ears - Google Patents
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Abstract
The invention provides a molecular identification method for transporting NAC2 gene mRNA between rootstock and scion of Cucurbitaceae, which comprises the following steps: (1) self-grafting the cucumber and the pumpkin and performing hetero-grafting on the cucumber/the pumpkin and the pumpkin/the cucumber; (2) analyzing the nucleotide sequence of the cucumber NAC2 gene, searching a difference fragment of the homologous gene of the cucumber NAC2 gene and designing a specific primer of the cucumber NAC2 gene; (3) carrying out PCR amplification on self-grafted cucumbers and pumpkins, hetero-grafted cucumbers and pumpkins which grow at a proper temperature and are treated at a low temperature by using the primer, and comparing the conditions of PCR products before and after grafting; (4) gel electrophoresis was used to determine whether the correct bands were present. The method provided by the invention is rapid, sensitive, high in accuracy, simple and convenient, and can be applied to other cucurbitaceae plants.
Description
Technical Field
The invention relates to the field of plant molecular biology, in particular to a molecular identification method for long-distance transmission of NAC2 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 the cucumber is identified, and the long-distance mRNA molecular regulation mechanism among the rootstocks and the 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 (Lucas 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).
NAC2 encodes a transcription factor protein, AtNAC2 in arabidopsis thaliana, which responds to salt stress and can also be induced by ABA and NAA, and which promotes or inhibits downstream genes and plays a regulatory role in lateral root development in arabidopsis thaliana (He et al, 2005). Overexpression of wheat TaNAC2 in arabidopsis resulted in increased tolerance of arabidopsis to drought, salt and cold stress (Mao et al, 2011). This suggests that long-distance transport of NAC2 mRNA may have potential implications for the regulation of development and resistance in engrafted plants.
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 transferability of genes in phloem more accurately, the procedures are complicated, the requirements are high, the operation is not easy, and the experimental difficulty is increased.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a molecular identification method for transporting NAC2 gene mRNA between cucurbitaceae scions.
In order to achieve the above purposes, the technical scheme adopted by the invention is as follows:
a cucumber NAC2 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.
The amino acid sequence of the protein coded by the cucumber NAC2 gene is shown in SEQ ID NO. 2.
A molecular identification method for transporting NAC2 gene mRNA between rootstocks and spikes of Cucurbitaceae comprises the following steps:
(1) respectively carrying out self-grafting on the cucumber and the pumpkin; cucumber is used as a scion, and pumpkin is used as a stock for grafting; pumpkin is used as a scion, cucumber is used as a stock for grafting, and the grafting modes are hypocotyl grafting;
(2) analyzing the nucleotide sequence of the cucumber NAC2 gene, searching a difference fragment of the gene and a homologous gene of the pumpkin, and designing a specific primer of the cucumber NAC2 gene to ensure that a strip can be obtained by amplification when the cucumber cDNA is taken as a template, the strip cannot be obtained by amplification when the pumpkin cDNA is taken as the template, and the strip cannot be generated by electrophoresis;
(3) the grafted plants are divided into two groups, one group is processed at low temperature, and the other group grows at proper temperature. Each group comprises self-grafting and hetero-grafting plants of cucumber and pumpkin, RNA of the rootstock and the scion after grafting survival is extracted, reverse transcription is carried out to obtain cDNA, and the gene segment of cucumber NAC2 is amplified by PCR;
(4) gel electrophoresis to detect whether there is a correct electrophoretic band: if the RNA is transferred in the grafting system, a scion specific strip can appear in the amplification product of the rootstock, and the rootstock specific strip can also appear in the amplification product of the scion; if no transmission occurs, the electrophoretogram of the amplified product shows the strip of the corresponding sample without other miscellaneous bands, namely, the product obtained by PCR amplification of the part of the cross-grafted pumpkin by using the specific primer of the cucumber NAC2 gene is transported if the specific strip exists, and the product without the specific strip is not transported.
On the basis of the scheme, the specific process of the step (1) comprises the following steps:
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 a knife at a position 1cm below the stock cotyledon, wherein the length of a cut plane is consistent with that of the inclined plane of the scion, aligning and tightly adhering the scion and the cut plane of the stock, and fixing the scion and the cut 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.
On the basis of the scheme, the specific primer sequence designed in the step (2) is as follows:
upstream primer 5'-CTCCGTCCCCAGAATGCACA-3'
The downstream primer 5'-CAAATGGAACGTCCTCTGGCATA-3'.
On the basis of the scheme, RNA of the root and the first true leaf of the grafted plant in the period of two leaves and one heart of the grafted plant which is processed at low temperature and grows at the proper temperature is extracted in the step (3) for reverse transcription.
Based on the above scheme, the PCR amplification reaction procedure in step (3) is as follows:
pre-denaturation at 95 ℃ for 3 min;
95℃ 30s；
60℃ 30s；
72℃ 1min；
39 cycles;
final extension at 72 ℃ for 5 min.
The invention protects the application of the molecular identification method in identification of inter-tassel NAC2 mRNA molecular transmission of cucurbitaceae plants.
Has the advantages that: the method provided by the invention is rapid, sensitive, high in accuracy, simple and convenient, and can be applied to other cucurbitaceae plants.
Drawings
The invention has the following drawings:
FIG. 1 is a schematic diagram showing the results of detection of transportability assay.
FIG. 2 shows a comparison of the amino acid sequence homology of NAC 2. Cucurbita moschata in the figure: pumpkin; cucurbita pepo: zucchini; cucurbita maxima: winter squash; cucumis sativus: cucumber; cucumismelo: muskmelon; momordia charrantia: bitter gourd is provided.
FIG. 3 shows a phylogenetic tree analysis of the amino acid sequence of NAC 2.
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 a knife at a position 1cm below the stock cotyledon by using the blade, wherein the length of a cut surface is consistent with that of the scion, aligning and tightly adhering the scion and the cut surface of the stock, and fixing the scion and the cut surface 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: test group cryo-treatment
Selecting grafted seedlings which are grafted to survive and grow consistently, culturing the grafted seedlings in an incubator at the temperature of 28 +/-2 ℃, and dividing the grafted seedlings into two groups after the grafted seedlings grow to two leaves and one heart, wherein each group comprises self-grafting and different grafted seedlings of cucumbers and pumpkins. One group was transferred to an incubator at 4 ℃ for 6 hours, and the other group was maintained under the same conditions, after which example 3 was carried out promptly.
Example 3: total RNA extraction
Total RNA was extracted using CTAB method, which comprises the following steps:
(1) 2g of the first true leaf or root is taken and ground into fine powder in liquid nitrogen.
(2) 10ml of 65 ℃ preheated CTAB extract and 500. mu.l of beta-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 1/3 volumes of 8M LiCl and 500. mu.l of beta-mercaptoethanol were added and precipitated overnight at-20 ℃.
(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, and after mixing, 5ml of phenol: chloroform: isopentanol (25: 24: 1), vortex and mix well, ice bath 5 min. Centrifuge at 12000rpm for 10min at 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 anhydrous ethanol into the rest samples, and storing at-70 ℃.
Example 4: reverse transcription of RNA into cDNA
Using TaKaRa Biochemical reagent, PrimeScriptTMRT 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.
(2) Reverse transcription reaction
The reaction solution 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 >
Example 5: transport assay
Specific primers are designed according to different fragments of NAC2 genes of cucumbers and pumpkins, so that the specific primers of the cucumbers cannot generate bands in pumpkin cDNA.
The designed specific primer sequences are as follows:
cucumber: upstream primer 5'-CTCCGTCCCCAGAATGCACA-3'
Downstream primer 5'-CAAATGGAACGTCCTCTGGCATA-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). Wherein S is the scion receiving portion and R is the anvil portion. Observing the bands, if the PCR product taking the cucumber cDNA as a template and the cucumber specific primer PCR product has bands, and the PCR product taking the cDNA of the heterograft pumpkin part as a template also has bands, the mRNA generated by NAC2 is proved to be transported in the phloem of the grafted plant, and the opposite is also true.
Example 6: bioinformatics analysis of pumpkin and cucumber NAC2 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 MEGA and the like is used for carrying out multiple sequence alignment analysis and evolutionary tree drawing analysis.
Blast alignment on NCBI showed: the amino acid sequence of the cucumber NAC2 gene has higher similarity with melon (Cucumis melo), winter squash (Cucurbita maxima), pumpkin (Cucurbita moschata) and bitter gourd (Momoradica charrantia). The amino acid sequence encoded by the cucumber NAC2 gene comprises 1 domain (fig. 2), which is identical to the NAM superfamily domain in genes known from other plants.
A phylogenetic tree of NAC gene amino acid sequences of 14 plants such as cucumber, pumpkin and the like is constructed through MEGA. The evolutionary tree shows that the amino acid sequence of cucumber NAC2 has a close relationship with melon (Cucumis melo) and a distant relationship with Nicotiana benthamiana (Nicotiana benthamiana) and tomato (Solanum lycopersicum). But most distant from Arabidopsis thaliana (Arabidopsis thaliana), barley (Hordeum vulgare), maize (Zea mays), Japonica rice (Oryza sativa Japonica Group) and Indica rice (Oryza sativa Indica Group) (FIG. 3). 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> molecular identification method for transporting NAC2 gene mRNA between rootstock and scion of Cucurbitaceae
<160>2
<170>PatentIn version 3.3
<210>1
<211>471
<212>DNA
<213> cucumber (Cucumis sativus)
<400>1
atgaatatgc agttggacga ctgggtgcta tgtcgtatct acaacaagaa aggttgtata 60
gaaaaacatt accaatccac cgacgacaag gcagccgaat tccctgattt cgaggatgag 120
aaacccaata ttaccaccaa caacgacatg gtacaactcc ctatccacaa ccagttgcaa 180
atggagacct ccgactccgt ccccagaatg cacaccgaat cctccagtgg ctcggatccc 240
gtgacgtcgc cggagctcac ctgggataag gaggtccaaa gtcaatccaa atgggaagga 300
gaaggaggag gagaaagagc agcagtagct gatttcgact tctttgagtt caattacatg 360
gattcgttct ctatgccaga ggacgttcca tttggaagcc aggttcagtt ccagatggac 420
catctgtcgc cattacagga catgttttct tatctacaga ggcagattta a 471
<210>2
<211>156
<212>PRT
<213> cucumber (Cucumis sativus)
<400>2
Met Asn Met Gln Leu Asp Asp Trp Val Leu Cys Arg Ile Tyr Asn Lys
1 5 10 15
Lys Gly Cys Ile Glu Lys His Tyr Gln Ser Thr Asp Asp Lys Ala Ala
20 25 30
Glu Phe Pro Asp Phe Glu Asp Glu Lys Pro Asn Ile Thr Thr Asn Asn
35 40 45
Asp Met Val Gln Leu Pro Ile His Asn Gln Leu Gln Met Glu Thr Ser
50 55 60
Asp Ser Val Pro Arg Met His Thr Glu Ser Ser Ser Gly Ser Asp Pro
65 70 75 80
Val Thr Ser Pro Glu Leu Thr Trp Asp Lys Glu Val Gln Ser Gln Ser
85 90 95
Lys Trp Glu Gly Glu Gly Gly Gly Glu Arg Ala Ala Val Ala Asp Phe
100 105 110
Asp Phe Phe Glu Phe Asn Tyr Met Asp Ser Phe Ser Met Pro Glu Asp
115 120 125
Val Pro Phe Gly Ser Gln Val Gln Phe Gln Met Asp His Leu Ser Pro
130 135 140
Leu Gln Asp Met Phe Ser Tyr Leu Gln Arg Gln Ile
145 150 155
Claims (8)
1. A cucumber NAC2 gene, the nucleotide sequence of which is shown in SEQ ID NO. 1.
2. Cucumber NAC2 gene encoding protein according to claim 1, having the amino acid sequence shown in SEQ ID No. 2.
3. A molecular identification method for transporting NAC2 gene mRNA between rootstocks and spikes of Cucurbitaceae is characterized in that: the method comprises the following steps:
(1) respectively carrying out self-grafting on the cucumber and the pumpkin; cucumber is used as a scion, and pumpkin is used as a stock for grafting; pumpkin is used as a scion, cucumber is used as a stock for grafting, and the grafting modes are hypocotyl grafting;
(2) analyzing the nucleotide sequence of the cucumber NAC2 gene, searching a difference fragment of the gene and a homologous gene of the pumpkin, and designing a specific primer of the cucumber NAC2 gene to ensure that a strip can be obtained by amplification when the cucumber cDNA is taken as a template, the strip cannot be obtained by amplification when the pumpkin cDNA is taken as the template, and the strip cannot be generated by electrophoresis;
(3) dividing grafted plants into two groups, wherein one group is processed at low temperature, and the other group grows at proper temperature; each group comprises self-grafting and hetero-grafting plants of cucumber and pumpkin, RNA of the rootstock and the scion after grafting survival is extracted, reverse transcription is carried out to obtain cDNA, and the gene segment of cucumber NAC2 is amplified by PCR;
(4) gel electrophoresis to detect whether there is a correct electrophoretic band: if the RNA is transferred in the grafting system, a scion specific strip can appear in the amplification product of the rootstock, and the rootstock specific strip can also appear in the 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.
4. The method for molecular characterization of NAC2 gene mRNA trafficking between cucurbitaceae tassels as claimed in claim 3, wherein: the specific process of the step (1) comprises the following steps:
1) cultivating scions and rootstocks: 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 a knife at a position 1cm below the stock cotyledon, wherein the length of a cut plane is consistent with that of the inclined plane of the scion, aligning and tightly adhering the scion and the cut plane of the stock, and fixing the scion and the cut plane of the stock by using a grafting clip; then grafting the cotyledon part of the rootstock to the section of the original scion, and fixing the grafting opening by using a grafting clamp;
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.
5. The method for molecular characterization of NAC2 gene mRNA trafficking between cucurbitaceae tassels as claimed in claim 3, wherein: the specific primer sequence designed in the step (2) is as follows:
upstream primer 5'-CTCCGTCCCCAGAATGCACA-3'
The downstream primer 5'-CAAATGGAACGTCCTCTGGCATA-3'.
6. The method for molecular characterization of NAC2 gene mRNA trafficking between cucurbitaceae tassels as claimed in claim 3, wherein: and (3) respectively extracting RNA of roots and first true leaves of the grafted plants which are subjected to low-temperature treatment and grow at a proper temperature in a one-heart period, and performing reverse transcription.
7. The method for molecular characterization of NAC2 gene mRNA trafficking between cucurbitaceae tassels as claimed in claim 3, wherein: the PCR amplification reaction procedure in step (3) is as follows:
pre-denaturation at 95 ℃ for 3 min;
95℃30s；
60℃30s；
72℃1min；
39 cycles;
final extension at 72 ℃ for 5 min.
8. Use of a molecular identification method according to any of claims 3 to 7 for the identification of inter-tassel NAC2 mRNA molecular delivery in cucurbits.
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