CN118726382B - Application of cucumber CsaTRM4 gene in promoting long-distance transport between pumpkin CmoCK1 rootstock and scion - Google Patents

Abstract

The present invention relates to an application of a cucumber CsaTRM4 gene in promoting long-distance transport between pumpkin CmoCK1 rootstocks and scions. The cucumber CsaTRM4 gene provided by the present invention can promote the long-distance transport of pumpkin rootstock CmoCK1 mRNA. The tobacco transient transformation system is used to co-express CsaTRM4 and CmoCK1, thereby improving the transport efficiency of CmoCK1. The new function of CsaTRM4 described in the present invention has good application potential, and provides gene resources and new ideas for rootstock breeding research. The CsaTRM4 gene can promote the long-distance transport of the key cold-resistant gene CmoCK1 mRNA, and has broad application prospects and high use value.

Description

Application of cucumber CsaTRM gene in promoting long-distance transportation of pumpkin CmoCK between ears

Technical Field

The field of plant molecular biology, in particular to application of cucumber CsaTRM gene in promoting long-distance transportation between scions of pumpkin rootstock CmoCK 1.

Background

Cucumber (cucumber sativus L.) is one of the main facility cultivated vegetable crops in China, is typical warm-loving vegetable, has poor low temperature tolerance, and grafting as a wide cultivation means has become one of the supporting measures of facility melon and solanaceous vegetable industrialized seedling cultivation and winter and spring cultivation. The pumpkin stock not only can maintain the excellent characters of cucumber cultivars, but also can improve disease resistance and stress resistance, especially low-temperature resistance. The cucumber scions regulated by different stocks have different properties, so that the molecular mechanism of the li qing stock scion interaction is always the research focus of the facility cucumber cultivation. The research on grafting to improve the stress resistance of cucumbers is focused on the aspects of phenotype change, nutrient absorption, hormone balance and the like, and with the development of molecular biology and bioinformatics, the discovery and deep research on the transportation of macromolecular substances such as mRNA, protein and the like as long-distance signals among the spikes provides a powerful basis for revealing the interaction mechanism of the spikes and accurately regulating and controlling the yield, quality and stress resistance. Previous studies have discovered the key gene CHOLINE KINASE (CHOLINE KINASE 1, cmock1) for low temperature resistance of cucumbers, and have clarified the function of CmoCK for improving low temperature resistance of cucumber scions by mRNA transport.

The interaction between stock and scion regulates and controls the high yield, disease resistance and stress resistance of the grafted plant. In recent years, bioinformatics analysis shows that a large amount of RNA signal communication exists between the stock ears of the grafted plants, and at present, m ⁵ C methylation modification is a relatively clear mRNA long-distance transport regulating factor, and the mRNA transport property of AtTCTP (Yang et al, 2019) is regulated in Arabidopsis thaliana. NOL1/NOP2/sun (Nsun) family proteins are responsible for "writing" m ⁵ C to RNA, during which these proteins catalyze the methyl transfer of S-adenosylmethionine to cytosine.

N6-methyladenosine (m ⁶ A) and 5-methylcytosine (m ⁵ C) are the most common RNA methylation modifications in plants. These modifications play a role in many developmental processes, such as embryonic development, stem cell fate, trichome branching, leaf morphogenesis, flower transformation, stress, fruit ripening, and root development. Cucume is a database previously established by the inventors, in which cucumber and pumpkin m ⁶ a and m ⁵ C RNA methylation sites can be queried at present.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide an application of cucumber CsaTRM4 gene in promoting long-distance transportation between the pumpkin CmoCK and the scion.

In order to achieve the above purpose, the invention adopts the following technical scheme:

the application of the cucumber CsaTRM gene is characterized in that the cucumber CsaTRM gene is overexpressed in plants, so that long-distance transportation of pumpkin CmoCK1 mRNA between the ears can be promoted;

The nucleotide sequence of the cucumber CsaTRM gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cucumber CsaTRM gene is shown as SEQ ID NO. 2.

The primer pair for amplifying the cucumber CsaTRM gene is characterized in that the nucleotide sequence of the primer pair is shown as SEQ ID NO. 3-4.

The method for obtaining the cucumber CsaTRM gene knockout mutant is characterized by comprising the following steps of:

Step 1, constructing a CsaTRM4 CRISPR/Cas9 vector, and performing four-primer PCR amplification by taking pCBC-DT1T2 plasmid as a template, wherein the four-primer sequence is shown as SEQ ID NO. 15-18;

The four primer sequences are specifically as follows:

DT1-F₀ 5‘-TGATGAAATCTCTTCAAGCCGGTTTTAGAGCTA GAAATAGC-3’;

DT1-R₀ 5‘-AACAACGTTCTCTCTGTTGTCGCAATCTCTTAGT CGACTCTAC-3’;

DT1-BsF 5‘-ATATATGGTCTCGATTGATGAAATCTCTTCAAG CCGGTT-3’;

DT1-BsR 5‘-ATTATTGGTCTCGAAACAACGTTCTCTCTGTTG TCGCAA-3’。

Performing enzyme digestion-connection on a gel recovery product and a p HSE401 plasmid;

step 2, carrying out genetic transformation on the cucumber by using the enzyme digestion-connection product obtained in the step 1;

And 3, selecting the genetic transformation explant obtained in the step 2 through Basta to obtain CsaTRM gene knockout plants, namely cucumber Csatrm mutant.

The application of the cucumber CsaTRM gene in promoting long-distance transportation between the pumpkin CmoCK1 and the scion has the beneficial effects that:

The CsaTRM gene provided by the invention can promote long-distance transportation of pumpkin stock CmoCK1 mRNA. The transient transformation system of tobacco is used for coexpression CsaTRM and CmoCK1, so that the transport efficiency of CmoCK is improved, and the knockout CsaTRM4 reduces the transport efficiency of CmoCK1 in the rooting transformed cucumber. The novel function of CsaTRM < 4 > has better application potential, provides gene resources and novel thought for stock breeding research, and the CsaTRM gene can promote long-distance transportation of the mRNA of the key low-temperature resistant gene CmoCK < 1 >, so that the novel function has wide application prospect and higher use value.

Drawings

The invention has the following drawings:

FIG. 1 is a diagram of CsaTRM4 gene cloning;

FIG. 2 is a CsaTRM amino acid phylogenetic tree analysis;

FIG. 3 is a diagram of a CsaTRM-4 promoter cis-acting element analysis;

FIG. 4 is a schematic diagram of CmoCK gene structures and methylation sites;

FIG. 5 is a schematic representation of transient transformed tobacco;

FIG. 6 is a schematic representation of a carriage analysis of CmoCK A mRNA in tobacco;

FIG. 7 is a graph showing the results of identification and phenotypic analysis of cucumber mutant Csatrm 4;

FIG. 8 is a schematic representation of identification of hairy root transformed positive plants;

FIG. 9 is a graph showing the results of the transportation analysis of CmoCK1 in cucumber WT and Csatrm.sup.4 mutants.

Detailed Description

The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.

Example 1 cucumber and pumpkin cultivation method

Cucumber and pumpkin seedling growing conditions are that seeds are soaked in warm water at 55 ℃ for 30 minutes, stirred twice, soaked in distilled water for 3-6 hours, then placed on wet filter paper, the filter paper is placed in a culture dish covered by a cover for preserving heat, the temperature is kept constant at 28 ℃ in a constant temperature box, and light-shielding germination acceleration is carried out for 24 hours. The germinated seeds are sown in 32-cavity trays, the substrate in the trays is prepared by the proportion of grass carbon to vermiculite=3:1 (v/v), then the films are covered for moisture preservation and placed in an illumination incubator for cultivation, the films are removed when subarch soil is planted after 2-3 days, the illumination intensity of the incubator is 600 mu mol.m ^-2·s^-1, the illumination is 16 hours/8 hours in the dark, and the illumination is 28 ℃/18 ℃ in the dark.

The greenhouse cultivation condition is that cucumber seedlings are planted in a sunlight greenhouse when two leaves and one heart are grown, and the cucumber seedlings are routinely managed.

Example 2 extraction of plant RNA

Total RNA extraction using TRIzol RNA extraction kit:

1) Fresh tissue within 100mg was placed in a 2mL round bottom centrifuge tube while adding 4mm diameter steel balls, then placed in liquid nitrogen for quick freezing, and the tissue was sufficiently finely ground by a tissue grinder.

2) Transferring the ground sample into a centrifuge tube, adding RNA lysate, standing, and continuously blowing until the lysate is transparent after the sample is completely melted.

3) Centrifuge at 12000rpm for 5 min at 4℃and aspirate the supernatant.

4) 1/5 Volume of chloroform was added to the above lysate. Shaking vigorously for 15 seconds to form emulsion, and standing at 4 ℃ for 5 minutes.

5) Centrifuge at 12000rpm at 4℃for 15 min.

6) The upper aqueous phase was carefully aspirated into a new enzyme-free centrifuge tube.

7) Adding an equal volume of precooled isopropanol, mixing uniformly upside down, and standing for 10 minutes at 4 ℃.

8) Centrifuging at 12000rpm and 4 ℃ for 10 minutes, and obtaining white precipitate which is RNA.

9) The supernatant was carefully discarded, and 1mL of 75% ethanol (prepared by RNase-free ddH 2O) was added. Flick the bottom of the tube, suspend and precipitate, reverse the tube upside down for several times, and stand at room temperature for 3-5 minutes.

10 Centrifugation at 12000rpm for 5 min at 4 ℃, the supernatant was discarded.

11 The precipitate was dried at room temperature for 2-5 minutes.

12 40-60. Mu.L of RNase-free ddH2O was added to dissolve the precipitate, and after complete dissolution, 1. Mu.L was taken and stored in a-80℃refrigerator after RNA concentration was detected by Nanodrop.

EXAMPLE 3 reverse transcription of RNA into cDNA

Press and vogueIIQ RT SuperMix for qPCR (+ GDNA WIPER) (R223-01) kit steps.

Genomic DNA removal:

the following systems were formulated in rnase-free centrifuge tubes:

gently beating and mixing by a pipette. 42 ℃ for 2min.

The reverse transcription reaction system was prepared as follows:

sucking and beating the mixture by a pipette, and uniformly mixing the mixture. 50 ℃ for 15min,85 ℃ and 5s.

Example 4 full Length cloning of cucumber CsaTRM4 and pumpkin CmoCK1 CDS

The sequence of Arabidopsis m ⁵ C methyltransferase AtTRM B is known, homologous genes thereof are searched in a melon genome database and named CsaTRM4, and cloning primers are designed according to the CDS sequence.

The designed primer sequences were as follows:

An upstream primer 5'-ATGGGTGGTAAGGGTAGGGG-3' (SEQ ID NO. 3);

A downstream primer 5'-TTACAAATCATTTTCAAGTC-3' (SEQ ID NO. 4).

Pumpkin stock CmoCK A mRNA is known to be transported to cucumber scions under low temperature stress, and the full length of the pumpkin stock is cloned by designing primers according to CDS sequences of the pumpkin stock.

The designed primer sequences were as follows:

An upstream primer 5'-ATGGCAATAAAGTCAAACGG-3' (SEQ ID NO. 5);

A downstream primer 5'-TTAGGAGGCATTCATCATTA-3' (SEQ ID NO. 6).

The primers were synthesized by Beijing Optimu Biotechnology Co.

PCR was performed using cDNA or plasmid as template and high fidelity DNA polymerase premix. The amplification system is as follows:

Amplification system (50. Mu.L)

After blowing and mixing, centrifuging to the bottom of the tube, and performing PCR (polymerase chain reaction) by the following procedures:

Reaction conditions

Electrophoresis of PCR products and (5) cutting and recycling after detection. PCR product detection, namely preparing 1% agarose gel according to the size of a target fragment, adding nucleic acid dye (ten thousandth), 1 xTAE electrophoresis buffer, and carrying out 110-120v voltage electrophoresis for about 25min, and detecting the size of the PCR product fragment under an ultraviolet lamp (figure 1). And (5) cutting the target strip and recycling.

Example 5 CsaTRM4 phylogenetic analysis

CsaTRM4 homology alignment and sequence acquisition the genome databases used for acquisition of sequences were Cu GenDB (http:// cucurbstgenomics. Org /), TAIR (https:// www.arabidopsi s. Org /) and NCBI (https:// www.ncbi.nlm.nih.gov /), respectively, nucleic acid and amino acid sequence alignment analysis was performed using MEGA-X after acquisition of cucumber, arabidopsis and human homology sequences, and the evolutionary tree was drawn using TBtools software. As a result, as shown in FIG. 2, it was found that there were 5 Nsun RNA methyltransferase family genes of cucumber, which belong to 5 subfamilies respectively, and there were only 1 cucumber genes in the same subfamily as the model plant Arabidopsis AtTRM B, namely CsaTRM.

Example 6 CssaTRM 4 promoter analysis

The gene promoter sequence (2000 bp upstream of the 5' end) was extracted using TBtools in the CsaTRM4 genome, and then possible cis-acting elements on the promoter were analyzed using the MEME insert on TBtools and pictures were generated. The CsaTRM4 promoter was found to contain cis-acting elements such as light response, abscisic acid response, gibberellin response, zein metabolic regulation, salicylic acid response, low temperature response, and drought induction (FIG. 3), and CsaTRM was presumed to be involved in the regulation of various biological processes in response to stress.

Example 7 CmoCK1 Gene Structure and m ⁵ C methylation site analysis

The exon and intron regions of the pumpkin CmoCK1 gene were extracted from gff3 file using Tbtools and mapped, and the methylation site on CmoCK mRNA was queried on CUCUME website, cmoCK1 was found to contain the m ⁵ C methylation site, located in the 3' region of CDS (906-1086 bp) (fig. 4).

Example 8 Agrobacterium-mediated transient transformation of tobacco leaves

Vector construction

The invention adopts Gateway method to construct plant expression vector, firstly constructs intermediate vector containing target gene, adopts nuuzan recombination cloning kit:

seamless cloning reaction system (10. Mu.L)

The system is blown and evenly mixed, the instant low-speed centrifugation is carried out, the reaction is carried out for 30 minutes at 37 ℃, and the temperature is reduced to 4 ℃ on ice, thus the competent escherichia coli can be transformed. And (3) picking the monoclonal, then sending the monoclonal to sequencing, and carrying out large-volume shake culture to extract plasmids after the sequencing is correct.

And connecting the pENTR4 vector with the target gene fragment in a seamless cloning mode to successfully construct an intermediate vector, and then carrying out LR reaction.

LR reaction system (5 μL)

The reaction was terminated by adding 1. Mu.L of proteinase K in a 25℃metal bath for 1 hour at 37℃for 10 minutes. And (5) completing Gateway vector construction.

Plasmid transformation Agrobacterium competence

The experiment adopts a unique biological agrobacterium competent cell GV3101, a Gateway carrier successfully constructed and GV3101 competent are dialed and mixed uniformly in a centrifuge tube, incubated on ice for 5 minutes, liquid nitrogen for 37 ℃ for 5 minutes and ice for 5 minutes, then a non-resistant LB liquid medium is added for 28 ℃ shaking table for 3 hours, and the culture medium is centrifuged at 5000rpm for 1 minute to collect the thalli, and the thalli is coated on a resistance medium plate containing kanamycin and rifampicin to screen positive clones. Namely, a 35S:: YFP-CsaTRM4 over-expression vector and a 35S:: YFP-CmoCK1 over-expression vector are successfully constructed.

Tobacco cultivation

The seeds of Nicotiana benthamiana (Nicotiana benthamiana) are spread on a substrate for film covering, and are placed in an illumination incubator, and after about 1 week, good tobacco seedlings are transplanted into a ten cm square nutrition pot for cultivation. The incubator temperature was 25 ℃/18 ℃ and the illumination and photoperiod were the same as for cucumber.

Transient transformation of tobacco lamina

After tobacco had grown to 4 weeks of age, agrobacterium GV3101 containing the gene fragment of interest was added to LB liquid medium containing the corresponding resistance, shake cultured to OD ₆₀₀ = 0.6-0.8, centrifuged at 4000rpm for 8 min to collect the cells, resuspended in an equal volume of invasive solution (10 mM MES, 100. Mu.M AS and 10mM MgCl ₂), and incubated at 28℃for 3 hours in the absence of light. 100. Mu.L of the infection solution containing the corresponding Agrobacterium was injected into the back of the 1 st, 3 rd and 5 th leaves by a 1mL syringe without a needle, the bacteria solution was not permeated onto the main vein, the excessive bacteria solution was washed out by spraying deionized water, and after 12 hours of light-shielding, fluorescence was observed or sampling was performed after growth for about 60 hours under normal conditions (FIG. 5). The 1 st, 3 rd and 5 th leaves are mixed and sampled as injection leaf samples, the 2 nd, 4 th and 6 th leaves are mixed and sampled as detection leaf samples (n is more than or equal to 6), and injection leaf and detection leaf RNA are respectively taken after 48h of tobacco injection and RT-PCR is carried out. Specific combinations of transient expression treatments are shown in the following table.

Transient expression tobacco treatment combinations

EXAMPLE 9 analysis of CmoCK mRNA trafficking in transient tobacco transformation System

The transportability of CmoCK1 was identified by transient expression in tobacco leaves under different treatment conditions. The 35S:: YFP-CsaTRM4 overexpression vector was successfully constructed and the 35S:: YFP-CmoCK1 overexpression vector was transformed into Agrobacterium and co-expressed in tobacco (CsaTRM 4+ CmoCK 1), and the long distance transportability of CmoCK mRNA in tobacco non-injected leaves was examined by RT-PCR compared to tobacco with only 35S: YFP-CmoCK1 transient overexpression (FIG. 6A). Specifically, the expression of YFP-CmoCK1, YFP-CsaTRM and Basta R gene fragments was detected by RT-PCR in the injection and detection leaves of the co-expressed (CsaTRM 4+ CmoCK 1) tobacco, respectively, and the expression of YFP-CmoCK, basta R gene fragments was detected by RT-PCR in the injection and detection leaves of the tobacco expressing CmoCK1 only. Basta R is a gene that is not transported on a vector and serves as a pollution control. NtActin is used as RNA extraction quality and loading control. The absence of expression of the Basta R gene in the test leaves indicates the elimination of contamination by the bacterial cells, and the expression of YFP-CmoCK1 in the test leaves indicates that it is transported from the injection leaves. Transport efficiency= (number of tobacco transported by YFP-CmoCK/number of tobacco treated by all transient expression) ×100%. The primers used were:

YFP-CmoCK1 upstream primer 5'-CCCGACAACCACTACCTGAG-3' (SEQ ID NO. 7);

YFP-CmoCK1 downstream primer 5'-TGGCCTTTCGTGAATCCGTT-3' (SEQ ID NO. 8).

Basta R upstream primer 5'-AAACCCACGTCATGCCAGTT-3' (SEQ ID NO. 9);

Basta R downstream primer 5'-CATCGAGACAAGCACGGTCA-3' (SEQ ID NO. 10).

YFP-CsaTRM4 upstream primer 5'-GAGCAAAGACCCCAACGAGA-3' (SEQ ID NO. 11);

YFP-CsaTRM4 downstream primer 5'-ACTCATTGAACGCGGGACTT-3' (SEQ ID NO. 12).

NtActin upstream primer 5'-CTGAGAGATTCCGCTGC-3' (SEQ ID NO. 13);

NtActin A downstream primer 5'-GAGGACAATGTTTCCGTAC-3' (SEQ ID NO. 14).

The results indicate that CsaTRM4 by itself is not transportable and that overexpression CsaTRM increases the transport efficiency of CmoCK1 (B in fig. 6). The CsaTRM4 has been shown to promote long-distance transport of CmoCK1 mRNA.

Example 10 construction of methyltransferase CsaTRM4 CRISPR/Cas9 vector

The method comprises the steps of designing CsaTRM gene editing targets by using a CRISPR-P2.0 website, constructing a CsaTRM-CRISPR gene editing vector, specifically searching a target gene sequence in a melon genome database (CuGenDB), selecting 2 targets by using the CRISPR-P2.0 website, and designing 2 pairs of primers DT1-BsF/R, DT1-F ₀/R₀. Four-primer PCR amplification was performed using pCBC-DT1T2 plasmid as template.

The designed primer sequences were as follows:

DT1-F₀ 5‘-TGATGAAATCTCTTCAAGCCGGTTTTAGAGCTA GAAATAGC-3’(SEQ ID NO.15)

DT1-R₀ 5‘-AACAACGTTCTCTCTGTTGTCGCAATCTCTTAGT CGACTCTAC-3’(SEQ ID NO.16)

DT1-BsF 5‘-ATATATGGTCTCGATTGATGAAATCTCTTCAAG CCGGTT-3’(SEQ ID NO.17)

DT1-BsR 5‘-ATTATTGGTCTCGAAACAACGTTCTCTCTGTTG TCGCAA-3’(SEQ ID NO.18)

Amplification system (50. Mu.L)

Blowing and mixing uniformly, and performing PCR reaction after instantaneous centrifugation.

PCR program

And (5) identifying and recovering the PCR product gel. The gel recovery product was subjected to the enzyme digestion-ligation treatment as follows:

enzyme cutting-connecting system (20 mu L)

After the system was mixed, the mixture was centrifuged to perform PCR.

PCR program

The PCR product was purified and recovered.

Example 11 cucumber genetic transformation

Preparation of relevant Medium

Germination Medium (GM) 6-Benzylaminopurine (BA) and 1mg/L abscisic acid (ABA) were added to MS solid medium at a final concentration of 0.5 mg/L.

Inoculating Medium (IM) 0.5mg/L BA,1mg/L ABA, 200. Mu.M Acetosyringone (AS) and 1.25M morpholinoethanesulfonic acid (MES) (pH 5.2) were added to MS liquid medium.

Co-culture Medium (COM) 0.5mg/L BA, 1mg/L ABA, 200. Mu.M AS, 1.25mM MES (pH 5.2) and 250. Mu.M LA were added to MS solid medium.

Bud induction Medium (SIM) 0.5mg/L BA, 2mg/L AgNO ₃, 1mg/L ABA and 200mg/L timentin were added to MS solid medium.

Root Induction Medium (RIM) 200mg/L Tim and 2mg/L AgNO ₃ were added to MS solid medium.

Sowing and germination

Seeding, namely taking a certain amount of cucumber seeds, soaking the cucumber seeds in distilled water at 55 ℃ for 30 minutes, standing the cucumber seeds at room temperature for 1-2 hours, and then peeling off the exocarp by using forceps. The cucumber seeds were sterilized in an ultra clean bench by adding 75% alcohol, shaking upside down for 30 seconds, discarding the alcohol and washing once with distilled water, adding 3% NaClO solution, shaking for 10 minutes in a shaker, washing 5 times with distilled water. After disinfection, the seeds are placed on a germination medium, and about 25 seeds are placed on each plate. Plates were incubated at 28℃for 36 hours in the dark.

Agrobacterium preparation

The successfully constructed CRISPR/Cas9 plasmid and GV3101 competent are stirred and mixed uniformly in a centrifuge tube, incubated on ice for 5 minutes, liquid nitrogen for 5 minutes, and at 37 ℃ for 5 minutes, and then added with a non-resistant LB liquid medium for 28 ℃ shaking culture for 3 hours, centrifuged at 5000rpm for 1 minute to collect the bacterial cells, and plated on a resistant medium plate containing kanamycin and rifampicin to screen positive clones.

Picking up monoclonal and culturing in LB liquid medium containing corresponding antibiotics under 28 ℃ with shaking, centrifuging at 4000rpm for 8 minutes when the bacterial liquid concentration is OD ₆₀₀ between 0.6 and 0.8, collecting agrobacterium, washing once with 10mL of MS liquid medium, centrifuging at 4000rpm for 8 minutes, re-suspending the bacterial bodies with MS liquid and diluting to OD ₆₀₀ value of 0.2-0.3, and standing for later use.

Explant preparation

The radicle of the germinated cucumber seed prepared previously is removed by forceps in an ultra-clean bench, the tissue beside the radicle is not destroyed as much as possible, then one third of the cotyledon far away from one end of the radicle is excised, the inner seed coat is removed, and two cotyledons are thoroughly separated and opened in MS liquid culture medium for standby.

Agrobacterium infection explant

And (3) carrying out infection twice by using a method of vacuumizing a syringe for 1.5 minutes each time, pouring out bacterial liquid after the infection is finished, and flatly placing the explant on filter paper and slightly sucking the attached bacterial liquid. And then placed on IMS differentiation medium in order. After sealing, co-cultivation was performed for 3 days under a dark condition at 25 ℃.

Explant differentiation culture

The explants were transferred to differentiation medium, approximately 9 explants per plate, and cultured in a tissue culture chamber for approximately 3 weeks.

Bud resistance screening and rooting culture

The buds newly grown on the cucumber explants in the last step are cut off and inserted into a screening culture medium (0.006% Basta is added to a differentiation culture medium) for three days, the buds grow well after three days, the buds which do not turn yellow possibly have Basta resistance, and the buds are transferred to a rooting culture medium and are subjected to secondary culture until a large number of roots are grown.

Domestication and transplantation

When the rooted buds continue to grow and develop into new plants, opening a culture bottle, taking out seedlings from the culture bottle after a plurality of days, washing off culture medium on roots, transplanting the seedlings into a culture pot with a sterilizing matrix, sleeving a fresh-keeping bag for moisturizing, growing in an illumination incubator until domestication is completed, planting the seedlings in a sunlight greenhouse after survival, and carrying out cultivation management normally.

Positive seedling identification

Extracting DNA from fresh leaves, carrying out PCR on sequences near gene editing sites, connecting the products to a T carrier to transform escherichia coli, plating a plate, picking 10-15 monoclonal sequencing, and detecting whether the plants are positive plants and mutation types.

3T 0 generation mutant plants are obtained by means of a cucumber stable transgenic technology system, and two T1 generation stable homozygous mutants (A and B in figure 7) of T1#1 and T1#2 are obtained through screening and identification, wherein the editing types are respectively the deletion of 30 bases and the addition of 1 base in a CsaTRM4 CDS region.

Example 12 measurement of cucumber plant height and stem thickness of Csatrm4 mutant

The T2 generation seeds obtained by T1 generation selfing are sown and cultivated in a greenhouse and are normally managed for observing Csatrm4 mutant phenotype changes, and the measurement of plant height and stem thickness is carried out on cucumber plants at 20 leaf ages. The plant height is the distance from the cotyledon to the plant growing point, the measuring tape is used for measuring, the stem thickness is the stem diameter of the position 1cm below the cotyledon, and the vernier caliper is used for measuring. No significant differences were found between either the plant height or the stem crude mutant and the wild type (C in FIG. 7).

EXAMPLE 13 hairy root transformation of CmoCK1 mRNA in WT and Csatrm4 mutant cucumber

The over-expression vector 35S constructed in example 8 was transformed into K599 Agrobacterium rhizogenes. The constructed K599 agrobacterium rhizogenes is utilized to respectively transform WT and Csatrm4 mutant cucumbers, and the specific method is as follows:

1) Sowing seed soaking and sterilizing the same as in example 10. The sterilized seeds were then placed into tissue culture flasks containing MS medium, each flask being incubated at about 25℃in the dark.

2) The explant is obtained by culturing for about 4 days, taking out the seedling on an ultra-clean bench when the cotyledon is not developed, cutting off one half of the cotyledon, reserving a growing point and the other half of the cotyledon connected with the growing point and about 0.5cm of hypocotyl, and transforming the part as the explant.

3) Infection, namely activating agrobacterium on the previous day, centrifuging at 4000rpm for 8 minutes to collect thalli when the bacterial liquid is subjected to shake culture at 28 ℃ until the OD 600=0.6-0.8, re-suspending the thalli to the OD 600=0.1 by using MS liquid culture medium, and adding 200 mu M acetosyringone. Cucumber explants were placed in the invasive dye solution and shaken for 20 minutes at 200 rpm.

4) Co-culturing, namely placing the explant on sterilized filter paper to suck redundant bacterial liquid after infection, placing a piece of sterilized filter paper on MS solid culture medium, and co-culturing for 4 days at 25 ℃ in dark condition.

5) Inducing rooting, namely inserting the hypocotyl of the explant into an MS solid culture medium containing 200mg/L of timentin after co-culture, and then placing the culture medium into a tissue culture chamber to induce hairy roots.

The hairy roots were incubated for about 1 week and fluorescence was observed under GFP channel using LUYOR-3415RG, and the fluorescing roots transformed into transgenic positive plants (FIG. 8). The non-fluorescent roots were removed and transferred to a flask containing fresh medium for cultivation.

EXAMPLE 14 analysis of the trafficking of CmoCK1 mRNA on WT and Csatrm4 mutant cucumber hairy roots-ground

The roots and aerial parts of the above hairy root transformed positive cucumber were extracted, RAN was extracted, and CsaActin, basa R and YFP-CmoCK1 gene fragments were amplified by RT-PCR, respectively, similarly to example 8, csaActin was used as a control, and Basa R was used as a pollution control. CsaActin the RT-PCR primer sequences are as follows:

CsaActin-F 5‘-ATGAGCTTCGTGTTGCACCT-3’(SEQ ID NO.19);

CsaActin-R 5‘-TCACACTTCATGATTGAGTTGTAGG-3’(SEQ ID NO.20)。

Basta R and YFP-CmoCK1 are constitutively expressed in roots of transgenic positive plants, basta R is not expressed but YFP-CmoCK1 is expressed in new leaves at the aerial parts, which means that the aerial parts of the cucumber are not polluted by thalli and YFP-CmoCK1 mRNA is transported from roots to aerial parts. Transport efficiency of YFP-CmoCK1 in WT (n=17), csatrm4#1 (n=16), and Csatrm4#2 (n=18), transport efficiency= (number of cucumber plants transported by YFP-CmoCK/number of cucumber plants positive for root transformation transgene) ×100% was calculated, respectively. As a result, the average transport efficiencies of YFP-CmoCK1 in WT, csatrm4#1 and Csatrm4#2 were 40.1%, 12.3% and 11.1%, respectively (FIG. 9). The CsaTRM4 mutation was demonstrated to reduce the transport efficiency of CmoCK1, laterally demonstrating the function of CsaTRM4 to enhance the transport properties of CmoCK.

What is not described in detail in this specification is prior art known to those skilled in the art.

Claims (1)

1. The application of the cucumber CsaTRM gene is characterized in that the cucumber CsaTRM gene is overexpressed in plants, so that long-distance transportation of pumpkin CmoCK1 mRNA between the ears can be promoted;

The nucleotide sequence of the cucumber CsaTRM gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by the cucumber CsaTRM gene is shown as SEQ ID NO. 2.