CN111321165B

CN111321165B - Method for promoting growth and development of tomatoes

Info

Publication number: CN111321165B
Application number: CN202010175241.5A
Authority: CN
Inventors: 周杰; 王开心; 潘燕婷; 喻景权
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-03-13
Filing date: 2020-03-13
Publication date: 2021-06-25
Anticipated expiration: 2040-03-13
Also published as: CN111321165A

Abstract

The invention discloses a method for promoting tomato growth and development, which inhibits expression of a base sequence in a tomato, wherein the base sequence is shown as SEQ ID NO: 1, miR171b gene. The expression of the tomato miR171b is inhibited through the CRISPR/Cas9 technology, the miR171b mutant plant is generated, the growth and development of the tomato can be promoted, the germination of tomato seeds is promoted, the growth of the tomato in a seedling stage is promoted, and the yield and the quality of the tomato are improved.

Description

Method for promoting growth and development of tomatoes

Technical Field

The invention relates to the technical field of biology, in particular to a method for promoting growth and development of tomatoes.

Background

MicroRNAs (miRNAs) are endogenous non-coding small RNAs widely existing in organisms, are 21-24 nt in length, and play an important role in plant growth and development, signal transduction, biotic or abiotic stress by regulating and controlling the expression of target genes of the RNAs at the level after transcription or transcription. miR171 has been widely studied in various species as one of the ancient and highly conserved miRNAs. miR171b is an important member in miRNAs family, plays an important role in regulating the balance of gibberellin and auxin in abiotic stress of plants, and has an influence on the growth and development of the plants.

Tomato (Solanum lycopersicum L.) is an annual or perennial herbaceous plant in Solanum of Solanaceae, namely tomato and persimmons, is the vegetable crop which is the widest cultivated and most consumed worldwide, is an important climacteric fruit due to the relatively short growth cycle and the fact that the facility cultivation technology is mature day by day, and gradually becomes one of important model plants for plant molecular research.

The previous research results show that the members in the same miRNAs family can play different regulation and control roles in different plants and different stresses. For example, overexpression of tomato miR169c enhances the drought resistance of plants, but in Arabidopsis, overexpression of miR169a increases the drought sensitivity of plants. Thus, no one can conclude about the same miRNA gene. In the early period, it has been reported that the gene miR171 is over-expressed in crops such as Arabidopsis, cucumber, rice and barley, and has a certain influence on the growth and development of plants. Specifically, Arabidopsis miR171 regulates the branching and chlorophyll biosynthesis of Arabidopsis by targeting GRAS family gene Scarechow-like (SCL); the over-expressed Csa-miR171a Arabidopsis strain is shown to inhibit main root elongation, increase the number of lateral roots and advance bolting time, the over-expressed cucumber Csa-miR171a obviously influences Arabidopsis chlorophyll accumulation, and the research result is the same as the research result of indirectly regulating and controlling plant chlorophyll synthesis by over-expressing miR171 targeting SCL transcription factor in Arabidopsis; after os-miR171b is over-expressed in rice, the ears of the over-expression strain are lengthened and the fruiting amount is increased compared with the wild type control; over-expression of barley miR171 can activate miR156 to inhibit expression of downstream target genes thereof, and results in transformation of vegetative growth to reproductive growth of plants.

Disclosure of Invention

Aiming at the defects in the field, the invention provides a method for promoting tomato growth and development, which inhibits the expression of tomato miR171b through a CRISPR/Cas9 technology to generate a miR171b mutant plant, can promote the tomato growth and development, and specifically can promote the tomato seed germination, promote the seedling growth and improve the tomato yield and quality.

A method for promoting tomato growth and development comprises inhibiting expression of a nucleotide sequence shown as SEQ ID NO: 1, miR171b gene.

The miR171b gene can be inhibited and expressed in the target tomato, and can be any method for reducing the expression of the miR171b gene in the target tomato. In the invention, the inhibition expression of the miR171b gene in the target tomato is realized by constructing a CRISPR/Cas9 vector of the gene and further infecting the gene by agrobacterium.

Specifically, a CRISPR/Cas9 vector of tomato miR171b gene can be introduced into a target tomato for inhibition expression, and a mutant plant of miR171b gene inhibition expression is obtained.

Preferably, the method for promoting tomato growth and development comprises the following steps:

(1) constructing an agrobacterium tumefaciens engineering bacterium A containing a CRISPR/Cas9 vector of a tomato miR171b gene;

(2) and (3) mediating and transforming the agrobacterium tumefaciens engineering bacteria A to a target tomato explant to prepare a mutant plant expressed by the miR171b gene inhibition.

In the step (1), the base sequence of the tomato miR171b gene is shown in SEQ ID NO: 2, respectively.

In the step (2), the explant is preferably a cotyledon with a seed germinating for 6-8 days.

Preferably, the mutant plant with miR171b gene suppression expression is subjected to normal growth management to obtain stably inherited transgenic F2 generation and later seeds.

In one experimental protocol, the specific steps are as follows:

1. respectively constructing an agrobacterium tumefaciens engineering bacterium A containing a CRISPR/Cas9 vector of a tomato miR171B gene and an agrobacterium tumefaciens engineering bacterium B containing a tomato miR171B gene overexpression vector;

2. respectively mediating and transforming the agrobacterium tumefaciens engineering bacteria A and B to target tomato explants to prepare miR171B gene mutant plants and overexpression plants;

3. carrying out normal growth management on the miR171b gene mutant plant and the overexpression plant to obtain stably inherited transgenic F2 generation and later seeds;

4. the transgenic F2 generation and later seeds are used for carrying out experiments, and the conditions of different growth and development stages such as the seed germination period, the seedling period, the flowering period, the fruiting period and the like of the tomato plant are observed.

The lower the expression level of the miR171b gene in the tomato, the faster the tomato seed germination in the growth and development process, the stronger the seedling growth, the increased yield and the excellent quality; the expression level of the miR171b gene in tomato is higher, the germination of tomato seeds is slowed down in the growth and development process, the germination rate is reduced, the seedling growth is slow, the yield is reduced, and the quality is poor.

The miR171b gene can be introduced into target tomatoes through a recombinant expression vector containing the gene, the recombinant expression vector can use the existing pFGC1008, pFGC5941, pCAMBIA1300, pBI121 and the like or other derivative plant expression vectors, and when the plant expression vector is used for constructing the recombinant vector, a constitutive, tissue-specific or inducible promoter can be used.

The tomato miR171b gene is concretely as follows:

a. the mature sequence of tomato miR171b is obtained from a miRbase (http:// www.mirbase.org /) database, and the base sequence is shown as SEQ ID NO: 1 is shown in the specification;

b. according to the chromosome position information, a 486bp fragment containing miR171b precursor sequence is obtained from tomato genome database (https:// solgenomics. net /), and the base sequence is shown as SEQ ID NO: 2, respectively.

Compared with the prior art, the invention has the main advantages that: the expression of the tomato miR171b is inhibited through the CRISPR/Cas9 technology, the miR171b mutant plant is generated, the growth and development of the tomato can be promoted, the germination of tomato seeds is promoted, the growth of the tomato in a seedling stage is promoted, and the yield and the quality of the tomato are improved.

Drawings

Fig. 1 is a schematic diagram of CRISPR/Cas9 vector construction for large fragment deletion. Wherein, FIG. 1A is the position structure diagram of vector gRNAs; fig. 1B shows the positions and sequences of two grnas on the tomato miR171B genome, with the gRNA specific recognition sequences underlined.

FIG. 2 is a simplified schematic diagram of the pFGC1008-3HA overexpression vector.

FIG. 3 shows sequencing results of tomato miR171b CRISPR/Cas9 knockout plants.

FIG. 4 is a validation of overexpressing transgenic positive plants. Detecting the over-expression transgenic positive plants by real-time fluorescent quantitative PCR. WT is non-transgenic wild type tomato Ailsa Craig; miR171b-OE is miR171b overexpression plant, wherein #1, #2, #3, #5, #6, #7 are different strains of miR171b overexpression plant.

FIG. 5 shows germination of tomato miR171b mutant and seeds of over-expressed plants. Wherein, FIG. 5A is a line graph of seed germination rate at different time points of 0, 24, 36, 48, 60, 72, 90h, etc.; FIG. 5B is a phenotype diagram of seed germination at 60h for each line; FIGS. 5C and 5D are graphs showing the statistics of radicle length and hypocotyl length of germinated seeds of each line at 90 h. The data shown in fig. 5C and 5D are the average of four replicates with standard error shown by the vertical line. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level. Wherein WT is a non-transgenic wild type tomato Ailsa Craig; miR171b-1 and miR171b-3 are miR171b mutant plants; miR171b-OE-1 and miR171b-OE-2 are miR171b overexpression plants.

FIG. 6 shows tomato miR171b mutant and overexpression plant seedling phenotype. Wherein, fig. 6A shows the seedling age of 15d, and the scale is 1 cm; FIG. 6B shows the age of the seedlings as 30 days old, with the scale being 5 cm.

FIG. 7 shows anthocyanin content in leaf blades of tomato miR171b mutant and over-expression plants in seedling stage. Wherein, fig. 7A is tomato leaf phenotype; fig. 7B shows the anthocyanin content. The data shown in fig. 7B is the average of four replicates with standard error shown by the vertical line. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level.

FIG. 8 shows pollen viability and pollen germination rate of tomato miR171b mutant and over-expressed plants. Wherein, fig. 8A is FDA staining and aniline blue staining for pollen viability and germination rate detection; FIGS. 8B and 8C are statistical results for the phenotype of FIG. 8A. The data shown in fig. 8A and 8B are the average of four replicates with standard error shown by the vertical line. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level.

Fig. 9 is a fruit phenotype plot of tomato miR171b mutant and overexpressed plants. Wherein, B +0, B +2, B +4, B +6 and B +8 respectively represent the 0 th, 2 th, 4 th, 6 th and 8 th days after the color of the tomato fruit is broken.

FIG. 10 shows the fruit ethylene release rate of tomato miR171b mutant and over-expressed plants. Wherein, B +0, B +1, B +2, B +3, B +4, B +5, B +6, B +7 and B +8 respectively represent the 0 th, 1 th, 2 th, 3 th, 4 th, 5 th, 6 th, 7 th and 8 th days after the color of the tomato fruit is broken. The data shown in the figure are the average of four replicates and the standard error is shown by the vertical line.

Detailed Description

The invention is further described with reference to the following drawings and specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The following examples are conducted under conditions not specified, usually according to conventional conditions, or according to conditions recommended by the manufacturer.

The test materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The experimental material selected tomato cultivar Ailsa Craig (Solanum lycopersicum L.cv).

Example 1

Cloning of tomato miR171b gene and vector construction

1. Tomato genomic DNA extraction

A CTAB method is adopted to quickly extract genome DNA of young and tender leaves of wild tomato in small quantity, and the method comprises the following steps:

(1) putting 50-100 mg of tomato leaves into a 1.5mL centrifuge tube, adding steel balls, quickly freezing in liquid nitrogen, grinding a sample into powder, and adding 500 mu L of CTAB buffer;

(2) water bath at 55 deg.C for 15min, and mixing by reversing for several times;

(3) add 500. mu.L chloroform: isoamyl alcohol (24:1), vortex mixing, centrifuging at 12000rpm for 5 min;

(4) transferring the supernatant into a new centrifuge tube, adding 1/10 volumes of sodium acetate (3M) and 2 times volumes of ice absolute ethyl alcohol, mixing uniformly by vortex, and precipitating for 1h at-20 ℃;

(5) centrifuging at 12000rpm for 3min to obtain bottom white precipitate as DNA, and discarding the supernatant;

(6) adding 1mL of precooled 70% ethanol, shaking up and down to wash, centrifuging at 12000rpm for 1min, and removing the supernatant;

(7) washing once again to remove residual liquid, drying on a clean bench, adding 50 μ L ddH₂Dissolving O, and storing at-20 ℃.

2. Gene cloning and Agrobacterium tumefaciens engineering bacterium construction

The tomato miR171b gene containing a precursor sequence obtained from a tomato genome database has the length of 486bp, in CRISPR-P website (http: cbi. hzau. edu. cn/cgi-bin/CRISPR) the target sequence of the tomato miR171b gene was designed, in order to perform deletion editing on a large fragment of the gene, primers sgRNA-miR171b-215-F were designed at 215bp and 312bp, respectively, starting from the 5' end (SEQ ID NO: 3) sgRNA-miR171b-215-R (SEQ ID NO: 4) sgRNA-miR171b-312-F (SEQ ID NO: 5) sgRNA-miR171b-312-R (SEQ ID NO: 6. the U6 promoter used in the present system, therefore, by adding linker sequences to both ends of the sgRNA recognition sequences, theoretically, when two grnas act simultaneously, a large fragment between them will be deleted.

Annealing the synthesized sgRNA forward and reverse primers to form double-stranded sgRNA containing a cohesive end joint, diluting by 200 times, and connecting with a BbsI-digested sgRNA-Cas9 framework vector at 16 ℃ overnight. The ligation product was transformed into E.coli DH 5. alpha. competent cells in the presence of ampicillin (Amp)⁺) After the LB solid culture medium is cultured overnight at 37 ℃, selecting a single clone, carrying out bacterial liquid PCR by using an M13F universal primer and an annealed downstream primer, and carrying out sequencing identification on a positive single clone to obtain positive sgRNA-miR171b-215 and sgRNA-miR171b-312 clones.

And (3) amplifying a second sgRNA sequence by taking AtU6-F-KpnI as a front primer and AtsgR-R-EcoRI as a rear primer and a positive vector sgRNA-miR171b-312 as a template, and then connecting a framework vector containing the sequences of sgRNA-miR171b-215 and Cas 9. The positive plasmid sgRNA-miR171b-215-sgRNA-miR171b-312 with correct sequencing and the plant expression vector pCAMBIA1300 are subjected to double enzyme digestion by HindIII and EcoRI respectively, the recovered sgRNA-miR171b-215-sgRNA-miR171b-312-Cas9 fragment and the linearized pCAMBIA1300 are subjected to overnight ligation, are transformed into DH5 alpha competence, are cultured in a solid LB culture medium containing 50mg/L kanamycin at 37 ℃ overnight, are subjected to single clone culture, are subjected to bacterial liquid PCR identification by using 1300-seq-R and AtUBQ-seq-R primers, and are subjected to sequencing after positive cloning. The positive plasmid was named pCAMBIA1300-sgRNA-miR171b-215-sgRNA-miR171b-312-Cas 9. Fig. 1 is a schematic diagram of CRISPR/Cas9 vector construction for large fragment deletion. Wherein, FIG. 1A is the position structure diagram of vector gRNAs; fig. 1B shows the positions and sequences of two grnas on the tomato miR171B genome, with the gRNA specific recognition sequences underlined.

Carrying out PCR amplification by taking the obtained genomic DNA as a template and SlmiR171b-OE-F (SEQ ID NO: 7) and SlmiR171b-OE-R (SEQ ID NO: 8) as primers (containing BamHI and AscI restriction enzyme sites) to obtain a tomato miR171b gene mature sequence-containing fragment with the length of 199bp, and carrying out enzyme digestion (BamHI and AscI); carrying out enzyme digestion on a plant transformation vector pFGC1008-3HA driven by a CaMV35S promoter by using BamHI and AscI restriction enzymes to linearize the vector; and then, connecting the fragment with a vector to construct an overexpression vector of the tomato miR171b gene, which is named as pFGC1008-SlmiR171b-3 HA. Fig. 2 is 35S: construction of miR171b overexpression vector. Wherein, FIG. 2 is a simple schematic diagram of an overexpression vector of pFGC1008-3 HA. The sequencing result is shown in SEQ ID NO: 9, the results show that the cloned sequence is identical to the sequence published in solgenomics.

Transferring the obtained CRISPR/Cas9 vector pCAMBIA1300-sgRNA-miR171B-215-sgRNA-miR171B-312-Cas9 into an agrobacterium-sensitive strain GV3101, transferring an overexpression vector pFGC1008-SlmiR171B-3HA into an agrobacterium-sensitive strain EHA105, and respectively obtaining an agrobacterium tumefaciens engineering strain A containing a CRISPR/Cas9 vector of a tomato miR171 gene 171B and an agrobacterium tumefaciens engineering strain B containing an overexpression vector of a tomato miR171B gene.

The primer sequences used were as follows:

sgRNA-miR171b-215-F:5′-TGATTGAGTAATACGTGATATTGGCA-3′(SEQ ID NO：3)，

sgRNA-miR171b-215-R:5′-AAACTGCCAATATCACGTATTACTCA-3′(SEQ ID NO：4)，

sgRNA-miR171b-312-F:5′-TGATTGTCTTTGCTTCCATATCATGC-3′(SEQ ID NO：5)，

sgRNA-miR171b-312-R:5′-AAACGCATGATATGGAAGCAAAGACA-3′(SEQ ID NO：6)，

SlmiR171b-OE-F:5′-CGCGGATCCTAGGAACTAATGGAAAAC-3′(SEQ ID NO：7)，

SlmiR171b-OE-R:5′-TTGGCGCGCCTGCCTAATCTTTGCTTCC-3′(SEQ ID NO：8)。

example 2

Construction of tomato miR171b gene mutant plant and overexpression plant

In general, the tomato cotyledon is infected through agrobacterium-mediated infection by using a leaf disc method, target vectors pCAMBIA1300-sgRNA-miR171b-215-sgRNA-miR171b-312-Cas9 and pFGC1008-SlmiR171b-3HA are transformed into the tomato cotyledon, and candidate transgenic plants are preliminarily screened by using hygromycin. The forward primer and the reverse primer of the hygromycin gene sequence are matched, and the obtained transgenic plant is screened by a PCR amplification method.

The method comprises the following specific steps:

1) preparation of culture Medium

Seeding culture medium: 2.15g/L MS powder +100mg/L inositol +10g/L sucrose +8g/L agar. The pH was 5.8.

A nursing culture medium: alcohol +1.3g/L thiamine hydrochloride +0.2 mg/L2, 4-D +200mg/L KH₂PO₄+0.1mg/L KT +7.5g/L agar. The pH was 5.8.

2Z selection of regeneration medium: 4.44g/L MS powder, 30g/L sucrose, 100mg/L inositol, 2mg/L ZR, 300mg/L timentin and 6mg/L hygromycin. The pH was 5.8.

0.2Z selection of regeneration Medium: 4.44g/L MS powder, 30g/L sucrose, 100mg/L inositol, 0.2mg/L ZR, 300mg/L timentin and 6mg/L hygromycin. The pH was 5.8.

Rooting culture medium: 4.44g/L MS powder, 30g/L sucrose, 100mg/L inositol, 300mg/L timentin and 6mg/L hygromycin. The pH was 5.8.

Liquid MS0.2 medium: 4.44g/L MS powder, 20g/L sucrose, 100mg/L inositol and 0.2mg/L thiamine hydrochloride. The pH was 5.8. For suspension infesting agrobacterium.

YEB Medium: 5g beef extract, 5g peptone, 1g yeast extract, 5g sucrose, 0.5g MgSO 5₄·7H₂And O, diluting to 1L with distilled water, adjusting the pH value to 7.0, and sterilizing at 121 ℃ for 20min for later use. 15g of agar powder is added into each liter of YEB solid culture medium, and other components are the same as liquid culture medium.

2) Cultivation of aseptic seedlings

Soaking tomato seeds in tap water (or shaking table at 28 ℃ for 200r/min) for 6-8 h, then sterilizing with 75% alcohol for 30sec, then sterilizing in 10% NaClO for 15min (shaking table at 28 ℃ for 200r/min), washing with sterilized distilled water for 3 times, transferring to a sterilized vessel, and inoculating to 1/2MS culture medium. Culturing at 25 deg.C in dark to expose white, transferring into light culture room, and culturing at 25 deg.C under 16h light/8 h dark with light intensity of 1800 lx.

3) Preparing explant and culturing agrobacterium

After the seeds germinate for about one week, when cotyledons are stretched and true leaves do not grow out, cutting the cotyledons of the aseptic seedlings into two sections by using a new scalpel, laying the cotyledons with a small leaf stalk in a nursing culture medium for pre-culture for 24 hours (the seedlings are protected from light and can stay overnight, and the too long nursing culture time easily causes over-infection). A single colony of Agrobacterium was picked on an antibiotic-containing LB plate, inoculated into 30mL of antibiotic-containing LB (150mL Erlenmeyer flask), and cultured overnight at 28 ℃ at 200r/min to mid-log phase (OD600 ≈ 1.0, about 16-24 h). Shaking the strain, and then cutting cotyledons (inoculating for 12-20 h).

4) Regeneration by transformation

Taking out the A bacterial liquid and B bacterial liquid of the Agrobacterium tumefaciens engineering bacteria containing target vector plasmids from a refrigerator at the temperature of minus 80 ℃, after activating on a YEB plate containing antibiotics, selecting single bacterial colonies of the Agrobacterium tumefaciens, inoculating the single bacterial colonies into 2mL of YEB containing the antibiotics, shaking at 28 ℃, 200rpm for overnight culture, then expanding and shaking 30mL of YEB according to the proportion of 1:100, culturing at 28 ℃, 200rpm for overnight culture until OD is reached₆₀₀0.8 to 1.0. Centrifuging the cultured agrobacterium at 4000r/min at 4 ℃ for 10 min; the supernatant was discarded, and 15mL of suspension medium MS0.2 was added to suspend the cells for further use. Transferring the pre-cultured cotyledon explants to a sterile culture dish poured with 15mL MS0.2, pouring the suspended bacterial liquid, infecting for 2-3 min in the dark, and slightly shaking the culture dish. Gently scooping the explant with forceps, transferring the explant to sterilizing filter paper, sucking off residual bacteria liquid, transferring to sterilizing filter paper, sucking off residual bacteria liquid, spreading the explant back to the original nursing culture medium with the reverse side facing upwards, and co-culturing for 48h in the dark at 22 ℃.

And transferring the explants subjected to co-culture to a 2Z culture medium with the right side facing upwards, culturing at 25 ℃ under 16h illumination/8 h dark light period, replacing the fresh 2Z culture medium every two weeks, cutting the browned explants after the buds are differentiated, and transferring the differentiated buds to a 0.2Z culture medium for selective culture. Fresh medium was changed every three weeks.

5) Rooting culture and transplantation

When the regeneration bud grows to about 1cm, the bud is cut off (optionally, the bud can not be cut so as to avoid damaging the rooting part), and the bud is put into a rooting culture medium for rooting. After 2 weeks, hardening the transformed seedlings with good roots and about 5cm long, and transplanting the transformed seedlings to grass carbon: and (3) obtaining tomato miR171b gene mutant plants and over-expression plants in a nutrition pot with a vermiculite-3: 1 substrate.

Example 3

Molecular detection of transgenic plants

Detection of transgenic plants at the DNA level by PCR

The transgenic tomato genome DNA extraction method was the same as example 1, and CTAB method was used.

1) PCR detection of miR171b CRISPR/Cas9 mutant plants

Specific primers are designed near the sequence position of sgRNAs of tomato miR171b gene to detect the change condition of a target gene sequence. The primers are as follows, and the fragment length is 486 bp. Homozygous mutants in miR171b knockout transgenic plants can be screened according to the size of a PCR product strip and a sequencing result. And (3) PCR products are sent for detection, and FIG. 3 shows the sequencing result of tomato miR171b CRISPR/Cas9 knockout plants.

CRISPR-miR171b-F:5′-GGCGCACACACATGATATAT-3′

CRISPR-miR171b-R:5′-AAGGCATAATATTCTTGATTAC-3′

2) PCR detection of tomato miR171b overexpression plants

The overexpression vector pFGC1008 contains a hygromycin resistance gene integrated into the plant genome, so that the primers for hygromycin screening genes can be used for identifying overexpression positive transgenic plants. The primers are as follows, and the fragment length is detected to be 812 bp.

HRH-F:5′-CGACAGCGTCTCCGACCTGA-3′

HRH-R:5′-CGCCCAAGCTGCATCATCGAA-3′

2. Detection of tomato miR171b overexpression positive plants at transcription level by utilizing qRT-PCR

Extracting total miRNA of tomato by using a miRcute miRNA extraction separation kit (Tiangen, DP501), and the detailed steps are described in the specification. Extracting the obtained miRNA, detecting the miRNA concentration of the sample by using a NanoDrop instrument, and directly using or storing at-80 ℃.

First strand synthesis reagent using miRcute enhanced miRNA cDNAThe cassette (Tiangen, KR211-02) reverse transcribes the extracted miRNA into cDNA. Real-time fluorescent quantitative PCR (qRT-PCR) Using Roche Light

480II real-time fluorescence detection system, and using a miRcute enhanced miRNA fluorescence quantitative detection kit (SYBR Green, Tiangen, FP411-02) to detect gene expression.

10 μ L of 2 × MIRcute Plus miRNA PreMix (SYBR) was included in a 20 μ L reaction&ROX), 0.4. mu.L of sense and antisense primers (10. mu.M), 1. mu.L of cDNA template and 8.2. mu.L of ddH₂And O. The procedure for quantitative PCR reactions was: 15min at 95 ℃; denaturation at 94 ℃ for 20S, annealing at 60 ℃ for 34S, and 40-45 cycles. Fluorescence data was collected at the end of extension of each cycle. After the PCR circulation reaction is completed, the melting curve shows that the generated PCR products are all single substances, and the specificity of the primer is judged according to the melting curve. The relative expression of the genes was calculated by the method of Livak and Schmitgen. The tests were all results of three replicates. Tomato U6 gene is used as an internal reference. The primer sequences are shown in Table 1. The results of qRT-PCR are shown in FIG. 4. Detecting the over-expression transgenic positive plants by real-time fluorescent quantitative PCR. WT is non-transgenic wild type tomato Ailsa Craig; miR171b-OE is miR171b overexpression plant, wherein #1, #2, #3, #5, #6, #7 are different strains of miR171b overexpression plant.

TABLE 1 real-time fluorescent quantitative PCR primers

Example 4

And carrying out growth and development observation on the obtained tomato miR171b gene mutant plant and overexpression plant, wherein the experimental materials are respectively as follows: wild type WT, mutant plants miR171b-1 and miR171b-3 (sequencing results of miR171b-1 and miR171b-3 correspond to M1: -103bp and M3: -4bp in a graph 3 respectively), and over-expressed plants miR171b-OE-1 and miR171b-OE-2 (all are over-expressed positive lines with stable inheritance). Wherein, the transgenic plants adopt stably inherited F2 generation seeds for subsequent experimental observation.

1. Seed germination stage

After fully wetting seeds of different strains, the seeds are paved in a culture dish with a diameter of 60cm and containing three layers of wet filter paper, and then the culture dish is placed in an environment with weak light, 25 ℃ and good ventilation for germination experiments, wherein the filter paper is kept wet in the period. At least three groups of biological replicates were set for each group of experiments, each group of biological replicates containing 30-50 test seeds.

The statistics of the seed germination conditions specifically comprise: and (3) paving the seeds on wet filter paper, recording the time for 0h, beginning to count the germination conditions of the seeds at 24h, and then counting the germination quantity every 12h to 90 h. In addition, phenotypes were recorded by photographing around 60h, and the radicle length and hypocotyl length of the germinated seeds were measured and counted at 90 h. The results are shown in FIG. 5. Wherein, FIG. 5A is a line graph of seed germination rate at different time points of 0, 24, 36, 48, 60, 72, 90h, etc.; FIG. 5B is a phenotype diagram of seed germination at 60h for each line; FIGS. 5C and 5D are graphs showing the statistics of radicle length and hypocotyl length of germinated seeds of each line at 90 h. The data shown in fig. 5C and 5D are the average of four replicates with standard error shown by the vertical line. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level. Wherein WT is a non-transgenic wild type tomato Ailsa Craig; miR171b-1 and miR171b-3 are miR171b mutant plants; miR171b-OE-1 and miR171b-OE-2 are miR171b overexpression plants.

As can be seen from fig. 5A, the miR171b mutant plant seeds germinated early compared to the wild type, while the miR171b overexpressed plant seeds germinated late, and the germination rate of the miR171b overexpressed plant seeds was also lower, approximately at 75%, and the germination rate of the miR171b mutant plant seeds was close to 100% compared to the wild type WT plant seeds at 90h final statistics. In addition, as can be seen from fig. 5C and 5D, when the seeds germinate for 90h, the radicle length of the miR171b mutant is 1.5-2 times that of the wild-type WT, and is 2.5-3 times that of the miR171b through over-expression; the length of the embryonic axis of the miR171b mutant is 2 times that of the wild-type WT and 4 times that of miR171b overexpression. In summary, from the measurement statistics of seed germination rate, post-germination hypocotyl length and radicle length, we believe that the miR171b gene can inhibit radicle and hypocotyl elongation during tomato seed germination, thereby affecting the germination period of the seed, even affecting the seed germination rate.

2. Seedling growth stage

After pregermination at 28 ℃ of seeds of different lines used for phenotype evaluation, the seeds were sown in 72-hole black plastic plug trays, and the matrix was a mixture of turf and vermiculite (2:1, v: v). Then the plug tray is placed in a climatic chamber, and the growth conditions are as follows: 600 μmol m^-2s^-1Light intensity (PPFD), 14h photoperiod, temperature 23/20 ℃ (day/night), relative humidity was maintained at 65%. The 1/2Hoagland nutrient solution is poured once every 2-3 days. When the first true leaf is completely unfolded, selecting the tomato seedlings with consistent growth vigor in each strain, transferring the tomato seedlings into a plastic nutrition pot with the diameter of 10cm, and continuing culturing.

(1) The 15d and 30d seedling age plants were photographed separately and the phenotype recorded. The results are shown in FIG. 6. Wherein, fig. 6A shows the seedling age of 15d, and the scale is 1 cm; FIG. 6B shows the age of the seedlings as 30 days old, with the scale being 5 cm. By observing the phenotype of miR171b mutant plants, over-expressed plants and wild WT, the over-expressed plants are found to have dwarfing performance, and compared with wild type and miR171b mutant plants, the over-expressed plants have the advantages of obviously slower growth speed and weaker growth vigor, while the miR171b mutant plants have the obviously faster growth vigor, and particularly the miR171b-3 mutant strain has the best growth vigor.

(2) Anthocyanin extraction and detection

And (4) taking 40d seedling-old tomato plants, and taking the 6 th mature and unfolded leaf from top to bottom for analyzing the accumulation of anthocyanin in the leaf. Accurately weighing 0.2g of plant leaf sample, grinding the plant leaf sample into powder at low temperature, adding 600 mu L of methanol solution containing 1% hydrochloric acid, and carrying out environmental protection under the following conditions: extract at 4 ℃ and protected from light overnight. At the 2 nd, after adding 400. mu.L of chloroform and water, the mixture is fully vortexed and mixed, and centrifuged at 10000g for 5min at 4 ℃. And absorbing 200 mu L of supernatant to a microplate reader plate, reading corresponding light absorption values at wavelengths of 530nm and 657nm, wherein the anthocyanin content is converted according to the light absorption value of fresh weight per gram, and the conversion formula is A530-0.33A 657. The results are shown in FIG. 7. Wherein, fig. 7A is tomato leaf phenotype; fig. 7B shows the anthocyanin content. The data shown in fig. 7B is the average of four replicates with standard error shown by the vertical line. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level.

The anthocyanin is mainly distributed in leaves, flowers and fruits of plants, and the leaves with higher anthocyanin content are darker and the back of the leaves are purplish red. The leaves of the miR171b mutant plant are darker and the color of the back of the leaves is purple when observed on the leaves among wild type, miR171b mutant and miR171b overexpression plants. By detecting the accumulation amount of anthocyanin in different plant lines, as shown in FIG. 7, the result shows that the accumulation amount of anthocyanin in mir171b mutant plants is obviously higher than that of wild-type and over-expressed plants and is about twice that of wild-type and over-expressed plants; there was no significant difference between miR171b overexpressing plants and wild type. The miR171b gene is thought to be capable of negatively regulating and participating in accumulation of anthocyanin in tomato leaves.

3. Flowering phase

The method for detecting the vitality of the tomato pollen and the pollen in vitro germination comprises the following steps:

tomato pollen viability was detected by staining with Fluorescein Diacetate (FDA). FDA (Aladdin, F109384) was dissolved in acetone to prepare 2 mg. multidot.mL^-1The mother liquor is stored at 4 ℃ in a dark place. When in use, the solution is diluted into 0.01 percent working solution by 0.5M sucrose solution. A drop of FDA working solution was dropped on a glass slide of a flower which had just opened at 9 AM, and the anthers were shaken with tweezers to disperse the pollen uniformly in the staining solution, and the flower was stained in a wet box at 28 ℃ in the dark for 1h, covered with a cover glass, observed under a fluorescence microscope (Leica, Germany) and photographed.

The pollen tube was stained with aniline blue (Sigma-aldrich, B8563) solution. 0.1% aniline blue solution was used as the working solution. Preparing a pollen in-vitro germination culture medium according to the table 2, adjusting the pH to 6.5 by using NaOH, adding agar powder with the final concentration of 0.1%, boiling in a microwave oven, and cooling to room temperature for later use. Dropping a drop of pollen germination culture medium on a glass slide, taking the flower which is just opened in the morning, shaking the anther with tweezers to make the pollen uniformly scatter in the culture medium, placing in a wet box, culturing for 1h at 28 ℃ in the dark, adding 20 μ L of 0.1% aniline blue solution, staining for 3min, covering a cover glass, observing with a fluorescence microscope (Leica, Germany) and taking pictures.

The results are shown in FIG. 8. Wherein, fig. 8A is FDA staining and aniline blue staining for pollen viability and germination rate detection; FIGS. 8B and 8C are statistical results for the phenotype of FIG. 8A. The data shown in fig. 8A and 8B are the average of four replicates with standard error shown by the vertical line. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level.

TABLE 2 pollen in vitro germination Medium formulation

As shown in FIG. 8, the fluorescence brightness of the pollen of the miR171b overexpression plant is obviously reduced compared with that of the wild type, and the fluorescence brightness of the pollen of the miR171b mutant plant is obviously improved compared with that of the wild type; for pollen germination, the pollen germination rate of miR171b overexpression plants is found to be the lowest, and the pollen germination rate of mutant plants is improved compared with that of wild plants. Overall, the miR171b gene affected tomato pollen viability and germination rate.

4. Period of fruiting

And (5) carrying out phenotype observation and physiological index determination on the plants in the fruit period. The plant seedling stage is managed in a growth chamber, and when wild WT grows to six leaves and one heart, the plants of each line are transplanted into a nutrition pot which contains the same matrix and is 35cm in diameter and 25cm in depth. Transferring the strain into an artificial greenhouse, wherein the culture conditions are as follows: 200 μmol m^-2s^-1The Hoagland nutrient solution is irrigated every 2-3 days under the conditions of light intensity (PPFD), 12h photoperiod and temperature of 25/20 ℃ (day/night).

(1) Fruit ripening time and yield statistics

Counting the days of sowing the plant to the first inflorescence, the number of red fruits per plant and the yield per plant after the fruits of the first inflorescence are mature, counting the real number of the fruits per plant and the yield per plant from the expanded green mature period fruits to the fully mature fruits, calculating the weight of the fruits per plant from the weight of the fruits at the fully mature period, counting the fruits at the color conversion period, and calculating the real number of the mature fruits per plant. The results are shown in Table 3.

TABLE 3 tomato fruit-related parameters

Note: the data shown in the table are the average of four replicates. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level.

As can be seen from the above table, the flowering time of the miR171b mutant two strains is advanced by 3-5 days compared with that of the wild type strain, and the flowering time of the miR171b over-expressed two strains is delayed by 3-8 days compared with that of the wild type strain. The leaf positions of the first inflorescence of all strains have no significant difference. We believe that this phenomenon appears to be due to slow prophytic growth affecting reproductive growth, thus indicating that miR171b overexpresses a phenomenon that delays flowering. Statistically, the results of the first inflorescence are different from the lines, the results of the miR171b-OE-2 overexpression line are the least, the fruiting number of a single plant is about 9.45, and the fruiting number of a single plant of the miR171b-3 mutant line is the highest, and the fruiting number of the single plant is about 16.53. From the statistics of the number of red ripens per plant of the entire plant at the time of complete maturation of the first inflorescence results, the mir171b mutant was also the most abundant in both lines, whereas the over-expressed lines were significantly less than the mutant and wild type. From the whole fruit yield of a single plant, the two mutant strains miR171b-1 and miR171b-3 are 21.3 percent and 22.4 percent respectively higher than the wild type, and the two overexpression strains miR171b-OE-1 and miR171b-OE-2 are 17.0 percent and 19.3 percent respectively lower than the wild type.

Marking the fully-swelled fruits in the green ripe period as Break +0, abbreviated as B +0, and correspondingly marking the fruits at 2d, 4d, 6d and 8d after color breaking as B +2, B +4, B +6 and B +8 respectively. Photographs were taken to record the fruit phenotype of the different lines of the invention. The results are shown in FIG. 9.

Phenotypic recordings were made by photographing fruits from green to red ripe as shown in fig. 9, and it was found that the fruit color transformation process of miR171b overexpression line was slower than that of mutant plants and wild type. After 7-8 days after color breaking, the mutant plant fruits turn red completely; the fruits of the over-expressed plants turned completely red 9d after the color breaking.

(2) Quality index measurement

Measuring the content of soluble sugar and organic acid in fruit

Grinding 0.2g of pulp into powder by liquid nitrogen, adding 1mL of chromatographic methanol, uniformly mixing by vortex, heating for 15min at 70 ℃ by rotating a metal bath at 950rpm, centrifuging for 10min at 10000g of a normal-temperature centrifuge, sucking supernatant and transferring the supernatant into a 1.5mL centrifuge tube, taking 100 mu L of supernatant from the centrifuge tube into a new 1.5mL centrifuge tube, adding 20 mu L of ribitol (0.2mg/mL) serving as an internal standard, drying for several hours at normal temperature in vacuum, then adding 400 mu L of freshly prepared methoxyamine hydrochloride solution (20mg/mL, pyridine solution), heating for 1.5h at 37 ℃ by rotating the metal bath at 950rpm, then adding 600 mu L of bis (trimethylsilyl) trifluoroacetamide (1% of trimethylchlorosilane), and continuing to heat for 30min at 950rpm and 37 ℃. After extraction was complete, the analytical instrument used was Shimadzu GC-MS-QP2010 plus and the column was VF-5MS (30m × 0.25mm × 0.25 μm). The carrier gas is helium (He), and the column flow is 1mL/min^-1. Weighing was done with a METTLER ten thousandth balance.

② extraction and content detection of fruit carotenoid

0.5g of tomato pulp is weighed, fully ground by liquid nitrogen and transferred into a 2mL centrifuge tube. Adding 350 μ L of methanol and 700 μ L, ddH2O 350 μ L of chloroform in sequence, then mixing by vortex, centrifuging 10min at 10000g of a 4 ℃ centrifuge, and collecting chloroform phase. The remaining residue was taken in a tube and 700. mu.L of chloroform was added thereto, and the extraction was repeated several times until colorless. The collected chloroform phases were combined and the collecting tube was blow dried with nitrogen. Then, 350. mu.L of a methanol solution (w/v) containing 6% KOH was added to dissolve the precipitate, and the precipitate was subjected to light-shielding treatment at 60 ℃ for 30min for derivatization. Adding 700 μ L chloroform and 350 μ L water, mixing by vortex, centrifuging at 4 deg.C for 5min at 10000g centrifuge, and collecting chloroform phase. Adding 700 μ L water into chloroform phase, extracting repeatedly until water layer is neutral. The collected chloroform phase was dried with nitrogen, finally dissolved in 100. mu.L chromatographic grade ethyl acetate, centrifuged at 14000g in a 4 ℃ centrifuge for 20min to ensure the precipitate completely settled, and 150. mu.L of the supernatant was taken for High Performance Liquid Chromatography (HPLC) analysis. The above operation should be set to more than 5 biological replicates.

The procedure for the introduction of carotenoids is described below. An Alliance2695 system (Waters Corporation, USA) was used, containing a 2695 separation module and a 2996PDA detector, equipped with a5 μm C30 reverse phase column (250 mm. times.4.6 mm) and a 20 mm. times.4.5 mm C30 pre-column (Waters Corp.). The temperature of the column is set to be 25 ℃, the flow rate is adjusted to be 1mL/min, the sample injection volume is set to be 20 mu L, and the wavelength range during detection is 220-600 nm. The mobile phase was gradient eluted using solvents A (methanol), B (80% methanol) and C (MTBE) at 0-6 min set to 95% A + 5% B, 7-11 min set to 80% A + 5% B + 15% C, 12-32 min mobile phase set to 30% A + 5% B + 65% C, 48-50 min set to 95% A + 5% B, then left unchanged to the end, the whole procedure was about 60 min. And then performing data analysis by using Waters Empower software, and performing systematic analysis and identification on each component of the carotenoid according to the retention time and the absorption spectrum curve of the standard sample. The results are shown in Table 4.

TABLE 4 tomato fruit quality-related indices

Note: the data shown in the table are the average of five replicates. Using the Tukey test, different lower case letters indicate that the difference between treatments was up to a 5% significance level.

The quality indexes of the fruits such as soluble sugar, organic acid content and carotenoid content are measured. As shown in table 4, the mir171b mutant has higher fructose and glucose contents, no significant difference in sucrose, and lower citric acid and malic acid contents in both strains compared to the wild type; the miR171b over-expresses that the contents of fructose, glucose and sucrose of the two strains are low, the contents of citric acid and malic acid are high, particularly the contents of miR171b-OE-2 are about twice of those of wild type.

(4) Measurement of ethylene Release amount in fruits

The ethylene release rate of the tomato fruits at different ripening stages is measured, the fruits at the green ripening stage which are completely swelled are marked as Break +0, abbreviated as B +0, and correspondingly, the fruits at 1d, 2d, 3d, 4d, 5d, 6d, 7d and 8d after color breaking are respectively marked as B +1, B +2, B +3, B +4, B +5, B +6, B +7 and B + 8.

At least 3 fruits at different periods are weighed and placed in a 550mL sealed preservation box for 1h at normal temperature in a dark place. More than 1mL of headspace gas in the box was withdrawn from the rubber tube above each crisper with a syringe marked with 1mL scale, 4 times per box, as 4 biological replicates. And sequentially inserting the needle heads on the prepared wooden plugs according to the corresponding labels, and injecting the needle heads into a gas chromatograph (Philips, UNICAM pro. GC) one by one for sample injection and measurement after the detection samples in each period are completely sucked. The analytical column used was a 1500X 4mm alumina glass column. The sample injector, detector and column temperatures were 130 deg.C, 130 deg.C and 200 deg.C, respectively. In the measurement, 1mL of gas was quantitatively injected for detection. According to the reference peak position of standard ethylene standard gas (10 muL/L), the corresponding peak-out time and peak-out area are obtained by utilizing the analysis software equipped in the gas chromatograph, the ethylene concentration in each sample is calculated, and the ethylene release value of each kilogram of sample per hour is calculated. The results are shown in FIG. 10. The data shown in the figure are the average of four replicates and the standard error is shown by the vertical line.

As can be seen from FIG. 10, the ethylene release peak of the two lines of the mutant appears 4-5 d after the fruit is broken, the ethylene release peak of the wild type appears 5-6 d after the fruit is broken, and the ethylene release peak of the miR171b overexpression appears 6d after the fruit is broken. The miR171b gene is shown to influence the release of fruit ethylene to some extent.

The research result of the invention shows that the lower the expression level of the miR171b gene in tomato, the faster the tomato seed germination in the growth and development process, the stronger the seedling growth, the increased anthocyanin content, the better the pollen quality, the increased fruit yield and the excellent quality.

Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the above description of the present invention, and equivalents also fall within the scope of the invention as defined by the appended claims.

Sequence listing

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Claims

1. A method for promoting tomato growth and development, which is characterized by comprising the following steps:

(1) constructing an Agrobacterium tumefaciens (Agrobacterium tumefaciens) engineering bacterium A containing a CRISPR/Cas9 vector of a tomato miR171b gene; the base sequence of the tomato miR171b gene is shown as SEQ ID NO: 2 is shown in the specification;

2. The method according to claim 1, wherein in the step (2), the explant is a cotyledon with a seed germinated for 6-8 days.

3. The method of claim 1 or 2, wherein the mutant plant with miR171b gene suppression expression is subjected to normal growth management to obtain stably inherited transgenic F2 generation and later seeds.