CN116254273A - Gene for regulating and controlling plant flowering phase, recombinant vector and application - Google Patents

Abstract

The invention belongs to the field of plant growth, relates to a gene for regulating and controlling plant flowering phase and disease resistance, and in particular relates to a gene for regulating and controlling plant flowering phase, a recombinant vector and application thereof. In the research process of disease-resistant breeding, the subject group clones NBS-LRR gene of Nicotiana benthamianaNbLOV1. The result of the belief analysis shows that the gene is closely related to the response of plants to fungal pathogens, and the gene is found in experiments for verifying the function of the gene by knocking out the gene through gene editing, and the gene is constructed by the methodNbLOV1The knockout vector of (2) and the agrobacterium-mediated transformation method create plants which are obviously different from the control: knock-outNbLOV1After that, the bud and flowering time of Benshi tobacco is obvious compared with the controlAdvanced. Knock-outNbLOV1The mutant plant of the gene shows stronger resistance to fusarium, and only leaves are slightly yellow and lightly wilted; the tobacco of the control has damaged vascular bundles, serious wilting and necrotic spots on leaf surfaces.

Description

Gene for regulating and controlling plant flowering phase, recombinant vector and application

Technical Field

The invention belongs to the field of plant growth, relates to a gene for regulating and controlling the flowering phase of plants, in particular to a gene, a recombinant vector and application for regulating and controlling the flowering phase of plants.

Background

Plant flowering is affected by a variety of intrinsic factors including photoperiod, endogenous hormones, etc., and the external environment. The flowering phase is one of key factors influencing the growth period of tobacco and determining the effective leaf number of tobacco, is an important link in the life cycle of flowering plants, and marks the transformation of plants from vegetative growth to reproductive growth. Flowers, fruits, seeds are the main products of many crops, and thus, the time to market and the economic value of the crops are determined by the flowers. The plants undergo long-term selective evolution to form a complex and fine regulation network to respond to various internal and external signals (such as photoperiod, temperature, age, gibberellin and the like), so that flowering is regulated, and the plants can bloom in the optimal time.

The flowering phase regulation is to artificially change the external environment of the plant or change the genes or internal components of the plant by biotechnology according to the flowering habit and growth and development rule of the plant so as to realize the early or late flowering. And the flowering phase is regulated, so that the economic value of the plant is improved. The application of the flowering phase regulation technology can prevent the early-flowering fruit trees from being frozen by early spring and late frost or be less damaged by the early-flowering fruit trees; can also supply vegetables, fruits, flowers and crops in reverse season, such as cucumber, tomato, strawberry in winter. In addition, for the cross breeding work, the flowering phase control technology can lead the hybrid parents which are not encountered in the original flowering phase to bloom in the same period, solve the contradiction between cross pollination and time, be beneficial to the development of the breeding work and have important agricultural and economic values in the regulation of the plant flowering phase. Patent 202110374691.1 discloses a transcription factor LbNAP for delaying the flowering phase of lily, which is used for prolonging the flowering phase of plants by reducing the expression level of the LbNAP transcription factor, and constructing a virus-induced gene silencing vector containing LbNAP transcription factor gene fragments based on tobacco brittle virus TRV; patent 201911317235.2 discloses a gene NtDUF599 related to tobacco low-temperature early flowering, which realizes the advance of flowering period under low-temperature stress; the gene for shortening the flowering period of plants is always a research of breeding work, and the method for shortening the growth period of germplasm materials by means of leading the flowering period to advance, and the like, thereby realizing more than one year and improving the breeding efficiency, and is a research direction of the subject group.

Disclosure of Invention

In order to solve the technical problems, the invention provides a gene, a recombinant vector and application for regulating and controlling the flowering phase of plants.

The technical scheme of the invention is realized as follows:

in the research process of disease-resistant breeding, the NBS-LRR gene in Nicotiana benthamiana is clonedNbLOV1，The sequence is shown as SEQ ID No. 1. The result of the biological analysis shows that the gene is closely related to the response of plants to fungal pathogens, and the gene is verified to be knocked out in experiments by gene editing and knocking outNbLOV1After that, the bud and flowering time of Benshi tobacco are obviously earlier than that of the control.

In one aspect, the present application claims a gene for regulating flowering phase in plants, the gene being a protein comprising a nucleotide binding site and a leucine-rich repeat.

Further, the amino acid sequence of the gene is shown as SEQ ID No. 2.

The nucleotide sequence of the gene is shown as SEQ ID No. 1.

Recombinant vector with the above gene as target gene.

Preferably, the recombinant vector is a CRISPR-based gene editing recombinant vector.

Further, the recombinant vector is a pORE-Cas9 editing vector or a pDC45 editing vector.

The application of the recombinant vector in shortening plant florescence.

The recombinant vector is applied to cultivation of new varieties for more than one year.

The recombinant vector is applied to enhancing the fusarium disease resistance of plants.

The application steps are as follows: transferring the recombinant vector into a sample plant through agrobacterium-mediated genetic transformation technology to prepare a variety with shortened flowering period and/or fusarium disease resistance.

Preferably, the plant is tobacco.

Further, the plant is nicotiana benthamiana.

The invention has the following beneficial effects:

1. genes newly discovered in the present applicationNbLOV1Not only responds to the gene related to diseases, but also shortens the flowering period by knocking out the gene, the disease resistance of the gene editing positive strain created by the application is obviously different from that of a control, and the gene editing positive strain is knocked out after constructionNbLOV1The tobacco plants transfected with the gene recombinant vector exhibit strong resistance to fusarium: only leaves were slightly yellowish and slightly wilted, while the control tobacco showed damage to vascular bundles, severe wilting and necrotic spots on leaf surfaces.

2. Knocking out using the methods of the present applicationNbLOV1Compared with the control, the new plant of the gene has obviously advanced flowering phase, and under the same culture condition, the control Benshi tobacco needs to grow to more than 20 leaves and plant height of more than 20 cm to flower budsNbLOV1When the number of leaves of the gene editing mutant is less than 10 and only more than 10 cm, the gene editing mutant can bloom, and the flowering period is shortened by about half.

Drawings

In order to more clearly illustrate the embodiments of the invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, it being obvious that the drawings in the following description are only some embodiments of the invention, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic view ofNbLOV1Electrophoresis gel diagram of gene CDS region.

FIG. 2 is a schematic view ofNbLOV1Schematic structural diagram of the gene.

FIG. 3 shows the results of PCR amplification and sequencing verification of the gene editing vector, wherein, the A.PCR electrophoretogram, M is DL2000 marker;1,2,3 are Cas9, U26 and gRNA fragments, respectively, in the P1 strain; B. the target site sequence of the sgRNA in the constructed vector is highlighted by comparison with the original vector sequence.

FIG. 4 is an electrophoretogram of a positive plant amplified by PCR, wherein M is DL2000 marker;1-11 in turn areNbLOV5、NbLOV-6、NbLOV-8、NbLOV9、NbLOV-10、NbLOV11、NbLOV15、NbLOV20、NbLOV28、NbLOV31、NbLOV45; 12 is a positive control and 13 is a negative control.

FIG. 5 is a physical diagram of the bud phase of the gene editing positive strain and the control strain under the same culture condition.

FIG. 6 is a graph showing results of editing of NbLOV28 and NbLOV31, wherein the underlined portions indicate target sites and the red color indicates the results of editing.

FIG. 7 is a control graph of bud phase under potting conditions.

FIG. 8 is a diagram showing a biological analysis of the structure of proteins.

FIG. 9 is a state diagram of leaves of different varieties after the petioles are immersed in crude toxin, wherein the left side is wild type and the right side is resistant plantNbLOV1。

FIG. 10 is a physical view of tobacco plants 30 days after Fusarium inoculation, the left sideNbLOV1(mutant) plants, exhibiting disease resistance; on the right are wild-type controls, presenting with a feeling of illness.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in conjunction with the embodiments of the present invention, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without any inventive effort, are intended to be within the scope of the invention.

Experimental material and reagent consumable

The present tobacco seeds are given benefit by the teaching of China university of agriculture Li Dawei. Sterile seedlings were cultured in the light incubator of the unit key laboratory.

The culture medium comprises 2.2g/LMS basic salt, 20g/L sucrose, gamborg's vitamin component and 8g/L agar. The culture conditions were 16 hours of light and 8 hours of darkness, and the temperature was set at 25-28 ℃.

The pORE-Cas9 editing vector was provided by southwest university and the pDC45 editing vector was given away by the stock Boer of the national academy of agricultural sciences, tobacco research institute (CN 113667689A is published).

Restriction enzyme Bsa I (NEB) was purchased from Gene Inc.

The plant genome extraction uses CTAB reagent and RNA extraction uses a kit, and the main reagent is purchased from Zhongkeruitai (Beijing) biotechnology Co.

Primers were done by Shanghai Bioengineering Co., ltd, sequencing was done by Prinsepia. PCR instrument, electrophoresis instrument and gel imager were purchased from Bio-Rad corporation, usa.

Example 1: gene cloning and structural analysis

NbLOV1Cloning of the Gene: the putative sequence information of the gene of Nicotiana benthamiana was obtained using the Solanaceae plant genome database (https:// solgenomics. Net) and the Nicotiana benthamiana genome, transcriptome database (https:// benthggenome. Qut. Edu. Au /) of Kunthphenom university (QUT), primers were designed, and their sequences were amplified with Nicotiana benthamiana DNA, cDNA templates and sequenced to verify sequence correctness. Primer sequence information is shown in Table 1.

The gene structure was drawn using an on-line tool. On-line tool: http:// gsds. Gao-lab. Org/index. Php.

TABLE 1 primers used herein and their sequence information

After the cloning of the gene,NbLOV1the position of the fragment on the electropherogram is shown in FIG. 1.NbLOV1The sequence of the gene is shown in the attached part. The CDS region of the gene has 2574bp and codes for a protein composed of 857 amino acids. By comparison of genomic information, the gene contained 2 introns, 3 exons, 2 UTR regions, as shown in fig. 2. As a result of analyzing the structure of a protein encoded by the gene, as shown in FIG. 8, it was revealed from FIG. 8 that the protein was a protein (NB-LRR) having a nucleotide binding site and a leucine-rich repeat.

EXAMPLE 2 sgRNA design, vector construction and validation

Based on the gene CDS sequence, the online tool CRISPRdirect (http:// crispr. Dbcls. Jp /) and CRISPR MultiTargeter (http:// www.multicrispr.net /) are utilized to design editing sites, and target sites with highest scores and lowest miss probability are selected through cross comparison. The DNA of the target site was obtained by means of an annealing reaction using 4. Mu. lAnnealing buffer for DNA Oligos reagent, 4. Mu.l each of the upstream and downstream primers, and 8. Mu.l each of ultra pure water, using a program cooling system (5 min at 95℃and 0.1℃every 8s until the temperature was reduced to 25 ℃).

Cleavage, ligation and validation of CRISPR/Cas9 vector: cleavage was performed using a system of NEB CutSmart buffer, bsaI, 50. Mu.l. The procedure of cleavage ligation was performed according to the kit instructions, and the cleavage product and annealing product were ligated with linearized pORE-Cas editing vector or pDC45 editing vector using T4 ligase. After DH5 alpha competent cells are transformed by the ligation product, the reverse complement of the target gene locus is used as an upstream primer and the reverse complement of the target gene locus is used as a downstream primer by using the sequence U26-jianceF on the vector, positive clones are screened by using a colony PCR method, and then sequencing verification is carried out to ensure that the target locus fragment is successfully inserted into the vector. The electrophoresis results are shown in FIG. 3, and the correctness of the vector is verified by PCR amplification and sequencing.

Example 3: genetic transformation of agrobacterium transformed with editing vector and Nicotiana benthamiana

After sequencing and verifying the vector, taking a monoclonal with completely correct sequence information, performing amplification culture, extracting plasmid DNA, and transforming the competent cells of the agrobacterium GV3101 by using a freeze thawing method. After shaking, the correctness of the plasmid in the bacteria is verified by bacterial liquid PCR and then the bacteria is used for genetic transformation experiments. Specific procedures were referred to the instructions for competent cell use.

The kanamycin-resistant T is obtained by agrobacterium-mediated genetic transformation technology ₀ Positive seedlings of Nicotiana benthamiana. PCR and electrophoresis prove that 11 transgenic lines of Benshiyan smoke containing Cas9 sequence are screened outNbLOV5、NbLOV-6、NbLOV-8、NbLOV9、NbLOV-10、NbLOV11、NbLOV15、NbLOV20、NbLOV28、NbLOV31、NbLOV45 WhereinNbLOV5、NbLOV-6、NbLOV-8、NbLOV9、NbLOV-10 with port-Cas as CRISPR/Cas9 editing vector, the remaining lines with pDC45 as CRISPR/Cas9 editing vector as shown in fig. 4;

under the same culture conditions, as shown in FIG. 5, the gene editing positive lines still survived in the later period and with good growth vigor in the two groups of experimentsNbLOV-5、NbLOV-6、NbLOV-8、NbLOV9、NbLOV-10、NbLOV20、NbLOV28、NbLOV31Bud and flower earlier than wild Nb-WT plants; in the culture flask, the liquid crystal is filled in the culture flask,NbLOV1the Benshi tobacco strain obtained after gene editing is flowering after about one month when the effective leaf number is only 5-7 and the plant height is only about 5 cm. Under the same culture conditions, the effective leaf of the control wild type Benshi tobacco has 10 leaves, and the bud is not found until the culture is carried out for about 2 months.NbLOV1The flowering period of the plant after gene editing is shortened by about half compared with that of the control wild type. Selecting NbLOV28 and NbLOV31 positive plants to perform hi-tom sequencing, and analyzingNbLOV1As shown in fig. 6, the red color is the result of editing, and it can be seen that both plants are new plants successfully edited by CRISPR.

After transplanting the tobacco plants, the same trend was also exhibited under potting conditions, as shown in fig. 7: the control Benshi cigarettes need to be as long as approximately 20 leaves and have a plant height of more than 20 cmFlower bud blossomNbLOV1The gene editing mutant NbLOV45 can bloom when the number of leaves is less than 10 and only more than 10 cm.NbLOV1The flowering period of the NbLOV45 plant after gene editing is shortened by half compared with that of a control wild type.

Example 4: disease resistance test of Positive plants

(1) Activation of pathogenic bacteria: taking strain preserved at-80deg.C (long-term preservation), inoculating into PDA (potato dextrose agar) culture medium, and culturing in 25-28deg.C incubator for 5-7 days in dark.

(2) Liquid culture of pathogenic bacteria: 1-3 pieces of mycelia with a diameter of about 5-10 mm are taken from the edge part of the mycelia in the above-mentioned activated culture dish ² Transferring into 250mL triangular flask containing 150mL sterile PDB culture medium (potato glucose liquid culture medium), placing into constant temperature shaking table, setting 25-28deg.C, shaking at 180 rpm for 3-5 days. Preferably 3 days.

(3) Preparation of crude toxin: pouring the liquid culture obtained in the step 2 into a funnel paved with 4 layers of sterile gauze, filtering to remove hyphae, transferring the filtrate into a centrifuge tube, and centrifuging at 8000rpm and 4 ℃ for 15min. The obtained supernatant is crude toxin, and is filtered by a filter membrane with the diameter of 0.22 microns for later use.

(4) Identification of resistance using crude toxin: a300 ml glass culture flask was used, about 30ml of the crude toxin solution was poured into the flask, fresh leaves were taken from the tobacco plant by rapidly cutting off the base of the leaf stalk with a sharp blade, the leaf stalk was placed in the toxin solution, and the leaf stalk state change was closely observed.

(5) Judgment of fusarium resistance: at 3-7 days after the petioles are immersed in the crude toxin, different leaf states of different varieties (strain) are observed, and as shown in FIG. 9, plants of different varieties respond differently to the toxin, wherein resistant plants generated after gene editingNbLOVLeaves of the wild control plants showed slight yellow leaf color and slight wilting, but the leaves of the wild control plants showed damaged vascular bundles, and the leaves showed severe wilting and necrotic spots.

(6) Tobacco plants 30 days after inoculation with Fusarium are shown in FIG. 10, with the left side onNbLOV1Gene editing positive plantsNbLOVRight, rightOn the side of the wild type control, the positive plants subjected to genetic editing show stronger resistance to fusarium, and the plants of the control group show serious wilting, dying and other symptoms, as clearly seen from fig. 10.

Examples of the effects

By editing Benz cigarettesNbLOV1After obtaining the edited strain, the gene is observed and inoculated with pathogenic bacteria for analysis,NbLOV1gene editing can result in: 1. the flowering phase of Benshi tobacco is advanced; 2. resistance to fusarium is improved.

Indicating thatNbLOV1The gene is involved in regulating flower development (growth period regulation) of Benshi tobacco and response of Benshi tobacco to fusarium. The results show that the gene has certain application value in the breeding work of regulating and controlling the growth period of crops, improving the resistance and the like.

The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, alternatives, and improvements that fall within the spirit and scope of the invention.

Claims (10)

1. A gene for regulating the flowering phase of a plant, characterized in that: the genes belong to proteins containing nucleotide binding sites and leucine rich repeats.

2. The gene for regulating flowering phase of plants of claim 1, wherein: the amino acid sequence of the gene is shown as SEQ ID No. 2.

3. The coding sequence of the nucleotide of the gene as set forth in claim 1 or 2 is shown in SEQ ID No. 1.

4. A recombinant vector comprising the gene according to claim 3.

5. The recombinant vector according to claim 4, wherein: the recombinant vector is a recombinant vector edited based on CRISPR genes.

6. Use of the recombinant vector of claim 4 for shortening the flowering phase of plants.

7. The use of the recombinant vector of claim 4 for enhancing fusarium disease resistance of plants.

8. The use according to claim 6 or 7, characterized in that the application steps are as follows: transferring the recombinant vector into a sample plant through agrobacterium-mediated genetic transformation technology to create a strain with shortened flowering phase and/or fusarium disease resistance.

9. The use according to claim 8, characterized in that: the plant is tobacco.

10. The use according to claim 9, characterized in that: the plant is Nicotiana benthamiana.

Images