CN113151353B

CN113151353B - Lily LlKnat13 gene clone and application method thereof in controlling formation of lily bulbil

Info

Publication number: CN113151353B
Application number: CN202110622521.0A
Authority: CN
Inventors: 杨盼盼; 明军; 徐雷锋; 何国仁
Original assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Current assignee: Institute of Vegetables and Flowers Chinese Academy of Agricultural Sciences
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2022-04-22
Anticipated expiration: 2041-06-04
Also published as: CN113151353A

Abstract

The invention discloses a lily Llknat13 gene clone and an application method thereof in controlling lily bulblet formation, wherein a primer is designed according to transcriptome data, cDNA is used as a template, a CDS region is obtained through PCR amplification, and an upstream Llknat13-F is obtained by amplifying CDS sequence primers as follows: 5'-ATGAGAGAGTCATACCACCCC-3', downstream Llknat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain the cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1. The invention provides a lily LlKnat13 gene clone and an application method thereof in controlling formation of lily bulbels, wherein a lily LlKnat13 gene cloned for the first time belongs to Class II KNOX, and the gene silencing of lily LlKnat13 is induced by VIGS technology, so that the bulbel induction rate is obviously reduced. Therefore, the invention not only enriches the research information of ClassII KNOX gene, but also utilizes the LlKnat13 gene to perform effective manual intervention on the formation of lily bulbil, and has wide application prospect in the aspect of improving the propagation efficiency of lily.

Description

Lily LlKnat13 gene clone and application method thereof in controlling formation of lily bulbil

Technical Field

The invention relates to the technical field of genetic engineering, in particular to lily LlKnat13 gene cloning and an application method thereof in controlling formation of lily bulbil.

Background

The bulbil refers to a bud with nutrient storage and large shape, also called bulbil, which grows at the axillary part of the plant, and is generally considered to be an overground metamorphosis stem or axillary bud metamorphosis formed by axillary buds. The bulblets grow depending on the plant of the parent, and the mature bulblets can develop into new and complete individuals after falling off from the parent, can propagate like seeds, and better retain the characteristics of the parent with less variation and degeneration. Therefore, the propagation of the bulbil has wide application prospect in production.

For lily, bulbil is an important reproductive organ for some of its species. The lily original seed only comprises the four species of the Chinese clary, the lilium brownii, the lilium nipponica and the bulbil lily, and the bulbil can form bulbil at the axillary of the stem under natural conditions. In the lilies with the bulbil propagation strategy, the lilies are the most widespread, highest economic value and most typical variety, have high ornamental value and strong resistance, have edible and medicinal values and are one of the main traditional varieties of edible and medicinal lilies in China. Lilium primitive species are generally diploid (2n ═ 2x ═ 24), whereas lilium tigrinum is the only primitive species with triploids, and china has only triploid forms of lilium tigrinum. Triploid lilium will not normally produce seeds, so bulbar reproduction is the most prominent natural reproduction strategy.

The bulbils are formed in the axilla of the plant, i.e. the inner region where the base of the leaf is connected with the stem. Research shows that the formation of axillary bulblet originates from axillary meristem, and the formation process comprises: (1) establishing axillary meristem; (2) starting bud primordium; (3) the bulbil primordium is differentiated to form bulbils. These processes are regulated by a series of genes. At present, more genes related to axillary meristem initiation have been cloned and subjected to primary functional verification. Such as: the LAS, REV and SPS genes of Arabidopsis, LS and BL genes of tomato and the MOC1 gene of rice are all key regulatory factors for the initiation of axillary meristems. However, related genes related to plant bead bud formation are isolated less, and mainly focus on gene cloning and expression analysis, and functional research is not reported. Therefore, how to clone and utilize genes related to bulblet formation in lily has important theoretical significance and practical application value for artificially regulating and controlling the bulblet formation of lily and obviously improving the propagation efficiency of lily.

The KNOX gene family is a transcription factor for coding homeobox protein, and plays an important role in the processes of plant growth, development and morphogenesis. In plants, the KNOX genes can be divided into two classes: the first is Class I KNOX, comprising STM1, BP/KNAT1, KNAT2 and KNAT6, which are expressed predominantly in the apical meristem, regulating the development of the apical meristem and the maintenance of stem cells. The Class I KNOX genes AtqKNOX1 and AtqKNOX2 in agave are associated with bulblet formation, with increased expression levels of AtqKNOX1 and AtqKNOX2 as bulblets are formed and matured. The second Class is Class II KNOX, which includes KNAT3, KNAT4, KNAT5 and KNAT7, which have more extensive expression sites, but their function is not yet known. Therefore, a lily LlKnat13 gene clone and an application method thereof in controlling lily bulblet formation are provided.

Disclosure of Invention

Based on the technical problems in the background technology, the invention provides lily LKnat13 gene cloning and an application method thereof in controlling lily bulblet formation, so as to solve the problems in the background technology.

The invention provides the following technical scheme: a lily LlKnat13 gene clone and an application method thereof in controlling lily bulblet formation comprise the following steps:

A. extracting RNA of axillary leaves of lilium tigrinum and lily, designing a primer according to transcriptome data, carrying out PCR amplification by taking cDNA as a template to obtain a CDS region of the lilium tigrinum and lily, wherein the CDS sequence primer is amplified as follows, and the upstream LlKnat 13-F: 5'-ATGAGAGAGTCATACCACCCC-3', downstream LlKnat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain a cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1;

B. extracting RNA of axillary axils of lilium brownii and lily; reverse transcription into cDNA, designing primer according to SEQ ID NO.1, using cDNA as template, making qRT-PCR amplification, adopting 2^-ΔΔCtThe method analyzes the relative expression level of the Llknat13 gene, and the amplification primers are as follows: upstream LlKnat 13-qRT-F: 5'-TCAGGGAAGAGATTCTACGCAAG-3', downstream LlKnat 13-qRT-R: 5'-TTCCTTCACCAATCGTGCCTT-3', respectively;

C. the application of the gene in controlling the formation of the bulblet of the lilium tigrinum and the lily is as follows: designing primers LlKnat13-V-F (5'-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3') and LlKnat13-V-R (5'-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3') with a joint for a target fragment (1bp-153bp) with a length of 153bp according to SEQ ID NO.1, connecting the LlKnat13 gene target fragment with the joint with a TRV2 linear vector after enzyme digestion by a homologous recombination method to obtain a TRV 2-LKnat 13 vector, transforming the TRV 2-LKnat 13 vector into an agrobacterium strain EHA105, activating and shaking the strain to OD600 ═ 0.8, vacuumizing for 5min to infect the Judan stem, cleaning the infected stem, inoculating the infected stem into a culture medium of MS +30g/L sucrose to induce formation of bulbels, observing and counting after two weeks, wherein compared with a control group, the bulbel induction rate of the bulbels is remarkably reduced.

Preferably, the travertine axilla is cut by a double-sided blade, total RNA is extracted according to the instruction of the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit of TianGen (Beijing), the extracted total RNA is used as a template, and TransGen (Beijing) is adopted

II One-step gDNA Removal and cDNA Synthesis SuperMix kit first strand cDNA was synthesized.

Preferably, in the step A, according to Unigene data obtained by sequencing of the subject group Voldahl transcriptome, a Primer LlKnat13-F (5'-ATGAGAGAGTCATACCACCCC-3') is designed through Primer Premier5.0 software; and LlKnat13-R (5'-CTATGTGTAGCGTTCGGAGT-3'), and PCR amplification was performed using the axillary cDNA of red sage root as a template.

Preferably, the material taking mode of the axilla of the lilium brownii in the step B is as follows: the method comprises the following steps of (1) respectively selecting bulbels from in-vitro cultured pennisetum crispum stem segments to form different stages: S0-S5, taking 5-7 axilla from 5 plant of red lead respectively, wrapping with tinfoil paper, quick freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C for use, wherein S0: underarm of uninduced culture; S1-S3: the bud primordium is started to form; S4-S5: differentiating the bulbil primordia into bulbils.

The invention provides a lily Lknat13 gene clone and an application method thereof in controlling lily bulbil formation, wherein the lilium tigrinum Lknat13 gene cloned for the first time belongs to Class II KNOX, and the lilium tigrinum Lknat13 gene silencing is induced by VIGS technology, so that the bulbil induction rate is obviously reduced, therefore, the invention not only enriches the research information of the Class II KNOX gene, but also utilizes the Lknat13 gene to perform effective manual intervention on lily bulbil formation, and has wide application prospect in the aspect of improving lily propagation efficiency.

Drawings

FIG. 1 is a schematic diagram of the steps of the present invention;

FIG. 2 is an electrophoretogram of CDS region amplification of LlKnat13 gene according to the present invention;

FIG. 3 is a diagram showing an alignment of amino acid sequences of LlKnat13 of the present invention and KNAT genes of other species;

FIG. 4 is a phylogenetic tree analysis of LlKnat13 and another species KNAT of the present invention;

FIG. 5 shows the expression of the LlKnat13 gene in different stages of formation of lily bulbil;

FIG. 6 is a drawing of TRV2-LlKnat13 inducing lily axillary LlKnat13 gene silencing.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Referring to fig. 1-6, the present invention provides a technical solution: a lily LlKnat13 gene clone and an application method thereof in controlling lily bulblet formation comprise the following steps:

A. extracting RNA of axillary leaves of lilium tigrinum, designing a primer according to transcriptome data, carrying out PCR amplification by taking cDNA as a template to obtain a CDS region of the lilium tigrinum, wherein the CDS sequence primer is amplified as follows, and the upstream LlKnat 13-F: 5'-ATGAGAGAGTCATACCACCCC-3', downstream LlKnat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain a cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1;

B. extraction rollRNA of axillary region of lilium davidii: reverse transcription into cDNA, designing primer according to SEQ ID NO.1, using cDNA as template, making qRT-PCR amplification, adopting 2^-ΔΔCtThe relative expression level of the LlKnat13 gene is analyzed by the method, and amplification primers are as follows: upstream LlKnat 13-qRT-F: 5'-TCAGGGAAGAGATTCTACGCAAG-3', downstream LlKnat 13-qRT-R: 5'-TTCCTTCACCAATCGTGCCTT-3', respectively;

C. the application of the gene in controlling the formation of the bulbil of lilium tigrinum comprises the following steps: designing primers LlKnat13-V-F (5'-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3') and LlKnat13-V-R (5'-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3') with a joint for a target fragment (1bp-153bp) with a length of 153bp according to SEQ ID NO.1, connecting the LlKnat13 gene target fragment with the joint with a TRV2 linear vector after enzyme digestion by a homologous recombination method to obtain a TRV 2-LKnat 13 vector, transforming the TRV 2-LKnat 13 vector into an agrobacterium strain EHA105, activating and shaking the strain to OD600 ═ 0.8, vacuumizing for 5min to infect the Judan stem, cleaning the infected stem, inoculating the infected stem into a culture medium of MS +30g/L sucrose to induce formation of bulbels, observing and counting after two weeks, wherein compared with a control group, the bulbel induction rate of the bulbels is remarkably reduced.

The first embodiment is as follows:

cutting the axilla of the leaf of herba Centellae with a double-sided blade, extracting total RNA according to the instruction of the RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit of TianGen (Beijing), extracting total RNA as a template, and adopting TransGen (Beijing) company

II, synthesizing first strand cDNA by using One-step gDNA Removal and cDNA Synthesis SuperMix kit; extracting RNA of axillary leaves of lilium tigrinum, designing a primer according to transcriptome data, carrying out PCR amplification by taking cDNA as a template to obtain a CDS region of the lilium tigrinum, wherein the CDS sequence primer is amplified as follows, and the upstream LlKnat 13-F: 5'-ATGAGAGAGTCATACCACCCC-3', downstream LlKnat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain the cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1. Extracting RNA from axillary region of Lilium tigrinum leaf according to SEQ IDNo.1 design primer, cDNA as template, qRT-PCR amplification, 2^-ΔΔCtThe relative expression level of the LlKnat13 gene is analyzed by the method, and amplification primers are as follows: upstream LlKnat 13-qRT-F: 5'-TCAGGGAAGAGATTCTACGCAAG-3', downstream Llknat 13-qRT-R: 5'-TTCCTTCACCAATCGTGCCTT-3', respectively; designing primers Llknat13-V-F (5'-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3') and Llknat13-V-R (5'-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3') with a joint for a target fragment (1bp-153bp) with a length of 153bp according to SEQ ID NO.1, connecting the Llknat13 gene target fragment with the joint with a TRV2 linear vector after enzyme digestion by a homologous recombination method to obtain a TRV2-Llknat13 vector, transforming the constructed TRV2-Llknat13 vector into an agrobacterium strain A105, activating and shaking to OD600 & lt 0.8, vacuumizing for 5min to infect a lilac stem segment, cleaning the infected stem segment, inoculating the cleaned infected stem segment into a culture medium of MS +30g/L sucrose to induce formation of a bulbule, observing and counting after two weeks, wherein compared with a control group, the induction rate of the bulbule is remarkably reduced.

Example two:

II, synthesizing first strand cDNA by using One-step gDNA Removal and cDNA Synthesis SuperMix kit; extracting RNA of lily leaf axillae, designing a primer according to transcriptome data, carrying out PCR amplification by taking cDNA as a template to obtain a CDS region of the lily leaf axillae, wherein the CDS sequence primer is amplified as follows, and the upstream LlKnat 13-F: 5'-ATGAGAGAGTCATACCACCCC-3', downstream LlKnat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain a cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1; according to Unigene data obtained by sequencing the Voronoi transcriptome of the subject group, a Primer LlKnat13-F (5'-ATGAGAGAGTCATACCACCCC-3') is designed through Primer Premier5.0 software; and LlKnat13-R (5'-CTATGTGTAGCGTTCGGAGT-3'),taking the axillary cDNA of the red sage root leaves as a template to carry out PCR amplification. Extracting RNA of lily axillary, designing primer according to SEQ ID NO.1, using cDNA as template, performing qRT-PCR amplification, adopting 2^-ΔΔCtThe relative expression level of the LlKnat13 gene is analyzed by the method, and amplification primers are as follows: upstream LlKnat 13-qRT-F: 5'-TCAGGGAAGAGATTCTACGCAAG-3', downstream Llknat 13-qRT-R: 5'-TTCCTTCACCAATCGTGCCTT-3', respectively; designing primers Llknat13-V-F (5'-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3') and Llknat13-V-R (5'-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3') with a joint for a target fragment (1bp-153bp) with a length of 153bp according to SEQ ID NO.1, connecting the Llknat13 gene target fragment with the joint with a TRV2 linear vector after enzyme digestion by a homologous recombination method to obtain a TRV2-Llknat13 vector, transforming the constructed TRV2-Llknat13 vector into an agrobacterium strain A105, activating and shaking to OD600 & lt 0.8, vacuumizing for 5min to infect a lilac stem segment, cleaning the infected stem segment, inoculating the cleaned infected stem segment into a culture medium of MS +30g/L sucrose to induce formation of a bulbule, observing and counting after two weeks, wherein compared with a control group, the induction rate of the bulbule is remarkably reduced.

Example three:

II, synthesizing first strand cDNA by using One-step gDNA Removal and cDNA Synthesis SuperMix kit; extracting RNA of lily leaf axillae, designing a primer according to transcriptome data, carrying out PCR amplification by taking cDNA as a template to obtain a CDS region of the lily leaf axillae, wherein the CDS sequence primer is amplified as follows, and the upstream LlKnat 13-F: 5'-ATGAGAGAGTCATACCACCCC-3', downstream LlKnat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain a cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1; according to Unigene data obtained by sequencing the Voronoi transcriptome of the subject group, a Primer LlKnat is designed through Primer Premier5.0 software13-F (5'-ATGAGAGAGTCATACCACCCC-3'); and LlKnat13-R (5'-CTATGTGTAGCGTTCGGAGT-3'), and PCR amplification was performed using the axillary cDNA of red sage root as a template. Extracting RNA of lily axillary, designing primer according to SEQ ID NO.1, using cDNA as template, performing qRT-PCR amplification, adopting 2^-ΔΔCtThe relative expression level of the LlKnat13 gene is analyzed by the method, and amplification primers are as follows: upstream LlKnat 13-qRT-F: 5'-TCAGGGAAGAGATTCTACGCAAG-3', downstream Llknat 13-qRT-R: 5'-TTCCTTCACCAATCGTGCCTT-3', respectively; according to the He and other (2020) material taking mode, the red leaf axils of the red lead which are formed into different stages (S0-S5) by the bulbels are respectively selected from the red lead stem segments cultured in vitro, 5-7 axils are taken from samples of each stage, are respectively obtained from 5 red leads, are wrapped by tinfoil paper, are quickly frozen by liquid nitrogen, and are stored in a refrigerator at minus 80 ℃ for later use, wherein S0: underarm of uninduced culture; S1-S3: the bud primordium is started to form; S4-S5: differentiating the bulbil primordium into bulbils; designing primers Llknat13-V-F (5'-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3') and Llknat13-V-R (5'-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3') with a joint for a target fragment (1bp-153bp) with a length of 153bp according to SEQ ID NO.1, connecting the Llknat13 gene target fragment with the joint with a TRV2 linear vector after enzyme digestion by a homologous recombination method to obtain a TRV2-Llknat13 vector, transforming the constructed TRV2-Llknat13 vector into an agrobacterium strain A105, activating and shaking to OD600 & lt 0.8, vacuumizing for 5min to infect a lilac stem segment, cleaning the infected stem segment, inoculating the cleaned infected stem segment into a culture medium of MS +30g/L sucrose to induce formation of a bulbule, observing and counting after two weeks, wherein compared with a control group, the induction rate of the bulbule is remarkably reduced.

Isolation of LlKnat13 Gene

According to Unigene data obtained by sequencing the Voronoi transcriptome of the subject matter group, primers LlKnat13-F (5'-ATGAGAGAGTCATACCACCCC-3') and LlKnat13-R (5'-CTATGTGTAGCGTTCGGAGT-3') are designed by PrimerPremier5.0 software, and PCR amplification is carried out by taking the Voronoi axillaris cDNA as a template.

PCR amplification System: 2 μ L cDNA, upstream and downstream primers (10 μmol. L)^-1) mu.L each, Super HF PCR Master Mix 10. mu.L, ddH₂And O is supplemented to 20 mu L. The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5 min;denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 1min, and performing 35 cycles; extension at 72 ℃ for 10 min.

The PCR product was detected by electrophoresis on a 1% agarose gel, and the band of interest was cut from the agarose gel using a clean scalpel and placed in a 1.5mL centrifuge tube

The target fragment was purified and recovered by the Quick Gel Extraction Kit (TransGen) agarose Gel recovery Kit, and then 5. mu.L of the recovered product was subjected to electrophoresis.

Ligation transformation of PCR products

According to

Instructions for the Blunt Cloning kit (TransGen) to ligate the recovered LlKnat13 fragment

On Blunt Vector, E.coli. After blue-white screening and bacterial liquid PCR detection, 10 positive clones are sent to Shanghai Biotechnology service company Limited for sequencing.

(1) Analytical method

An open reading frame of LlKnat13 and an amino acid sequence coded by the open reading frame are analyzed by using an online software ORF Finder, physicochemical properties and conserved structural domains of proteins are analyzed by using an ExPASy and SMART online tool, homologous sequences are searched in an NCBI database BLASTx, DNAMAN software is used for carrying out homologous sequence alignment, a Neighbor-Joining phylogenetic tree is constructed by MEGA6.06, and the number of times of bootstrap repetition is 1000.

(2) Analysis results

The open reading frame of LlKnat13 has a length of 1353bp (figure 2), the sequence is shown in SEQ ID NO.1, 450 amino acid residues are coded, the sequence is shown in SEQ ID NO.2, the molecular weight of the coded protein is 48.78kDa, the theoretical isoelectric point (pI) is 5.57, and the LlKnat13 is TALE/KNOX Homeobox family protein and comprises KNOX1, KNOX2, ELK and Homeobox KN structural domains (figure 3). Is highly similar to related homologous protein (> 65.78%), and has the closest relationship with dendrobium officinale (figure 4). Evolutionary analysis was performed with members of the KNOX family of arabidopsis thaliana and rice, and LlKnat13 grouped together with AtKNAT3, AtKNAT4, AtKNAT5, and AtKNAT7, belonging to Class II KNOX (fig. 4).

Real-time fluorescent quantitative PCR method

The method comprises the steps of detecting the expression conditions of LlKnat13 at different stages of lily bulbil formation by utilizing a real-time fluorescent quantitative PCR (qRT-PCR) method, wherein the fluorescent quantitative primers used for LlKnat13 expression analysis are LlKnat13-qRT-F (5'-TCAGGGAAGAGATTCTACGCAAG-3') and LlKnat13-qRT-R (5'-TTCCTTCACCAATCGTGCCTT-3'), and the LilyActin (GenBank Accession Number: JX826390) is used as an internal reference gene (lyActin-F: 5'-GCATCACACCTTCTACAACG-3', LilyActin-R: 5'-GAAGAGCATAACCCTCATA-3'), so that the PCR reaction of the internal reference gene and a target gene is completed in the same batch.

The reaction system of qRT-PCR is 20. mu.L, and comprises 2. mu.L cDNA (diluted 3 times) and upstream and downstream primers (10. mu. mol. L)^-1) 0.5 μ L each, 2 in

Premix Ex Taq TM(Tli RNaseH Plus)(TaKaRa)10μL，ddH₂O7. mu.L. The reaction procedure is as follows: pre-denaturation at 95 ℃ for 3 min; 95 ℃ for 20s, 60 ℃ for 10s, 72 ℃ for 20s, 40 cycles. By using 2^-ΔΔCtThe method analyzes the relative expression level of the LlKnat13 gene, 3 biological replicates are arranged in each sample, and the PCR reaction of the reference gene and the target gene is completed in the same batch.

Expression of LlKnat13 gene in different stages of bulbar formation

As shown in FIG. 5, LlKnat13 always maintains a high expression level during the formation of the bulbil of Toddalia juba, wherein the LlKnat13 gene is significantly up-regulated in the initial stage of the establishment of the bulbil meristem (S1), and then its expression level is maintained at a high level during the initiation of the bulbil primordia (S1-S3), while it is down-regulated in the differentiation of the bulbil primordia into bulbil (S4-S5), but the expression level is still higher than that of the S0 stage.

VIGS induces gene silencing of lilyturf LlKnat13

TRV vector construction

Primers for amplifying a target fragment (1bp-153bp) with the length of 153bp with a joint are designed, an upstream primer Llknat13-V-F (5'-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3') and a downstream primer LlKnat13-V-R (5'-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3'). Taking the axillary cDNA of the red sage root leaf as a template, amplifying a target band by using the primer, and recovering the gel.

The TRV2 carrier plasmid is cut by restriction enzyme EcoR I, and the cutting system is as follows: TRV2 vector plasmid 8. mu.L, EcoRI 1. mu.L, 10 XFast Digest 1. mu.L, ddH₂O10. mu.L. Enzyme cutting time: incubate at 37 ℃ for 20min, and stop the reaction at 80 ℃ for 5 min.

By using

Basic Seamless Cloning and Assembly Kit (TransGen) the desired fragment was ligated to the digested TRV2 linear vector by homologous recombination. Reaction system: TRV2 Linear vector 3. mu.L, target fragment 2. mu.L, 2 XOneStep Cloning Mix 5. mu.L. The connection reaction conditions are as follows: reacting at 50 deg.C for 30min, and storing at-20 deg.C after connection.

The TRV2 empty vector and the constructed TRV2-LlKnat13 vector are transformed into escherichia coli DH5 alpha competence through a heat shock method, whether an inserted target fragment is correct or not is detected through PCR and sequencing, an upstream primer is LlKnat13-V-jc-F (5'-TAGATAATGGTTTGGTGGTC-3') and a downstream primer is LlKnat13-V-jc-R (5'-TAGTTTAATGTCTTCGGGAC-3') are detected through PCR. Reaction system: bacterial liquid 1. mu.L, upstream and downstream primers (10. mu. mol. L)^-1) mu.L of each, 2 XTaq enzyme 5. mu.L, ddH₂O3. mu.L. Reaction procedure: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 1min for 30s, and performing 35 cycles; extension at 72 ℃ for 10 min. Detecting correct positive clone to extract plasmid, and transforming agrobacterium EHA105 by a freeze-thaw method.

VIGS mediated Agrobacterium infection

Agrobacterium EHA105 containing the different plasmids was inoculated in YEB liquid medium (containing 25mg/L rifampicin and 50mg/L kanamycin) and shaken at 200rpm at 28 ℃ overnight. 1mL of the bacterial suspension was added to 100mL of LYEB liquid medium (containing 25mg/L rifampicin and 50mg/L kanamycin), and the mixture was cultured at 28 ℃ and 200rpm with shaking until OD600 became 0.8. Centrifuge at 5000rpm for 10min, discard the supernatant, resuspend with permeation buffer (10mM MES +10mM MgCl2+ 200. mu.M acetosyringone), and adjust OD600 to 1.0. TRV1 and TRV2 bacterial liquid are mixed in a ratio of 1:1, and incubated for 3 hours in the dark at room temperature.

Selecting and sterilizing the surfaces of the lilium tigrinum stems, cutting the long stems into 1.5cm single stems with petioles under the aseptic condition, putting the single stems into an aseptic wide-mouth bottle, pouring the single stems into suspended mixed bacteria liquid, wrapping the bottle mouth with aseptic gauze wetted by 75% absolute ethyl alcohol, vacuumizing for 5min at 50kpa, slowly deflating, and repeatedly vacuumizing once again. Then washing the infected stem segments with sterile water, inoculating into a culture medium of MS +30g/L sucrose, culturing for 2d in the dark at 15 ℃, and then culturing at 22 ℃ under the light/dark condition of 16/8 h. Each treatment contained 3 replicates, each replicate 30 axils.

3. Molecular and phenotypic identification

Molecular and phenotypic identification was performed after two weeks of culture. qRT-PCR analysis shows that the expression of LlKnat13 gene is obviously reduced compared with the control group in the silent group. The statistics of the induction conditions of the bulblets are carried out, and the result shows that the induction rate of the bulblets in the silencing group is 76.72%, the induction rate of the bulblets in the control group is 90.92%, the induction rate of the bulblets in the silencing group is obviously lower than that of the control group, and the induction rate of the bulblets in the silencing group is reduced by 14.2% (fig. 6). From the VIGS-induced silencing result of the LlKnat13 gene, it can be concluded that the LlKnat13 gene plays an important role in the formation of lily bulbels, and that the LlKnat13 gene can be applied to the control of the formation of lily bulbels.

According to the invention, the lily LKnat13 gene cloned for the first time belongs to Class II KNOX, and the lily LKnat13 gene silencing is induced by VIGS technology, so that the bulbil induction rate is obviously reduced, therefore, the invention not only enriches the information of the research of the Class II KNOX gene, but also can perform effective manual intervention on lily bulbil formation by using the LKnat13 gene, and has wide application prospect in the aspect of improving the lily propagation efficiency.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Sequence listing

<110> vegetable and flower institute of Chinese academy of agricultural sciences

<120> clone of lily LlKnat13 gene and application method thereof in controlling formation of lily bulbil

<141> 2021-06-02

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1353

<212> DNA

<213> Lilium lancifolium

<400> 1

atgagagagt cataccaccc ccacctcccg cacgatatgg ccctgccatc gtactccgac 60

cagcatttgg cgatgatcgg cggtgactcc aacaccgctg gcggcaccga cggcggcgcc 120

ggcaaggagg ccaaggacat ccagcgcctc atgatcggtg ccgccggtaa gcagccgccg 180

cagctcggcg gcggcgggcc gaattggctc aacaacgccg tcctccgcca gcagggccac 240

cagtacggcg acggcagctt cctccatctc cagaccgcct ccgactcgtc cacctccccg 300

gtcgccggcg gtcgccagtg gctcccgcgc acggcgttcc ggagcgccag cgacgacgag 360

gtcccggcgt cgagcgactc gatgatcgcc gcggcgatgt cgcacgggtc cgccggcggc 420

ggacacggtg gagaggggga tgacggcggc ggaggcggtg gcggcgtcgg catgggaggg 480

gagggggcgg agatacaggt gggggaggtg gcctggcaga gtgctaggta caaggcggag 540

atactggccc acccgctgta cgagcagctg ctgtcggcgc acgtggcatg tctccgtatc 600

gcgacgccgg tggatcagct gccgaggatc gacgcgcagc tcgcgcagtc acagcaggtc 660

gttgcgaagt actccgtcct cggtgggggt cagatgattg gagatgatga taaggagctt 720

gatcagttca tgacacacta tgtactgtta ctgtgttcct tcaaagagca actacaacaa 780

catgtaagag tccatgcaat ggaagcagtg atggcttgct gggaacttga acaatctctt 840

caaagcctga caggagtctc tcctggagag ggaactggtg caactatgtc cgatgacgac 900

gatgatcagg ccgatagtga aactaacttc tacgatggaa gcatggatgc acaggatggc 960

ctgggtatgg gatttggtct tccaactgag agtgaacggt ccttgatgga gcgtgtcagg 1020

cacgagctga agcatgaact gaaacagggc tacaaagaga agattgtcga catcagggaa 1080

gagattctac gcaagcgaag agctgggaaa ctcccaggag ataccacttc taccttgaag 1140

gcatggtggc agtcccactc caaatggcct tacccgacgg aggatgataa ggcacgattg 1200

gtgaaggaaa cgggattgca actcaagcag ataaataact ggttcattaa tcaaaggaaa 1260

cgaaactggc acaacaatcc atcgtcatcc actgtaacaa agagcaaacg gaagagcaat 1320

gcaggtgaca ccaactccga acgctacaca tag 1353

<210> 2

<211> 450

<212> PRT

<213> Lilium lancifolium

<400> 2

Met Arg Glu Ser Tyr His Pro His Leu Pro His Asp Met Ala Leu Pro

1 5 10 15

Ser Tyr Ser Asp Gln His Leu Ala Met Ile Gly Gly Asp Ser Asn Thr

20 25 30

Ala Gly Gly Thr Asp Gly Gly Ala Gly Lys Glu Ala Lys Asp Ile Gln

35 40 45

Arg Leu Met Ile Gly Ala Ala Gly Lys Gln Pro Pro Gln Leu Gly Gly

50 55 60

Gly Gly Pro Asn Trp Leu Asn Asn Ala Val Leu Arg Gln Gln Gly His

65 70 75 80

Gln Tyr Gly Asp Gly Ser Phe Leu His Leu Gln Thr Ala Ser Asp Ser

85 90 95

Ser Thr Ser Pro Val Ala Gly Gly Arg Gln Trp Leu Pro Arg Thr Ala

100 105 110

Phe Arg Ser Ala Ser Asp Asp Glu Val Pro Ala Ser Ser Asp Ser Met

115 120 125

Ile Ala Ala Ala Met Ser His Gly Ser Ala Gly Gly Gly His Gly Gly

130 135 140

Glu Gly Asp Asp Gly Gly Gly Gly Gly Gly Gly Val Gly Met Gly Gly

145 150 155 160

Glu Gly Ala Glu Ile Gln Val Gly Glu Val Ala Trp Gln Ser Ala Arg

165 170 175

Tyr Lys Ala Glu Ile Leu Ala His Pro Leu Tyr Glu Gln Leu Leu Ser

180 185 190

Ala His Val Ala Cys Leu Arg Ile Ala Thr Pro Val Asp Gln Leu Pro

195 200 205

Arg Ile Asp Ala Gln Leu Ala Gln Ser Gln Gln Val Val Ala Lys Tyr

210 215 220

Ser Val Leu Gly Gly Gly Gln Met Ile Gly Asp Asp Asp Lys Glu Leu

225 230 235 240

Asp Gln Phe Met Thr His Tyr Val Leu Leu Leu Cys Ser Phe Lys Glu

245 250 255

Gln Leu Gln Gln His Val Arg Val His Ala Met Glu Ala Val Met Ala

260 265 270

Cys Trp Glu Leu Glu Gln Ser Leu Gln Ser Leu Thr Gly Val Ser Pro

275 280 285

Gly Glu Gly Thr Gly Ala Thr Met Ser Asp Asp Asp Asp Asp Gln Ala

290 295 300

Asp Ser Glu Thr Asn Phe Tyr Asp Gly Ser Met Asp Ala Gln Asp Gly

305 310 315 320

Leu Gly Met Gly Phe Gly Leu Pro Thr Glu Ser Glu Arg Ser Leu Met

325 330 335

Glu Arg Val Arg His Glu Leu Lys His Glu Leu Lys Gln Gly Tyr Lys

340 345 350

Glu Lys Ile Val Asp Ile Arg Glu Glu Ile Leu Arg Lys Arg Arg Ala

355 360 365

Gly Lys Leu Pro Gly Asp Thr Thr Ser Thr Leu Lys Ala Trp Trp Gln

370 375 380

Ser His Ser Lys Trp Pro Tyr Pro Thr Glu Asp Asp Lys Ala Arg Leu

385 390 395 400

Val Lys Glu Thr Gly Leu Gln Leu Lys Gln Ile Asn Asn Trp Phe Ile

405 410 415

Asn Gln Arg Lys Arg Asn Trp His Asn Asn Pro Ser Ser Ser Thr Val

420 425 430

Thr Lys Ser Lys Arg Lys Ser Asn Ala Gly Asp Thr Asn Ser Glu Arg

435 440 445

Tyr Thr

450

Claims

1. A lily LlKnat13 gene clone and an application method thereof in controlling lily bulbil formation are characterized in that: the method comprises the following steps:

A. the method for cloning lilium tigrinum LlKnat13 gene comprises the following steps: extracting RNA of axillary leaves of lilium tigrinum, designing a primer according to transcriptome data, carrying out PCR amplification by taking cDNA as a template to obtain a CDS region of the lilium tigrinum, wherein the CDS sequence primer is amplified as follows, and the upstream Llknat 13-F: 5'-ATGAGAGAGTCATACCACCCC-3', downstream Llknat 13-R: 5'-CTATGTGTAGCGTTCGGAGT-3', sequencing the CDS sequence amplified, and comparing with the transcriptome sequence to obtain a cDNA sequence of LlKnat13 gene containing complete ORF, wherein the base sequence is shown as SEQ ID NO. 1;

B. relative expression analysis method of lilium tigrinum LlKnat13 gene: extracting RNA of lilium tigrinum leaf axillary, designing a primer according to SEQ ID NO.1, carrying out qRT-PCR amplification by using cDNA as a template, and adopting 2^-ΔΔCtThe method analyzes the relative expression level of the Llknat13 gene, and the amplification primers are as follows: upstream LlKnat 13-qRT-F: 5'-TCAGGGAAGAGATTCTACGCAAG-3', downstream LlKnat 13-qRT-R: 5'-TTCCTTCACCAATCGTGCCTT-3', respectively;

C. the application of the gene in controlling the formation of the bulbil of lilium tigrinum comprises the following steps: primers LlKnat13-V-F5 '-TTCTGTGAGTAAGGTTACCGATGAGAGAGTCATACCACCCCCA-3' and LlKnat13-V-R5 '-CCCATGGAGGCCTTCTAGAGATCATGAGGCGCTGGATGTCCTT-3' with joints are designed according to SEQ ID NO.1, a target segment of the LlKnat13 gene with the joints is connected with a TRV2 linear vector after enzyme digestion through a homologous recombination method to obtain a TRV 2-LKnat 13 vector, the constructed TRV 2-LKnat 13 vector is transformed into an agrobacterium strain EHA105, the vector is activated and shaken to reach OD600 ═ 0.8, then vacuum pumping is carried out for 5min to infect the lilac stalk segment, then the infected stalk segment is cleaned and inoculated into a culture medium of MS +30g/L sucrose to induce formation of bulbels, observation and statistics are carried out after two weeks of culture, and compared with a control group, the bulbel induction rate is remarkably reduced.

2. The lily LlKnat13 gene clone and the application method thereof in controlling lily bulblet formation according to claim 1, wherein the gene clone comprises the following steps: the leaf axils in the step A and the step B are obtained by cutting the leaf axils of the clanis bilineata with a double-sided blade, extracting total RNA by adopting the instruction of an RNAprep Pure polysaccharide polyphenol plant total RNA extraction kit, and adopting Trans as a template and adopting the extracted total RNA

3. The lily LlKnat13 gene clone and the application method thereof in controlling lily bulblet formation according to claim 1, wherein the gene clone comprises the following steps: the material taking mode of axilla of lilium tigrinum leaf in the step B is as follows: the method comprises the following steps of (1) respectively selecting bulbels from in-vitro cultured pennisetum crispum stem segments to form different stages: S0-S5, taking 5-7 axilla from 5 plant of red lead respectively, wrapping with tinfoil paper, quick freezing with liquid nitrogen, and storing in a refrigerator at-80 deg.C for use, wherein S0: underarm of uninduced culture; S1-S3: the bud primordium is started to form; S4-S5: differentiating the bulbil primordia into bulbils.