CN114058630B

CN114058630B - Litchi MADS-box transcription factor LcMADS1 and application thereof in inhibiting plant organ abscission

Info

Publication number: CN114058630B
Application number: CN202111415537.0A
Authority: CN
Inventors: 李彩琴; 宋雯佩; 李娟�; 张宪智; 李芳�; 赵明磊; 温珍熹; 马兴帅; 李建国
Original assignee: Zhongkai University of Agriculture and Engineering
Current assignee: Zhongkai University of Agriculture and Engineering
Priority date: 2021-11-25
Filing date: 2021-11-25
Publication date: 2022-06-07
Anticipated expiration: 2041-11-25
Also published as: CN114058630A

Abstract

The invention discloses a litchi MADS-box transcription factor LcMADS1 and application thereof in inhibiting plant organ abscission. The complete open reading frame 834bp of litchi LcMADS1 gene is cloned, 277 amino acids are coded, the gene is heterogeneously and over-expressed in an Arabidopsis wild type, the falling of plant floral organs is obviously inhibited, even the floral organs are not fallen when the pod is dried and cracked, and the litchi LcMADS1 transcription factor is shown to have the function of inhibiting the falling of the plant organs. The invention can obtain the strain for inhibiting plant organs by regulating the expression of litchi LcMADS1 gene, and the provided litchi LcMADS1 gene can provide direction and technical support for variety improvement and breeding of plants, especially litchi.

Description

Litchi MADS-box transcription factor LcMADS1 and application thereof in inhibition of plant organ abscission

Technical Field

The invention relates to the field of plant genetic engineering, in particular to a litchi MADS-box transcription factor LcMADS1 and application thereof in inhibiting plant organ abscission.

Background

Litchi (lichi chinensis Sonn.) belongs to the sapinduceae (Sapindaceae) Litchi (lichi), is a characteristic fruit tree in China, frequently appears in the production field of 'flowers but not fruits', 'full flowers and half fruits, even no grains', and brings difficulties to the stable, high-quality, healthy and sustainable production of the Litchi industry.

MADS-box is a transcription factor widely distributed in eukaryotes and plays an important role in the process of plant organ abscission. Over-expressing Arabidopsis MADS-box family members AGL15, AGL18 and AtFYF can delay the shedding and the senescence of Arabidopsis floral organs, and the shedding of the floral organs of the double mutant AGL15/AGL18 is earlier than that of the wild type; STK is an important MADS-box family member for controlling the development of the stipe of arabidopsis thaliana and the shedding of seeds, the volume of the pods and the seeds of the STK mutant is reduced, but the division degree of the cells of the stipe is improved, the characteristics of the isolated cells at the joint of the stipe and the seeds are lost, and the mature seeds cannot shed freely. Tomato MADS-box family members J, MC and SlMBP21 may regulate the differentiation of tomato flower (fruit) stalk abscissas by forming the J-MC-SlMBP21 complex, e.g., in J mutants, MC antisense transgenic plants and SlMBP21-RNAi plants, tomato flower (fruit) stalk abscissas cannot be formed; after the SLMBP21 is over-expressed, the near axial end of the tomato stalk is shortened and ectopic tissue structures similar to the abscission area appear; j, MC and SlMBP21 are co-transferred to wild tomatoes, the carpopodium is obviously shortened and is converted into an isolated tissue structure, and the near axial end, the isolated area and the far axial end of the carpopodium are not obviously differentiated; J. the MC protein and the SlMBP21 protein can interact pairwise, and the J protein and the SlMBP21 protein can form a dimer; downstream genes that are regulated together by gene expression analysis surface, such as LATERAL SUPPRESSOR (Ls), WUSCHEL (LeWUS), GOBLET (GOB), Blind (Bl); the over-expression plant of the tomato Sl-AGL11 gene which is highly homologous with the Arabidopsis STK shows the phenomenon that the carpopodium abscission area is abnormally enlarged and the fruit does not fall off when being mature. The J homologous gene MdJb of the apple can recover the structure and the function of a flower (fruit) stalk abscission region after being heterogeneously and over-expressed in a tomato jointless mutant; heterologous overexpression of the J homologous gene psjoins of 'kohler bergamot' in the wild type tomato can lead to significant increases in petiole abscission rate and cellulase activity, thinning of the petioles, increased numbers of ectopic cells and disorganization of the arrangement. Therefore, the MADS-box transcription factor plays an important role in regulation and control in the process of plant organ abscission, and research results on the litchi MADS-box transcription factor provide important theoretical guidance for breeding varieties with few fruit drops, provide effective breeding means for yield improvement, and have important significance for agricultural production practice.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a litchi MADS-box transcription factor LcMADS 1.

The invention also aims to provide the application of the litchi MADS-box transcription factor LcMADS1 in inhibiting the abscission of plant organs.

The purpose of the invention is realized by the following technical scheme:

a litchi MADS-box transcription factor LcMADS1 has an amino acid sequence shown in SEQ ID NO. 2.

The biological material related to the litchi MADS-box transcription factor LcMADS1 is any one or combination of more of the following biological materials:

1) nucleic acid molecules encoding the litchi MADS-box transcription factor LcMADS 1;

2) an expression cassette comprising the nucleic acid molecule of 1);

3) a recombinant vector comprising the nucleic acid molecule of 1) or a recombinant vector comprising the expression cassette of 2);

4) a recombinant microorganism comprising the nucleic acid molecule of 1), or a recombinant microorganism comprising the expression cassette of 2), or a recombinant microorganism comprising 3) the recombinant vector;

5) transgenic plant tissue comprising the nucleic acid molecule of 1) or transgenic plant tissue comprising the expression cassette of 2).

Further, the sequence of the nucleic acid molecule in 1) is shown as SEQ ID NO. 1.

Further, the recombinant vector in 3) is obtained by constructing the sequence shown in SEQ ID NO. 1 into a pCAMBIA1302 plant expression vector.

Further, the recombinant microorganism described in 4) is obtained by transforming the recombinant vector described in 3) into Agrobacterium GV 3101.

The application of the biological material related to the litchi MADS-box transcription factor LcMADS1 or litchi MADS-box transcription factor LcMADS1 in inhibiting the shedding of plant organs is provided.

The application of the biological material related to the litchi MADS-box transcription factor LcMADS1 or litchi MADS-box transcription factor LcMADS1 in culturing transgenic plants with delayed organ abscission.

Further, the plant is a dicotyledonous plant or a monocotyledonous plant.

Still further, the plants include, but are not limited to, litchi, Arabidopsis, tomato.

Further, the organ includes, but is not limited to, fruit, petals, calyx, filaments, leaves.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides a litchi MADS-box transcription factor LcMADS1 with the function of inhibiting the shedding of plant organs, the expression quantity of the gene in a stalk separation area for promoting the fruit dropping treatment is obviously lower than that of a control and the fruit dropping treatment inhibition, and the gene is closely related to the shedding of litchi fruits.

Further experiments show that the litchi LcMADS1 gene heterologous overexpression can obviously inhibit the shedding of arabidopsis flower organs, and the litchi LcMADS1 gene plays an important role in regulating and controlling the shedding of plant organs, so the litchi LcMADS1 gene provided by the invention can provide direction and technical support for the variety improvement and breeding of plants, particularly litchi.

Drawings

FIG. 1 is a diagram showing the analysis of the gene expression level of LcMADS1 in the abscission zone of litchi carpopodium; wherein, A is the influence of different treatments on the drop of the young fruits of the Feizixiao litchi; b, analyzing the gene expression level of LcMADS1 in the litchi carpopodium abscission area by using a qRT-PCR technology; GPD is the treatment of girdling and leaf removing, cutting off the nutrition supply of the tree body and inducing fruit drop; ETH is ethephon treatment and fruit drop induction; GPDD is prepared by girdling and removing leaves and carrying out 2,4-D treatment to inhibit fruit drop; data are presented as mean ± standard deviation; different lower case letters represent significant differences at the level of P <0.05 using the LSD test.

FIG. 2 is a graph showing the observation result of heterologous overexpression of litchi LcMADS1 gene for inhibiting the abscission of arabidopsis flower organs; wherein, A is the wild type (Col-0) and heterologous overexpression LcMADS1(35S:: LcMADS1) strain inflorescence; b is comparison of inflorescences in Col-0 and 35S LcMADS1 strains; c is 35S, namely, LcMADS1 strain is matured and dried to crack fruit pods; the number represents the position of the flower counted from top to bottom from the top of the inflorescence from which the white petal flower emerges; arrows indicate non-shedding floral organs.

FIG. 3 is the cross-section scanning electron microscope observation image of the flower organs of LcMADS1 strain Col-0 and 35S; wherein A is a Col-0 strain; b is 35S, LcMADS1 strain; the observed flower was the P8 flower; the arrow indicates 35S, the fracture surface of the LcMADS1 strain after the flower organ is forcibly pulled out.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

Example 1LcMADS1 Gene acquisition and analysis of expression Pattern during litchi fruit shedding

The experimental material is Feizixiao litchi. Selecting ears with basically consistent growth vigor in different directions of plants about 25 days after blooming. Since fruit shedding is closely related to ethylene and nutrient supply, we designed 3 different treatments for this, 6 per ear treated, 3 replicates: (1) ethephon Treatment (ETH), soaking the ears with 250mg/L Ethephon for 1 min; (2) girdling Plus Defomation (GPD), performing Girdling with a width of about 2cm and a depth reaching xylem at a distance of about 10cm from the base of the cluster to remove phloem, and simultaneously picking off leaves above the Girdling opening to completely block the supply of young fruit carbohydrates by the tree; (3) removing leaves by girdling and treating with 2,4-D (GPDD), and soaking the ears with 20 mg/L2, 4-D for 1min after girdling and removing leaves. Ears without any treatment were used as controls (controls). Fruit drop was recorded daily after treatment and observed continuously for 3 days. The results show that both ETH and GPD treatment can remarkably induce a large amount of shedding of litchi young fruits, and GPDD can remarkably inhibit shedding of litchi young fruits (fig. 1A).

Collecting litchi carpopodium departure zone samples (namely cutting materials at 0.2cm above and below the carpopodium departure position) at 0, 1, 2 and 3d after treatment, extracting RNA, and detecting the expression level of LcMADS1 gene by qRT-PCR (quantitative reverse transcription-polymerase chain reaction), wherein the expression level of the gene in the carpopodium departure zone of the fruit-drop promotion treatment (ETH and GPD) is obviously lower than that of a Control (Control) and a fruit-drop inhibition treatment (GPDD) (figure 1B). Therefore, we guess that the gene is closely related to litchi fruit shedding. The specific steps of analyzing the expression difference of the LcMADS1 gene in different carpopodium abscission areas are as follows:

1. extraction of litchi RNA

The total RNA is extracted from the litchi carpopodium by a columniform plant RNAout 2.0 extraction kit (Tiannuze) according to the instruction. The extracted RNA was checked for integrity by electrophoresis on a 1.2% agarose gel and RNA concentration and OD by UV spectrophotometer (BioSpec-nano).

2. Reverse transcription of cDNA

The extracted RNA was reverse-transcribed into cDNA in equal amounts using One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransScript), and diluted 20-fold for use.

3、qRT-PCR

Specific primers of LcMADS1 gene are designed by using Primer 5.0 software, and litchi LcEF-1 alpha and LcGAPDH are used as reference genes. The fluorescent dye used was GoTaq qPCR Master Mix and the instrumentation used was a Bio-Rad CFX96 PCR instrument. Amplification program setupComprises the following steps: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 5 s; annealing at 55 ℃ for 30 s; extension at 72 ℃ for 30 s; the number of cycles was 40. After completion of the reaction, data were exported according to 2^-△△CTThe expression level of the gene is calculated. The primer sequences are shown in Table 1.

TABLE 1LcMADS1 qRT-PCR primer sequences

The CDS sequence of the LcMADS1 gene is shown as SEQ ID NO. 1, the open reading frame is 834bp, 277 amino acids are coded, as shown as SEQ ID NO. 2, the gene is named as LcMADS 1:

LcMADS1 CDS sequence (SEQ ID NO: 1):

ATGCAACTTATTATTAGGTACTTCAAGATGGGAAGAGGGAAGATCGAGATCAAGAGAATCGAAAACACGACGAATCGTCAGGTCACCTTCTGCAAGAGGAGAAATGGACTCTTGAAGAAAGCTTATGAACTATCAGTTCTTTGTGATGCTGAAGTTGCCCTCATCGTCTTCTCCAGCCGCGGTCGCCTCTATGAGTACTCTAATAACAACAACATAAGGTCAACCATAGAGAAGTACAAGAAGGCAGCCTCTGATAATACAAATGCAACATCTGTTACAGAAATCAATGCTCAGTATTATCAGCAGGAATCAGCTAAGCTGCGTCAACAGATTCAAATGTTGACGAATTCCAACAGGCACTTAATGGGAGATTCCCTGAGCTCTTTGTCTGTGAAGGAGCTGAAGCAGCTGGAGAATAGGCTTGAACGAGGCATTACTAGAATCAGATCAAAGAAGCATGAAATGTTGCTAGCTGAAATTGAGTACTTGCAGAAAAGGGAGATGGAGCTGGAAAATGAAAGTGTGACTCTTAGATCAAGGATTGCGGACATTGAGAGGTTTCAGCAGTCTAACATGGTAACAGGCCCAGAGCTGAATGCAATTCATGCGTTGGCTTCTCGAAATTTCTTCAATCCAGCTTTCATTGAGGATGCAAGTGGAAGCTCATATTCCCATCCTGACAAGAAACTGCTCCATCTTGGGTATTTTCTCTACATATTTCATTTCATTGCATTTATATATTTCCATAGTTATTTATTGGAGGCTAATCGGGTGTGGGACAAAATTTCGTGGGGAATCAGATTTAGTATCATTACAAAACCAACCGTCAGATGA

LcMADS1 (SEQ ID NO: 2):

MQLIIRYFKMGRGKIEIKRIENTTNRQVTFCKRRNGLLKKAYELSVLCDAEVALIVFSSRGRLYEYSNNNNIRSTIEKYKKAASDNTNATSVTEINAQYYQQESAKLRQQIQMLTNSNRHLMGDSLSSLSVKELKQLENRLERGITRIRSKKHEMLLAEIEYLQKREMELENESVTLRSRIADIERFQQSNMVTGPELNAIHALASRNFFNPAFIEDASGSSYSHPDKKLLHLGYFLYIFHFIAFIYFHSYLLEANRVWDKISWGIRFSIITKPTVR

example 2 Arabidopsis thaliana heterologous overexpression identification of litchi LcMADS1 gene function

1. LcMADS1 gene full-length amplification primer design

Performing sequence amplification by using KOD-Plus-Neo enzyme by using a PCR method, wherein the cDNA is the litchi carpopodium sample in the step. Full-length amplification primers are designed by using Primer 5.0 software, and primers containing proper enzyme cutting sites are designed at an initiation codon ATG and a termination codon respectively according to a CDS sequence of LcMADS1, wherein the enzyme cutting sites are Nco I. The primer sequences are shown in Table 2, and the square frame part is the enzyme cutting site.

TABLE 2 LcMADS1 full-Length amplification primer sequences

2. PCR reaction system, program and product detection for gene cloning

(1) PCR reaction system, as shown in table 3:

TABLE 3 PCR reaction System

(2) PCR reaction procedure

Pre-denaturation at 94 deg.C for 2min, denaturation at 94 deg.C for 15s, annealing at 58 deg.C for 30s, extension at 68 deg.C for 2min, 35 cycles, extension at 68 deg.C for 5min, and storage at 12 deg.C.

(3) Detection and recovery of PCR products

The PCR product was detected by 1.2% agarose gel electrophoresis at 135V and 110A for 30 min. And then, placing the agarose Gel into a Gel imaging system for imaging, determining whether a fragment containing the target gene exists, if a bright band appears and the size is correct, quickly cutting a Gel block containing the target fragment by using a scalpel under an ultraviolet Gel cutting instrument, and then recovering by using a DNA Gel recovery Kit (FastPugel DNA Extraction Mini Kit, Novosa), wherein the method refers to the Kit instruction, and the recovered product is stored in a refrigerator at the temperature of-20 ℃.

3. LcMADS1-pCAMBIA1302 plant expression vector construction

The LcMADS1 clone fragment was inserted into pCAMBIA1302 vector (promoter CaMV35S, selection marker for transformed plants hygromycin) by using In-fusion ligation Kit (Clonexpress II One Step Cloning Kit, Novonza), the ligation system was as shown In Table 4, mixed well, and reacted at 37 ℃ for 30 min.

TABLE 4 In-fusion ligation System

After the reaction is completed, Escherichia coli DH5 alpha is transformed, positive clones are identified and screened by PCR, and the single clones which are verified to be correct are sent to Guangzhou Egypti Biotechnology Limited for sequencing.

4. Transformation of Agrobacterium

After sequencing identification, the recombinant plasmid is transformed into agrobacterium GV3101, and positive clones are identified and screened by PCR.

5. Agrobacterium-mediated Arabidopsis genetic transformation (floral dip method) and screening of positive transgenic lines

The obtained agrobacterium containing the recombinant plasmid is cultured in LB liquid culture medium (with 100mg/L Kan and 50mg/L Rif added) until OD is 0.8, centrifuged at 5000rpm at room temperature for 10min to collect thalli, prepared staining solution (5% sucrose, 0.05% (v/v) silwet L-77) is slowly added to the thalli sediment, and the OD of the bacterial solution is about 1. Placing the wild arabidopsis thaliana Col-0 inflorescence into the infection solution for 1min, culturing the arabidopsis thaliana plant overnight under the condition of keeping out of the sun after infection, and taking out the plant from the next morning to continue culturing under the normal condition. In order to improve the infection efficiency, the arabidopsis thaliana needs to be infected once every 4-5 days, and the arabidopsis thaliana is normally cultured until seeds are mature after being infected for 3-4 times. After the seeds are matured, the arabidopsis seeds are harvested, namely the transgenic T0 generation seeds.

6. Phenotypic identification of transgenic Arabidopsis plants

After drying mature T0 generation seeds, the seeds were treated with 0.5% NaClO₃After disinfection, the seedlings are sown on an MS screening culture medium containing hygromycin B (Hyg, 50mg/L) antibiotics, placed at 4 ℃ for vernalization for 2-3d, then moved to a light incubator for culture, positive seedlings are screened when the seedlings grow roots and 2 cotyledons, and the seedlings are transferred to nutrient soil for continuous culture (false positive seedlings do not grow roots, cotyledons are yellowed or do not grow under the condition of Hyg resistance). And when about 8 leaves grow out from the positive plant seedlings, collecting the leaves and carrying out qRT-PCR detection.

The plants of each generation were tested for positive lines until propagation to T3 generations to obtain homozygous transgenic Arabidopsis lines.

The transgenic T3 generation strain and the wild type strain are planted under the same conditions, and the phenotype observation shows that the wild type Arabidopsis thaliana Col-0 strain starts from the 9 th flower/fruit pod (P9) and the flower organs (calyx, petals and stamen) are completely shed, while 35S:: the flower organs of the LcMADS1 strain do not show obvious shedding phenomenon, which indicates that the heterologous overexpression of the LcMADS1 can inhibit the shedding of the flower organs, the calyx does not show obvious senescence phenomenon, even the mature fruit pod still keeps fresh green and has no yellowing phenomenon, and the calyx still does not shed after the fruit pod is dried up (figure 2).

EXAMPLE 3 Arabidopsis thaliana floral organ abscission surface scanning electron microscope observation

Collecting Col-0 and 35S respectively, namely, collecting flowers/fruit pods (P8) at the 8 th position of the LcMADS1 strain, removing flower organs by using forceps, fixing by using FAA fixing solution (50% absolute ethyl alcohol, 5% glacial acetic acid and 1% formaldehyde), and storing at 4 ℃ after air extraction. And drying the sample at a critical point, spraying a gold coating film, and observing and photographing under a scanning electron microscope. The results (FIG. 3) show that 35S:LcMADS 1 strain flower organ (P8 flower/fruit pod) is difficult to pull out, obvious cell damage phenomenon (obvious fracture and arrow) is left at the abscission area after the flower organ is pulled out by force, and Col-0 strain P8 position flower organ abscission area cells have smooth surfaces, the surface cells are loosened early and fall off at once.

In conclusion, the expression of the litchi LcMADS1 gene in the carpopodium abscission area is obviously reduced along with the aggravation of litchi fruit abscission, the abscission of arabidopsis flower organs can be obviously inhibited by carrying out heterologous overexpression on the LcMADS1 gene by a genetic transformation means, and the litchi LcMADS1 gene is presumed to have the function of inhibiting the abscission of plant organs.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

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Claims

1. A litchi MADS-box transcription factor LcMADS1 is characterized in that: the amino acid sequence is shown in SEQ ID NO. 2.

2. The litchi MADS-box transcription factor LcMADS 1-related biological material as claimed in claim 1, which is characterized in that: is any one or more combination of the following biological materials:

2) an expression cassette comprising the nucleic acid molecule of 1);

4) a recombinant microorganism comprising the nucleic acid molecule of 1), or a recombinant microorganism comprising the expression cassette of 2), or a recombinant microorganism comprising the recombinant vector of 3).

3. The litchi MADS-box transcription factor LcMADS 1-related biomaterial as claimed in claim 2, wherein: 1) the sequence of the nucleic acid molecule is shown as SEQ ID NO. 1.

4. The litchi MADS-box transcription factor LcMADS 1-related biomaterial as claimed in claim 2, wherein: 3) the recombinant vector is obtained by constructing a sequence shown in SEQ ID NO. 1 into a pCAMBIA1302 plant expression vector.

5. The litchi MADS-box transcription factor LcMADS 1-related biomaterial as claimed in claim 2, wherein: 4) the recombinant microorganism described in (3) above is obtained by transforming the recombinant vector described in (3) above into Agrobacterium GV 3101.

6. The use of the litchi MADS-box transcription factor LcMADS1 in claim 1 or the litchi MADS-box transcription factor LcMADS 1-related biomaterial in any one of claims 2 to 5 for inhibiting the abscission of plant organs, wherein:

the plant is litchi or arabidopsis thaliana;

the organs are fruits, petals, calyx and/or filaments.

7. The use of the litchi MADS-box transcription factor LcMADS1 in claim 1 or the litchi MADS-box transcription factor LcMADS 1-related biomaterials in claim 2 to 5 for breeding transgenic plants with delayed organ abscission, wherein the biomaterials comprise:

the plant is litchi or arabidopsis thaliana;

the organs are fruits, petals, calyx and/or filaments.