阅读说明：本技术 芝麻蛋白SiLLR在调控植物根系发育中的应用 (Application of sesamin SiLLR in regulation and control of plant root system development ) 是由 游均 山姆 黎冬华 苏如齐 张秀荣 周瑢 刘爱丽 于 2020-07-08 设计创作，主要内容包括：本发明阐述了芝麻蛋白SiLLR在调控植物根系发育中的应用。通过将芝麻蛋白SiLLR的编码基因转入拟南芥中进行过表达,可显著增加植株的侧根长度和根鲜重,并最终可以增加植株根系总量。依此表明,该基因是控制植株根系生长的关键基因之一,在提高植物抗性及产量等性能上有很好的应用前景。(The invention discloses application of sesamin SiLLR in regulation and control of plant root system development. The coding gene of the sesamin SiLLR is transferred into arabidopsis thaliana for overexpression, so that the length of lateral roots and the fresh weight of the roots of a plant can be remarkably increased, and the total amount of the root system of the plant can be finally increased. Therefore, the gene is one of the key genes for controlling the growth of the plant root system, and has good application prospect in improving the performances of plant resistance, yield and the like.)

1. The application of the sesamin SiLLR or the related biological material thereof in regulating and controlling the development of plant roots; the related biological material is a nucleic acid molecule capable of expressing the sesamin SiLLR or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

2. The use of claim 1, wherein the sesamin SiLLR or the gene encoding the same is expressed in the plant in an increased amount and/or activity to enhance plant root development.

3. Use as claimed in claim 1 or claim 2 wherein the plant root development is manifested as at least one of: average lateral root length, fresh root weight.

4. The use as claimed in claim 1 or 2, wherein the sesamin SiLLR is an amino acid sequence having the amino acid sequence shown in SEQ ID No.1 or an amino acid sequence with the same function formed by replacing, deleting or adding one or more amino acids in the sequence.

5. The use as claimed in claim 1 or 2, wherein the encoding gene of the sesamin SiLLR is a DNA sequence as shown in SEQ ID NO.2 or a DNA sequence with equivalent function generated by adding, substituting, inserting or deleting one or more nucleotides in the sequence.

6. A method for producing a plant variety having an increased mean lateral root length and/or an increased fresh root weight, comprising the step of increasing the expression level and/or activity of the sesamin sirllr as defined in claim 1 in a recipient plant.

7. A method of breeding transgenic plants with increased mean lateral root length and/or increased fresh root weight comprising the steps of: introducing into a recipient plant a nucleic acid molecule capable of expressing a sesamin SiLLR as defined in claim 1, to obtain a transgenic plant; the transgenic plant has an increased average lateral root length and/or an increased fresh root weight as compared to the recipient plant.

8. The method of claim 7, wherein said introducing into a recipient plant a nucleic acid molecule capable of expressing sesamin SiLLR is effected by introducing into said recipient plant a recombinant expression vector comprising a gene encoding said sesamin SiLLR.

9. The method of claim 7, wherein the recipient plant is Arabidopsis thaliana.

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to application of sesamin SiLLR in regulation and control of plant root system development.

Background

Sesame (Sesamum indicum L.) is one of important high-quality oil crops in the world, the annual planting area of Chinese sesame is 80 ten thousand hectares, the total yield is about 75 ten thousand tons, and the sesame occupies the top of the world. Meanwhile, sesame is a typical oil crop planted in summer, is warm and bright, and can complete the whole growth period within 3-4 months. Despite the many advantages of sesame, the problems of low and unstable sesame yield have been significant problems limiting the development of the sesame industry. The main reason for the limited sesame production is that sesame is mainly distributed in developing countries, usually in small farmers. Although sesame has a high yield potential, the actual yield is rather low and unstable due to the combined action of biotic and abiotic stress, which is also a significant bottleneck problem for the development of the sesame industry.

Sesame is a shallow root crop, the sesame root system is not only an organ for supporting the plant body to carry out vegetative growth, but also a main organ for absorbing water and mineral elements, and is also a metabolic circulation organ, and the sesame is sensitive to the response of external environmental conditions. The sesame root system is divided into a straight root system and a branch root system, wherein the straight root system is characterized in that the main root is very long and the side roots are few; the branch root system has less developed main root and more lateral roots. Generally, sesame varieties in arid areas are mostly straight root systems, and sesame varieties in sandy soil or humid areas are mostly branch root systems. However, the root system of the sesame is an underdeveloped root system, the total amount of the root system is less, and the improvement of the yield of a single sesame plant is indirectly influenced, so that the high and stable yield is promoted by enhancing the total amount of the root system of the sesame, and the sesame cultivation method is necessary for promoting the development of the sesame industry in China and solving the problem of insufficient total amount self-supply.

Disclosure of Invention

The invention aims to provide application of sesamin SiLLR in regulation and control of plant root system development. The sesame protein SiLLR and the coding gene thereof have very low homology with arabidopsis thaliana and other crops, and the gene sequence has no functional annotation, the invention proves that the gene and the expression protein thereof have the function of increasing the total amount of plant root systems for the first time, and the gene can be transferred into a receptor plant through a gene engineering technology to achieve the aim of improving the plant biomass and the yield.

In order to solve the technical problems, the technical scheme adopted by the invention is as follows:

the application of the sesamin SiLLR or the related biological material thereof in regulating and controlling the development of plant roots; the related biological material is a nucleic acid molecule capable of expressing the sesamin SiLLR or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

A method for producing a plant variety having an increased mean lateral root length and/or an increased fresh root weight, comprising the step of increasing the expression level and/or activity of said sesamin SiLLR in a recipient plant.

A method of breeding transgenic plants with increased mean lateral root length and/or increased fresh root weight comprising the steps of: introducing a nucleic acid molecule capable of expressing the sesamin SiLLR into a receptor plant to obtain a transgenic plant; the transgenic plant has an increased average lateral root length and/or an increased fresh root weight as compared to the recipient plant.

Compared with the prior art, the invention has the advantages that:

the invention can transfer the nucleic acid molecule capable of expressing the sesamin SiLLR into a receptor plant, and the overexpression of the nucleic acid molecule in the receptor plant can enhance the root development of the receptor plant. According to the method provided by the invention, the total root system amount of the plant can be increased by using the overexpression of the sesamin SiLLR so as to be used for breeding high-yield varieties of oil crops and ensure the high and stable yield of the crops.

Drawings

FIG. 1 is a diagram of the construction of a plant recombinant expression vector pCAMBIA1301S-SiLLR containing a SiLLR gene provided in the embodiment of the present invention;

FIG. 2 is a diagram showing the results of the verification of the quantitative expression of T2 generation plants of SiLLR transgenic Arabidopsis provided by the embodiments of the present invention; in the figure, WT represents a wild-type control group, and SiLLR-1, SiLLR-2 and SiLLR-3 represent three groups of SiLLR transgenic Arabidopsis T2 generation plants, respectively;

FIG. 3 is a scanned image of the root system of plants of T2 generation of SiLLR transgenic Arabidopsis provided by the embodiment of the present invention;

FIG. 4 is a scanned view of a root system of a wild type Arabidopsis plant according to an embodiment of the present invention;

FIG. 5 is a graph comparing the average lateral root length (cm) results of the SiLLR transgenic Arabidopsis T2 generation plant groups (SiLLR-1 and SiLLR-2) and the wild type Arabidopsis plant group (WT) provided in the example of the present invention;

FIG. 6 is a comparison of the root fresh weight (mg) results of the SiLLR transgenic Arabidopsis T2 generation plant groups (SiLLR-1 and SiLLR-2) and the wild type Arabidopsis plant group (WT) provided in the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Molecular cloning is generally performed according to conventional conditions such as Sambrook et al: a Laboratory Manual (New York: Cold spring Harbor Laboratory Press,1989), or Draper et al (Blackwell scientific Press, 1988), or according to the conditions recommended by the manufacturer of the reagents used.

Acquisition of sesame gene SiLLR

The invention relates to a sesame gene SiLLR which can regulate and enhance the root development of a target plant, and an obtaining method comprises the following steps:

(1) according to the detection results of phenotype, geographical source and genetic diversity of drought-resistant related characters, a step-by-step sampling strategy is adopted from 7910 sesame germplasm resources at home and abroad stored in a national sesame middle-stage library, and 327 sesame samples with different drought resistances are selected for re-sequencing analysis;

(2) carrying out whole genome re-sequencing on 327 sesame samples with low coverage by using a2 x 76 double-end sequencing method by using an Illumina Hiseq2500 sequencing platform to obtain a genome sequence with 2.6 times coverage;

(3) combining 327 parts of root system and drought resistance (PEG mediated) related character data, genotype data and group structure of a germplasm resource group of a sesame sample under a water culture condition, performing whole genome association analysis on sesame related characters by adopting an EMMAX software package and a Peal program, and detecting 1 marker site which is positioned on 5028931 on the No. 15 linkage group and is obviously associated with the dry weight of the root system of the sesame and the total number of the roots;

(4) through sesame reference genome (http:// ocri-genomics.org/Sinbase/index. html) comparison and gene annotation analysis, the marker locus is found to fall inside the SIN _1025575 gene and within the CDS sequence of the gene, and the gene has no homologous gene with Arabidopsis and other crops through homologous comparison, so the gene is a newly discovered sesame gene with unknown functions and is presumed to be possibly related to the enhancement of the root system development of sesame, and the gene is named as SiLLR;

(5) primers were designed using Primer5.0 to amplify the entire CDS sequence of the SiLLR gene, based on its CDS sequence (including modified bases) and the names:

SiLLR-F: 5'-gctttcgcgagctcggtaccatgagagaaaaacccattta-3', as shown in SEQ ID NO. 3;

SiLLR-R: 5'-cgactctagaggatcctcagatctgaaccttgaccc-3', as shown in SEQ ID NO. 4;

(6) taking the root system of the sesame material G340 in the seedling stage, extracting total RNA of the root system, carrying out reverse transcription to generate cDNA, carrying out RT-PCR amplification by using the primer SiLLR-F/SiLLR-R in the step S5 by using the reversed cDNA as a template, and sequencing the amplified fragment to obtain a SiLLR gene sequence for enhancing the development of the root system of the sesame, wherein the SiLLR gene sequence is shown as SEQ ID NO. 2.

Construction of SiLLR transgenic Arabidopsis thaliana

1. Construction of recombinant expression vector of SiLLR gene

The sesame gene SiLLR cloned in the embodiment and used for enhancing root development of plants is connected with pCAMBIA1301S (provided by the laboratory) plasmid by a homologous recombination method to construct a plant recombinant expression vector, which is named as pCAMBIA1301S-SiLLR (shown in figure 1). The specific operation is as follows:

(1) firstly, obtaining a linearized vector of pCAMBIA1301S by using a double enzyme digestion (BamHI and KpnI) (Takara) method, and then purifying the linearized vector by using an agarose gel electrophoresis and gel recovery kit (Tiangen Biochemical technology Co., Ltd.) to obtain a high-purity pCAMBIA 1301S;

(2) adding the target fragment DNA and a linearized vector pCAMBIA1301S into a centrifugal tube of 1.5ml according to the molar ratio of 3:1 for recombination reaction, uniformly mixing, placing at 37 ℃ for about 30min, adding 10 mu l of reaction solution into 50 mu l of DH5a competent cells, gently mixing by using a pipette, incubating on ice for 20min, thermally shocking in a water bath at 42 ℃ for 45 seconds, and rapidly placing on ice for cooling for 2 min;

(3) adding 300. mu.l LB liquid medium, and incubating at 37 ℃ for 45-60 min. Centrifuging at 5,000rpm for 2min, collecting thallus, discarding part of supernatant, re-suspending thallus with the rest culture medium, lightly spreading on LB solid culture medium containing Kan resistance with sterile spreading rod, and culturing in 37 deg.C incubator by inversion for 16-24 hr;

(4) selecting a plurality of clones on the recombinant reaction conversion plate to carry out colony PCR identification, identifying as positive colonies, selecting corresponding single colonies to culture in a liquid LB culture medium containing Kan antibiotics at 37 ℃ and 200rpm for overnight, extracting plasmids or directly sequencing bacterial liquid, and identifying the carrier accuracy through enzyme digestion electrophoresis.

Any recombinant vector, transgenic cell line and recombinant bacterium containing the related protein coding gene SiLLR for enhancing plant root development belong to expression systems of sesame gene SiLLR.

The existing plant recombinant expression vector can be used for constructing the plant recombinant expression vector containing the SiLLR gene. The plant recombinant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-Ubin or other derivative plant recombinant expression vectors.

When the gene SiLLR is used for constructing a plant recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin (Ubiquitin) gene promoter (pUbi) and the like, can be added in front of a transcription initiation nucleotide, and can be used independently or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant recombinant expression vectors, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

2. Expression of

The specific expression steps in the embodiment of the invention are as follows:

the vector pCAMBIA1301S-SiLLR prepared in the example was transferred to Agrobacterium tumefaciens LBA4404 (Shanghai Weidi Biotechnology Co., Ltd.), and introduced into Arabidopsis thaliana plants for expression. The specific operation is as follows:

(1) the recombinant vector is transferred into agrobacterium LBA 4404:

adding 2 μ g of recombinant vector pCAMBIA1301S-SiLLR into each 100 μ l of LBA4404 Agrobacterium tumefaciens competent cells, uniformly mixing by dialing the tube bottom with hands, and standing on ice for 5min, liquid nitrogen for 5min, water bath at 37 ℃ for 5min and ice bath for 5min in sequence;

700. mu.l of LB liquid medium without antibiotics were added and cultured at 28 ℃ for 5 hours with shaking. And (3) centrifuging at 6000rpm for 1min to collect thalli, reserving about 100 mu l of supernatant, slightly blowing and beating the resuspended thalli, uniformly coating the thalli on an LB solid culture medium containing Kan and Rif, inversely placing the thalli in an incubator at 28 ℃ for 2 days, and picking a plurality of positive clones to simply verify the result by utilizing colony PCR.

(2) Plate culture of Arabidopsis thaliana:

1) counting a certain amount of arabidopsis seeds according to experiment requirements, and filling the arabidopsis seeds into a sterile 1.5mL centrifuge tube;

2) 1mL of 75% ethanol was added, the mixture was inverted and mixed, and the supernatant was discarded and repeated 1 time. Placing into a shaker at 37 deg.C and 200rpm, and shaking for 10min for surface sterilization;

3) discard 75% ethanol, add 1mL 95% ethanol, mix by inversion, discard the supernatant, and repeat 1 time. Adding 300-;

4) after the ethanol is volatilized, dibbling the arabidopsis seeds on a prepared flat plate by using toothpicks;

5) sealing the flat plate, performing vernalization at 4 ℃ for 48h under the dark condition, after vernalization, vertically culturing the flat plate in an illumination incubator, and transplanting after seedling emergence for one week;

6) the seedlings were planted in soil of a small pot with tweezers, first kept wet for 24h with a preservative film, placed in the plant growth room and cultured until the growth of Arabidopsis thaliana bolting (about one month) for transformation experiments.

(3) Genetic transformation:

in order to facilitate identification and screening of transgenic plant cells or plants, the plant recombinant expression vectors used may be processed, for example, by adding genes expressing an enzyme or a luminescent compound which produces a color change in plants (GUS gene, GFP gene, luciferase gene, etc.), antibiotic markers having resistance (gentamicin marker, kanamycin marker, etc.), or chemical-resistant marker genes (e.g., herbicide-resistant gene), etc. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

The plant recombinant expression vector carrying the coding gene SiLLR of the protein related to plant root development can be transformed into receptor plant cells or tissues by Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation and other conventional biological methods. The gene SiLLR is introduced into the recipient plant through the plant recombinant expression vector.

The genetic transformation procedure for the SiLLR gene in this example is as follows:

1) activating agrobacterium: respectively adding 20 mu L of Rif and Kan (Sigma company) into 20mL of LB liquid culture medium, shaking uniformly, inoculating bacteria, and performing shaking activation at 28 ℃ and 220rpm for 8-10h to obtain activated bacteria liquid of agrobacterium;

2) and (3) agrobacterium tumefaciens enlarged culture: respectively adding 200 mul of Rif and Kan into 200mL of LB liquid culture medium, adding 5-10mL of activated bacterium liquid, shake-culturing at 28 ℃ and 220rpm for 14-16h until OD value is 1.6-2.0, centrifuging at 4500rpm for 10min, depositing thalli, discarding supernatant, and naturally drying;

3) adding 100mL of 5% sucrose solution into the precipitated thalli to resuspend the thalli, and blowing and beating the thalli uniformly by a pipette to resuspend the thalli;

4) adding the bacterial liquid in the centrifugal bottle into a plate, adding 100mL of 5% sucrose solution, adding 40 mu of LSilwet-L-77 (0.02%) before conversion, shaking the plate and uniformly mixing;

5) the Arabidopsis inflorescences were closed, immersed in a plate, and gently shaken for 15 s. After the conversion is finished, uniformly stirring the bacterial liquid;

6) sleeving the plants with black bags, keeping out of the sun and preserving moisture for 24 hours;

7) the transformation was repeated once more after one week.

(4) Screening positive plants of T1 generation

Seeds harvested from T0 generation of Arabidopsis are planted, the seeds from T0 generation are disinfected, inoculated with MS screening culture medium containing 30mg/L hygromycin (added with 25mg/L of cefamycin for bacteriostasis) and cultured for 7-10 days under illumination at 22 ℃, and screened to obtain positive plants (plants with normal growth of seedlings and roots) from T1 generation. The experiment obtains 6 positive strains, the positive seedlings are transplanted into soil, the film is uncovered after the soil is covered by a preservative film for 2 to 3 days, and then the seedlings grow normally. And extracting DNA from leaves of the screened T1-generation positive plants, identifying that the leaves contain SiLLR genes by using a PCR method, performing molecular verification on target genes of transgenic plants, and finally confirming that the genes are transferred into T1-generation positive plants.

(5) Positive detection of transgenic plant T2 generation

Carrying out single plant seed collection on the T1 generation positive plants to obtain T1 generation seeds, continuously carrying out hygromycin screening to obtain T2 generation positive plants, transplanting the obtained positive plants to grow, extracting leaf genome DNA (deoxyribonucleic acid) to carry out PCR (polymerase chain reaction) molecular identification, and determining the T2 generation positive plants;

(6) quantitative expression verification of transgenic T2 positive plants

Respectively taking young leaves of a transgenic SiLLR gene T2 generation positive arabidopsis plant and a wild arabidopsis plant in the growth period, extracting total RNA of the leaves by using an RNA extraction kit (Beijing Adela biotechnology limited), then obtaining cDNA by using a reverse transcription kit (Nanjing NuoWeizan biotechnology limited), taking respective cDNA as templates, and taking arabidopsis beta-actin as an internal reference (aF: 5'-cccgctatgtatgtcgcca-3' shown as SEQ ID NO. 5; aR: 5'-aaccctcgtagattggcacag-3' shown as SEQ ID NO. 6); the target gene quantitative primer sequence is as follows: LLRF: 5'-acaaggctgaatcaagtt-3', as shown in SEQ ID NO. 7; LLRR: 5'-ccgctttcgtagaagaat-3', as shown in SEQ ID NO.8, qRT-PCR expression validation (qRT-PCR Mix: Nanjing Nodezam Biotech Co., Ltd.; Instrument: Roche LightCyclerR480) was performed.

FIG. 2 shows a graph of the expression level of the trans-SiLLR gene in leaves of Arabidopsis T2 generation plants, wherein the WT group represents a control group of non-transgenic wild type Arabidopsis, and the SiLLR-1 group, the SiLLR-2 group and the SiLLR-3 group represent leaf expression groups of 3 transgenic Arabidopsis lines, respectively. As shown in the result of FIG. 2, the expression level of the sesame gene SiLLR in the leaves of the 3 Arabidopsis transgenic lines tested was significantly increased (as in the SiLLR-1 group, the SiLLR-2 group and the SiLLR-2 group in FIG. 2), while in the leaves of the transgenic control Arabidopsis, the expression of the sesame SiLLR gene was not detected.

Root system assay of arabidopsis positive plants

Transgenic plants of the T2 generation were grown vertically in square plastic dishes and the dishes were scanned with EPSONV800 as measured at the seedling stage (10 days) and the results are shown in fig. 3 and 4. Mean lateral root length was measured with ImageJ software (results as in fig. 5) and root fresh weight was weighed (results as in fig. 6).

The root development of the mutant SiLLR transgenic Arabidopsis T2 strain group is more vigorous as compared with the mutant Arabidopsis T2 strain group in FIG. 3 and the mutant Arabidopsis (WT) strain group in FIG. 4. Fig. 5 shows that the mean lateral root length is significantly higher for both the sirr-1 and the sirr-2 groups than for the WT group. FIG. 6 shows that the root fresh weights of both the SiLLR-1 and SiLLR-2 groups are significantly higher than the WT groups. Therefore, the lateral root length and the fresh root weight of the transgenic SiLLR arabidopsis strain obtained in the research are obviously higher than those of a wild arabidopsis control, and the expression of the sesame SiLLR gene is shown to increase the total root system amount of a receptor plant.

The related gene SiLLR for regulating and controlling the growth and development of the plant root system is cloned and utilized in production, so that a more characteristic and effective gene resource is provided for the research of the high-efficiency absorption and utilization of the nutrients of main crops, the gene plays an important role in the research of the high-efficiency absorption and utilization of the nutrients of the genetically engineered improved plants, and the gene has important practical value and direct economic benefit.

The above-described embodiments of the present invention should not be construed as limiting the scope of the present invention. Any other corresponding changes and modifications made according to the technical idea of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<110> institute of oil crop of academy of agricultural sciences of China

Application of <120> sesamin SiLLR in regulation and control of plant root system development

<141>2020-06-05

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