阅读说明：本技术 Lsa-MIR408基因及其在调控生菜产量及种子大小中的应用 (Lsa-MIR408 gene and application thereof in regulation of lettuce yield and seed size ) 是由 杨效曾 杨攀 于 2020-04-24 设计创作，主要内容包括：本发明公开了一种Lsa-MIR408基因及其在调控生菜产量及种子大小中的应用。本发明利用高通量测序技术对生菜进行了系统地小RNA测序,通过基因工程手段将候选基因Lsa-MIR408过表达到生菜中,观察了生菜的表型,研究结果表明,过表达Lsa-MIR408可促进生菜营养生长、提高生菜产量及改变生菜种子大小。本发明对于生菜产量提高及生菜育种有重要意义。(The invention discloses a Lsa-MIR408 gene and application thereof in regulation and control of lettuce yield and seed size. According to the invention, the lettuce is subjected to systematic small RNA sequencing by using a high-throughput sequencing technology, the candidate gene Lsa-MIR408 is overexpressed into the lettuce by a genetic engineering means, the phenotype of the lettuce is observed, and research results show that the overexpression of Lsa-MIR408 can promote the vegetative growth of the lettuce, improve the yield of the lettuce and change the seed size of the lettuce. The invention has important significance for increasing the yield of the lettuce and breeding the lettuce.)

1. A method of breeding a transgenic plant comprising the steps of: introducing the specific DNA molecule into the starting plant to obtain transgenic plant with increased yield; the specific DNA molecule is any one of the following (1) to (5):

(1) DNA molecule shown in SEQ ID No. 2;

(2) DNA molecule shown in SEQ ID No. 4;

(3) DNA molecule shown in SEQ ID No. 5;

(4) DNA molecules which can be hybridized with the DNA sequences defined in (1) or (2) or (3) under strict conditions and have the same functions;

(5) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and have the same functions.

2. A method of breeding a transgenic plant comprising the steps of: introducing specific DNA molecules into a starting plant to obtain a transgenic plant with an accelerated growth speed; the specific DNA molecule is any one of the following (1) to (5):

(1) DNA molecule shown in SEQ ID No. 2;

(2) DNA molecule shown in SEQ ID No. 4;

(3) DNA molecule shown in SEQ ID No. 5;

(4) DNA molecules which can be hybridized with the DNA sequences defined in (1) or (2) or (3) under strict conditions and have the same functions;

(5) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and have the same functions.

3. An miRNA shown as SEQ ID No. 1.

4. A DNA molecule encoding the miRNA of claim 3 which is (a1) or (a2) or (a 3):

(a1) DNA molecule shown in SEQ ID No. 2;

(a2) a DNA molecule which hybridizes with the DNA sequence defined in (a1) under stringent conditions and has the same function;

(a3) and (c) a DNA molecule having 90% or more homology with the DNA sequence defined in (a1) and having the same function.

5. An RNA shown as SEQ ID No. 3.

6. A DNA molecule encoding the RNA of claim 5, which is (b1) or (b2) or (b3) as follows:

(b1) DNA molecule shown in SEQ ID No. 4;

(b2) a DNA molecule which hybridizes with the DNA sequence defined in (b1) under strict conditions and has the same function;

(b3) and (b) a DNA molecule having 90% or more homology with the DNA sequence defined in (b1) and having the same function.

7. A recombinant vector, expression cassette, transgenic cell line or recombinant bacterium comprising the specific DNA molecule of claim 1.

8. The miRNA of claim 3 or the DNA molecule of claim 4 for use as (c1), (c2), (c3) or (c 4):

(c1) regulating and controlling the growth speed of the plants;

(c2) regulating and controlling the yield of the plant;

(c3) promoting the growth of plants;

(c4) the yield of the plant is improved.

9. The RNA of claim 5 or the DNA molecule of claim 6 for use as (c1), (c2), (c3) or (c 4):

(c1) regulating and controlling the growth speed of the plants;

(c2) regulating and controlling the yield of the plant;

(c3) promoting the growth of plants;

(c4) the yield of the plant is improved.

10. Use of any of the following in plant breeding;

(A) the method of claim 1 or 2;

(B) the miRNA of claim 3;

(C) the DNA molecule of claim 4;

(D) the RNA of claim 5;

(F) the DNA molecule of claim 6;

(G) the recombinant vector, expression cassette, transgenic cell line, or recombinant bacterium of claim 7.

Technical Field

The invention relates to a Lsa-MIR408 gene and application thereof in regulation and control of lettuce yield and seed size.

Background

Lettuce (Lactuca sativa L.) is one of the most widely consumed leaf vegetables in the world and is widely cultivated in most regions. The lettuce meets the current favor of the public on pollution-free vegetables and is popular with the public due to the characteristics of low probability of plant diseases and insect pests, less pesticide residue and the like. In recent years, the demand of lettuce in our country is increased sharply, the planting area is enlarged continuously, the yield of lettuce is low, and the market supply is insufficient. Therefore, it is important to find key genes for improving lettuce yield.

Disclosure of Invention

The invention aims to provide an Lsa-MIR408 gene and application thereof in regulation and control of lettuce yield and seed size.

The invention provides miRNA named Lsa-MIR408, which is derived from American lettuce and is shown as SEQ ID No. 1. The invention also protects RNA (precursor of Lsa-MIR408, abbreviated as Lsa-MIR408 precursor), which is shown as a sequence SEQ ID No.3 in a sequence table.

The DNA molecule encoding the Lsa-MIR408 also belongs to the protection scope of the invention.

The DNA molecule may specifically be (a1) or (a2) or (a3) as follows:

(a1) DNA molecule shown in SEQ ID No. 2;

(a2) a DNA molecule which hybridizes with the DNA sequence defined in (a1) under stringent conditions and has the same function;

(a3) and (c) a DNA molecule having 90% or more homology with the DNA sequence defined in (a1) and having the same function.

DNA molecules encoding the Lsa-MIR408 precursor are also within the scope of the invention.

The DNA molecule may specifically be (b1) or (b2) or (b3) as follows:

(b1) DNA molecule shown in SEQ ID No. 4;

(b2) a DNA molecule which hybridizes with the DNA sequence defined in (b1) under strict conditions and has the same function;

(b3) and (b) a DNA molecule having 90% or more homology with the DNA sequence defined in (b1) and having the same function.

The stringent conditions can be hybridization and washing with 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS solution at 65 ℃ in DNA or RNA hybridization experiments.

The recombinant expression vector, the expression cassette, the transgenic cell line or the recombinant strain containing any one of the DNA molecules belong to the protection scope of the invention.

The recombinant vector can be obtained by inserting any one of the DNA molecules into a multiple cloning site or a recombination site of a plant expression vector. The recombinant vector may be specifically a recombinant plasmid obtained by substituting a small fragment between XbaI and XhoI of the plant expression vector pJIM19 into SEQ ID No. 5.

The invention also protects the application of the Lsa-MIR408, the Lsa-MIR408 precursor or any one of the DNA molecules, and the application is as follows (c1), (c2), (c3) or (c 4):

(c1) regulating and controlling the growth speed of the plants;

(c2) regulating and controlling the yield of the plant;

(c3) promoting the growth of plants;

(c4) the yield of the plant is improved.

The modulation of plant yield may in particular be embodied as modulation of plant leaf area and/or petiole length and/or seed length and/or fresh weight. The improvement in plant yield may in particular be embodied as an improvement in plant leaf area and/or petiole length and/or seed length and/or fresh weight. The regulation of the plant growth rate can be embodied as regulation of the increase rate of leaf area and/or petiole length and/or seed length and/or fresh weight of the plant at the same time. The promotion of plant growth can be embodied in the way that the leaf area and/or the leaf stalk and/or the seed length and/or the fresh weight of the plant growing at the same time are large.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: introducing the specific DNA molecule into the starting plant to obtain transgenic plant with increased yield; the specific DNA molecule is any one of the following (1) to (5):

(1) DNA molecule shown in SEQ ID No. 2;

(2) DNA molecule shown in SEQ ID No. 4;

(3) DNA molecule shown in SEQ ID No. 5;

(4) DNA molecules which can be hybridized with the DNA sequences defined in (1) or (2) or (3) under strict conditions and have the same functions;

(5) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and have the same functions.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: introducing specific DNA molecules into a starting plant to obtain a transgenic plant with an accelerated growth speed; the specific DNA molecule is any one of (1) to (5):

(1) DNA molecule shown in SEQ ID No. 2;

(2) DNA molecule shown in SEQ ID No. 4;

(3) DNA molecule shown in SEQ ID No. 5;

(4) DNA molecules which can be hybridized with the DNA sequences defined in (1) or (2) or (3) under strict conditions and have the same functions;

(5) and (3) DNA molecules which have more than 90% of homology with the DNA sequences defined in (1) or (2) or (3) and have the same functions.

Any of the above DNA molecules may be introduced into the starting plant by means of a recombinant expression vector. The recombinant expression vector can be a recombinant plasmid obtained by inserting any one of the DNA molecules into a multiple cloning site or a recombination site of a plant expression vector. The recombinant vector may be specifically a recombinant plasmid obtained by substituting a small fragment between XbaI and XhoI of the plant expression vector pJIM19 into SEQ ID No. 5.

The invention also protects the application of any one of the following in plant breeding;

(A) the method of any of the above;

(B) the Lsa-MIR 408;

(C) a DNA molecule encoding said Lsa-MIR 408;

(D) a precursor of Lsa-MIR 408;

(E) a DNA molecule encoding said Lsa-MIR408 precursor;

(F) any of the above recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria.

The breeding may in particular be aimed at growing plants with a fast growth rate and/or high yield. The high yield can be embodied in particular by a high value of the leaf area and/or petiole length and/or seed length and/or fresh weight of the plant. The high growth speed can be embodied as large leaf area and/or large petiole and/or large seed length and/or large fresh weight of plants growing at the same time.

Any of the above plants is a dicotyledonous plant or a monocotyledonous plant. The dicotyledonous plant may be a plant of the family Compositae. The Compositae plant can be lettuce, more specifically American lettuce.

According to the invention, the lettuce is subjected to systematic small RNA sequencing by using a high-throughput sequencing technology, the candidate gene Lsa-MIR408 is overexpressed into the lettuce by a genetic engineering means, the phenotype of the lettuce is observed, and research results show that the overexpression of Lsa-MIR408 can promote the vegetative growth of the lettuce, improve the yield of the lettuce and change the seed size of the lettuce. The invention has important significance for increasing the yield of the lettuce and breeding the lettuce.

Drawings

FIG. 1 is the genetic transformation process of lettuce. A, sowing disinfected lettuce seeds on an MS culture medium; 7, seedling cotyledon of the natural vegetable; c, callus induction; d, bud induction; e: rooting induction; f, domesticating the plants.

FIG. 2 shows a phenotypic comparison of over-expressed Lsa-MIR408 plants with wild-type. (A) Overexpression lines and wild type 12 days after germination; (B) comparing the over-expressed strain with leaves of wild type; (C) comparing the over-expressed lines of lettuce grown to 50 days with the wild type phenotype; (D) carrying out statistical analysis on the length of a petiole; (E) counting the fresh weight of the lettuce which grows for 50 days.

FIG. 3 shows the result of PCR assay of positive transgenic seedlings. M is DNA molecular marker; 1-18: positive seedlings; p is an overexpression vector as a positive control; WT lettuce DNA as negative control.

FIG. 4 shows a comparison of the morphology of transgenic lettuce seeds with wild type lettuce seeds. (A) Comparing the shapes of the transgenic lettuce seeds and wild lettuce seeds; (B) and (4) carrying out length statistical analysis on the transgenic lettuce seeds and wild lettuce seeds.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

American big lettuce: purchased from vegetable institute of agriculture and forestry, Beijing.

Plant expression vector pJIM 19: purchased from Beijing Quanjin Biotechnology Ltd.

Agrobacterium EHA 105: jinghua Vietyol Biotech Ltd.

Example 1 discovery of Lsa-MIR408 Gene

Extracting total RNA of the American lettuce variety, constructing an sRNA sequencing library, sequencing, and performing a large amount of sequence analysis and functional verification to obtain Lsa-MIR408, wherein the Lsa-MIR408 is shown as SEQ ID No.1, and the coding sequence of the Lsa-MIR408 is shown as SEQ ID No. 2. The precursor sequence of Lsa-MIR408 is shown as SEQ ID No.3, and the coding sequence of the precursor sequence is shown as SEQ ID No. 4.

Example 2 application of Lsa-MIR408

First, construction of overexpression vector

The DNA molecule shown by SEQ ID No.5 (160 bp extended before and after SEQ ID No. 4) was substituted with the fragment between the XbaI and XhoI sites of the plant expression vector pJIM19 to obtain an overexpression vector (which was sequenced).

Second, preparation of recombinant bacterium

And (3) introducing the overexpression vector obtained in the step one into agrobacterium EHA105 to obtain a recombinant bacterium.

Genetic transformation of lettuce

1. Seeds of the American lettuce were taken in a 2mL centrifuge tube, sterilized with 75% ethanol for 45s, and then quickly washed with sterile water for 2-3 times.

2. Adding 15% sodium hypochlorite, placing in a vortex instrument, and slowly vortex for cleaning for 20 min.

3. The seeds are washed 5-6 times with sterile water.

4. Seeds were picked up from the centrifuge tubes with sterilized forceps and planted on MS minimal medium.

MS minimal medium: MS 4.4g/L, sucrose 30g/L and agar powder 8 g/L.

5. The seeds are placed in a light incubator for culturing for 7d (the light is 16h, the dark is 8h, and the light intensity is 2000 Lux).

6. The cotyledons of 7d lettuce seedlings were cut with a scalpel, each leaf was cut two times in the transverse direction, and cultured in MS1 medium for 24 h.

MS1 medium: MS minimal medium +0.2mg/LNAA +0.2 mg/L6-BA.

7. Shaking the recombinant bacteria prepared in the step two until OD600 is between 0.6 and 0.8, centrifuging at 5000rpm for 10min, pouring out supernatant, suspending the bacteria by using MS0 culture medium, and adjusting OD600 to be between 0.6 and 0.8.

MS0 medium: MS 4.4g/L + sucrose 30 g/L.

8. And (4) soaking the lettuce cotyledons in the bacterial liquid prepared in the step (7) for 30min, and shaking for several times at intervals of 5 min.

9. After infection, the lettuce cotyledons are clamped out by using forceps, wiped dry by using filter paper, and placed in an MS1 culture medium for dark culture for 12 h.

10. After the dark culture is finished, transferring the lettuce cotyledons to an MS2 callus and bud induction culture medium, and normally culturing in a tissue culture room until callus formation appears for about 10 days and buds appear for 15-20 days.

MS2 callus and shoot induction medium: MS minimal medium +0.2mg/L NAA +0.2 mg/L6-BA +300mg/L timentin.

11. When the bud grows about 1cm, the bud is cut off by a scalpel, and the bud is transferred to an MS3 rooting culture medium to induce rooting, and roots appear about 15 days later.

MS3 rooting medium: 1/2MS minimal medium +300mg/L timentin.

12. When the root grows to 3-5cm, the tissue culture seedling is gently removed from the tissue culture bottle, the root is washed by distilled water without culture medium residue, and then the seedling is transplanted in mixed nutrient soil. (nutrient soil: vermiculite: perlite: 9:3: 1).

13. When the plants grow to a certain degree, a small amount of leaves are taken to extract genome DNA, a primer pair consisting of a primer HYG-F and a primer HYG-R is adopted to carry out PCR positive seedling detection, and the amplification product of the positive seedling is about 400 bp.

HYG-F：5’-TACACAGGCCATCGGTCCAGA-3’；

HYG-R：5’-TAGGAGGGCGTGGATATGTC-3’。

The results are shown in FIG. 3.

The above genetic transformation process is shown in FIG. 1.

Selfing T0 transgenic plants identified as positive seedlings by PCR to obtain T1 plants, selfing T1 plants to obtain T2 plants, and selfing T2 plants to obtain T3 transgenic lines for subsequent experiments.

Fourthly, detecting the phenotype of the over-expression plant

And (3) the plant to be detected: wild type American big-speed lettuce (WT), T3 generation transgenic lines (OE-2, OE-5, OE-6).

5 plants were tested per line.

After sowing seeds of a plant to be detected, culturing the seeds under the conditions of 20 ℃, 16 h/8 h of illumination/darkness and illumination intensity of about 200 mu mol.m < -2 > s < -1 >, and observing the phenotype of the plant. The results are shown in FIG. 2. The results show that the transgenic lettuce leaves over-expressing the Lsa-MIR408 promote the vegetative growth of lettuce, improve the yield of lettuce and change the seed size of the lettuce, compared with the wild type, the transgenic lettuce leaves over-expressing the Lsa-MIR408 are enlarged, the leaf stalks are elongated, the leaf stalk lengths of three transgenic lines (OE-2, OE-5 and OE-6) are respectively increased by 15.7 percent, 11.7 percent and 12.9 percent, the fresh weight is respectively increased by 27.1 percent, 26.7 percent and 28.3 percent, and the seed length is respectively increased by 8.7 percent, 9.3 percent and 10.0 percent.

Sequence listing

<110> research center of agricultural biotechnology in Beijing

<120> Lsa-MIR408 gene and application thereof in regulation of lettuce yield and seed size

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