CN113652434B - Gorgon fruit DNA molecule with function of promoting rice grain enlargement and application thereof - Google Patents

Abstract

The invention relates to a Gorgon fruit DNA molecule with a function of promoting rice grain enlargement and application thereof, belonging to the technical field of genetic engineering. The DNA molecule is easy to obtain, the molecular sequence is definite, and the rice plant with larger grain can be obtained through simple infection steps after molecular cloning. The invention verifies that EuSAUR gene is a key gene for regulating and controlling seed size in the growth process of gorgon euryale seed for the first time; meanwhile, the function of the EuSAUR gene for promoting rice grain enlargement is further confirmed based on a heterologous expression method. The invention promotes the development of the industrialization of Gorgon fruit resources to a certain extent; meanwhile, a DNA molecule for improving the grain size of rice is provided for the grain field.

Description

Gorgon fruit DNA molecule with function of promoting rice grain enlargement and application thereof

Technical Field

The invention relates to a Gorgon fruit DNA molecule with a function of promoting rice grain enlargement and application thereof, belonging to the technical field of genetic engineering.

Background

Gorgon fruit is a mature seed of annual aquatic herb Gorgon fruit (Euryale ferox Salisb.) of Nymphaeaceae, and is a medicinal and edible product. The edible parts of the gorgon euryale seed and the rice are kernels, and belong to aquatic crops, and the growth process is similar. Therefore, the regulation and control method for the growth and the yield of the rice and the gorgon euryale seed is similar.

Genetic background is one of the determining factors regulating plant growth. With the deep research, more and more functional genes are excavated in recent years and applied to the improvement of the yield of crops. For example: the Os10g37880 gene in the rice found by the academy of agricultural science of Jiangxi province rice research can increase the rice yield by increasing the spike number of small spikes (CN 110923245A); the Wuhan bioengineering institute finds that the OsNPF5.11 gene can increase the tillering number of rice and the number of grouted grains per plant, and improve the yield of rice (CN 107056909B); the research institute of genetics and developmental biology of the national academy of sciences discovers that a gene LGY3 capable of controlling grain length, grain weight, yield and grain appearance of rice can realize the synergistic improvement of the yield and the quality of the rice (CN 107384937B); the national academy of sciences Shanghai life sciences has obtained a nucleotide that can promote an increase in the number of primary branches, the number of secondary branches per ear, the number of small ears per ear, etc., and has been found to promote an increase in the yield of gramineous plants (CN 105695478B).

In plants, the SAUR gene family is reported to have related functions to regulate auxin synthesis and transport, but the effect of the SAUR gene family on crop grain size has not been clarified. Meanwhile, the SAUR gene family has the characteristics of short sequence, almost no introns and the like, and is easier to obtain from genes or apply. In addition, the gene for controlling the size of the gorgon euryale seed is never reported, and the development of the gorgon euryale industry is severely limited. Therefore, the functional genes for promoting the grain growth are excavated from the gorgon euryale seed and applied to crops, so that the industrialized development of the gorgon euryale seed and the solution of the grain problem can be promoted.

Disclosure of Invention

The invention solves the technical problems that: provides a Gorgon fruit DNA molecule with the function of promoting rice grain enlargement. The DNA molecule is easy to obtain, the molecular sequence is definite, and the rice plant with larger grain can be obtained through simple infection steps after molecular cloning. At present, the sequence and the function of SAUR family genes in gorgon euryale plants are not clear, and an acquisition method of DNA molecules with seed size regulation function in the family has not been developed yet.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a Gorgon fruit DNA molecule with the function of promoting rice grain enlargement has a nucleotide sequence which is a sequence 1 in a sequence table.

In order to solve the technical problems, another technical scheme provided by the invention is as follows: the method for obtaining the gorgon euryale seed DNA molecule with the function of promoting rice grain enlargement comprises the following specific preparation steps:

(1) Weighing fresh and tender gorgon euryale seed samples, extracting the DNA of the gorgon euryale seed according to the instruction of an Ezup column type plant tissue genome DNA extraction kit of the biological engineering Co., ltd, wherein the specific steps are as follows:

a) Placing a sample in liquid nitrogen, fully grinding the sample into powder, and taking a proper amount of uniformly mixed powder to be placed in a 1.5mL centrifuge tube;

b) Adding 600 μl of preheated PCB Buffer and 12 μl of beta-mercaptoethanol, shaking, mixing, placing in water bath at 65deg.C for 25min, and mixing;

c) Adding 600 mu L of chloroform, shaking and mixing uniformly, centrifuging at 12000rpm for 1min, and transferring the upper water phase into a new 1.5mL centrifuge tube;

d) Adding a proper volume of BD Buffer, uniformly mixing, adding a proper volume of absolute ethyl alcohol, fully uniformly mixing, fully transferring into an adsorption column by using a pipettor, standing at room temperature for 2min, centrifuging at 12000rpm for 10min after full adsorption, and pouring out waste liquid;

e) Adding 500 mu L of PW Solution into an adsorption column, centrifuging at 12000rpm for 1min, and pouring out waste liquid;

f) Adding 500 mu L of Wash Solution into the adsorption column, centrifuging at 12000rpm for 1min, and pouring out the waste liquid;

g) Centrifuging the adsorption column 12000rpm for 2min, and drying ethanol in a fume hood;

h) Transferring the adsorption column into a new centrifuge tube, adding appropriate amount of TE Buffer, standing for 5min, heating appropriately, centrifuging at 12000rpm for 10min, and collecting filtrate;

i) The concentration and purity are detected by a spectrophotometer, 1 mu L of OD value is taken, OD 260/280 is 1.7-2.0, which indicates that DNA quality is good, protein pollution is less than 1.7, and RNA pollution is more than 2.0;

(2) Using the DNA extracted in step (1) as a template, and designing a primer pair (primer F ₁ Sequence 2, primer R ₁ Performing PCR amplification for sequence 3), the amplification procedure is as follows; taking 5 mu L of PCR product, adopting 1% agarose gel, carrying out 150V constant-pressure electrophoresis for 20min, and observing on a gel imager by contrast with a DNA marker;

reaction system and reaction procedure:

(3) The product obtained by PCR amplification was recovered according to the SanPrep column PCR product purification kit of Biotechnology Co., ltd, comprising the following steps:

a) Cutting out the target DNA fragment on the agarose gel as much as possible by using a clean scalpel, putting the cut target DNA fragment into a 1.5mL centrifuge tube, and weighing;

b) Adding 500 μl of B2 Buffer per 100mg agarose, and placing in 55deg.C water bath, mixing thoroughly for 10min to melt the gel block completely;

c) Sucking all the completely dissolved peptized solution into an adsorption column by a pipetting gun, centrifuging at 12000rpm for 1min, and pouring out waste liquid;

d) Adding 300 mu L of B2 Buffer into an adsorption column, centrifuging at 12000rpm for 1min, and pouring out waste liquid;

e) Adding 500 μl of Wash Solution into the adsorption column, centrifuging at 12000rpm for 1min, pouring off the waste liquid, repeating the steps for 1 time, and drying the adsorption column at normal temperature in a ventilation kitchen;

f) Absorbing a proper amount of the absorption Buffer, adding the absorption Buffer into an absorption column, incubating for 5min in a water bath at 55 ℃, centrifuging for 10min at 12000rpm, and collecting filtrate;

g) The concentration and purity are detected by a spectrophotometer, 1 mu L of OD value is taken, OD 260/280 is 1.7-2.0, which indicates that DNA quality is good, protein pollution is less than 1.7, and RNA pollution is more than 2.0;

(4) Through sequencing, the PCR product has the nucleotide shown in a sequence 1 in a sequence table, and is named EuSAUR.

In order to solve the technical problems, another technical scheme provided by the invention is as follows: the application of the gorgon euryale seed DNA molecule with the function of promoting rice grain enlargement further comprises the following specific steps:

and 4, taking the PCR product of the step 3 after sequencing and confirming in the step 4, respectively carrying out double enzyme digestion on the target gene fragment and the pCambia1301 vector by using KpnI/XbaI, fusing GFP at the 3' end of the target fragment, constructing the GFP on the expression vector, and transforming agrobacterium GV3101 to obtain the expression vector pCambia1301-EuSAUR and the cloning agrobacterium GV3101-EuSAUR.

In order to solve the technical problems, another technical scheme provided by the invention is as follows: the recombinant expression vector containing the DNA molecule, and the starting vector is pCambia1301.

In order to solve the technical problems, another technical scheme provided by the invention is as follows: gram Long Nong bacillus GV3101-EuSAUR of the recombinant expression vector obtained according to the above method.

In order to solve the technical problems, another technical scheme provided by the invention is as follows: a method for culturing transgenic plant features that the recombinant expression carrier containing said DNA molecule is introduced into plant cells or tissue to obtain transgenic plant.

Preferably, the plant is rice.

In order to solve the technical problems, another technical scheme provided by the invention is as follows: the application of the DNA molecule, the recombinant expression vector or the cloning agrobacterium in the cultivation of transgenic plants.

The invention provides a Gorgon fruit DNA molecule with the function of promoting rice grain enlargement, comprising an expression vector of the DNA fragment and gram Long Nong bacillus.

(1) Extracting genomic DNA of young gorgon euryale seed plant tissues to obtain gorgon euryale seed genomic DNA;

(2) Carrying out PCR amplification on the obtained genome DNA by using a designed specific primer, and preserving a product for later use;

the sequence of one primer in the primer pair used in the PCR reaction in the step (2) is sequence 2, and the sequence of the other primer is sequence 3.

(3) The resulting PCR product was subjected to agarose gel electrophoresis, the bands were excised against a DNA marker for purification, and sequencing verified. The obtained purified product is a DNA fragment of an EuSAUR gene open reading frame, and is shown in a sequence 1;

(4) The resulting DNA fragment and pCambia1301 vector were double digested with KpnI/XbaI, the fragment of interest was constructed on an expression vector, and Agrobacterium GV3101 was transformed. The expression vector pCambia1301-EuSAUR and the clone Agrobacterium GV3101-EuSAUR were obtained.

The invention has the beneficial effects that:

(1) The invention proves that EuSAUR gene in plant gorgon euryale is a key yield gene for controlling the size of gorgon euryale for the first time, and provides a gene capable of improving the yield of rice. The gene is overexpressed in japonica rice variety Nipponbare (Oryza sativa ssp. Japonica cv. Nipponbare), so that the growth of rice can be remarkably accelerated, the formation of dominant characters related to the yield of the rice is promoted, and the main manifestations are remarkably accelerated plant height increase and remarkably more dispersed plant types. And, over-expression of the gene in rice can significantly increase the size of the produced grain. Through further observation, the gene is found to promote the formation of dominant properties of rice by promoting cell elongation, and finally, the yield of the rice is improved.

(2) The invention promotes the improvement of crop yield and economic value and relieves the pressure brought by the food feeding problem in the life of common people; meanwhile, the industrialized development and reasonable resource utilization of the Gorgon fruit are promoted, the medicinal and edible cost of the Gorgon fruit is relatively reduced, and each common people can eat and use the Gorgon fruit.

Drawings

The invention is further described below with reference to the accompanying drawings.

FIG. 1 is a seed size comparison of the seed sizes of the gorgon and hybrid gorgon euryales.

FIG. 2 is a microscopic observation of the breeding organs of the gorgon euryale and hybrid gorgon euryale.

FIG. 3 is a comparison of length of cells near the ovary of Gorgon and hybrid Gorgon.

FIG. 4 is a transcriptome analysis of the breeding organs of gorgon and hybrid gorgon euryales.

FIG. 5 shows the PCR detection results of EuSAUR gene transferred rice.

FIG. 6 is a view showing plant height observation during the growth of transgenic and control plants.

FIG. 7 shows the length of rice leaf blebs of transgenic and control plants on day 10 of growth.

FIG. 8 shows the observation of plant types and leaf angles of transgenic and control plants on day 60 of growth.

FIG. 9 shows the length of transgenic and control plant rice leaf occipital cells on day 60 of growth.

FIG. 10 is a view of grain size of transgenic and control plants.

Detailed Description

Example 1

1. Instrument and materials

1.1 instruments

Table 1 instrument

1.2 reagents

TABLE 2 reagents

2. Experimental method

2.1 different cultivars of Gorgon fruit quality difference observation

2.1.1 seed size comparison of three cultivars of Gorgon fruit

Taking semen euryales stored in a refrigerator at-20deg.C, and photographing to record seed diameter, kernel diameter and seed coat thickness of three cultivated species of semen euryales. The seed diameter, kernel diameter and seed thickness of different cultivars of gorgon euryale were measured using imagej1.52 software.

2.1.2 microscopic observations of the breeding organs of the Gorgon fruit and hybrid Gorgon fruit

a) Taking the hybrid gorgon euryale seed breeding organ samples in different growth periods, and observing and recording morphological and anatomical characteristics in the growth process.

b) Paraffin section:

fixing and dehydrating the breeding organ samples of the gorgon euryale seed and the hybrid gorgon euryale seed in different growth periods, cutting the breeding organ samples by hands, putting the breeding organ samples into a tissue embedding box, numbering the breeding organ samples, putting the breeding organ samples into a tissue dehydrator, performing gradient dehydration (50% ethanol soaking for 10min, 70% ethanol soaking for 10min, 90% ethanol soaking for 10min, 100% ethanol soaking for 10min, 50% ethanol+50% xylene soaking for 10min, 100% xylene soaking for 10 min) by using a set program, and keeping the gas in the plant samples to be exhausted in the dehydration process.

After impregnating the plant samples with xylene, the samples were immersed in melted paraffin, the paraffin was replaced every 2 hours, and a total of 3 paraffin changes were made.

Embedding, namely heating and dissolving paraffin in a paraffin embedding machine, dripping the paraffin into a mould in which plant tissues are placed, and placing the paraffin on a precooling workbench together with the mould to quickly solidify the paraffin and embed the plant tissues.

And (3) placing the paraffin blocks embedded with the samples on an objective table of a paraffin slicer, adjusting the slicing angle, cutting off redundant paraffin blocks, and slicing the paraffin blocks with the slicing thickness of 35 mu m.

The water bath temperature of the sheet dryer is adjusted to 42 ℃ by the patch, the sheet is carefully placed on the water surface, and the sheet is unfolded by the temperature. The slice on the water surface is then fished out with a clean slide and adjusted to the appropriate position.

The temperature of a drying groove of the sheet dryer is adjusted to 60 ℃, the glass slide after the surface mounting is inserted into the drying groove of the sheet dryer, and the glass slide is dried for more than 2 hours, but the sheet can not be baked for too long, so that the sample is prevented from being damaged.

The slide glass after baking the slice is inserted into a slide glass frame, placed into an automatic dyeing instrument and dyed by a set program (100% dimethylbenzene soaking for 20min, 50% ethanol+50% dimethylbenzene soaking for 10min, 100% ethanol soaking for 10min, 90% ethanol soaking for 10min, 70% ethanol soaking for 10min, 50% ethanol for 10min, 50% safranine solution soaking for 4h, 50% ethanol soaking for 10min, 70% ethanol soaking for 10min, 90% ethanol soaking for 10min, 100% ethanol soaking for 10min, 50% ethanol+50% dimethylbenzene soaking for 10min, 100% dimethylbenzene soaking for 10 min).

The slide was removed from the xylene and stained, and a 30% neutral gum in xylene solution was quickly added dropwise and covered with a coverslip. The air bubbles should be removed in the sealing process.

Observation the slides were placed under a bulk microscope or a fluorescence microscope and the plant sections were observed.

2.2 transcriptomic analysis of the organs bred with the Gorgon fruit and hybrid Gorgon fruit

2.2.1 extraction of Total RNA and QC

Grinding the gorgon euryale seed sample with liquid nitrogen, uniformly mixing, taking a proper amount of tissue homogenate, adding the TRI Reagent, and carrying out shake extraction. Extracting with chloroform, centrifuging, collecting supernatant, adding isopropanol to precipitate total RNA, centrifuging, washing precipitate with 75% ethanol, drying precipitate, and dissolving precipitate with buffer. Total RNA of acceptable quality (RIN. Gtoreq.7 and ratio of 28S to 18S. Gtoreq.1.5) was selected as a stock sample for mRNA sequencing by Agilent 2100BioAnalyzer detection, and the RNA was quantified using QUBIT RNA ASSAY KIT.

2.2.2 library construction

a) mRNA purification and fragmentation mRNA from total RNA was purified using beams with oligo (T). mRNA fragmentation was performed using the following reaction system (Table 3).

TABLE 3 fragmentation system and reaction procedure

b) 1st Strand cDNA and 2 ^nd Strand cDNA Synthesis 1st Strand cDNA synthesis was performed using the following reaction system (Table 4).

Table 4 1st Strand cDNA Synthesis System and reaction procedure

Then the reaction was carried out with the following reaction system 2 ^nd Strand cDNA synthesis and purification with 144. Mu.L of AMPure XP Beads, and finally re-suspension with 60mL of Nuclease free water, were taken out for appropriate use (Table 5).

Table 5 2nd Strand cDNA Synthesis System and reaction procedure

c) End Repair and linker the End Repair/dA-tail was performed using the following reaction system (Table 6).

TABLE 6 End Repair/dA-tail reaction System and reaction procedure

Adaptor Ligation was performed using the following reaction system (Table 7).

Table 7 Adaptor Ligation reaction system and reaction program

Subsequently, 3. Mu.L (red) of USER Enzyme was added to the product and reacted at 37℃for 15 minutes. Nuclease-free Water was added to make the total volume 100. Mu.L, and purification was performed using AMPure XP Beads.

d) PCR amplification was performed according to the following reaction system (Table 8).

TABLE 8 transcriptome PCR amplification System and reaction procedure

Purifying with appropriate amount of AMPure XP Beads, and finally re-suspending with Resuspension Buffer, and storing for use.

e) The library mixture was adjusted to a molar concentration of 2nM for each library based on library results. Mixing library in equal amount according to experimental group, ensuring that the final concentration after mixing is 2nM, and storing in-80 ℃ refrigerator with volume not less than 10 μl.

2.2.3 sequencing

a) Sample treatment NaOH was mixed with the library by shaking, left at room temperature for 5min, transferred into pre-chilled Hybridization buffer, adjusted to a final concentration of 20pM and diluted appropriately as required.

b) Clustering, namely clustering Flowcell and the processed library sample on cBet, combining molecules in the library with primers fixed on Flowcell, performing bridge PCR reaction, and finally sequencing on a sequencing platform.

c) Sequencing while synthesizing, selecting proper Recipe to sequence the cluster generation product, judging whether the A/T/C/G base signals on each lane are normal according to First Base Report, and determining that the primer is combined without error; and the generated data can meet the sequencing requirement according to the cluster quantity estimation.

2.2.4 cDNA Synthesis

cDNA synthesis was performed using the TaKaRa reverse transcription kit, and the reaction system was as shown in Table 9.

TABLE 9 cDNA Synthesis System and reaction procedure

2.2.5 real-time quantitative PCR detection

Specific primer pairs (primer F) ₂ For sequence 4, primer R ₂ For sequence 5), qRT-PCR was performed on a fluorescent quantitative PCR apparatus using the NovoStart SYBR qPCR Supermix Plus kit, and the reaction system is shown in Table 10.

TABLE 10 qRT-PCR reaction system and reaction program

2.3 amplification of EuSAUR Gene of Gorgon fruit

2.3.1DNA extraction

Extracting the DNA of gorgon euryale according to the instruction of the Ezup column type plant tissue genome DNA extraction kit:

a) The sample is placed in liquid nitrogen and fully ground into powder, and a proper amount of the uniformly mixed powder is taken in a 1.5mL centrifuge tube.

b) 600. Mu.L of preheated PCB Buffer and 12. Mu.L of beta-mercaptoethanol are added, mixed by shaking, placed in a water bath at 65 ℃ for 25min, and mixed uniformly.

c) 600. Mu.L of chloroform was added, mixed by shaking, centrifuged at 12000rpm for 1min, and the upper aqueous phase was transferred to a new 1.5mL centrifuge tube.

d) Adding a proper volume of BD Buffer, uniformly mixing, adding a proper volume of absolute ethyl alcohol, and fully and uniformly mixing. Transferring the waste liquid into an adsorption column by using a liquid transfer device, standing for 2min at room temperature, centrifuging at 12000rpm for 10min after the waste liquid is fully adsorbed, and pouring the waste liquid.

e) 500. Mu.L of PW Solution was added to the column, centrifuged at 12000rpm for 1min, and the waste liquid was decanted.

f) 500. Mu.L of Wash Solution was added to the column, centrifuged at 12000rpm for 1min, and the waste liquid was decanted.

g) Centrifuging the adsorption column 12000rpm for 2min, and drying ethanol in a fume hood.

h) Transferring the adsorption column into a new centrifuge tube, adding appropriate amount of TE Buffer, standing for 5min, heating appropriately, centrifuging at 12000rpm for 10min, and collecting filtrate.

i) The concentration and purity are detected by a spectrophotometer, 1 mu L of detection OD value is taken, OD 260/280 is 1.7-2.0, and the DNA quality is good, the protein pollution is less than 1.7, and the RNA pollution is more than 2.0.

2.3.2PCR reaction

a) Primer F was designed according to sequence 2 and sequence 3 ₁ Primer R ₁ PCR was performed, and the amplification system and reaction procedure are shown in Table 11.

Table 11. Reaction system and reaction procedure:

b) The electrophoresis detection strip takes 5 mu L of PCR products, adopts 1% agarose gel, and is subjected to 150V constant voltage electrophoresis for 20min, and is observed on a gel imager by contrast with a DNA marker.

2.3.3PCR product recovery

a) The target DNA fragment on agarose gel was cut out as much as possible with a clean scalpel, placed in a 1.5mL centrifuge tube, and weighed.

b) Adding 500 μl of B2 Buffer per 100mg agarose, and mixing in 55deg.C water bath for 10min to completely melt the gel.

c) Completely dissolved peptized solution was all sucked into the adsorption column by pipetting gun, centrifuged at 12000rpm for 1min, and the waste liquid was decanted.

d) The B2 Buffer was added to the column by pipetting 300. Mu.L, and the column was centrifuged at 12000rpm for 1min to remove the waste liquid.

e) Adding 500 μl of Wash Solution into the adsorption column, centrifuging at 12000rpm for 1min, removing the waste liquid, repeating the steps for 1 time, and drying the adsorption column at normal temperature in a ventilated kitchen.

f) An appropriate amount of the Elutation Buffer was sucked up and added to the adsorption column, incubated in a 55℃water bath for 5min, centrifuged at 12000rpm for 10min, and the filtrate was collected.

g) The concentration and purity are detected by a spectrophotometer, 1 mu L of detection OD value is taken, OD 260/280 is 1.7-2.0, and the DNA quality is good, the protein pollution is less than 1.7, and the RNA pollution is more than 2.0.

2.3.4 sequencing assays

To a 96-well plate, 2. Mu.L of 125mM EDTA and 2. Mu.L of 3M NaAc were sequentially added, followed by 50. Mu.L of absolute ethanol, covered, shaken at room temperature for 15min, and centrifuged at 3000g for 30min. 70. Mu.L of 70% ethanol was added and centrifuged at 3000g for 15min at low temperature. The mixture was placed in a fume hood, dried with ethanol at room temperature, and 10. Mu.L of Hi-Di Formamide was added to dissolve DNA. Denaturation was carried out on a PCR apparatus at 95℃for 4min. Samples were sequenced using a 3730XL sequencer and analyzed by Sequence Analysis software after the results were obtained.

2.4 functional verification of EuSAUR Gene of Gorgon fruit

2.4.1 establishment of Rice over-expressed EuSAUR Gene

a) Construction of Rice expression vector the target gene fragment and pCambia1301 vector were digested with KpnI/XbaI, respectively, and the target fragment was constructed on the expression vector to transform Agrobacterium GV3101.

b) Transformation and cultivation of Agrobacterium to construct competent cells of Agrobacterium GV3101, transforming vector plasmids into competent cells of Agrobacterium by freeze thawing (simultaneously transforming pCambia1301 empty vector AS a negative control), coating transformed Agrobacterium with a YEP resistant medium plate containing 25mg/L Rif+50mg/L Kana+25mg/L Gen, picking up a single clone in a YEB liquid medium with a shaking table at 28℃to identify correct gene bands, continuing shaking cultivation for 2d, centrifuging the thalli at 4000rpm for 4min, resuspending the thalli with a suspension of 10mM MgCl2 (AS containing 120 uM) after removal of the supernatant, and adjusting OD600 to about 0.5.

c) Taking full rice seeds from the callus culture of rice, removing the shells, soaking and sterilizing for three times by using 75% ethanol, soaking and washing the seeds for 10min by using 10% sodium hypochlorite solution each time, washing for 10 times by using sterile water, drying by suction by using sterile filter paper, inoculating the seeds on an NBD callus induction culture medium for culturing for about 15d at 26 ℃, stripping by using a sterile knife when the callus grows out, and transferring to a new NBD culture medium for culturing; selecting a small amount of compact callus with better growth vigor, and inoculating the callus to a secondary culture medium for culturing for 15d at 26 ℃; appropriate amount of callus is cut and inoculated on a culture medium for light-shielding culture for 3d.

d) Placing the pre-cultured callus of the agrobacterium-mediated transformation rice in a conical flask, adding a proper amount of agrobacterium liquid which is successfully transformed, shaking and culturing to fully infect the callus, and standing; pouring out the bacterial liquid, placing on sterile filter paper, sucking to dry, transferring to a new culture medium, and culturing for 3d at the temperature of 26 ℃ in a dark place; the infected calli were then placed on hygromycin-resistant medium for resistance screening. Selecting non-blackened callus, and culturing for 10d on a new pre-culture medium at 26 ℃ for pre-differentiation; and (3) selecting compact callus to transfer into a differentiation medium for culture until the callus is differentiated into seedlings.

e) And (3) hardening off the transgenic rice seedlings, taking out the differentiated seedlings, cutting off surrounding callus, and transferring to a rooting medium for culturing for 10 days. When plants grow by 10 cm to 15cm, opening a bottle cap, adding 3mL of sterile water into a culture bottle, culturing for 48h in an open mode, then transplanting into non-fertilizer soil, adding pure water, skipping the soil surface, and culturing in an illumination incubator under the conditions of: 16h of illumination, 100% of light intensity, 28 ℃ of temperature and 60% of humidity; 8 hours of darkness, 24 ℃ of temperature and 50% of humidity, and a proper amount of compound fertilizer is added every week after 20 days of culture.

2.4.2 identification of transgenic Rice

a) DNA extraction transgenic rice leaves were extracted according to the method of "2.3.1DNA extraction".

b) The PCR amplification uses rice leaf DNA as a template, the EuSAUR gene sequence in transgenic rice is amplified according to a 2.3.2PCR reaction method, the PCR product is subjected to agarose gel electrophoresis, the electrophoresis result is observed in a gel imager, positive rice plants are identified according to the displayed strip, and the primer F ₃ For sequence 6, primer R ₃ Sequence 7.

2.4.3 phenotypic observations of Rice over-expressing the EuSAUR Gene

After hardening and transplanting, the plant height of the rice seedlings is 10-15cm, each 2d of the rice seedlings are photographed and recorded, and the plant height and leaf included angle phenotype of the transgenic rice are measured by using imageJ1.52 software.

2.4.4 Effect of over-expressed EuSAUR Gene on Rice cells

The leaf and leaf sheath samples of rice were taken, slit and then immersed in 4% (w/v) paraformaldehyde solution overnight at 4 ℃. Samples were removed, rinsed 3 times with 10mM PBS buffer for 5min each, followed by rinsing with distilled water. The sample was placed on a sample support, embedded with OCT embedding medium and allowed to set at-20 ℃ for 20min. And after the embedding agent is completely solidified, rapidly transferring the embedding agent to a precooled objective table for slicing, wherein the slice thickness is 60 mu m. The sections were placed in the appropriate position on the slide and the sections were unfolded by melting the OCT embedding medium with finger temperature. Washing with clear water until OCT embedding agent is completely eluted, and loading into water for observation.

3. Experimental results

3.1 observing the sizes of Gorgon fruit seeds of different cultivars

The hybrid gorgon euryale seed has higher yield, the dominant character is highlighted year by year from the breeding, and the area test per mu yield can reach 355kg in the first town of Jiangsu Gaoyou in 2018. In the same year, the yield per mu of the production test carried out in Siyang of Jiangsu and Huainan of Anhui can reach 330kg, and the yield is improved by about 35% compared with 250kg per mu of the gorgon euryale seed. By observing the seeds of the gorgon euryale seed and the hybrid gorgon euryale seed, the diameter of the hybrid gorgon euryale seed is about 1.4cm, and the diameter of the seed of the gorgon euryale seed is 1.0cm; measuring the thickness of seed coats, hybridizing 1.8mm, and thickening 0.7mm; the diameter of the seed kernel of the hybrid gorgon euryale is 1.1cm, which is obviously larger than that of the gorgon euryale seed. The seed number of each gorgon euryale seed is not obviously different from that of each gorgon euryale seed, so that the larger seed of the gorgon euryale seed is the main reason for the high yield of the gorgon euryale seed. (FIG. 1)

3.2 microscopic observations of the Coneurium spinosum and hybrid Gorgon fruit

The ovary development of the hybrid gorgon euryale is found to be significantly earlier than that of the gorgon euryale in the same growth period by microscopic observation and comparison of longitudinal cuts of the breeding organs of the gorgon euryale and the hybrid gorgon euryale collected at week 12 and week 13. Moreover, as the seed numbers of the two cultivated species of gorgon euryale are not obviously different from each other, the fact that the seeds of the hybrid gorgon euryale are larger is probably caused by the fact that the ovary development is earlier, the development period is longer, and endosperm can be fully filled is indicated. (FIG. 2)

The growth of cells near the ovary of the plant directly affects the size and development speed of the ovary, so that microscopic observation of hybrid Gorgon fruit and cells near the ovary of the Gorgon fruit is carried out in the experiment. By observing the red autofluorescence of the sample slice, the cell length near the ovary of the gorgon euryale is found to be about 16-18 mu m, the cell length near the ovary of the gorgon euryale is concentrated to 18-22 mu m, and the cell length around the ovary of the gorgon euryale is proved to be obviously longer than the cell length around the ovary of the gorgon euryale in the same period. Thus, it is concluded that: the faster growth of the ovary of hybrid Gorgon fruit is caused by the faster elongation of cells in the vicinity of its ovary, since the elongation of cells contributes to the development of plant organs. (FIG. 3)

3.3 transcriptome analysis of the breeding organs of Gorgon fruit and hybrid Gorgon fruit

By performing KEGGPathway analysis on differential genes between the breeding organs of the gorgon euryale and the hybrid gorgon euryale, the differential genes of the breeding organs of the two cultivars of gorgon euryale in the plant hormone signal transmission path are mainly enriched in SAUR family genes. The SAUR gene sequence of the model plant is compared by a local blast method, the SAUR gene in gorgon euryale is identified, and the gene expression quantity obtained by combining with transcriptome sequencing is screened for researching the key genes. The results of e-value <10-10 and bit score >100 were screened and CD-Hit was used to remove redundancy, ultimately identifying SAURs of 31 gordon euryales. Wherein, a sequence is screened to have obvious expression difference in the growth process of two cultivated species gorgon euryale, the expression is higher in hybrid gorgon euryale, and the expression is hardly carried out in gorgon euryale, and the gene is named EuSAUR gene. (FIG. 4)

3.4 Amplification of EuSAUR Gene

The full length of the open reading frame of the EuSAUR gene is obtained by PCR amplification using the primer pair of sequence 2 and sequence 3. The open reading frame sequence (432 bp) of EuSAUR gene is obtained by sequencing; as a result of sequence comparison using Blastx from NCBI, it was found that the EuSAUR gene belongs to the auxin-induced superfamily. (sequence 1)

3.5 Functional verification of EuSAUR genes

3.5.1 Effect of overexpression of EuSAUR Gene on Rice plant height and leaf blebs

By observing the rice plant height characters of the over-expressed EuSAUR genes, positive rice plant heights growing for 10d and 20d after transplanting are found to be higher than that of wild rice, but the plant heights growing to 30d have no obvious difference; until 30d after transplanting, the tiller number and the internode spacing have obvious advantages in plants over-expressing EuSAUR genes, but the leaf number and the internode number have no obvious difference. The result shows that the EuSAUR gene can rapidly improve the plant height of rice. (FIGS. 5 and 6)

In the experiment, 10d rice leaves are cut after transplanting through microscopic observation, and the length of the foam cells in the leaves is measured and calculated by utilizing red autofluorescence. The results showed a significant increase in the number of blebs in rice leaves over-expressing the EuSAUR gene. By measuring the length of the foam cells, it was found that the wild type foam cell length was concentrated around 22. Mu.m, whereas the foam cell length of the positive seedlings was distributed over 34-42. Mu.m. The result shows that EuSAUR gene can promote the elongation of transgenic rice leaf vesicular cells, and finally lead to early rapid elevation of rice. (FIG. 7)

3.5.2 Effect of over-expressed EuSAUR Gene on Rice plant type and leaf occipital cell

Through phenotypic observation of transgenic rice, the rice strain type of the over-expressed EuSAUR gene is found to be loose, and the rice strain type is considered to be possibly caused by the increase of leaf included angles. The leaf angle of rice refers to the angle between the leaf and the upright stem, is an important agronomic trait of monocotyledonous plants, and is related to the sunlight capturing efficiency of the plants. The experiment shows that the leaf included angle of the inverted leaf is obviously increased after the rice over-expressing EuSAUR gene grows for 60 days after transplanting. (FIG. 8)

The leaf pillow cell elongation at the rice included angle can be controlled to regulate the size of the rice leaf included angle. The experiment prepares the sections of leaf pillow at the included angle of the inverted leaf of the rice by a frozen section method, carries out microscopic observation, and adopts imagej1.52 software to measure the length of leaf pillow cells. As a result of observing red autofluorescence of the cells under 100-fold and 400-fold mirrors, respectively, it was found that leaf pillow cells of rice over-expressing EuSAUR gene were significantly longer than those of wild-type rice, the leaf pillow cell length of the wild-type rice was concentrated around 16. Mu.m, and the leaf pillow cell length of rice over-expressing EuSAUR gene was distributed over a range of 19-25. Mu.m. The result shows that EuSAUR gene can promote the elongation of rice leaf occipital cells and lead to the increase of rice leaf included angles, so that the strain type is loose. (FIG. 9)

3.5.3 Effect of over-expressed EuSAUR Gene on Rice grain size

The grain size of the rice directly influences the yield of the rice, and the rice grain which is over-expressed with EuSAUR genes is found to be obviously larger than that of wild rice through the phenotypic observation of the grain of the transgenic rice in the experiment. The result proves that the over-expression of EuSAUR gene in rice can obviously improve the size of rice seeds, and also reveals that EuSAUR gene plays an important role in regulating the size of Gorgon seeds in Gorgon fruits. (FIG. 10)

The invention is not limited to the specific technical scheme described in the above embodiments, and all technical schemes formed by adopting equivalent substitution are the protection scope of the invention.

SEQUENCE LISTING

<110> university of Nanjing traditional Chinese medicine

<120> Gordon euryale seed DNA molecule with function of promoting rice grain enlargement and application thereof

<160> 7

<170> PatentIn version 3.5

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<211> 432

<212> DNA

<213> Gordon Euryale ferox Salisb)

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Claims (8)

1. A Gorgon fruit DNA molecule with the function of promoting rice grain enlargement is characterized in that the nucleotide sequence is SEQ ID N0:1.

2. The method for obtaining the gorgon fruit DNA molecule with the function of promoting rice grain enlargement according to claim 1, characterized in that: the preparation method comprises the following specific steps:

(1) Weighing fresh and tender gorgon euryale seed samples, extracting the DNA of the gorgon euryale seed according to the instruction of an Ezup column type plant tissue genome DNA extraction kit of the biological engineering Co., ltd, wherein the specific steps are as follows:

a) Placing a sample in liquid nitrogen, fully grinding the sample into powder, and taking a proper amount of uniformly mixed powder to be placed in a 1.5mL centrifuge tube;

b) Adding 600 μl of preheated PCB Buffer and 12 μl of beta-mercaptoethanol, shaking, mixing, placing in water bath at 65deg.C for 25min, and mixing;

c) Adding 600 mu L of chloroform, shaking and mixing uniformly, centrifuging at 12000rpm for 1min, and transferring the upper water phase into a new 1.5mL centrifuge tube;

d) Adding a proper volume of BD Buffer, uniformly mixing, adding a proper volume of absolute ethyl alcohol, fully uniformly mixing, fully transferring into an adsorption column by using a pipettor, standing at room temperature for 2min, centrifuging at 12000rpm for 10min after full adsorption, and pouring out waste liquid;

e) Adding 500 mu L of PW Solution into an adsorption column, centrifuging at 12000rpm for 1min, and pouring out waste liquid;

f) Adding 500 mu L of Wash Solution into the adsorption column, centrifuging at 12000rpm for 1min, and pouring out the waste liquid;

g) Centrifuging the adsorption column 12000rpm for 2min, and drying ethanol in a fume hood;

h) Transferring the adsorption column into a new centrifuge tube, adding appropriate amount of TE Buffer, standing for 5min, heating appropriately, centrifuging at 12000rpm for 10min, and collecting filtrate;

i) The concentration and purity are detected by a spectrophotometer, 1 mu L of OD value is taken, OD 260/280 is 1.7-2.0, which indicates that DNA quality is good, protein pollution is less than 1.7, and RNA pollution is more than 2.0;

(2) 1-2 mu L of DNA extracted in the step (1) is used as a template, and the primer F is matched according to the designed primer ₁ SEQ ID No. 0:2, primer R ₁ For SEQ ID N0:3, 2. Mu.L of each of the forward and reverse primers and 5. Mu.L of MgCl-containing primer were taken ₂ Taq Buffer, 2. Mu.L dNTP, 0.5. Mu.L Taq enzyme, and pure water to 50. Mu.L of the total system, and carrying out PCR amplification under the following conditions: 5min at 95 ℃,30 s at 94 ℃,30 s at proper annealing temperature, 50s at 72 ℃,35 cycles, and final extension at 72 ℃ for 8min; taking 5 mu L of PCR product, adopting 1% agarose gel, carrying out 150V constant-pressure electrophoresis for 20min, and observing on a gel imager by contrast with a DNA marker;

(3) The product obtained by PCR amplification was recovered according to the SanPrep column PCR product purification kit of Biotechnology Co., ltd, comprising the following steps:

a) Cutting out the target DNA fragment on the agarose gel as much as possible by using a clean scalpel, putting the cut target DNA fragment into a 1.5mL centrifuge tube, and weighing;

b) Adding 500 μl of B2 Buffer per 100mg agarose, and placing in 55deg.C water bath, mixing thoroughly for 10min to melt the gel block completely;

c) Sucking all the completely dissolved peptized solution into an adsorption column by a pipetting gun, centrifuging at 12000rpm for 1min, and pouring out waste liquid;

d) Adding 300 mu L of B2 Buffer into an adsorption column, centrifuging at 12000rpm for 1min, and pouring out waste liquid;

e) Adding 500 μl of Wash Solution into the adsorption column, centrifuging at 12000rpm for 1min, pouring off the waste liquid, repeating the steps for 1 time, and drying the adsorption column at normal temperature in a ventilation kitchen;

f) Absorbing a proper amount of the absorption Buffer, adding the absorption Buffer into an absorption column, incubating for 5min in a water bath at 55 ℃, centrifuging for 10min at 12000rpm, and collecting filtrate;

g) The concentration and purity are detected by a spectrophotometer, 1 mu L of OD value is taken, OD 260/280 is 1.7-2.0, which indicates that DNA quality is good, protein pollution is less than 1.7, and RNA pollution is more than 2.0;

(4) The PCR product was sequenced with the nucleotide shown in SEQ ID No. 0:1, designated EuSAUR.

3. The method for promoting rice grain growth by using Gordon euryale seed DNA molecules according to claim 2, wherein: the method also comprises the following specific steps:

and (3) taking the PCR product of the step (3) after sequencing and confirming in the step (4), respectively carrying out double enzyme digestion on the target gene fragment and the pCambia1301 vector by using KpnI/XbaI, fusing GFP at the 3' end of the target fragment, constructing the target fragment on the expression vector, and transforming agrobacterium GV3101 to obtain the expression vector pCambia1301-EuSAUR and the cloned agrobacterium GV3101-EuSAUR.

4. A recombinant expression vector comprising the DNA molecule of claim 1, wherein: the starting vector was pCambia1301.

5. Gram Long Nong bacillus GV3101-EuSAUR of the recombinant expression vector obtained according to the method of claim 3.

6. A method for breeding transgenic plant, which comprises introducing a recombinant expression vector comprising the DNA molecule of claim 1 into a plant cell or tissue to obtain a transgenic plant.

7. The method of growing a transgenic plant according to claim 6, characterized in that: the plant is rice.

8. Use of the DNA molecule of claim 1, the recombinant expression vector of claim 4 or the cloned agrobacterium of claim 5 for the cultivation of transgenic plants.

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