CN117965557A - Application of rice plant height gene and preparation method thereof - Google Patents
Application of rice plant height gene and preparation method thereof Download PDFInfo
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Abstract
The present disclosure provides application of rice plant height genes and a preparation method thereof, and belongs to the technical field of biology. The application comprises: the rice plant height gene is used for reducing the height of rice plants, and is the gene OsNAC073. The embodiment of the invention provides an application of a rice plant height gene and a preparation method thereof, wherein the application comprises the step of using the rice plant height gene for reducing the height of a rice plant, the rice plant height gene is gene OsNAC073, a receptor plant can effectively reduce the plant height, the effect is obvious, and a coding region of the rice plant height gene can be accurately obtained by the preparation method for reducing the height of the rice plant.
Description
Technical Field
The present disclosure relates to biotechnology, and is especially application of rice plant height gene and its preparation process.
Background
Rice is the main food crop in the world, and more than half of the population takes rice as main food. The rice plant height is an important agronomic trait and is obviously related to rice yield. Moderate dwarfing of the rice plant height can improve the lodging resistance of crops, improve the light energy utilization efficiency, improve the yield, facilitate mechanical harvesting and reduce the probability of being infected by microorganisms in soil. The rice plant height is subjected to multiple effects of genetics and external environment. Genetic factors such as the internode length and the number of rice stems, the number of intervening meristematic cells, the cell length and the like have important influences on the rice plant height. Soil in the growing environment, climatic conditions, planting modes and the like can also influence the plant height of the rice. The research shows that the silicon-calcium-potassium-magnesium soil conditioner can obviously reduce the plant height of rice, enhance the lodging resistance and obviously improve the yield indexes such as effective spike number, grain number, thousand grain weight and the like. Premature use of the lodging-resistant agent can reduce the plant height of the rice in the mature period, and can also lead to yield reduction. The proportion of different nutrient elements in the fertilizer and different fertilization modes can also cause great influence on the plant height and the yield of the rice. It is also considered that the plant height and yield of rice in the mature period are obviously positively correlated. From the above results, it is clear that the influence of the external environment on the rice plant height is very complex.
With the development of modern biotechnology, genes and gene loci (Quantitative Trait Locus, QTL) related to rice plant height traits are discovered. For example, a commonly used rice lodging-resistant gene (Shortened basal internod, SBI) is positioned on a fifth chromosome of rice, codes GA-2 oxidase, and regulates the plant height of the rice by inhibiting the activity of gibberellin (Gibberellins, GAs), so that the lodging-resistant capability of the rice is improved, and further, the yield indexes such as effective spike number, grain number, thousand grain weight and the like are remarkably improved. Further analysis shows that SBI is an incomplete dominant gene and has different allelic types, and the different allelic types correspond to different product activities, so that the influence degree on the rice plant height is different. Therefore, the gene which controls the rice plant height trait is not good.
Although a plurality of related genes are detected at present, the genes cannot be well applied due to the defects of the genes in the improvement of the rice plant height. Therefore, more rice plant height genes need to be mined, and more gene resources are provided for rice breeding.
BRIEF SUMMARY OF THE PRESENT DISCLOSURE
In order to solve the problems of the prior art, the embodiment of the disclosure provides an application of a rice plant height gene and a preparation method thereof. The technical scheme is as follows:
in one aspect, the present disclosure provides an application of the rice plant height gene, the application comprising: the rice plant height gene is used for reducing the height of rice plants, and is the gene OsNAC073.
Specifically, the application includes: and connecting the coding region of the rice plant height gene with an over-expression promoter to reduce the height of rice plants.
Specifically, adding an A tail at the blunt end of the amplification product, and connecting with a pCXUN-MYC carrier subjected to enzyme digestion to obtain a connecting product;
the ligation products are transformed into acceptor material by invasion.
In another aspect, the present disclosure provides a method of preparing a rice plant height gene, the method comprising:
extracting total RNA in rice materials;
performing reverse transcription on the total RNA to obtain cDNA;
Amplifying by using the cDNA as a template and adopting a forward primer and a reverse primer to obtain an amplification product, namely a coding region of the rice plant height gene, wherein the sequence of the amplification product is shown as SEQ ID NO:1, wherein the sequence of the forward primer is shown as SEQ ID NO:2, the reverse primer is shown as SEQ ID NO: 3.
Specifically, the rice material is Japanese sunny.
Specifically, the amplification system comprises, per 50. Mu.L: 10 Xbuffer 5. Mu.L; 2mM dNTP 5. Mu.L; 1.5. Mu.L of forward primer at a concentration of 10. Mu.m; 1.5. Mu.L of reverse primer at a concentration of 10. Mu.m; 1 μl of cDNA; 1U/. Mu.L KOD-Plus-Neopolymerase. Mu.L; ddH 2 O35. Mu.L.
Specifically, the amplification procedure includes: pre-denaturation at 98℃for 2min; further 30 amplification cycles were performed, each cycle comprising denaturation at 98℃for 10sec, annealing at 55℃for 15sec, and extension at 68℃for 40sec.
The technical scheme provided by the embodiment of the disclosure has the beneficial effects that: the embodiment of the invention provides an application of a rice plant height gene and a preparation method thereof, wherein the application comprises the step of using the rice plant height gene for reducing the height of a rice plant, the rice plant height gene is gene OsNAC073, a receptor plant can effectively reduce the plant height, the effect is obvious, and a coding region of the rice plant height gene can be accurately obtained by the preparation method for reducing the height of the rice plant.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present disclosure, the drawings required for the description of the embodiments will be briefly introduced below, and it is obvious that the drawings in the following description are only some embodiments of the present disclosure, and other drawings may be obtained according to these drawings without inventive effort for a person of ordinary skill in the art.
FIG. 1 is a comparative seedling plot of control group N, test group material 73OE-9, and test group 73OE-12 provided in example two of the present disclosure, where N represents control group Nippon and 73OE-9 and 73OE-12 represent two test group materials, respectively;
FIG. 2 is a comparative plot of maturity of control group N, experimental group materials 73OE-9 and experimental group 73OE-12 provided in example two of the present disclosure, where N represents control group Nippon and 73OE-9 and 73OE-12 represent two experimental group materials, respectively;
FIG. 3 is a statistical plot of maturity plant heights for control group N, experimental group material 73OE-9, and experimental group 73OE-12 provided in example two of the present disclosure, the data representing mean value.+ -. Standard error, the asterisks on the error bars representing significant differences compared to the control group (single factor analysis of variance calculation p-value,.+ -. P < 0.01);
FIG. 4 is a graph comparing internode length at maturity for the highest two tillers in control group N versus plants of experimental group 73OE-9 and experimental group 73OE-12 provided in example two of the present disclosure;
FIG. 5 is a graph of internode length statistics for control group N, experimental group material 73OE-9, and experimental group 73OE-12 provided by example two of the present disclosure.
Detailed Description
For the purposes of clarity, technical solutions and advantages of the present disclosure, the following further details the embodiments of the present disclosure with reference to the accompanying drawings.
Example 1
The embodiment of the invention provides a preparation method of rice plant height genes, which comprises the following steps:
extracting total RNA in rice materials;
Specifically, the rice material of this example was the whole plant of rice material Nipponbare grown for 15 days. In other embodiments, the rice material may be other varieties of rice, and the process may refer to this embodiment. Total RNA of rice material was extracted with RNAiso Plus kit (TaKaRa Code: D9108A), and the specific procedure was described in the specification of RNAiso Plus kit. The specific process is as follows:
1. Fresh rice material was ground to a powder in a mortar containing liquid nitrogen.
2. 1MLTrizol solution was added to a 1.5mL centrifuge tube and then the appropriate amount of powder was transferred to the 1.5mL centrifuge tube. And (3) carrying out intense shaking to uniformly mix the powder and the Trizol, thereby obtaining homogenate. The homogenate was placed on ice for 5min.
3. 200. Mu.L of chloroform was added to the homogenate, and the mixture was inverted and mixed. Centrifugation was performed at 16363rcf for 10min at 4℃to give supernatant.
4. The supernatant was transferred to a new 1.5mL centrifuge tube, and an equal volume of pre-chilled isopropyl alcohol was added to the new centrifuge tube, mixed upside down and precipitated for 1h at-20 ℃.
5. After the precipitation, the centrifuge tube was removed and centrifuged at 16363rcf for 10min at 4 ℃. At this point, a white precipitate was seen at the bottom of the centrifuge tube, and the supernatant was decanted, leaving the precipitate.
6. 750 Μl of absolute ethanol was added to the centrifuge tube, 250 μl LRNase-Free H 2 O was added, mixed upside down, and the precipitate was washed.
7. The supernatant in the centrifuge tube was pipetted off and the pellet was dried on a super clean bench. An appropriate amount of RNase-Free H 2 O was added to the pellet to solubilize the total RNA.
8. 1.5 Mu L of the product obtained in the step 7 is taken, the concentration of total RNA is measured by a nucleic acid concentration measuring instrument (Thermo), 1 mu L of the product is taken, agarose gel electrophoresis with the concentration of 1% is used for detecting whether the RNA is complete and has no degradation, and the detection shows that the total RNA of the embodiment is complete and has no degradation.
Carrying out reverse transcription on the total RNA to obtain cDNA; specifically, PRIMESCRIPTTMRTREAGENTKITWITHGDNAERASER (Lot#AK4901) kit from TAKARA was used. Genomic DNA was removed sequentially and reverse transcribed according to the steps of the kit instructions. Finally, the mixture is preserved at 4 ℃.
PCR amplification is carried out by taking cDNA as a template and adopting a forward primer and a reverse primer, so that an amplification product with the length of 1008bp, namely a coding region of a rice plant height gene, can be seen in Rice GenomeAnnotation Project (uga. Edu) database, and the sequence of the amplification product is shown as SEQ ID NO:1, the rice plant height gene is a gene OsNAC073, and the sequence of the forward primer is shown as SEQ ID NO:2, the reverse primer is shown as SEQ ID NO: 3.
Specifically, each 50. Mu.L of the amplification system comprises: 10 Xbuffer 5. Mu.L; 2mM dNTP 5. Mu.L; 1.5. Mu.L of forward primer at a concentration of 10. Mu.m; 1.5. Mu.L of reverse primer at a concentration of 10. Mu.m; 1 μl of cDNA; 1U/. Mu.L KOD-Plus-Neo polymerase 1. Mu.L; ddH 2 O35. Mu.L.
Specifically, the amplification procedure is shown in table 1.
Table 1 shows the amplification procedure
And (3) recovering and purifying the amplified product, adding an A tail at the flat tail end of the purified amplified product, and connecting with pCXUN-MYC carrier subjected to XcmI digestion to obtain a connecting product. And after the connection product is verified to be correct by sequencing, the construction of the OsNAC073 over-expression transgenic vector for driving expression by the UBI promoter is completed.
Example two
The embodiment of the invention provides an application of rice plant height genes, which comprises the following steps: the rice plant height gene is used for reducing the height of rice plants, and is the gene OsNAC073.
Specifically, the application includes: the coding region of the rice plant height gene is connected with the over-expression promoter to reduce the height of rice plants.
The ligation product was first transformed into Agrobacterium EHA105 by electrotransformation, and Agrobacterium EHA105 was purchased from Shanghai Biotechnology Inc. The ligation product was then transformed into the acceptor material Nippon-Qing by Agrobacterium-mediated methods, comprising the following steps:
1. Induction of callus:
seed sowing is carried out by a seed sowing machine, broken, shelled and relatively black seeds are removed, mature and full seeds are selected, 14g are weighed according to thousand seed weight in the embodiment, and the seeds are filled into a 50mL centrifuge tube.
Washing with tap water for at least 5 times, washing with 30mL of distilled water for 3 times, soaking with 70% ethanol solution for 5min, washing with distilled water for 3 times, adding 0.15% HgCl 2, and oscillating at 100rpm for 15-20 min. During the shaking period, the sterilized filter paper on the super clean bench is prepared, and the sterilized spoon is burned on an alcohol lamp for a few minutes.
The centrifuge tube was opened on a sterile bench, hgCl 2 was poured out, the seeds were washed 4 to 5 times with sterile water, poured onto sterilized filter paper, transferred to fresh dry filter paper after several minutes, and left to stand for at least 1 hour until the seeds were dry.
Placing the dried seeds on an N6 solid culture medium, sealing with a sealing film, and culturing in dark at 28 ℃ for 1 month.
2. And (3) subculture:
When the induced callus is observed, the callus with good state can fall off automatically, the pale yellow and compact embryogenic callus on the N6 solid culture medium is transferred to a new N6 solid culture medium, and the callus is subjected to dark culture at 28 ℃ for 2 weeks.
3. Pre-culture:
compact and relatively dry calli were selected for transfer to fresh N6 medium and dark cultured at 28℃for 3 days.
4. Culturing and suspending agrobacterium:
Appropriate amounts of Agrobacterium were plated on YEP solid medium including kanamycin and rifampicin antibiotics and incubated overnight at 28℃in dark.
10ML of 1/2N6 liquid culture solution with the final concentration of 150uMAS is added into a 50mL centrifuge tube, the cultured agrobacterium is scraped by a sterilization small spoon and placed into the centrifuge tube, and the mixture is repeatedly blown and evenly mixed by a pipetting gun to obtain suspension. The suspension was transferred to a 1/2N6 liquid culture medium containing a final concentration of 150uMAS, and the concentration was adjusted so that the OD600 of the suspension reached about 0.8.
5. Infection and co-cultivation:
infection: the callus after the pre-culture is concentrated into a sterilized culture dish, and is transferred into a glass bottle containing bacterial liquid at one time, and is infected for about 15 to 20 minutes on a shaking table at a low speed.
Co-cultivation: pouring out bacterial liquid, pouring the callus into a plate with sterile filter paper, standing for 2 hours, turning the plate once in the middle, finally scooping the callus directly onto a 1/2N6 solid culture medium with the final concentration of 150uMAS by a spoon, and culturing the callus in dark at 20 ℃ for 1.5-2 days to obtain co-cultured callus.
6. Removal of agrobacteria
Co-cultured calli were filled into 200mL glass bottles and washed at least 3 times with sterile water until the liquid was clear.
Pouring out sterile water, adding N6 liquid culture solution containing 500mg/L cephalosporin, shaking at low speed on a shaking table for 15-20 min, and repeating for 2-3 times.
Pouring the callus into a plate containing sterile filter paper, turning the plate once in the middle, placing the plate on an ultra-clean bench for more than 3 hours to enable the callus to be completely dried, and turning the callus with a sterile spoon from time to accelerate drying.
7. Screening of callus
The dried calli were transferred to N6 solid medium containing 250mg/L cephalosporin and 50mg/L hygromycin and dark cultured at 28℃for 30 days.
The callus which is not contaminated by agrobacterium and has better growth condition is further transferred to fresh N6 solid medium containing 250mg/L cephalosporin and 50mg/L hygromycin, and dark-cultured at 28 ℃ for about 15 days. The new culture medium can increase the activity of the callus and make the callus differentiated better.
8. Differentiation and transplantation
The callus from the last selection was transferred to a triangle flask containing MS medium and dark cultured at 28℃for about 10 days.
The flask was changed from dark culture to light culture, and light culture was performed at 28℃for about 20 days. During which observation was noted.
When green buds grow in the calli and the length reaches about 1-2 cm, the calli are transferred into a test tube containing 1/2MS rooting culture medium. And when the root system of the callus grows out and the height of the seedling reaches the height of the test tube, obtaining the T 0 -generation plant.
The T 0 generation plant is selfed for two generations to obtain a T 2 generation plant line for subsequent experiments.
Soaking seeds of the generation T 2 and seeds of a control material Japanese sunny for germination acceleration, respectively taking the seeds as an experimental group and a control group, selecting 300 seeds of two groups of seeds which are normal in germination and similar in germination speed, sowing the seeds in a seedling tray, transplanting the seeds into a field according to a row spacing of 26.7cm and a plant spacing of 16.7cm when the seedling age is 25 days, planting the seeds according to a rule that 10 plants are multiplied by 5 rows are taken as a cell, planting 3 cells in each material, and carrying out normal field water and fertilizer management. After transplanting for one month, 5 rice plants of the control group and the experimental group are respectively selected at the middle position to be removed from the field, roots of the rice plants are not damaged as much as possible when the rice plants are removed, and photographing is carried out, the photographing result is shown in figure 1, and compared with the control group N, the plant heights of the experimental groups (73 OE-9 and 73 OE-12) are obviously reduced, wherein the 73OE-9 and 73OE-12 are plants of two different positive transgenic lines as shown in figure 1.
When the plants of the experimental group and the control group grew for about 110 days to enter the maturity, the plant heights of the experimental group and the control group were again compared, and as shown in fig. 2, it is apparent from fig. 2 that the plant heights of the experimental group (73 OE-9 and 73 OE-12) were significantly lower than those of the control group N. All plant heights of the control group and the experimental group were measured at the same time, and the statistical measurement results are shown in fig. 3. As can be seen from fig. 3, the plant heights of the experimental group (73 OE-9 and 73 OE-12) were significantly reduced compared with the control group N.
The highest two tillers in the plants of the control group N and the experimental group (73 OE-9 and 73 OE-12) are completely removed, leaf sheaths are peeled off, the nodes of rice stalks are exposed, and the length of each node is measured. As shown in fig. 4, the results of 15 plants per material were shown, and it can be seen from fig. 4 that the length of each internode of the experimental group was shorter than that of the control group N. Statistical analysis of the length of each internode between control group N and experimental groups (73 OE-9 and 73 OE-12), as shown in FIG. 5, it is seen from FIG. 5 that the height of each internode of the experimental group is significantly reduced compared to control group N.
The foregoing description of the preferred embodiments of the present disclosure is provided for the purpose of illustration only, and is not intended to limit the disclosure to the particular embodiments disclosed, but on the contrary, the intention is to cover all modifications, equivalents, alternatives, and alternatives falling within the spirit and principles of the disclosure.
Claims (7)
1. An application of a rice plant height gene, which is characterized in that the application comprises: the rice plant height gene is used for reducing the height of rice plants, and is the gene OsNAC073.
2. The application according to claim 1, characterized in that it comprises: and connecting the coding region of the rice plant height gene with an over-expression promoter to reduce the height of rice plants.
3. The use according to claim 1, wherein the blunt end of the amplification product is added with an A tail and then ligated with the enzyme-digested pCXUN-MYC vector to obtain a ligation product;
the ligation products are transformed into acceptor material by invasion.
4. A method for producing a rice plant height gene according to claim 2, comprising:
extracting total RNA in rice materials;
performing reverse transcription on the total RNA to obtain cDNA;
Amplifying by using the cDNA as a template and adopting a forward primer and a reverse primer to obtain an amplification product, namely a coding region of the rice plant height gene, wherein the sequence of the amplification product is shown as SEQ ID NO:1, wherein the sequence of the forward primer is shown as SEQ ID NO:2, the reverse primer is shown as SEQ ID NO: 3.
5. The method according to claim 4, wherein the rice material is Nipponbare.
6. The method of claim 4, wherein each 50 μl of the amplification system comprises: 10 Xbuffer 5. Mu.L; 2mM dNTP 5. Mu.L; 1.5. Mu.L of forward primer at a concentration of 10. Mu.m; 1.5. Mu.L of reverse primer at a concentration of 10. Mu.m; 1 μl of cDNA; 1U/. Mu.L KOD-Plus-Neo polymerase 1. Mu.L; ddH 2 O35. Mu.L.
7. The method of claim 4, wherein the amplification procedure comprises: pre-denaturation at 98℃for 2min; a further 30 cycles were performed, each cycle comprising: denaturation at 98℃for 10sec, annealing at 55℃for 15sec, elongation at 68℃for 40sec.
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