Application of calcium hydrogen transporter OsCAX1a in regulating stomatal development and / or photosynthesis capacity in rice

Abstract

The present invention discloses the application of calcium hydrogen transporter OsCAX1a in regulating rice stomatal development and / or photosynthesis ability. The present invention constructs cax1a random knockout mutant material using CRISPR-Cas9 technology. After the OsCAX1a gene mutation, the stomatal width does not change, the length is significantly reduced, and the stomatal density is also significantly reduced. This directly leads to the entry and exit of photosynthetic products panicle and carbon dioxide, which directly leads to a decrease in photosynthetic efficiency.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and relates to the application of calcium-hydrogen antiporter OsCAX1a in regulating rice stomatal development and / or photosynthesis ability. Background Art

[0002] Currently, six calcium-hydrogen antiporters CAXs have been found in rice, namely CAX1a, CAX1b, CAX1c, CAX2, CAX3 and CAX4. In rice, except for OsCAX2, other OsCAXs genes in rice are sensitive to low concentrations of Ca 2+ OsCAX1a and OsCAX1c can promote Cd 2+ OsCAX4 can reduce the accumulation of Cd 2+ The accumulation of (Zou W, Chen J, Meng L, Chen D, He H, Ye G. The Rice Cation / H + Exchanger Family Involved in CdTolerance and Transport. International Journal of Molecular Sciences. 2021; 22(15): 8186). In addition, pH has a crucial influence on the cation transport activity of OsCAXs (Pittman et al., 2005), 2+ OsCAX1a can convert Ca 2+ Transported to the vacuole, involved in maintaining high Ca concentrations 2+ Intracellular Ca 2+ OsCAX1a has at least two physiological functions: one is to protect companion cells and endothelial cells, which are affected by the Ca 2+ The influx of Ca2+ causes calcium ion toxicity; one is to convert Ca 2+ Active transport into the vacuole as a second signal molecule for mineral nutrition and signal transduction (Takehiro Kamiya; Taro Akahori; Motoyuki Ashikari; Masayoshi Maeshima Expression of the Vacuolar Ca 2+ / H + Exchanger,OsCAX1a,in Rice:Cell and Age Specificity of Expression,and Enhancement by Ca 2+Plant and Cell Physiology, 2006, 47(1): 96-106). The chlorophyll content in leaves lacking OsCAX1a was significantly reduced. Histochemical staining and quantitative detection of ROS-related indicators showed that there was obvious accumulation of superoxide anions at the leaf tip accompanied by cell death. Element content detection showed that the content of multiple metal elements in the upper part of the leaves was significantly reduced, and the metal elements were unbalanced. The abnormal development of pedicels and vascular bundles led to the obstruction of nutrient transport and spikelet degeneration. The leaves lost water and curled, and the fresh weight of seedlings was significantly reduced. In response to high concentrations of Ca 2+ Sensitive to calcium salt stress (Wang Zhonghao, Shi Yongfeng Map-based cloning and functional analysis of rice calcium hydrogen ion exchange protein gene CAX1a. Master. Chinese Academy of Agricultural Sciences). However, the existing technology has not yet disclosed the relationship between OsCAX1a and stomatal morphology and photosynthetic efficiency. Summary of the invention

[0003] The purpose of the present invention is to address the above-mentioned deficiencies in the prior art and to provide an application of the calcium hydrogen transporter OsCAX1a in regulating the stomatal development and / or photosynthesis capacity of rice.

[0004] The purpose of the present invention can be achieved through the following technical solutions:

[0005] Application of rice OsCAX1a gene in regulating stomatal development and / or photosynthesis of plant leaves, wherein the sequence of rice OsCAX1a gene is shown in SEQ ID NO.1.

[0006] As a preferred embodiment of the present invention, knocking out the rice OsCAX1a gene or inhibiting the expression of the gene can reduce stomatal length, stomatal density and photosynthetic efficiency.

[0007] The application of a substance for knocking out or silencing the rice OsCAX1a gene in regulating stomatal development and / or photosynthesis in plant leaves, wherein the rice OsCAX1a gene sequence is shown in SEQ ID NO.1.

[0008] As a preferred embodiment of the present invention, knocking out the rice OsCAX1a gene or inhibiting the expression of the gene can reduce stomatal length, stomatal density and photosynthetic efficiency.

[0009] As a preferred embodiment of the present invention, the substance for knocking out or silencing the rice OsCAX1a gene is a gene editing system or siRNA targeting the rice OsCAX1a gene.

[0010] As a further preference of the present invention, the gene editing system is selected from a CRISPR-CAS9 vector or a CRISPR-CAS9 vector composition.

[0011] Beneficial effects:

[0012] The present invention uses CRISPR-Cas9 technology to construct cax1a random knockout mutant materials. We found that after the OsCAX1a gene mutation, the stomatal width did not change, the length was significantly reduced, and the stomatal density was also significantly reduced. This directly led to a decrease in the inflow and outflow of photosynthetic products and carbon dioxide, and directly led to a decrease in photosynthetic efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Construction and identification of mutants

[0014] Figure 2 X-ray cold spectrometer in situ detection of calcium content in rice. Bright field (left) and fluorescence field (right) Figure 3 Comparison of stomatal density and stomatal morphology on leaf surface under microscope (red triangles indicate stomata within the field of view)

[0015] Figure 4 Comparison of pore length and width

[0016] Figure 5 Comparison of photosynthesis capacity: light response curve (left) and CO2 response curve (right)

[0017] Figure 6 Comparison of net photosynthetic rate, transpiration rate and stomatal conductance

[0018] Figure 7 Comparison of transpiration water potential DETAILED DESCRIPTION

[0019] Example 1 Construction and identification of gene knockout materials

[0020] (1) Vector construction

[0021] The accession number of the OsCAX1a gene in the Rice Genome Annotation Project (RGAP7, MSU) (http: / / rice.plantbiology.msu.edu / index.shtml) is LOC_Os01g37690. The CRISPR-P (http: / / crispr.hzau.edu.cn / CRISPR / ) website was used to search for the corresponding gene-specific Spacers according to the gene number, and several Spacers with a low off-target rate (green) and located near the 5' end of the exon were randomly selected; restriction sites were added to both ends of the screened Spacers, and the forward and reverse chains were synthesized separately by Nanjing GenScript Biotechnology Co., Ltd.; the two synthesized chains were diluted to 100 μM and then mixed in equal amounts, and double-stranded synthesis was performed by gradient annealing. The annealing process is as follows;

[0022] 95°C for 2 minutes;

[0023] 72°C for 2 minutes;

[0024] 55°C for 2 minutes;

[0025] 37°C for 2 minutes;

[0026] 25°C for 2 minutes;

[0027] Maintain constant temperature at 10℃.

[0028] The above products were connected to the corresponding intermediate vector pYLgRNA-OsU6a / LacZ which had been linearized with BsaⅠ in advance using T4 ligase; after the connection was completed, the two pairs of primers B1' / BL were used to amplify and recover the amplified products; the recovered products were connected to the expression vector pYLCRISPR / Cas9-MH using the efficient "cutting and ligating" method and sequenced for verification.

[0029] The usage method is disclosed in: Ma

[0030] (2) Genetically modified organisms:

[0031] Take 100 μL of competent cells of Agrobacterium tumefaciens (EHA105) and 2 μL of the verified expression vector, and gently mix them in a pre-cooled sterile Ep tube. Slowly transfer the mixture into a -20℃ pre-cooled electroporation cup to avoid bubbles. Carefully wipe the outer wall of the electroporation cup, set the electrode voltage of the Bio-Rad electroporator to 2500V, and perform high-voltage electroporation transformation. After electroporation, use pre-cooled YEP culture solution for pre-cultivation in turn, and YEP plate culture containing corresponding antibiotics to screen the successful transfer of single clones. After centrifugation and precipitation of 1mL of the above bacterial solution with OD600 reaching 0.8-1.0, use the bacterial solution (30mL AAM with 200μmol / L As) to make a suspension. Take an appropriate amount of light yellow, round and solid high-quality rice callus tissue, pour it into the suspension for infection for 5 minutes, drain or air-dry on sterile filter paper, and transfer to the co-culture medium for dark culture for 3 days.

[0032] (3) Plant tissue culture:

[0033] Select white and full rice seeds for peeling, soak in water for 30 minutes after disinfection, air-dry on sterile filter paper and place in induction medium, 12-14 seeds per culture dish, and culture in the tissue culture room for 4 weeks. Then place on subculture medium and continue to culture for 2 weeks to obtain healthy rice callus. Transfer the infected callus to a sterile tube, wash with sterile water for many times, and soak in sterile water containing 500mg / L carbenicillin (Car) for 30-60 minutes, transfer to selection medium for screening after drying, and move the screened resistant callus into a differentiation tank containing differentiation medium, culture at a constant temperature, wait for differentiation into seedlings (30-60 days) and then place in rooting medium for rooting.

[0034] (4) Knockout effect identification:

[0035] The transgenic rice genome was sequenced from 1:21071497 to 21076720 and compared with the wild type. Figure 1 shown.

[0036] Example 2

[0037] (1) In situ detection of calcium content in rice using X-ray cold spectrometer

[0038] The wild-type rice "Nipponbare" and the OsCAX1a mutant seedlings constructed in Example 1 were cultured in appropriate culture medium at 28°C for 2 weeks, and the abundance of metal ions in various parts of the rice was measured in situ using an X-ray cold spectrometer (XRF). The rice seedlings were laid flat, pasted on the XRF special tape, and fixed flat in the test tray. The test tray was placed in the radiometer, evacuated and filled with liquid helium for protection, and the scanning area and step length were set for scanning. After the scan, the helium was recovered and the vacuum was released. The results are shown in Figure 2 ,Depend on Figure 2 It can be seen that after the OsCAX1a gene mutation, the calcium content in the body decreased and the plant height became shorter.

[0039] (2) Determination of plant photosynthetic traits

[0040] The determination of plant photosynthetic indicators was carried out with the help of LI-6800 from LI-COR, USA. A sunny noon was selected to conduct self-inspection and temperature and light intensity correction on the instrument in the field. A positive pressure carbon dioxide cylinder was installed and checked for leaks. The flag leaf to be tested was selected, and its width was measured with a ruler and entered into the instrument. The instrument's built-in photosynthetic rate detection program, light response curve detection program, and carbon dioxide curve detection program were used for measurement. After measurement, the supporting software was used to directly read and calculate each characteristic value. The changes in photosynthesis of mutants and wild types were dynamically monitored. Figure 5 , 6 , 7. The results showed that after the OsCAX1a gene mutation, the photosynthetic rate decreased ( Figure 5), the light response curve and the carbon dioxide response curve are low ( Figure 6 ). This directly affects the photosynthesis and growth of rice.

[0041] (3) Fixation and observation of stomata

[0042] In order to explore the cause of the decline in photosynthesis, the stomatal development of mature rice leaves was observed: the stomatal morphology was determined using the cell blotting method, and the rice stomatal cells were fixed and preserved in situ with the help of a fixative (nail polish, etc.), and then brought back to the laboratory for observation under a microscope. The specific operation is that when the stomata are open at noon on the third day of continuous sunny weather, the fixative is applied to the back of the flag leaf to be observed, blow dried, and the flag leaf is removed and clamped in a fixing clip. After returning to the laboratory, use tweezers or tape to carefully tear off the fixed epidermis, place it on a glass slide to make a temporary mount for observation, and what is observed is the morphological imprint of the stoma when it is fixed. The pictures are measured and calculated using ImageJ and other software. See the results. Figure 3 and 4 ,Depend on Figure 3 and 4 It can be seen that after the OsCAX1a gene mutation, the stomatal width did not change, but the length was significantly reduced, and the stomatal density was also significantly reduced. This directly led to the inflow and outflow of photosynthetic products and carbon dioxide, and directly led to a decrease in photosynthetic efficiency.

[0043] (4) Leaf water potential measurement

[0044] The leaf water potential is measured with the help of the WP4C dew point water potential meter. The specific operation is to place the WP4C dew point water potential meter in the test environment for 2 hours to make the water potential meter test chamber consistent with the outside temperature. Take the flag leaf of the rice seedling to be tested, cut it into pieces and spread it flat on the tray, trying to cover it as much as possible without overlapping. Run the test program, and wait for the green light to light up, indicating that the test is completed. Record the data and the results are shown in Figure 7 .Depend on Figure 7 It can be seen that the mutation of the OsCAX1a gene affects the water potential of rice, reduces the transpiration pull, and further affects the transport of calcium ions from the underground to the aboveground part, aggravating the calcium deficiency of rice.

Claims

1. Rice OsCAX1a Application of genes in reducing stomatal density in rice leaves, wherein the application is to knock out or silence rice OsCAX1a Gene.

2. The use according to claim 1, characterized in that: The knockout or silencing rice OsCAX1a Rice OsCAX1a Gene editing system, siRNA.

3. The use according to claim 2, characterized in that: The gene editing system is selected from a CRISPR-CAS9 vector or a CRISPR-CAS9 vector combination.

Images