Application of Ghd7 gene in regulation and control of photosynthetic efficiency and light protection capability of rice leaves

Abstract

The invention relates to application of a Ghd7 gene in regulation and control of photosynthetic efficiency and light protection capability of rice leaves. The effect of the Ghd7 is proved by using a near-isogenic line and a transgenic material, how the Ghd7 enhances the rice light protection capability in a light protection regulation related pathway is researched, and the gene is found to improve the rice light protection capability and the photosynthetic efficiency by up-regulating the expression quantity of an NPQ related gene. Therefore, the gene has an important breeding value in high-photosynthetic-efficiency breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to the application of the Ghd7 gene in regulating the photosynthetic efficiency and light protection ability of rice leaves. Background Art

[0002] Photosynthesis is the basis of all life activities of crops and the main source of crop yield. Most of the light energy absorbed by crops is used for photosynthesis, but it is still consumed in the form of heat dissipation and chlorophyll fluorescence. The amount of dissipation through chlorophyll fluorescence is very small and can be mostly ignored. When the light is too strong, the plant absorbs excess light energy, which will cause photodamage and poison the cells. At this time, the excess light energy must be dissipated through the non-photochemical quenching (NPQ) process. This is the photoprotection mechanism evolved by plants to consume excessive light energy in the form of heat dissipation when faced with excessive light absorption. Therefore, NPQ is an important indicator reflecting the photoprotection mechanism. By optimizing the NPQ process, the plant can be protected from light inhibition to the maximum extent, and the utilization efficiency of absorbed light energy can be increased, thereby increasing the biomass and yield of crops.

[0003] Ghd7 is a pleiotropic gene that controls the heading period, plant height, and number of spikelets per panicle of rice. It is located near the centromere of rice chromosome 7, and its encoded protein contains a CCT conserved domain. Ghd7 is regulated by the photoperiod. Long-day conditions induce an increase in its expression, thereby inhibiting the expression of downstream genes, leading to a delay in the heading period of rice, an increase in plant height, and an increase in yield. The pleiotropic gene Ghd7 is also involved in the period conversion, plant type regulation, stress response, and hormone regulation of rice. However, the Ghd7 gene has not yet been reported in regulating rice photosynthetic efficiency and light protection ability. Summary of the invention

[0004] The present invention fully confirmed the effect of Ghd7 by using near-isogenic lines and transgenic materials, and studied how Ghd7 enhances the photoprotection ability of rice in pathways related to photoprotection regulation. It was found that the gene improved the photoprotection ability of rice by upregulating the expression of NPQ-related genes, indicating that the gene has important breeding value in high photosynthesis breeding.

[0005] The present invention provides the use of the Ghd7 gene in any one of the following A1)-A4):

[0006] A1) regulating plant photosynthetic efficiency and / or photoprotection ability;

[0007] A2) preparing products for regulating plant photosynthetic efficiency and / or photoprotection ability;

[0008] A3) Cultivating plant varieties with high photosynthetic efficiency and / or photoprotection ability;

[0009] A4) Preparation and cultivation of plant varieties with high photosynthetic efficiency and / or photoprotection ability.

[0010] Furthermore, the plant includes rice, and the nucleotide sequence of the Ghd7 gene is shown in SEQ ID NO:1.

[0011] Furthermore, the plant exhibits all or part of the following:

[0012] B1) Increased photosynthetic efficiency;

[0013] B2) Enhanced photoprotection ability.

[0014] Furthermore, the period for measuring the photosynthetic efficiency is the tillering stage of rice.

[0015] The present invention also provides a method for enhancing the photosynthetic efficiency and / or light protection ability of plants, which involves overexpressing the Ghd7 gene in the plant.

[0016] Furthermore, the plant includes rice, and the nucleotide sequence of the Ghd7 gene is shown in SEQ ID NO:1.

[0017] Furthermore, the period for measuring the photosynthetic efficiency is the tillering stage of rice.

[0018] The present invention also provides a plant breeding method, which comprises introducing an expression cassette, an expression vector, and a recombinant microorganism for overexpressing the Ghd7 gene into a recipient plant to obtain a transgenic plant, wherein the transgenic plant exhibits all or part of the following:

[0019] B1) Increased photosynthetic efficiency;

[0020] B2) Enhanced photoprotection ability;

[0021] Furthermore, the plant includes rice, and the nucleotide sequence of the Ghd7 gene is shown in SEQ ID NO:1.

[0022] Furthermore, the period for measuring the photosynthetic efficiency is the tillering stage of rice.

[0023] Compared with the prior art, the present invention has the following beneficial effects: using near-isogenic lines and transgenic materials, it is confirmed for the first time in rice that the Ghd7 gene regulates rice photosynthetic efficiency and light protection ability. Further research finds that the gene improves the rice light protection ability by upregulating the expression of two NPQ-related genes. The gene has important breeding value in rice high-light-efficiency breeding; the present invention also provides a technical reference for cloning related genes in other crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0025] Figure 1 :Comparison of chlorophyll fluorescence parameters of different genotypes of Ghd7. Figure A shows the chlorophyll fluorescence parameters of leaves at the tillering stage, and Figure B shows the chlorophyll fluorescence parameters of leaves at the heading stage. "*" indicates a p value < 0.05 in the t test, indicating a significant difference; "**" indicates a p value < 0.01 in the t test, indicating an extremely significant difference.

[0026] Figure 2 :Net photosynthetic rate of Ghd7 near-isogenic lines at heading stage. “*” indicates p value < 0.05 in t test, indicating significant difference.

[0027] Figure 3 :Changes in the expression of OsPsbS1 and OsbZIP72 in Ghd7 transgenic rice leaves under strong and weak light. A: Changes in the expression of OsPsbS1 in Ghd7 transgenic rice leaves under strong and weak light. B: Changes in the expression of OsbZIP72 in Ghd7 transgenic rice leaves under strong and weak light. In the figure: "*" indicates a p value < 0.05 in the t test, indicating a significant difference. "**" indicates a p value < 0.01 in the t test, indicating an extremely significant difference. DETAILED DESCRIPTION

[0028] The following examples are only used to more clearly illustrate the technical scheme of the present invention, and are therefore only used as examples, and cannot limit the scope of protection of the present invention. It should be noted that, unless otherwise stated, the technical terms or scientific terms used in this application should be the usual meanings understood by those skilled in the art to which the present invention belongs. Unless otherwise stated, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art. Unless otherwise stated, the reagents and materials used in the following examples are commercially available.

[0029] Example 1: Using near-isogenic lines and transgenic materials to confirm the regulatory effect of the Ghd7 gene on rice photoprotection ability

[0030] NIL(mh7) and NIL(zs7) are a pair of near-isogenic lines constructed in our laboratory with Zhenshan 97 as the genetic background and Minghui 63 as the donor parent. The main difference between the two is the Ghd7 gene segment. NIL(mh7) carries a fully functional allele from Minghui 63, while NIL(zs7) completely lacks it. ZH11This is a transgenic material that overexpresses Ghd7 cultivated in our laboratory (Zhonghua 11 is used as the transformation receptor, overexpressing the Ghd7 cDNA from Minghui 63, and Zhonghua 11 is known to carry a weaker allele). RNAi-Ghd7 ZH11 This is the transgenic material of RNA interference Ghd7 cultivated in our laboratory (a CDS fragment of appropriate length in the Ghd7 genome sequence was selected to construct the RNAi vector, and the same specific fragment was connected to the RNAi expression vector in the forward and reverse directions through BamHⅠ / KpnⅠ and SacⅠ / SpeⅠ restriction endonucleases, and Zhonghua 11 was selected as the transformation receptor). WT-Ghd7 ZH11 It is a wild-type Zhonghua 11 material. The CDS sequence of the Ghd7 gene is shown in SEQ ID NO: 1, and the protein sequence is shown in SEQ ID NO: 2.

[0031] The present invention measures the chlorophyll fluorescence parameters of leaves of Ghd7 near-isogenic lines and transgenic lines during the tillering and heading stages. The specific process of chlorophyll fluorescence parameter determination is as follows: select the latest fully expanded leaves of the main stem during the tillering stage and the sword leaves of the main stem during the heading stage, repeat each material three times, put the leaves into 50mL of 0.1% agarose solution, bring them back to the laboratory, put the taken leaves in a dark room, darken them overnight, and use a Dual-PAM-100 dual-channel modulated chlorophyll fluorescence instrument to measure the chlorophyll fluorescence parameters of the leaves during the tillering and heading stages at 8 am the next day. The actinic light parameter is set to 1000μmol·m -2 ·s -1 The results obtained are described below.

[0032] At the tillering stage, the NPQ values ​​of the Ghd7 near-isogenic line NIL (mh7) were 2.601±0.11, the NPQ values ​​of the Ghd7 near-isogenic line NIL (zs7) were 2.347±0.13, and the NPQ values ​​of RNAi-Ghd7 ZH11 The NPQ value of WT-Ghd7 was 2.532±0.17. ZH11 The NPQ value is 3.113±0.11 (see Figure 1 At the heading stage, the NPQ values ​​of NIL(mh7) and NIL(zs7) were 2.621±0.11 and 2.003±0.20, respectively. ZH11 The NPQ value of WT-Ghd7 was 2.003±0.13. ZH11 The NPQ value is 2.216±0.13 (see Figure 1Figure B). It can be seen that the NPQ value of the functional Ghd7 material (NIL(mh7)) is higher than that of the non-functional Ghd7 material (NIL(zs7)), and there is a significant difference between the two. The NPQ value of the Ghd7 overexpression material is higher than that of the wild-type material and the RNAi material, and there is a significant difference between the RNAi material and the wild type. The above phenotypic results of the near-isogenic lines and transgenic materials indicate that the Ghd7 gene can promote the photoprotection of rice.

[0033] Example 2: Using near-isogenic line materials to confirm the regulatory effect of the Ghd7 gene on photosynthetic efficiency during the tillering stage of rice

[0034] The present invention investigates the photosynthetic efficiency of Ghd7 near-isogenic lines NIL (mh7) and NIL (zs7). The light response curves of NIL (mh7) and NIL (zs7) plants in the tillering stage were measured using a Li-6400 photosynthetic instrument. The latest fully expanded leaves of the main stem in the tillering stage were measured from 09:00 to 16:30 on a sunny day, and each material was repeated 3 times. The middle of the tillering stage was defined as 20 days after transplanting. The results showed that as the light intensity gradually increased, the net photosynthetic rate of rice also gradually increased. When the light intensity reached 1000 μmol m -2 s -1 The net photosynthetic rate of the functional Ghd7 material (NIL(mh7)) was higher than that of the non-functional Ghd7 material (NIL(zs7)), and there was a significant difference between the two (see Figure 2 ), indicating that the Ghd7 gene can improve the photosynthetic efficiency of rice during the tillering stage.

[0035] The specific steps for measuring the light response curve are:

[0036] (1) Check whether the chemicals (desiccant and soda) on the right side of the instrument are available, connect and install the hardware according to the instructions (pay special attention to the correct connection of the D-type connector, which is wide at the top and narrow at the bottom; the red mark of the round plug and the red mark of the analyzer should be aligned and pushed in horizontally without leaving any gap), install the LED light source, connect the buffer bottle, and place it more than 5 meters away from people. If there is wind, place it upwind.

[0037] (2) After confirming that the instrument is connected properly and the power supply is sufficient, press the power button on the right side of the instrument to turn it on and off. After the system is running, select the 2*3 LED light source configuration on the configuration interface. After the system displays "All OK", press "Y" to confirm the connection status, then enter the main menu interface and preheat for about 20 minutes.

[0038] (3) During the warm-up period, perform daily inspections. First, press F4 (New Msmnts) to enter the measurement menu and fully tighten the two chemical tubes on the left side of the machine to the Bypass position.

[0039] ① Temperature check: Press the pgup key to jump to the third page and call up line h, check the machine air conditioning temperature, atmospheric temperature and leaf chamber temperature. The difference between these three temperature values ​​should be within 1°C. If it is out of range, the machine should be repaired before the experiment. And check the position of the leaf temperature thermocouple in the analyzer leaf chamber, and its node should be about 1mm higher than the leaf chamber gasket.

[0040] ② Check the light source, photon sensor, and atmospheric pressure sensor: Press the number key 2 to call out function row 2, then press the F5 (Lamp=) function key to set the light intensity, check whether the ParIn value on row g is consistent with the set light intensity, cover the external photon sensor with your hand, check whether the ParOut value changes, and check whether the Prss_kPa value is reasonable (generally 100kPa).

[0041] ③ Check the mixing fan in the leaf chamber: Press F5 (Lamp=), then press the letter O to turn off the light source. Then press F1 (Leaffan), then press the letter O to turn off the mixing fan in the leaf chamber. Put the analyzer next to your ear (you should not hear the fan sound at this time). Then press F1 (Leaffan), the number 5 to turn on the mixing fan in the leaf chamber. If you hear the fan sound at this time, it is working normally (its working state is generally FAST state).

[0042] ④ Check for gas line blockage: Press the home button to jump to the first page, press the number key 2 to call out the function row, press F2 (Flow), set the flow rate Target to 1000, and check whether the flow value of row b can reach above 650 (under standard atmospheric pressure). Then, completely rotate the adjustment knob of the chemical tube containing Na2CO3 from the bypass side to the scrub side, and observe that the flow value of row b should drop by less than 20. Similarly, completely rotate the adjustment knob of the chemical tube containing desiccant from the bypass side to the scrub side, and observe that the flow value of row b should drop by less than 20 (note that the knobs must be adjusted one by one).

[0043] ⑤ Flow meter zero check: turn off the air pump F2 (Flow), letter O and leaf chamber mixing fan F1 (Leaffan), and observe whether the change of Flow in line b is between ±2. If it is, it is normal; if it is not in this range, press F3 (Calib Menu) and select "Flow meter zero" mode for calibration. After completion, adjust the flow rate back to 500 and set the leaf chamber mixing fan to FAST state.

[0044] ⑥ Check for air leakage in the leaf chamber: Keep the drug tube in the scrub state, tighten the leaf chamber, blow air around the leaf chamber, and observe whether the change in the CO2 reading of row a (CO2 concentration in the sample chamber) is less than 2. If the change is large, it means that the leaf chamber is leaking.

[0045] ⑦CO2 and H2O IRGAs zero point check: Ensure that the leaf chamber is in a leak-free state, the drug tube is in a scrub state, and observe whether the CO2 reading in row a is within ±5; H2O is within ±0.5mmol. If the value fluctuates beyond this range, find the cause of interference with the zero point, such as chemical failure, leakage, etc. If the data cannot meet the requirements after stabilization, enter the calibration menu to perform IRGAszero. After completion, completely screw the drug tube to Bypass.

[0046] ⑧ Zero point check of leaf temperature thermocouple: unplug the purple plug on the IRGA analyzer (left side of the analyzer), call up line h, and check whether the difference between the air conditioning temperature (Tblock) and the leaf chamber temperature (Tleaf) is within 0.1℃. If it is greater than 0.1℃, use a flat-blade screwdriver to calibrate the temperature of the potentiometer at the bottom of the analyzer so that the leaf chamber temperature and the air conditioning temperature are equal or within 0.1℃. Turn the screwdriver clockwise to increase the leaf chamber temperature, and vice versa to reduce it. After completion, re-insert the purple plug.

[0047] ⑨ Matching valve check: Press the number key 1 to call up function row 1, then press F5 (Match) to observe whether the matching valve at the bottom of the analyzer is working properly.

[0048] (4) After ensuring that the machine is working properly, prepare to start measuring. At this time, the chemical tube is in the full Bypass position and the leaf chamber is completely closed. Press the number key 2, press F4 (Temp=), move the cursor, click "Edit" to set the Block-Target temperature to within 7°C of the ambient temperature, and press F5 (keep). Press F5 (Lamp=) again, click "Edit" to set the PAR-Target to "1200μmol·m -2 ·s -1 ”, press F5 (keep).

[0049] (5) After setting the conditions, press the number key 1, then press F1 (Open LogFile) to create a folder for data storage, set the folder name, select the storage path as Flash (CF card), press "Enter", and then add a remark, "Enter".

[0050] (6) Open the leaf chamber and clamp the plant leaves to be measured, taking care not to break the leaves.

[0051] (7) Press the number key 3, then press F1 (area) to input the actual leaf area of ​​the measured leaf in the leaf chamber. After completion, press the number key 1, return to the recording interface, and press F5 (Match) to match.

[0052] (8) Wait for the parameters of each row to stabilize. This process usually takes 2-3 minutes in outdoor field experiments. The main purpose is to wait for the CO2 fluctuation in row b to be within 0.2 μmol·mol -1 The Photo in row c is stable within one decimal place (with fluctuation of 0.5); other parameters in row c are also within the normal range (0 <Cond<1、Ci> 0, Tr>0).

[0053] (9) After stabilization, press F1 (Log) or directly press the shortcut button on the analyzer to record the data. Repeat the recording 3-5 times.

[0054] (10) After the measurement, change another blade, press F4 (Add remark), add a number, and repeat steps 7-10 to measure. Perform a Match each time you change a blade.

[0055] (11) After all data measurements are completed, press F3 (Close file) to save the data file. Set the machine to sleep mode and pack the instrument back to the laboratory.

[0056] (12) Connect the machine to the computer via a data cable and open the LI-6400XTerm software installed on the computer. Press any key to wake up the machine, then press F5 (Utility Menu), select "fileexchange mode" under communication, click connect on the computer, and import the data in the machine into the computer software. Disconnect after completion (if there is a card reader, directly remove the data card and store the data on the computer).

[0057] (13) Turn off the machine and remove the power supply to charge it for the next experiment. At the same time, turn the two chemical tube knobs to the middle loose state; rotate the leaf chamber fixing screw to keep the leaf chamber in the open state.

[0058] Example 3: Analysis of the expression of NPQ-related genes regulated by Ghd7 in transgenic lines

[0059] The present invention utilizes a light incubator to design strong light and weak light alternately to treat OX-Ghd7 ZH11 and RNAi-Ghd7 ZH11 The materials were placed in a growth incubator to detect the expression of OsPsbS1 in the materials. Rice seedlings were grown under stable light conditions (14 h light / 10 h dark, light intensity 300 μmol·m -2 ·s -1 , 30°C, 70% humidity) in an incubator until the 6-leaf stage, and then strong and weak light treatments were started. -2 ·s -1After 2 h of illumination, the first high light sample of rice leaves was taken and 120 μmol·m -2 ·s -1 2 h of illumination, take the first low light sample, and repeat 1200 μmol·m -2 ·s -1 and 120 μmol·m -2 ·s -1 Expose once to obtain the second high light sample and the second low light sample. The results show that under strong light, OX-Ghd7 ZH11 The expression level of OsPsbS1 was significantly higher than that of RNAi-Ghd7 ZH11 ( Figure 3 Figure A); The expression trend of OsbZIP72 was consistent with that of OsPsbS1, and OX-Ghd7 ZH11 Significantly higher than RNAi-Ghd7 ZH11 ( Figure 3 Figure B). This indicates that OsPsbS1 and OsbZIP72 are involved in the Ghd7 light protection regulation pathway.

[0060] The expression level is mainly detected by RT-PCR (for methods, see: J. Sambrook, EF Fritsch, T. Maniatis, translated by Huang Peitang, Wang Jiaxi, etc., Molecular Cloning Experiment Guide (3rd edition), Beijing, Science Press, 2002 edition). The specific steps are as follows:

[0061] (1) Leaf RNA was extracted using the Trizol extraction kit from Invitrogen (for specific steps, see the kit's instructions);

[0062] (2) The steps of RT-PCR reverse transcription synthesis of cDNA first strand are as follows: ① Prepare mixed solution 1: 3 μg total RNA, 2U of DNaseI, 1 μl of 10×DNaseI buffer, add DEPC (diethylpyrocarbonate, a strong inhibitor of RNase) treated water (0.01% DEPC) to 10 μl, mix well and place mixed solution 1 at 37°C for 15 minutes to remove DNA; ② Add 10 μl of EDTA and place in a 65°C water bath for 10 minutes to remove DNAse I activity, then place on ice for 5 minutes; ③ Add 1 μl of 500 μg / ml oligo(dT) and 1 μl of 10 μg / ml dNTP to mixed solution 1 and place in a 65°C water bath for 10 minutes to completely denature the RNA, then immediately place on ice for 5 minutes; ④ Prepare 7 μl of mixed solution 2: 4 μl of 5×first strand buffer, 0.1M ⑤ After the reaction is completed, the mixture is inactivated at 70℃ for 15 minutes; ⑥ The final reaction product is stored at -20℃.

[0063] The above specific embodiments describe the implementation of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the claims and technical concept of the present invention, the technical solution of the present invention can be modified and changed in many simple ways, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. Use of the Ghd7 gene in any of the following A1)-A4): A1) regulating plant photosynthetic efficiency and / or photoprotection ability; A2) preparing products for regulating plant photosynthetic efficiency and / or photoprotection ability; A3) Cultivating plant varieties with high photosynthetic efficiency and / or photoprotection ability; A4) Preparation and cultivation of plant varieties with high photosynthetic efficiency and / or photoprotection ability.

2. The use according to claim 1, characterized in that: The plant includes rice, and the nucleotide sequence of the Ghd7 gene is shown as SEQ ID NO:

1.

3. The use according to any one of claims 1-2, characterized in that: The plant exhibits all or part of the following: B1) Increased photosynthetic efficiency; B2) Enhanced photoprotection ability.

4. The use according to claim 3, characterized in that: The photosynthetic efficiency is measured during the tillering period of rice.

5. A method for enhancing plant photosynthetic efficiency and / or light protection ability, characterized in that: Overexpression of the Ghd7 gene in plants.

6. The method according to claim 5, characterized in that The plant includes rice, and the nucleotide sequence of the Ghd7 gene is shown as SEQ ID NO:

1.

7. The method according to any one of claims 5-6, characterized in that: The photosynthetic efficiency is measured during the tillering period of rice.

8. A plant breeding method, characterized in that: The expression cassette, expression vector, and recombinant microorganism for overexpressing the Ghd7 gene are introduced into a recipient plant to obtain a transgenic plant, wherein the transgenic plant exhibits all or part of the following: B1) Increased photosynthetic efficiency; B2) Enhanced photoprotection ability.

9. The method according to claim 8, characterized in that The plant includes rice, and the nucleotide sequence of the Ghd7 gene is shown as SEQ ID NO:

1.

10. The method according to any one of claims 8-9, characterized in that: The photosynthetic efficiency is measured during the tillering period of rice.

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