Use of genes to enhance photosynthetic efficiency in rice

Abstract

The present invention relates to a gene for improving photosynthetic efficiency in rice and a method for using the same, which belongs to the field of genetic breeding of crops. The present invention discloses the use of a gene for improving photosynthetic efficiency in rice, i.e., overexpressing the Os07g0101400 gene in rice to increase the photosynthetic efficiency of rice leaves and increase the yield, and has potential application value in high-yield rice breeding. The base sequence of the Os07g0101400 gene is shown in SEQ ID NO: 1.

Description

[Technical Field]

[0001] The present invention relates to a gene that enhances photosynthetic efficiency in rice and a method for using the same, and belongs to the field of genetic breeding of crops. [Background technology]

[0002] Rice is an important food crop, and high-yield breeding is an effective guarantee for ensuring China's food security and sustainable agricultural development. After rice emergence, the efficiency of photosynthetic compound accumulation in functional leaves (the flag leaf, the second leaf from the top, and the third leaf from the top) and the duration of high light efficiency play a crucial role in grain yield. Research has shown that 60% to 80% of the nutrients required for rice grain ripening are obtained through photosynthesis in functional leaves after heading. Photosynthesis utilizes chlorophyll in chloroplasts to convert carbon dioxide and water into organic matter under visible light irradiation. Research has shown that chlorophyll content is positively correlated with photosynthetic rate within a certain range (Sarkar et al., 1998). During leaf senescence, photosynthetic rate decreases due to chlorophyll degradation; however, if leaves can maintain a relatively high chlorophyll content for a long period of time, they can absorb more light energy, thereby promoting biomass synthesis (Liu et al., 2016). For example, natural mutations in rice SNU-SG1 significantly increase chlorophyll content, photosynthetic rate, and light conversion efficiency, leading to increased dry matter yield during the grain filling stage and increased rice yield (Fu et al., 2008; 2011). The chlorophyll b content and photosynthetic rate of the dark green rice Gc mutant leaf increased significantly throughout the growing season, resulting in a 17% and 16% increase in plant biomass and grain yield, respectively (Wang et al., 2008). The rice OsNAP gene encodes a NAC transcriptional activator. OsNAP directly targets genes involved in chlorophyll degradation, senescence, and nutrient transport, positively regulating leaf senescence. Transgenic rice plants expressing OsNAP have a relative increase in chlorophyll content, delayed senescence, and increased grain yield by 6.3–10.3%. It is clear that one effective way to increase rice yields is to thoroughly search for new genes that have high photosynthetic efficiency in plants and promote the efficient use of light energy in crops.

[0003] Although the sequence of the Os07g0101400 gene has been published in the rice database (https: / / www.ricedata.cn / gene / ), the function of this gene has not been reported. Summary of the Invention [Problem to be solved by the invention]

[0004] The technical problem that the present invention aims to solve is how to effectively improve the photosynthetic efficiency of rice plants. [Means for solving the problem]

[0005] To solve the above technical problems, the present invention provides a gene that can effectively improve rice photosynthesis and a method for using the gene, i.e., overexpression of the Os07g0101400 gene in rice can effectively increase the photosynthetic efficiency of rice leaves. The nucleotide sequence of this gene is shown in SEQ ID No: 1.

[0006] The present invention also provides a method for improving photosynthesis in rice leaves, i.e., a method for transforming rice (Nipponbare) with an Os07g0101400 gene overexpression vector to obtain genetically modified rice, thereby increasing the photosynthetic efficiency of rice leaves.

[0007] In this study, we PCR-amplified the Os07g0101400 gene (SEQ ID No. 1) from rice cDNA, constructed an overexpression vector for this gene, and genetically transformed it into the wild-type rice cultivar Nipponbare using Agrobacterium-mediated transformation. Two overexpression lines, OE1 and OE2, were identified, and RT-qPCR demonstrated that the expression levels of Os07g0101400 in these two lines were significantly higher than those of the wild-type control Nipponbare (Figure 1). The leaf chlorophyll content (Figure 2) and photosynthetic efficiency (Figure 3) of these two overexpression lines were significantly higher than those of the wild-type control Nipponbare, indicating that increasing the expression level of the Os07g0101400 gene in rice can effectively improve photosynthesis, thereby increasing yield and potentially being useful for high-yield rice breeding. [Effects of the Invention]

[0008] The present invention discloses for the first time that the Os07g0101400 gene has the effect of enhancing photosynthetic efficiency. [Brief explanation of the drawings]

[0009] In Figures 1 to 3 below, WT is the wild-type control Nipponbare, and OE1 and OE2 are the two overexpression lines of the Os07g0101400 gene, respectively. ** indicates a highly significant difference (P<0.01) in the t-test. [Figure 1] Expression analysis of the rice Os07g0101400 gene is shown. [Figure 2] The photosynthetic pigment content in leaves of rice Os07g0101400 gene overexpression lines and their wild-type controls is shown. [Figure 3] The photosynthetic efficiency of leaves of rice Os07g0101400 gene overexpression lines and their wild-type controls is shown. [Figure 4] FIG. 1 is a diagram of the Os07g0101400 gene overexpression vector. DETAILED DESCRIPTION OF THE INVENTION

[0010] Specific embodiments of the present invention will be described in more detail below with reference to the drawings.

[0011] (Step 1. Rice total RNA extraction and cDNA synthesis) Total RNA was extracted from leaves of wild-type rice cultivar Nipponbare using the RNeasy Plant Mini Kit (QIAGEN) according to the manufacturer's instructions, and analyzed using PrimeScript. TM Reverse transcribe into cDNA using the 1st Strand cDNA Synthesis Kit (TaKaRa) according to the manufacturer's instructions.

[0012] (Step 2. PCR amplification of the Os07g0101400 gene) Synthesize upstream and downstream PCR primers. F1: 5'-ggggacaagtttgtacaaaaaagcaggctccATGACGGCGGCGGCGTCGTGGT-3'; R1: 5'-ggggaccactttgtacaagaaagctgggtcCTAAACCCTCCTGCGGATGCGCC-3'. The sequence in uppercase letters is the specific sequence of the Os07g0101400 gene, and its amplification allows the full-length coding region sequence of the gene to be obtained. The sequence in lowercase letters is the adapter sequence of the entry vector constructed using Gateway technology.

[0013] Using the cDNA obtained in step 1 as a template, PCR amplify the Os07g0101400 gene using PrimeSTAR® HS DNA Polymerase (TaKaRa). The reaction system was configured according to the manufacturer's instructions. Specifically, the reaction mixture was as follows: 10 μl 5x PrimeSTAR Buffer, 4 μl dNTP Mixture (2.5 mM each), 2 μl upstream and downstream F1 and R1 primers (10 μM each), 1 μl template cDNA (<200 ng), 0.5 μl PrimeSTAR HS DNA Polymerase, and ddH2O to a volume of 50 μl.

[0014] The PCR amplification program was as follows: pre-denaturation at 95°C for 5 minutes, denaturation at 98°C for 10 seconds, annealing at 65°C for 15 seconds, extension at 72°C for 90 seconds, repeated 30 times, and extension at 72°C for 5 minutes. After the reaction was completed, 2 μl of the PCR product was sampled and detected by 1% agarose gel electrophoresis.

[0015] (Step 3. Construction of Os07g0101400 gene overexpression vector (Figure 4)) (1) Use the AxyPrep PCR Clean-up Kit (Axygen) to purify the PCR product obtained in step 2. Follow the product instructions to obtain the purified target fragment (SEQ ID NO: 1).

[0016] (2) Invitrogen Gateway TM BP Clonase TM The purified target fragment is inserted into the entry vector using the II Enzyme Mix Kit. The reaction system is as follows: 1 μl purified PCR product (50-80 ng / μl), 1 μl entry vector plasmid pDONR / Zeo (100-150 ng / μl), 0.5 μl BP Clonase. TM II enzyme; after incubation at 25°C for 1 hour, transform E. coli DH5α using the conventional heat shock method for 1 hour.

[0017] (3) Several clonal colonies were collected and cultured overnight in a shaker at 37°C and 220 rpm. 2x Taq PCR premix reagent (Tiangen) was used for PCR verification of the bacterial suspension. The primer sequences for PCR amplification were as follows: F2: 5'-ATGACGGCGGCGGCGTCGTGGT-3'; R2: 5'-CTAACCCTCCTGCGGATGCGCC-3'.

[0018] The PCR reaction system consisted of 1 μl of bacterial suspension, 10 μl of 2x Taq PCR MasterMix II, 1 μl of F2+R2 primers (10 μM each), and ddH2O to a total volume of 20 μl. The PCR amplification program consisted of 35 cycles of pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30 seconds, annealing at 65°C for 30 seconds, and extension at 72°C for 30 seconds, followed by extension at 72°C for 5 minutes. After the reaction, 5 μl of the reaction product was sampled and subjected to 1% agarose gel electrophoresis to identify positive clones.

[0019] (4) For positive clones, extract the plasmid using the Rapid Mini Plasmid Kit (Tiangen) to obtain the entry vector plasmid containing the target fragment. Take 1 μl of this plasmid (100-150 ng / μl), add 1 μl of pCAMBIA2300-Actin final vector plasmid (approximately 100-150 ng / μl) and 0.5 μl of LR Clonase II enzyme, incubate at 25°C for 1 hour, and then transform E. coli DH5α using the conventional heat shock method.

[0020] (5) Multiple clonal colonies were collected and cultured overnight in a shaker at 37°C and 220 rpm. 2x Taq PCR premix reagent (Tiangen) was used for PCR verification of the bacterial suspension. The method for identifying positive clones was the same as in (3) above.

[0021] (6) For positive clones (i.e., colonies from which the target fragment can be amplified by PCR), the plasmid was extracted using the Rapid Mini Plasmid Kit (Tiangen) and sent to a biotechnology company for sequencing. Primers F3: 5'-CCCTCAGCATTGTTCATCG-3' and R3: 5'-TAGGCGTCTCGCATATCTCA-3' were used to perform bidirectional sequencing to identify the sequence of the inserted fragment, ultimately obtaining the Os07g0101400 gene overexpression vector (Figure 4). The Os07g0101400 gene overexpression vector contains the nucleotide sequence shown in SEQ ID NO: 1.

[0022] (Step 4. Genetic transformation of rice with the Os07g0101400 gene overexpression vector) Use the Os07g0101400 gene overexpression vector constructed in step 3 to transform the wild-type rice cultivar Nipponbare using the method described by Nishimura et al. (Nishimura et al., Nat Protoc, 2006) to obtain transgenic rice plants.

[0023] Step 5. Identification of transgenic plants overexpressing the Os07g0101400 gene Following the method in step 1, extract total RNA from the leaves of the transformed plants obtained in step 4 and synthesize cDNA. The expression level of the Os07g0101400 gene is measured using conventional RT-qPCR method.

[0024] The Os07g0101400 gene-specific primers F4: 5'-ATGCCCGTCCCTTGCTATCTGA-3' and R4: 5'-ATTCGCTCCTTCGTTACCACCG-3' were used. The rice actin gene was used as an internal reference, and the PCR primers were F5: 5'-TGGCATCTCTCAGCACATTCC-3' and R5: 5'-TGCACAATGGATGGGTCAGA-3' (Chen et al., Rice, 2013). SYBR® Premix Ex Taq (TaKaRa) was used. TM The reaction system used was the following: 10 μl SYBR® Premix Ex Taq® II, 2 μl cDNA template, 1 μl upstream and downstream F4 and R4 primers (10 μM each), 0.4 μl ROX reference dye, and ddH2O to 20 μl. The PCR program was 40 cycles of pre-denaturation at 95°C for 30 seconds, denaturation at 95°C for 5 seconds, annealing and extension at 65°C for 30 seconds. Each sample was repeated three times. -ΔΔCT The data were processed using the method (Livak et al., 2001) and significant differences were analyzed using the t-test. In the transgenic rice plants, the expression levels of the Os07g0101400 gene were found to be 20-fold and 15-fold higher, respectively, than in the wild-type Nipponbare lines (Figure 1). In this way, plants overexpressing the Os07g0101400 gene were obtained.

[0025] Step 6. Measurement of photosynthetic pigment content in leaves of Os07g0101400 gene-overexpressing plants The Os07g0101400 gene-overexpressing lines OE1 and OE2 identified in step 5 and wild-type Nipponbare were planted in paddy fields, with each plant spaced 30 cm x 15 cm apart. At the tillering stage, six plants were randomly selected from each variety. Fully expanded flag leaves were removed from each plant, the major veins were removed, and the leaves were cut into small pieces. 0.1 g of each leaf was weighed and immersed in 3 ml of 80% (v / w) acetone for 48 hours at 28°C in the dark. The chlorophyll a (Chla), chlorophyll b (Chlb), and carotenoid (Car) contents were measured according to the method of Amon (1949). Significant differences were analyzed using a t-test. The results showed that the chlorophyll a, chlorophyll b, and carotenoid contents of the Os07g0101400 gene-expressing lines OE1 and OE2 were higher than those of the wild-type Nipponbare (Figure 2).

[0026] Step 7. Measurement of photosynthetic efficiency in leaves of Os07g0101400 gene-overexpressing plants Plant according to the method in step 6. At the tillering stage, select six plants at random from each variety and measure the flag leaf photosynthetic efficiency parameters: net photosynthetic rate, intercellular CO2 concentration, stomatal conductance, and transpiration rate using a Li-6400 (Li-COR, USA) photosynthesis meter. The measurement method should follow the general instructions for use with the instrument. Significant differences will be analyzed using a t-test. The results show that the net photosynthetic rate, intercellular CO2 concentration, stomatal conductance, and transpiration rate of the Os07g0101400 gene overexpression lines OE1 and OE2 are significantly higher than those of the wild-type Nipponbare (Figure 3). Finally, it should be noted that the above lists are only some specific embodiments of the present invention. It goes without saying that the present invention is not limited to the above embodiments, and various modifications are possible. All modifications that a person skilled in the art can directly derive or associate with the disclosure of the present invention should be considered within the protection scope of the present invention.

Claims

[Claim 1] A method for increasing photosynthetic efficiency of rice leaves by overexpressing the Os07g0101400 gene in rice, comprising: The nucleotide sequence of the Os07g0101400 gene is shown in SEQ ID NO: 1, A method for increasing the photosynthetic efficiency of rice leaves by constructing an overexpression vector for the Os07g0101400 gene, genetically transforming rice using Agrobacterium-mediated transformation, and obtaining genetically modified rice.

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