WO2022257601A1

WO2022257601A1 - Use of gene for improving photosynthesis efficiency of rice

Info

Publication number: WO2022257601A1
Application number: PCT/CN2022/087090
Authority: WO
Inventors: 陈析丰; 许以灵; 陆喆晓; 马季荣; 马伯军
Original assignee: 浙江师范大学
Priority date: 2021-06-08
Filing date: 2022-04-15
Publication date: 2022-12-15
Also published as: CN113265420A; CN113265420B

Abstract

The present invention relates to the field of crop genetic breeding and relates to a gene for improving the photosynthesis efficiency of rice and a use method thereof. Disclosed in the present invention is a use of a gene for improving the photosynthesis efficiency of rice: Os07g0101400 gene is over-expressed in rice, such that the photosynthesis efficiency of rice leaves is improved, the yield is increased, the gene has potential application value in rice high-yield breeding, and the nucleotide sequence of the Os07g0101400 gene is shown as SEQ ID NO: 1.

Description

Use of Genes to Improve Photosynthetic Efficiency in Rice

technical field

The invention relates to a gene for increasing photosynthetic efficiency of rice and its application method, belonging to the field of crop genetics and breeding.

Background technique

Rice is an important food crop, and high-yield breeding is an effective guarantee to ensure food security and sustainable agricultural development in my country. After rice heading, the accumulation efficiency of photosynthetic compounds in the functional leaves (flap leaf, inverted second leaf, and inverted third leaf) and the duration of high light efficiency period have a decisive effect on grain yield. Studies have shown that 60% of the nutrients required for rice grain filling ~80% comes from photosynthesis of functional leaves after heading. Photosynthesis is the use of chlorophyll (Chlorophyll) in chloroplasts to convert carbon dioxide and water into organic matter under the irradiation of visible light. Studies have shown that chlorophyll content is positively correlated with photosynthetic rate within a certain range (Sarkar et al, 1998). During the aging process of leaves, the photosynthetic rate decreases due to the degradation of chlorophyll. If the leaves can maintain a relatively high chlorophyll content for a long time, it is possible to absorb more light energy, thereby promoting the synthesis of biomass (Liu et al. 2016). For example, the natural variation of SNU-SG1 in rice can significantly increase chlorophyll content, photosynthetic rate, and photoconversion efficiency, and can increase dry matter production at the filling stage to increase rice yield (Fu et al, 2008; 2011). The chlorophyll b content and photosynthetic rate of the leaves of the dark green Gc mutant of rice increased significantly throughout the growth period, which increased the biomass and grain yield of the plant by 17% and 16% respectively (Wang et al, 2008); the rice OsNAP gene encodes A NAC transcriptional activator, OsNAP directly targets genes related to regulation of chlorophyll degradation, senescence and nutrient transport, positively regulates leaf senescence, and inhibits the relative increase of chlorophyll content in leaves of transgenic rice plants expressing OsNAP gene, delays senescence, and increases grain yield by 6.3 ~10.3% (Liang et al, 2014). It can be seen that fully exploring the new genes of high photosynthetic efficiency in plants and promoting the efficient use of light energy in crops is one of the effective ways to increase rice yield.

The sequence of the Os07g0101400 gene has been published in the rice database (https://www.ricedata.cn/gene/), but the function of the gene has not been reported.

Contents of the invention

The technical problem to be solved by the invention is how to effectively improve the photosynthesis efficiency of rice.

In order to solve the above technical problems, the present invention provides a gene that can effectively improve the photosynthesis of rice and its use method: overexpressing the Os07g0101400 gene in rice can effectively increase the photosynthesis efficiency of rice leaves; the nucleotide sequence of the gene is as follows: Shown in SEQ ID NO: 1.

The invention also provides a method for improving photosynthesis of rice leaves: transforming the Os07g0101400 gene overexpression vector into rice (Nipponbare) to obtain transgenic rice plants, thereby increasing the photosynthesis efficiency of rice leaves.

The present invention PCR-amplifies the Os07g0101400 gene (SEQ ID NO: 1) from rice cDNA, constructs an overexpression vector of the gene, and uses the Agrobacterium-mediated method to genetically transform the vector into the wild-type rice variety Nipponbare, and identifies Two overexpression lines OE1 and OE2 were obtained, and RT-qPCR was used to prove that the expression level of Os07g0101400 gene in these two lines was significantly higher than that of the wild-type control Nipponbare (Figure 1). The chlorophyll content (Figure 2) and photosynthetic efficiency (Figure 3) of the leaves 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 the photosynthesis of rice leaves Efficiency, thereby increasing yield, has potential application value in high-yield breeding of rice.

The present invention discloses for the first time that the Os07g0101400 gene has the effect of promoting photosynthesis efficiency.

Description of drawings

The specific implementation manners of the present invention will be described in further detail below in conjunction with the accompanying drawings.

Figure 1 shows the expression level analysis of rice Os07g0101400 gene.

Figure 2 shows the leaf photosynthetic pigment content of the rice Os07g0101400 gene overexpression line and its wild-type control.

Fig. 3 shows the leaf photosynthesis efficiency of the rice Os07g0101400 gene overexpression line and its wild-type control.

In Figures 1 to 3, WT is the wild-type control Nipponbare; OE1 and OE2 are two overexpression lines of the Os07g0101400 gene, respectively. ** indicates that there is a very significant (P<0.01) difference by t test.

Fig. 4 is a map of Os07g0101400 gene overexpression vector.

Detailed ways

Step 1, rice total RNA extraction and cDNA synthesis

RNeasy Plant Mini Kit (QIAGEN) was used to extract total RNA from leaves of rice wild-type variety Nipponbare according to the product instructions; and PrimeScript ^TM 1st Strand cDNA Synthesis Kit (TaKaRa) was used to reverse transcribe into cDNA, and the method was operated according to the product instructions.

Step 2, PCR amplification of Os07g0101400 gene

Synthesize upstream and downstream PCR primers:

F1: 5'-ggggacaagtttgtacaaaaaagcaggctccATGACGGCGGCGGCGTCGTGGT-3';

R1: 5'-ggggaccactttgtacaagaaagctgggtcCTAAACCCTCCTGCGGATGCGCC-3'.

Among them, the uppercase sequence is the specific sequence of the Os07g0101400 gene, which can be amplified to obtain the full-length coding region sequence of the gene; the lowercase sequence is the linker sequence for the construction of the entry vector using Gateway technology.

Using the cDNA obtained in step 1 as a template, adopt

HS DNA Polymerase (TaKaRa), PCR amplified Os07g0101400 gene, the configuration of the reaction system was operated according to the product instructions, specifically: 10 μl 5× PrimeSTAR Buffer, 4ul dNTP Mixture (each 2.5mM), 2 μl upstream and downstream F1 and R1 primers (each 10 μM), 1 μl template cDNA (<200ng), 0.5 μl PrimeSTAR HS DNA Polymerase, and ddH ₂ O to make up to 50 μl.

The PCR amplification program was: pre-denaturation at 95°C for 5 min; denaturation at 98°C for 10 sec, annealing at 65°C for 15 sec, extension at 72°C for 90 sec, 30 cycles; extension at 72°C for 5 min. After the reaction, 2 μl of the PCR product was taken and detected by 1% agarose gel electrophoresis.

Step 3, construction of Os07g0101400 gene overexpression vector (Fig. 4)

(1) Purify the PCR product obtained in step 2 with the AxyPrep PCR Cleaning Kit (Axygen), the method is operated according to the product instructions, and the purified target fragment (SEQ ID NO: 1) is obtained;

(2 ^{) Use Invitrogen’s Gateway BP Clonase TM} ^II Enzyme mix kit to construct the purified fragment of interest on the entry vector. The reaction system is: 1 μl of the purified PCR product (50-80 ng/μl), 1 μl of the entry vector Plasmid pDONR/Zeo (100-150ng/μl), 0.5μl BP Clonase ^TM II enzyme; after incubation at 25°C for 1 hour, transform Escherichia coli DH5α by conventional heat shock method.

(3) Multiple clonal colonies were picked and cultivated overnight at 37° C. on a shaker at 220 rpm. Use 2×Taq PCR premix reagent (Tiangen) to verify the bacterial liquid by PCR. The primer sequences for PCR amplification are:

F2: 5'-ATGACGGCGGCGGCGTCGTGGT-3';

R2: 5'-CTAAACCCTCCTGCGGATGCGCC-3'.

The PCR reaction system was: 1 μl bacterial solution, 10 μl 2×Taq PCR MasterMix II, 1 μF2+R2 primers (10 μM each), and ddH ₂ O to make up to 20 μl. The PCR amplification program was: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 sec, annealing at 65°C for 30 sec, extension at 72°C for 30 sec, 35 cycles; extension at 72°C for 5 min. After the reaction, 5 μl of the reaction product was taken and detected by 1% agarose gel electrophoresis to identify positive clones.

(4) For the positive clone bacterial liquid, use the rapid plasmid extraction kit (Tiangen) to carry out plasmid extraction to obtain the entry vector plasmid containing the target fragment, take 1 μl of the plasmid (100-150ng/μl), and add pCAMBIA2300-Actin 1 μl of final vector plasmid (about 100-150ng/μl), 0.5 μl of LR Clonase ^TM II enzyme; after incubation at 25°C for 1 hour, transform Escherichia coli DH5α by conventional heat shock method.

(5) Multiple clonal colonies were picked and cultured overnight at 37° C., 220 rpm on a shaker. Use 2×Taq PCR premix reagent (Tiangen) to verify the bacterial liquid by PCR. The method is the same as above (3), and positive clones are identified.

(6) For the positive clone bacterial liquid (that is, the colony where the target fragment can be amplified by PCR), the plasmid is extracted by using the rapid plasmid mini-extraction kit (Tiangen), and sent to a biotechnology company for sequencing, using primer F3: 5' -CCCTCAGCATTGTTCATCG-3' and R3:5'-TAGGCGTCTCGCATATCTCA-3' were bidirectionally sequenced to identify the sequence of the inserted fragment, and finally an overexpression vector of the Os07g0101400 gene was obtained ( FIG. 4 ). That is, the Os07g0101400 gene overexpression vector contains the nucleotide sequence shown in SEQ ID NO: 1.

Step 4, rice genetic transformation of Os07g0101400 gene overexpression vector

The Os07g0101400 gene overexpression vector constructed in step 3 was transformed into the wild-type rice variety Nipponbare by the method of Nishimura et al. (Nishimura et al, Nat Protoc, 2006) to obtain transgenic rice plants.

Step 5, identification of Os07g0101400 gene overexpression transgenic plants

According to the method of step 1, the total RNA of the leaves of the transgenic plants obtained in step 4 was extracted, and cDNA was synthesized. The expression level of Os07g0101400 gene was detected by conventional RT-qPCR method:

Os07g0101400 gene-specific primers were used: F4: 5'-ATGCCCGTCCCTGCTATCTGA-3' and R4: 5'-ATTCGCTCCTTCGTTACCACCG-3'. The rice Actin gene was used as an internal reference, and the PCR primers were F5: 5'-TGGCATCTCTCAGCACATTCC-3', R5: 5'-TGCACAATGGATGGGTCAGA-3' (Chen et al., Rice, 2013). Using TaKaRa's

Premix Ex Taq ^TM II kit, reaction system: 10μl

Premix Ex

II. 2 μl cDNA template, 1 μl upstream and downstream F4 and R4 primers (10 μM each), 0.4 μl ROX Reference Dye, and ddH ₂ O to make up to 20 μl. The PCR program was: pre-denaturation at 95°C for 30 sec; denaturation at 95°C for 5 sec, annealing and extension at 65°C for 30 sec, 40 cycles. Each sample was replicated three times, and the data were processed by the 2 ^-ΔΔCT method (Livak et al., 2001), and the significant difference was analyzed by the t-test method. In the transgenic rice, it was found that the Os07g0101400 gene expression levels of the OE1 and OE2 lines were 20 times and 15 times higher than that of the wild-type Nipponbare (Figure 1), thus obtaining the overexpression plants of the Os07g0101400 gene.

Step 6, determination of photosynthetic pigment content in Os07g0101400 gene overexpression plant leaves

The Os07g0101400 gene overexpression lines OE1 and OE2 identified in step 5) and wild-type Nipponbare were planted in a rice field, and each rice plant was planted at a distance of 30 cm×15 cm. At the tillering stage of rice, randomly select 6 plants for each variety, take a fully expanded flag leaf from each plant, remove the main vein, cut into small pieces, take 0.1g and soak in 3ml 80% (v/w) acetone, 28 °C dark extraction for 48 hours. The contents of chlorophyll a (Chl a), chlorophyll b (Chl b) and carotenoid (Car) were determined according to the method of Amon (1949). The t-test method was used to analyze the significant difference. The result obtained was that the chlorophyll a, chlorophyll b, and carotenoid contents of the Os07g0101400 gene expression lines OE1 and OE2 were significantly higher than those of the wild type Nipponbare (Fig. 2).

Step 7, Determination of Photosynthetic Efficiency of Os07g0101400 Gene Overexpression Plant Leaves

Plant according to the method of step 6. At the tillering stage of rice, 6 plants of each variety are randomly selected, and the photosynthetic efficiency parameters of its flag leaves are measured with a Li-6400 (Li-COR, USA) photosynthetic measuring instrument: net photosynthetic rate, intercellular CO ₂ concentration, stomatal conductance and transpiration rate. The determination method is in accordance with the routine instructions of the instrument. The t-test method was used to analyze the significant difference. The results obtained showed that the net photosynthetic rate, intercellular CO ₂ concentration, stomatal conductance and transpiration rate of the Os07g0101400 gene overexpression lines OE1 and OE2 were significantly higher than those of the wild type Nipponbare (Figure 3).

Finally, it should be noted that the above examples are only some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments, and many variations are possible. All deformations that can be directly derived or associated by those skilled in the art from the content disclosed in the present invention should be considered as the protection scope of the present invention.

Claims

The gene application for improving the photosynthetic efficiency of rice is characterized in that: Os07g0101400 gene is overexpressed in rice, thereby increasing the photosynthetic efficiency of rice leaves; the nucleotide sequence of the Os07g0101400 gene is shown in SEQ ID NO:1.
The method for improving the photosynthesis of rice leaves is characterized in that: the Os07g0101400 gene overexpression vector is transformed into rice to obtain transgenic rice plants, thereby increasing the photosynthesis efficiency of rice leaves; the nucleotide sequence of the Os07g0101400 gene is shown in SEQ ID NO: 1 .