Method for increasing tiller number and effective ear number of rice

Abstract

The invention belongs to the field of gene engineering, and particularly relates to a method for increasing the tiller number and the effective ear number of rice. The glycollic acid dehydrogenase gene and the malic acid synthase gene are transferred, and the PLGG1 gene is inhibited, so that the tiller number and the effective panicle number of the rice are increased.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for increasing the number of rice tillers and the number of effective panicles. Background Art

[0002] Photorespiration is the process by which plants absorb atmospheric oxygen (O 2 ) and carbon dioxide (CO 2 ) ratio increases, allowing plants to adapt to environmental changes and survive in high oxygen concentrations. It starts with the oxygenation reaction of ribulose-1,5-bisphosphate (RuBP) by ribulose-1,5-bisphosphate carboxylation / oxidase (RuBisco). At the same time, this enzyme is also a key enzyme involved in carbon fixation in photosynthesis, that is, it catalyzes the carboxylation reaction of RuBP. When the oxygen concentration is high, RuBP will react with oxygen to produce 2-phosphoglycolic acid (2-PG), which is toxic to plants. In order to alleviate the toxic effects of 2-PG on themselves, plants degrade 2-PG through photorespiration and recycle it into 3-phosphoglyceric acid (3-PGA), but this is accompanied by a large loss of carbon and energy. In C 3 If the carbon and energy losses can be reduced and the carbon and nitrogen utilization rate can be improved while removing the toxic effects of 2-PG in plants (such as rice), plant photosynthesis can be enhanced, and biomass and yield can be increased. Introducing alternative photorespiration pathways can reduce the disadvantages of photorespiration while retaining the photorespiration pathway.

[0003] Since some non-photosynthetic microorganisms can use glycolate as the sole carbon source and convert it into glycerate or malate, the glycerate pathway and the malate pathway can be introduced into C3 plants as a strategy to replace photorespiration. Among them, the first step of the glycerate alternative pathway is the dehydrogenation of glycolate into glyoxylate by glycolate dehydrogenase (GlyDH) of Escherichia coli, and the cofactor of GlyDH is an organic compound. However, the GlyDH of Escherichia coli is composed of multiple subunits, which is more complicated to implement and the effect after replacement is limited.

[0004] Chlamydomonas reinhardtii also contains a glycolate dehydrogenase (GDH) that can catalyze glycolate into glyoxylate. Under the action of this enzyme, no byproduct hydrogen peroxide is generated, and the enzyme structure is not as complex as that in Escherichia coli, and only includes one domain. Therefore, the Escherichia coli GlyDH in the malic acid substitution pathway can be replaced with GDH in Chlamydomonas reinhardtii. This strategy has been successfully introduced into tobacco and ultimately increased the biomass of tobacco in the field by about 40% (South et al., Science, 2019, 363: eaat9077). It is not clear whether this strategy can be successfully applied in rice, what technical obstacles will be encountered during application, and what traits of rice will be affected.

[0005] In order to solve the above problems, the present invention provides a method for improving rice photosynthesis by introducing a malic acid photorespiration alternative pathway. Summary of the invention

[0006] The object of the present invention is to provide a method for increasing rice yield.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention provides a method for increasing the number of rice tillers and / or the number of effective panicles, characterized in that it comprises any of the following:

[0009] (1) simultaneously expressing the proteins described in SEQ ID NO. 6 and SEQ ID NO. 8 or their encoding genes in rice, and selecting rice plants with increased tiller numbers and / or effective panicle numbers;

[0010] (2) The proteins described in SEQ ID NO. 6 and SEQ ID NO. 8 or their encoding genes are simultaneously expressed in rice, and the expression of the Os01t0511600-02 gene in the RAP-DB database is inhibited, and rice plants with increased tiller numbers and / or effective panicle numbers are selected.

[0011] In some embodiments, the method for simultaneously expressing the proteins described in SEQ ID NO.6 and SEQ ID NO.8 or their encoding genes is to deliver the CTPGDH expression cassette and the CTPMS expression cassette into rice cells;

[0012] The CTPGDH expression cassette is sequentially connected by the nucleic acid molecule of SEQ ID NO.9, the nucleic acid molecule of SEQ ID NO.10, the nucleic acid molecule of SEQ ID NO.5, and the nucleic acid molecule of SEQ ID NO.11;

[0013] The CTPMS expression box is connected in sequence by the nucleic acid molecule of the sequence shown in SEQ ID NO.12, the nucleic acid molecule of the sequence shown in SEQ ID NO.10, the nucleic acid molecule of the sequence shown in SEQ ID NO.7, and the nucleic acid molecule of the sequence shown in SEQ ID NO.13.

[0014] In some embodiments, the method for inhibiting the expression of the Os01t0511600-02 gene is a method using RNAi inhibition.

[0015] In some embodiments, the interference fragment used in the above RNAi inhibition is shown as SEQ ID NO. 15 and its complementary sequence.

[0016] In some embodiments, the above-mentioned RNAi inhibition method is to deliver an expression cassette composed of a nucleic acid molecule with a sequence as shown in SEQ ID NO.14, a nucleic acid molecule with a sequence as shown in SEQ ID NO.15, a nucleic acid molecule with a sequence as shown in SEQ ID NO.16, a nucleic acid molecule with a sequence as shown in SEQ ID NO.17, and a nucleic acid molecule with a sequence as shown in SEQ ID NO.11 into rice cells in sequence.

[0017] In some embodiments, the rice is japonica rice Kitaake.

[0018] The innovation and beneficial effects of the present invention are as follows: the present invention introduces the modified glycolate dehydrogenase gene and malate synthase gene and inhibits the PLGG1 gene, which can increase the number of rice tillers and the number of effective panicles. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Subcellular localization results of CTPGDH and CTPMS in rice protoplasts. A: CTPGDH, scale bar: 8 μm; B: CTPMS, scale bar: 5 μm.

[0020] Figure 2 Agronomic traits of transgenic plants. Error bars represent SD (n=3), *: P<0.05, **: P<0.01, t-test was used for statistical analysis. The numbers on the horizontal axis represent the plant number. DETAILED DESCRIPTION

[0021] The following definitions and methods are provided to better define this application and guide those of ordinary skill in the art in the practice of this application. Unless otherwise specified, terms are understood according to the conventional usage of those of ordinary skill in the relevant field. All patent documents, academic papers, industry standards and other public publications cited herein are incorporated herein by reference in their entirety.

[0022] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, the modifications or substitutions made to the methods, steps or conditions of the present invention are within the scope of this application. Unless otherwise specified, the examples are based on conventional experimental conditions, such as Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 2001), or the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0023] Example

[0024] Example 1 Effect of Transferring CrGDH and CmMS Genes in Rice

[0025] According to publicly available data, after the introduction of CrGDH (SEQ ID NO.2) and Cucurbita maxima (pumpkin) malate synthase (MS) (SEQ ID NO.4) into tobacco and the inhibition of the PLGG1 gene by RNAi, the biomass of tobacco was greatly increased, but the effect of increasing tobacco seed weight was not obvious (South et al., Science, 2019, 363: eaat9077). 3 For crops, increasing rice yield requires increasing seed weight. Excessive biomass will have a negative effect on yield. It is uncertain whether expressing CrGDH and CmMS genes can achieve the effect of increasing yield.

[0026] The inventors first tested the specific effects of transforming the CrGDH gene (SEQ ID NO.1) and the CmMS gene (SEQ ID NO.3) into rice. The promoter and terminator of CrGDH are Rbcs (SEQ ID NO.9) and 35S (SEQ ID NO.11), respectively, and the promoter and terminator of CmMS are Ubiquitin (SEQ ID NO.12) and NOS (SEQ ID NO.13), respectively. The OsPLGG1 RNA interference fragment selected a 300bp fragment (SEQ ID NO.15) and a complementary fragment (SEQ ID NO.17) in the exon of the Os01t0511600-02 gene in the RAP-DB database to form an interference fragment, and a Guslinker connection fragment (SEQ ID NO.16) was added between the two sequences. The promoter and terminator of the OsPLGG1 interference fragment are 35S (SEQ ID NO.14) and 35S (SEQ ID NO.11), respectively.

[0027] The three expression cassettes were constructed into two vectors, 3EdGM and 3EdGMG. 3EdGM includes the CrGDH expression cassette and the CmMS expression cassette, as well as the selection marker EPSPS gene expression cassette; 3EdGMG has an additional OsPLGG1 expression cassette compared to 3EdGM. The 3EdGM and 3EdGMG vectors were transformed into the japonica rice variety Kitaake using the Agrobacterium-mediated method to obtain rice transformed seedlings. The positive transformed seedlings were selected for breeding and further investigation of the agronomic traits of rice, including the total number of panicles, the number of effective panicles, the number of filled grains, the number of empty shells, the weight of filled grains, the weight of empty shells, and the panicle length. The results showed that the above traits of multiple lines transformed with 3EdGMG and 3EdGM vectors were not significantly different from those of the wild-type control.

[0028] The inventors also attempted to optimize the codons of CrGDH and CmMS genes to increase the usage frequency of rice preferred codons to further test their effects on rice agronomic traits. However, the results of the trait survey showed that this still did not lead to improvements in the traits.

[0029] Example 2 Modification of CrGDH and CmMS genes

[0030] The inventors tested the localization of CrGDH protein and CmMS protein in rice organelles, and the results showed that CrGDH protein was localized in mitochondria, CmMS protein was localized in peroxisomes, and the content of these two proteins in chloroplasts was very low, which may be the main reason why the yield of transgenic rice did not change. Therefore, the inventors replaced part of the sequence at the N-terminus of CrGDH protein with a chloroplast localization signal peptide, added this signal peptide to the N-terminus of CmMS protein, and added an actin untranslated region before the coding sequence of this signal peptide to enhance protein expression.

[0031] The inventors first tested the expression of the modified CrGDH and CmMS proteins in rice chloroplasts. Using the protoplasts of rice material Zhonghua 11 that had germinated for 12 days, the constructed transient expression vectors pCAMBIA1301GFP-CTPGDH and pCAMBIA1301GFP-CTPMS were transferred into the rice protoplasts by the PEG method. After incubation for 12 hours, they were aspirated onto a glass slide and observed under a confocal microscope. The results of the confocal microscope showed that CTPGDH (SEQ ID NO.6) and CTPMS (SEQ ID NO.8) could be successfully localized in rice chloroplasts ( Figure 1 ).

[0032] The results showed that the transgenic rice obtained by transforming 3EdGMG and 3EdGM vectors had no significant difference in panicle length, number of grains per panicle, thousand-grain weight, and fruit setting rate compared with the control, but the aboveground dry weight, yield per plant, number of tillers, and number of effective panicles were significantly improved compared with the control ( Figure 2 ), which shows that the increase in the yield per plant of transgenic plants is due to the increase in the number of tillers and the number of effective panicles. In addition, the rice transformed with the 3EdGMG vector is slightly better than the rice transformed with the 3EdGM vector in the above traits. It may be that the interference with OsPLGG1 limits the large amount of glycolic acid in the chloroplasts from entering the peroxisomes, thereby further reducing the endogenous photorespiration pathway and increasing the role of the alternative pathway, so that 2-PG is recycled into 3-PGA faster, and the carbon cycle is completed faster in the chloroplasts. Therefore, the transformation of 3EdGMG and 3EdGM vectors can achieve the technical effect of increasing the number of rice tillers and the number of effective panicles, and ultimately increasing the yield per plant.

[0033] The structure of the CTPGDH expression cassette is Rbcs promoter (SEQ ID NO.9)-UTR (SEQ ID NO.10)-CTPGDH gene (SEQ ID NO.5)-35S terminator (SEQ ID NO.11); the structure of the CTPMS expression cassette is Ubiquitin promoter (SEQ ID NO.12)-UTR (SEQ ID NO.10)-CTPMS gene (SEQ ID NO.7)-NOS terminator (SEQ ID NO.13); the structure of the OsPLGG1 RNAi expression cassette is 35S promoter (SEQ ID NO.14)-interference fragment (SEQ ID NO.15)-connection fragment (SEQ ID NO.16)-complementary fragment (SEQ ID NO.17)-35S terminator (SEQ ID NO.11).

[0034] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements may be made thereto based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A method for increasing the number of rice tillers and / or effective panicles, characterized in that: Includes any of the following: (1) simultaneously expressing the proteins described in SEQ ID NO. 6 and SEQ ID NO. 8 or their encoding genes in rice, and selecting rice plants with increased tiller numbers and / or effective panicle numbers; (2) The proteins described in SEQ ID NO. 6 and SEQ ID NO. 8 or their encoding genes are simultaneously expressed in rice, and the expression of the Os01t0511600-02 gene in the RAP-DB database is inhibited, and rice plants with increased tiller numbers and / or effective panicle numbers are selected.

2. The method according to claim 1, characterized in that: The method for simultaneously expressing the proteins described in SEQ ID NO.6 and SEQ ID NO.8 or their encoding genes is to deliver the CTPGDH expression cassette and the CTPMS expression cassette into rice cells; The CTPGDH expression cassette is sequentially connected by the nucleic acid molecule of SEQ ID NO.9, the nucleic acid molecule of SEQ ID NO.10, the nucleic acid molecule of SEQ ID NO.5, and the nucleic acid molecule of SEQ ID NO.11; The CTPMS expression box is connected in sequence by the nucleic acid molecule of the sequence shown in SEQ ID NO.12, the nucleic acid molecule of the sequence shown in SEQ ID NO.10, the nucleic acid molecule of the sequence shown in SEQ ID NO.7, and the nucleic acid molecule of the sequence shown in SEQ ID NO.

13.

3. The method according to claim 1, characterized in that: The method for inhibiting the expression of the Os01t0511600-02 gene is a method using RNAi inhibition.

4. The method according to claim 3, characterized in that: The interference fragment used in the RNAi inhibition is shown in SEQ ID NO. 15 and its complementary sequence.

5. The method according to claim 4, characterized in that: The RNAi inhibition method is to deliver an expression cassette composed of a nucleic acid molecule with a sequence shown in SEQ ID NO.14, a nucleic acid molecule with a sequence shown in SEQ ID NO.15, a nucleic acid molecule with a sequence shown in SEQ ID NO.16, a nucleic acid molecule with a sequence shown in SEQ ID NO.17, and a nucleic acid molecule with a sequence shown in SEQ ID NO.11 to a rice cell in sequence.

6. The method according to any one of claims 1 to 5, characterized in that: The rice is Kitaake japonica rice.

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