阅读说明：本技术 一种提高光呼吸改良植物耐旱性的方法 (Method for improving drought tolerance of plants through light respiration ) 是由 王东芳 张先文 沈志成 于 2019-08-13 设计创作，主要内容包括：本发明公开了一种提高光呼吸改良植物耐旱性的方法,所述方法为：抑制或敲除植物中胆汁酸钠协同转运蛋白基因,同时过表达乙醛酸脱氢酶基因和苹果酸合酶基因。本发明创造性的发现抑制植物中BASS6基因的表达,结合在叶绿体中过表达乙醇酸脱氢酶基因、苹果酸合酶基因,能显著减少光呼吸,提高植物的光合效率,提高植物生物量或产量,更重要的是这种转基因植物的耐旱性显著高于抑制PLGG1基因表达的植株,耐旱性提高5％-50％。(The invention discloses a method for improving drought tolerance of plants improved by light respiration, which comprises the following steps: inhibiting or knocking out sodium bile cotransporter gene in plant, and over-expressing glyoxylate dehydrogenase gene and malic acid synthase gene. The invention creatively discovers that the expression of BASS6 gene in the plant is inhibited, and the overexpression of glycollic dehydrogenase gene and malic acid synthase gene in chloroplast can obviously reduce the light respiration, improve the photosynthetic efficiency of the plant and improve the biomass or yield of the plant, more importantly, the drought tolerance of the transgenic plant is obviously higher than that of the plant inhibiting the expression of PLGG1 gene, and the drought tolerance is improved by 5-50%.)

1. A method for improving drought tolerance of plants modified by light respiration, which is characterized by comprising the following steps: inhibiting or knocking out sodium bile cotransporter gene in plant, and over-expressing glyoxylate dehydrogenase gene and malic acid synthase gene.

2. The method of claim 1, wherein the amino acid sequence of the sodium bile acid cotransporter is as set forth in one of SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3.

3. The method of claim 1, wherein the method of inhibiting a sodium bile acid cotransporter gene in a plant comprises: introducing into a plant a double-stranded RNA nucleotide sequence forming a hairpin structure of a targeted bile acid sodium cotransporter gene.

4. The method of claim 3, wherein the double stranded RNA has the nucleotide sequence set forth in SEQ ID No.4 and SEQ ID No. 5.

5. The method of claim 1 wherein the nucleotide sequence of said glycolate dehydrogenase gene is set forth in SEQ ID No. 6.

6. The method of claim 1, wherein the nucleotide sequence of the malate synthase gene is set forth in SEQ ID No. 8.

7. The method according to claim 3, wherein the method is carried out by constructing a T-DNA vector and introducing the vector into a plant; the construction method of the T-DNA vector comprises the following steps: the method is characterized in that a pCambia1300 binary vector containing a glufosinate ammonium bar resistant gene is taken as a basic vector, and a glycollic acid dehydrogenase gene expression frame, a malic acid synthase gene expression frame and a bile acid sodium cotransporter gene RNAi expression frame are respectively connected into the basic vector.

8. The method of claim 7, wherein the signal peptide for mediating overexpression of the glycolate dehydrogenase gene or the malate synthase gene in chloroplasts has the amino acid sequence shown in SEQ ID No.10 or SEQ ID No. 11.

9. The method of claim 7, wherein the promoters used to mediate overexpression of the glycolate dehydrogenase gene, the malate synthase gene in the chloroplasts comprise the maize Ubi promoter and the 35S promoter of cauliflower mosaic virus CaMV, and the terminator is ter1 or ter 2.

10. The method of claim 7, wherein the plant is rice or soybean.

(I) technical field

The invention relates to a method for improving drought resistance of plants by improving light respiration, which improves the photosynthetic efficiency, biomass or yield of the plants and plant resources by a transgenic method.

(II) background of the invention

The increase in the number of human beings and the improvement of living standard require more food and feed to be consumed, which requires more food to be harvested on a limited land. Therefore, it is very important to breed new high-yielding plant varieties.

The photosynthesis products of the whole plant are all derived from enzyme-catalyzed CO₂Is converted into organic carbon compounds. Ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) is a carboxylase in the Calvin cycle (Calvin-Benson (CB) cycle). Due to RubisCO and CO₂Or O₂All can react, RubisCO and O₂The reaction produces glycolic acid phosphate, entering the photorespiration cycle, which results in the waste of fixed carbon and nitrogen in the plant. This process releases approximately 29 GT of fresh assimilated carbon into the atmosphere each year, worldwide (Anav a, ethyl. spatial patterns of terrestrial gross production: a review. rev Geophys 2015,

53:785-818.)。

In order to reduce the loss caused by light respiration and improve the photosynthetic efficiency of plants, the common method at present is to recover CO in glycolic acid by a new light respiration branch₂thereby achieving the purpose of reducing light respiration and improving photosynthetic efficiency (Peterhansel C, Blume C, Offermann S.Photorespiratory bypass: how can the work].Journal of Experimental Botany,2013,64(3):709-715.)。

Glycolate Dehydrogenase (GDH) can convert glycolate into glyoxylate. The glycolate dehydrogenase currently used for plant transgenic research and application is mainly derived from lower plant green algae (Chlamydomonaseinharmtii) or Escherichia coli. Glycolate dehydrogenase in green algae is encoded by one gene, while glycolate dehydrogenase in large bacillus is composed of D, E, F three subunits encoded by 3 genes, respectively. It has been reported that by overexpressing fusion genes encoding genes for the D, E, F subunits in potato, DEFP fusion protein expression in plants is increased, and sugars such as glucose, fructose, and sucrose are multiplied, and biomass is also significantly increased (Nolke G, Houdel M, Kreuzaler F, et al. the expression of a recombinant glucose dehydrogenase polypeptide amino acids (Solanumtuberosum) plants are expressed in biomass and thus synthesized and tuberyeld [ J ] Plant Biotechnology Journal,2014,12(6): 734-742.). However, since there is a significant difference in the function and activity between glycolate dehydrogenases derived from Escherichia coli and those derived from green algae, there is a large difference in the expression in transgenic plants.

Malate Synthase (MS) catalyzes the conversion of acetyl-CoA and glyoxylate into malate and CoA. The malate synthase participates in the glyoxylate cycle and is widely present in different plants. It has been reported that MS overexpressing GDH from green algae and squash (C.maxima) in tobacco can increase photosynthetic efficiency and biomass (PF South, APCavanagh, HW Liu, ethyl. synthetic glycerol methyl cellulose protein yield and productivity in the field, Science,2019:363(6422): eat 9077.).

Sodium Bile cotransporter (BASS) and plastidial Glycolate/glycerate transporter 1 (PLGG 1) are key proteins in photophoresis for transporting Glycolate in chloroplasts to peroxisomes (South P F, Walker B J, Cavanagh AP, et al. Bile Acid Sodium Symporter BASS6 Can Transport Glycolate and insulin in Photobacterium Metabolism [ J ]. The Plant Cell,2017: tpc.00775.2016). The BASS and PLGG1 genes have the function of transporting glycolic acid, but the PLGG1 also has the function of transporting glycolic acid and glyceric acid. Previous studies showed that overexpression of GDH and MS genes in tobacco chloroplasts, together with suppression of the expression of PLGG1 gene, can significantly increase the biomass of tobacco (PF South, AP Cavanagh, HW Liu, equivalent. synthetic glycerol metabolism pathway protein yield and productivity in the field, Science,2019:363(6422): eat 9077.).

Drought tolerance is a very important characteristic of plants. The plants have certain drought tolerance, are favorable for resisting drought stress and are suitable for different geographical environments. Under the condition of increasingly tense water resources, the cultivation of new varieties of crops with strong drought resistance is very important.

However, we found that plants overexpressing GDH and MS genes in chloroplasts, while suppressing the expression of PLGG1 gene, had significantly reduced drought tolerance compared to controls. In contrast, plants overexpressing GDH and MS genes in the chloroplast, while suppressing expression of BASS6 gene, had significantly higher drought tolerance than plants suppressing expression of PLGG1 gene.

Disclosure of the invention

The present invention aims to provide a method for improving plant biomass or yield by reducing plant light respiration while maintaining or improving plant drought tolerance, and improving plant photosynthetic efficiency.

The technical scheme adopted by the invention is as follows:

The invention provides a method for improving drought tolerance of plants improved by light respiration, which comprises the following steps: suppressing or knocking out a bile acid sodium cotransporter (BASS) gene in a plant, and overexpressing a Glyoxylate Dehydrogenase (GDH) gene and a Malate Synthase (MS) gene.

Furthermore, the encoding gene of the bile acid sodium cotransporter is derived from plants (table 1), and the amino acid sequence of the encoding gene is shown in one of SEQ ID No.1, SEQ ID No.2 or SEQ ID No. 3. When the plant is rice, the amino acid sequence of the BASS gene is SEQ ID NO.1, and when the plant is soybean, the amino acid sequence of the BASS gene is SEQ ID NO.2 or 3.

Further, the method for inhibiting the sodium cholate cotransporter gene in the plant is an RNA interference method, and specifically, a double-stranded RNA nucleotide sequence forming a hairpin structure of the targeted sodium cholate cotransporter gene is introduced into the plant. Preferably OsBASS-RNAi and GmBASS-RNAi sequences which target rice OsBASS genes and soybean GmBASS genes to form hairpin structures, and the sequences are shown as SEQ ID NO.4 and SEQ ID NO. 5.

Further, the glycolate dehydrogenase GDH gene (Table 2) can be derived from prokaryotes or eukaryotes, including but not limited to the genes shown in Table 2, with the nucleotide sequence shown as SEQ ID NO.6 (with the amino acid sequence shown as SEQ ID NO. 7).

Further, the Malate Synthase (MS) (table 3) can be derived from prokaryotes or eukaryotes, including but not limited to the genes shown in table 3, preferably the nucleotide sequence of MS is shown as SEQ ID No.8, and the amino acid sequence thereof is shown as SEQ ID No. 9.

The method is completed by constructing a T-DNA vector and introducing the T-DNA vector into a plant; the construction method of the T-DNA vector comprises the following steps: taking a pCambia1300 binary vector containing a glufosinate-ammonium-resistant bar gene as a basic vector, and respectively connecting a glycollic acid dehydrogenase gene expression frame, a malic acid synthase gene expression frame and a bile acid sodium cotransporter gene RNAi expression frame; the pCambia1300 binary vector containing the glufosinate-ammonium-resistant bar gene is obtained by replacing the hygromycin-resistant gene hptII with the glufosinate-ammonium-resistant bar gene.

In the present invention, the BASS gene RNAi expression cassette, the GDH gene overexpression cassette, and the MS gene overexpression cassette can be realized by molecular polymerization or hybrid polymerization. The molecular polymerization is that a BASS gene RNAi expression frame, a GDH gene and an MS gene expression frame are constructed on T-DNA of the same vector, and the T-DNA is transferred into a receptor plant genome by a transgenic method, so that the expression of the BASS gene is inhibited in a target plant at the same time, and the GDH gene and the MS gene are overexpressed. The hybrid polymerization is to obtain plants containing BASS gene suppression expression frame, GDH overexpression frame and MS overexpression frame, and then cross the plants containing one or two expression frames by conventional breeding method to obtain transgenic plants containing 3 expression frames.

The signal peptide for mediating the overexpression of GDH and MS genes in chloroplasts is derived from plant RuBisCO small subunit (RbcS) or phosphoglucomutase transit peptide sequence, preferably, the amino acid sequence of the chloroplast signal peptide is shown as SEQ ID NO.10 or SEQ ID NO.11, and the chloroplast signal peptide sequence is fused at the N end of the GDH or MS protein. The promoter for mediating the overexpression of GDH and MS can be derived from eukaryote or prokaryote, can also be obtained by artificial synthesis, and can be a constitutive promoter or a specific promoter. The promoter comprises p35S (NCBI ACCESSION: MG719235 REGION:848-1628), a maize UBI promoter (NCBI ACCESSION: KR297238 REGION:4879-6876) and a rice Actin1 promoter (NCBI ACCESSION: AY452735 REGION: 2428-3797).

The terminator for mediating GDH and MS overexpression in the present invention can be derived from eukaryotes or prokaryotes, or can be obtained by artificial synthesis, and the preferred terminators are ter1(NCBI ACCESS: KJ716235REGION:3962-4158) and ter2(NCBI ACCESS: MG733984 REGION: 2092-2314).

The plant of the invention is a carbon-three plant, and is CO₂The primary products of assimilation are plants of the three-carbon compound 3-phosphoglycerate in the photosynthetic carbon cycle, including mainly rice and soybean.

The invention provides a method for improving drought tolerance of plants, which comprises the steps of carrying out RNA interference on BASS genes of the plants, and combining with overexpression of glyoxylate dehydrogenase Genes (GDH) and malate synthase genes (MS) in chloroplasts of the plants, converting glycollic acid into glyoxylate and further converting the glyoxylate into malic acid in the chloroplasts, converting the malic acid into pyruvic acid under the action of the malic enzyme in the chloroplasts of the plants, and converting the pyruvic acid into acetyl coenzyme A under the action of pyruvate dehydrogenase, thereby achieving the purposes of reducing photorespiration and improving photosynthetic efficiency and yield. RNA interference is carried out on BASS genes in rice and soybean chloroplasts, glyoxylate dehydrogenase genes and malate synthase are overexpressed, so that the yield of rice is increased by 3% -50%, the yield of soybean is increased by 3% -50%, and the drought tolerance is equivalent to or better than that of a non-transgenic control.

Table 1: bile acid sodium cotransporter (BASS) gene

Numbering	species of origin	NCBI Accession Number
			1	Arabidopsisthaliana	NP 567671
2	Zeamays	NP 001158917
			3	Sorghumbicolor	XP 021308938
4	Oryzasativa	XP015612294
			5	Glycinemax	XP 003538535/XP 003517442

Table 2: glycolate Dehydrogenase (GDH) genes from different species

Numbering	Species of origin	NCBI Accession Number
			1	Chlamydomonas reinhardtii	XP 001695381.1
2	Volvox carteri f.nagariensis	XP002946459.1
			3	Gonium pectorale	KXZ46746.1
4	Chlamydomonas eustigma	GAX77289.1
			5	Escherichia coli K-12	NP 417453.1、YP 026191.1、YP 026190.1

Table 3: malate Synthase (MS) genes from different species

Compared with the prior art, the invention has the following beneficial effects:

The invention creatively discovers that the expression of BASS6 gene in the plant is inhibited, and the combination of the overexpression of glycollic dehydrogenase Gene (GDH) and malic acid synthase gene (MS) in chloroplast can obviously reduce the light respiration, improve the photosynthetic efficiency of the plant and improve the biomass or yield of the plant, more importantly, the drought tolerance of the transgenic plant is obviously higher than that of the plant inhibiting the expression of PLGG1 gene, and the drought tolerance is improved by 3-50%.

(IV) detailed description of the preferred embodiments

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto: