CN110564760B

CN110564760B - Method for improving drought tolerance of plants through light respiration

Info

Publication number: CN110564760B
Application number: CN201910744576.1A
Authority: CN
Inventors: 王东芳; 张先文; 沈志成
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2019-08-13
Filing date: 2019-08-13
Publication date: 2021-12-21
Anticipated expiration: 2039-08-13
Also published as: CN110564760A

Abstract

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 the drought tolerance of plants by photorespiration. The method comprises the steps of inhibiting or knocking out a sodium bile co-transporter gene in a plant, and simultaneously overexpressing a glyoxylate dehydrogenase gene and a malate synthase. enzyme gene. The invention creatively finds that inhibiting the expression of BASS6 gene in plants, combined with overexpression of glycolate dehydrogenase gene and malate synthase gene in chloroplast, can significantly reduce photorespiration, improve plant photosynthetic efficiency, and increase plant biomass or yield , and more importantly, the drought tolerance of this transgenic plant was significantly higher than that of the plants with suppressed PLGG1 gene expression, and the drought tolerance was increased by 5%-50%.

Description

Method for improving drought tolerance of plants through light respiration

(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. On a global scale, this process will be about 29GT fresh each yearThe assimilating carbons were released back into the atmosphere (Anav A, ethyl. spatial porous patterns of aqueous gross primary production: a review. Rev Geophys 2015,53: 785-.

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 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 in protein fertilizer to Plant and tube yield [ J ] Plant Biotechnology Journal,2014,12(6): 734-. 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 the overexpression of GDH from green algae and the MS from pumpkin (C.maxima) in tobacco can increase the photosynthetic efficiency and biomass (PF South, AP Cavanagh, HW Liu, equivalent. synthetic glycerol metabolism pathway protein yield and productivity in the field, Science,2019:363(6422) and 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 glycorates Is and invented in a phoplastic Metabolism assay for The Metabolism of The enzyme. 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) may 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: MG719235REGION:848 & 1628), a maize UBI promoter (NCBI ACCESSION: KR297238REGION:4879 & 6876) and a rice Actin1 promoter (NCBI ACCESSION: AY452735REGION:2428 & 3797).

The terminator mediating the overexpression of GDH and MS in the present invention can be derived from eukaryote or prokaryote, or can be obtained by artificial synthesis, and the preferred terminators are ter1(NCBI ACCESS: KJ716235REGION:3962-4158) and ter2(NCBI ACCESS: MG733984REGION: 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)

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 Dehydrogenases (GDH) 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) 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:

example 1 construction of vectors

The GDH gene may be derived from a prokaryote or a eukaryote, and the GDH gene provided by the present invention includes, but is not limited to, the genes shown in table 2. In order to construct a transformation vector, an escherichia coli-derived GDH gene and a corresponding terminator sequence were artificially synthesized, including a chloroplast signal peptide, a GDH coding gene and a terminator, the nucleotide sequence was as shown in SEQ ID No.6 (amino acid sequence is shown in SEQ ID No.7), and BamHI and KpnI sites were provided at the 5 'end and 3' end, respectively.

The MS gene can be derived from prokaryotes or eukaryotes, and includes, but is not limited to, the genes shown in table 3. The rice MS gene is artificially synthesized, and comprises chloroplast signal peptide, an MS coding gene and a terminator, wherein the nucleotide sequence is shown as SEQ ID NO.8 (the amino acid sequence is shown as SEQ ID NO. 9), and BamHI and HindIII sites are respectively arranged at the 5 'end and the 3' end.

In order to achieve the suppression or knock-out of expression of BASS genes, the present invention provides BASS genes of plants themselves (table 1), the amino acid sequence of which is SEQ ID No.1 when the plants are rice, and SEQ ID No.2 or 3 when the plants are soybean. In order to construct BASS gene interference expression cassettes, OsBASS-RNAi and GmBASS-RNAi sequences which can form hairpin structures and target rice OsBASS genes and soybean GmBASS genes are artificially synthesized respectively, and the sequences are shown as SEQ ID NO.4 and SEQ ID NO. 5. As a control, OsPGGL1-RNAi and Gm PGGL1-RNAi sequences which target rice OsPGGL1 gene and soybean GmPGGL1 gene and can form hairpin structures are synthesized respectively, and are shown as SEQ ID NO.12 and SEQ ID NO. 13. Terminators ter1 are added to the 3' ends of the above sequences, respectively, to finally constitute OsBASS-RNAi-ter, GmBASS-RNAi-ter, Os PGGL1-RNAi-ter, and Gm PGGL 1-RNAi-ter. BglII and HindIII sites are respectively arranged at the 5 'end and the 3' end.

Meanwhile, 35S of cauliflower mosaic virus (CaMV) and a maize Ubi promoter sequence are synthesized artificially. EcoRI and BamHI sites are respectively arranged at the 5 'end and the 3' end of the 35S promoter, and HindIII and BamHI sites are respectively arranged at the 5 'end and the 3' end of the Ubi promoter and are used for mediating the transcription of RNAi sequences.

In order to construct a binary vector which can be used for transforming plants by the Agrobacterium method, a commercial binary vector pCambia1300 is used as a basis, the prior hptII (hygromycin resistance) gene is replaced by a glufosinate-resistant bar gene (NCBI ACCESSION: MG719235REGION:287-835) through an XhoI cleavage site, and the replaced vector is named as pCambia 1300-bar.

The Ubi promoter was ligated into the pCambia1300-bar vector with the GDH gene shown in SEQ ID NO.6 via EcoRI and KpnI sites to obtain the over-vector pCambia 1300-bar-GDH. The 35S promoter and the MS gene shown in SEQ ID NO.10 were ligated into the over-vector pCambia1300-bar-GDH through KpnI and HindIII sites to obtain the over-vector pCambia 1300-bar-GDH-MS. Finally, pCambia1300-bar-GDH-MS was digested with HindIII, and then OsBASS-RNAi-ter or GmBASS-RNAi-ter digested with BglII and HindIII and Ubi promoter digested with BamHI and HindIII were ligated to construct final vectors, which were named pCambia1300-bar-GDH-MS-OsBASS-RNAi (GMOsBi) and pCambia1300-bar-GDH-MS-GmBASS-RNAi (GMGmGmBi), respectively.

As a control, vectors containing the expression cassette for inhibiting PGGL1 gene were constructed in the same manner and named pCambia1300-bar-GDH-MS-OsPGGL1-RNAi (GMOsPi) and pCambia1300-bar-GDH-MS-GmPGGL1-RNAi (GMGmPi), respectively.

Finally, the T-DNA plasmid was transferred to Agrobacterium LB4404 by electrotransfer, and positive clones were selected by YEP solid medium containing 15. mu.g/mL tetracycline and 50. mu.g/mL kanamycin and maintained for the subsequent plant transformation.

Example 2 transformation of Rice

The transgenic rice is obtained by adopting the prior art (Luzhong, Gong ancestor Xun (1998) Life sciences 10: 125-. Mature and full 'Xishui-134' seeds are selected to be hulled, and callus is generated by induction and is used as a transformation material. Agrobacterium plates containing plasmids pCambia1300-bar-GDH-MS-OsBASS-RNAi (GMOsBi) and pCambia1300-bar-GDH-MS-OsPGGL1-RNAi (GMOsPi), respectively, constructed in example 1 were taken. A single colony is selected and inoculated, and agrobacterium for transformation is prepared. The callus to be transformed is placed into an Agrobacterium tumefaciens liquid with an OD of about 0.6 (preparation of Agrobacterium tumefaciens liquid: Agrobacterium tumefaciens is inoculated into a culture medium to be cultured until the OD is about 0.6, the culture medium consists of 3g/L K₂HPO₄、1g/L NaH₂PO₄、1g/L NH₄Cl、0.3g/L MgSO₄·7H₂O、0.15g/L KCl、0.01g/L CaCl₂、0.0025g/L FeSO₄·7H₂O, 5g/L sucrose, 20mg/L acetosyringone, water as solvent, pH 5.8), allowing Agrobacterium to bind to the callus surface, and transferring the callus to co-culture medium (MS +2 mg/L2, 4-D (2, 4-dichlorophenoxyacetic acid) +30g/L glucose +30g/L sucrose +3g/L agar (sigma7921) +20mg/L acetosyringone) for co-culture for 2-3 days. The transformed calli were rinsed with sterile water, transferred to selection medium (MS +2 mg/L2, 4-D +30g/L sucrose +3g/L agar (Sigma7921) +20mg/L acetosyringone +2mM glyphosate (Sigma)), and cultured for two months with selection (intermediate subculture). After screening, transferring the callus with good growth activity to a pre-differentiation culture medium (MS +0.1g/L inositol +5mg/L ABA (abscisic acid) +1mg/L NAA (naphthylacetic acid) +5 mg/L6-BA (6-benzylamino adenine) +20g/L sorbitol +30g/L sucrose +2.5g/L plant gel (gelrite)), culturing for about 20 days, then transferring the pre-differentiated callus to the differentiation culture medium, and irradiating for differentiation and germination for 14 hours every day. After 2-3 weeks, transferring the resistant regenerated plants to a rooting culture medium (1/2MS +0.2mg/L NAA +20g/L sucrose +2.5g/L gelrite), strengthening and rooting the strong seedlings, finally washing the regenerated plants and removing agar, transplanting the washed regenerated plants to a greenhouse, selecting transgenic lines with high yield, large seeds or high biomass and the like which can improve the rice yield, and culturing new varieties. Obtaining the transgenic rice plant containing the transformation vector.

Example 3 transformation of Soybean

The procedure used here to obtain transgenic soybeans is known from the prior art (Deng et al, 1998, Plant Physiology Communications 34: 381-387; Ma et al, 2008, Scientia Agricutural national informa 41: 661-668; Zhou et al, 2001, Journal of northern Agricultural University 32: 313-319). Healthy, full and mature soybeans of 'Tianlong No. 1' are selected, sterilized by 80% ethanol for 2 minutes, washed by sterile water and then placed in a dryer filled with chlorine (generated by the reaction of 50ml of NaClO and 2ml of concentrated HCl) for sterilization for 4-6 hours. The sterilized semen glycines is sowed in B5 culture medium in clean bench, and cultured at 25 deg.C for 5 days with optical density of 90-150 μmol photon/m²S level. Chinese angelica rootThe leaves turn green and burst the seed coat, and aseptic bean sprouts grow. The bean sprouts with the hypocotyl removed were cut into five-five pieces in length so that both explants had cotyledons and epicotyls. The explants are cut at about 7-8 of the node of the cotyledon and epicotyl and can be used as the target tissue to be infected.

Monoclonal agrobacteria containing the vectors pCambia1300-bar-GDH-MS-GmBASS-RNAi (GMGmBi) and pCambia1300-bar-GDH-MS-GmPGGL1-RNAi (GMGmPi), respectively, constructed by example 1 were separately cultured for use. The prepared explants are immersed in the agrobacterium suspension and co-cultured for about 30 minutes. Then, the excess cell suspension on the infected tissue was absorbed up with absorbent paper and transferred to 1/10B5 co-culture medium for 3-5 days at 25 ℃ in the dark.

The co-cultured plant tissue was washed with B5 liquid medium to remove excess Agrobacterium, and then placed in B5 solid medium for 5 days at 25 ℃ until it germinated. The induced germ tissue was transferred to B5 selection medium containing 0.2mM glyphosate and incubated at 25 ℃ with light for 4 weeks, during which the medium was changed every two weeks. Transferring the selected embryo tissue to a solid culture medium, culturing at 25 deg.C, and growing into plantlet. Subsequently, the transgenic plants were transferred to 1/2B5 medium for rooting induction. Finally, the grown plantlets are washed to remove agar and planted in a greenhouse.

Example 4: identification of transgenic Rice

Transgenic rice plants were obtained from the vectors pCambia1300-bar-GDH-MS-OsBASS-RNAi (GMOsBi) and pCambia1300-bar-GDH-MS-OsPGGL1-RNAi (GMOsPi), respectively, by example 2. The above transgenic plants had increased biomass and yield compared to the non-transgenic controls, and the GMOsBi plants had the greatest magnitude of increase in biomass or yield. In order to further identify the expression change of GMOsBi transgenic plants, the biomass and seed yield of the transgenic plants are evaluated and compared, normal water conditions, light drought conditions and medium drought conditions are set according to index grades based on farmland and crop drought shapes, which are formulated by the agricultural rural area part in Table 6, and the results are shown in Table 4. Under normal conditions, the biomass or yield of the GMOsBi transgenic plant and the GMOsPi transgenic plant is obviously increased by 5-50 percent compared with that of a non-transgenic control; but under the drought conditions, the biomass or yield of the GMOsBi transgenic plant is still significantly increased compared to the control, while the biomass or yield of the GMOsPi transgenic plant is increased very little compared to the non-transgenic control; under the drought condition, the biomass or yield of the GMOsBi transgenic plant is still remarkably increased compared with that of the control, and the biomass or yield of the GMOsPi transgenic plant is not increased or even reduced compared with that of the non-transgenic control. The results show that the drought resistance of the GMOsBi transgenic plant is obviously superior to that of the transgenic plant in the comparison scheme under the drought condition.

TABLE 4 determination of the yield of transgenic Rice under different drought conditions

Example 5: identification of transgenic Soybean

Transgenic soybean plants of the vectors pCambia1300-bar-GDH-MS-GmBASS-RNAi (GMGmBi) and pCambia1300-bar-GDH-MS-GmPGGL1-RNAi (GMGmPi), respectively, were obtained by example 3. The above transgenic plants had increased biomass and yield compared to the non-transgenic controls, and the transgenic plants had the greatest magnitude of increase in biomass or yield. In order to further identify the expression changes of the transgenic plants, the biomass and seed yield of the transgenic plants were evaluated, and normal moisture conditions, mild drought conditions and moderate drought conditions were set according to the index rating based on the drought morphology of farmland and crops, which was formulated by the rural part of agriculture in table 6, and the results are shown in table 5. Under normal conditions, the biomass or yield of the GMGmBi transgenic plant and the GMGmPi transgenic plant is remarkably increased by 5-50% compared with that of a non-transgenic control; but under the drought conditions, the biomass or yield of the gmgmgmbi transgenic plants is still significantly increased compared to the control, while the biomass or yield of the GMGmPi transgenic plants is increased very little compared to the non-transgenic control; under the drought condition, the biomass or yield of the GMGmBi transgenic plant is still remarkably increased compared with that of the control, and the biomass or yield of the GMGmPi transgenic plant is not increased or even reduced compared with that of the non-transgenic control. The results show that the drought resistance of the GMGmBi transgenic plant is obviously superior to that of the transgenic plant in the comparison scheme under the drought condition.

TABLE 5 yield determination of transgenic Soybean under different drought conditions

TABLE 6 drought form index rating based on farmland and crops (GB/T32136-

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Arg Ile Cys Asn Val Ile Arg Pro Phe Leu Pro Pro Leu Ser Val Leu

275 280 285

Val Ala Ser Ile Cys Ala Gly Ala Pro Leu Ala Phe Asn Val Glu Thr

290 295 300

Met Lys Ser Pro Leu Gly Val Val Ile Leu Leu Leu Val Val Ala Phe

305 310 315 320

His Leu Ser Ser Phe Ile Ala Gly Tyr Ile Leu Ser Gly Phe Val Phe

325 330 335

Arg Asp Ser Leu Asp Val Lys Ala Leu Gln Arg Thr Ile Ser Phe Glu

340 345 350

Thr Gly Leu Gln Ser Ser Leu Leu Ala Leu Ala Leu Ala Asn Lys Phe

355 360 365

Phe Glu Asp Pro Lys Val Ala Ile Pro Pro Ala Ile Phe Thr Ser Ile

370 375 380

Met Ser Leu Met Gly Phe Val Leu Val Leu Ile Trp Thr Arg Arg Gly

385 390 395 400

Lys Arg Asp Ile Lys His Ser Ser

405

<210> 3

<211> 416

<212> PRT

<213> Unknown (Unknown)

<400> 3

Met Ile Ser Ser Gly Leu Lys Pro Lys His Phe Asn Asn Val His Ser

1 5 10 15

Leu Phe Asn Leu Ser Lys Ser Gln Gln Pro Pro Asn Pro Ile Ile Val

20 25 30

Pro Cys Cys Arg Thr Asn Thr Asn Asn Asn Ile Ser Ser Pro Phe Ser

35 40 45

Ile Arg Phe Asn Ser Pro Phe Pro Tyr Arg Ser Pro Lys Ile Pro Leu

50 55 60

Lys Cys Ala Pro Leu His Ser Ser Asp Ser Leu Pro Pro Asp Pro Ser

65 70 75 80

Ser Ala Ser Thr Gln Met Glu Gln Asn Ser Met Ser Ile Leu Glu Ile

85 90 95

Leu Lys Gln Ser Asn Ser Tyr Leu Pro His Ala Leu Ile Ala Ser Ile

100 105 110

Leu Leu Ala Leu Ile Tyr Pro Arg Ser Leu Thr Trp Phe Thr Ser Arg

115 120 125

Phe Tyr Ala Pro Ala Leu Gly Phe Leu Met Phe Ala Val Gly Val Asn

130 135 140

Ser Asn Glu Asn Asp Phe Leu Glu Ala Phe Lys Arg Pro Ala Glu Ile

145 150 155 160

Val Thr Gly Tyr Phe Gly Gln Phe Ala Val Lys Pro Leu Leu Gly Tyr

165 170 175

Leu Phe Cys Met Ile Ala Val Thr Val Leu Gly Leu Pro Thr Thr Val

180 185 190

Gly Ala Gly Ile Val Leu Val Ala Cys Val Ser Gly Ala Gln Leu Ser

195 200 205

Ser Tyr Ala Thr Phe Leu Thr Asp Pro Gln Met Ala Pro Leu Ser Ile

210 215 220

Val Met Thr Ser Leu Ser Thr Ala Ser Ala Val Phe Val Thr Pro Leu

225 230 235 240

Leu Leu Leu Leu Leu Ile Gly Lys Lys Leu Pro Ile Asp Val Lys Gly

245 250 255

Met Val Tyr Asn Ile Thr Gln Ile Val Val Val Pro Ile Ala Ala Gly

260 265 270

Leu Leu Leu Asn Arg Phe Phe Pro Arg Ile Cys Asn Val Ile Arg Pro

275 280 285

Phe Leu Pro Pro Leu Ser Val Leu Val Ala Ser Ile Cys Ala Gly Ala

290 295 300

Pro Leu Ala Leu Asn Val Glu Thr Met Lys Ser Pro Leu Gly Val Ala

305 310 315 320

Ile Leu Leu Leu Val Val Ala Phe His Leu Ser Ser Phe Ile Ala Gly

325 330 335

Tyr Ile Leu Ser Gly Phe Val Phe Arg Asp Ser Leu Asp Val Lys Ala

340 345 350

Leu Gln Arg Thr Ile Ser Phe Glu Thr Gly Met Gln Ser Ser Leu Leu

355 360 365

Ala Leu Ala Leu Ala Asn Lys Phe Phe Glu Asp Pro Lys Val Ala Ile

370 375 380

Pro Pro Ala Ile Ser Thr Ser Ile Met Ser Leu Met Gly Phe Val Leu

385 390 395 400

Val Leu Ile Trp Thr Arg Arg Gly Lys Ser Glu Ile Lys Asn Ser Ser

405 410 415

<210> 4

<211> 856

<212> DNA

<213> Unknown (Unknown)

<400> 4

gcttttgatg gaaagacaga catcataccg aatttataaa aggaaaagaa ataaattcaa 60

aactttacat tttttatgcc accaaccaaa ggtgaatcaa agatatgaac aagagtttct 120

taagactatt agcccccccc cccccccccc aacgacctcc aactccaatc ctccttaatc 180

gccaacccac acagctataa aaaggggata tttcagatcg gatcaagcag agcacctacg 240

ccgtgaaaac ggcggcgaga ccgcctgggg aggagccaga cggggcagtc gccggccggt 300

gggcagatgg tggacgccgg cgatgtgggg ccgcaccagg acggcggcgt tgggggccat 360

tcgagcgccg gcgaccgcga gggtgggtgg gttttggttt cagagtttca gagctgatga 420

cgcaacgcag cgaaagagac gattcagatt tcagtgagaa gttgggagtt tcgacaagga 480

acgaacaatc agtcgaatgg cccccaacgc cgccgtcctg gtgcggcccc acatcgccgg 540

cgtccaccat ctgcccaccg gccggcgact gccccgtctg gctcctcccc aggcggtctc 600

gccgccgttt tcacggcgta ggtgctctgc ttgatccgat ctgaaatatc ccctttttat 660

agctgtgtgg gttggcgatt aaggaggatt ggagttggag gtcgttgggg gggggggggg 720

ggggctaata gtcttaagaa actcttgttc atatctttga ttcacctttg gttggtggca 780

taaaaaatgt aaagttttga atttatttct tttcctttta taaattcggt atgatgtctg 840

tctttccatc aaaagt 856

<210> 5

<211> 733

<212> DNA

<213> Unknown (Unknown)

<400> 5

atgactgaaa ttgaagtcta agtggagggg gaacttacag aaattgctgg aggaatagcc 60

acttttggat cttcaaagaa cttattagca agagccaagg caagcaggct actttgcatt 120

cctggtatta gataaatgaa gttattgaaa caacatgcta ttacaggcat ttgatgtatg 180

aaaatggaga aactttgtac ctgtctcaaa ggaaattgtt cgttgcagtg ccttcacatc 240

aagagaatca cggaagacaa atccactgag gatataacca gctataaaag atgacaaatg 300

aaaagcaaca acaagcaaca agatagcaac tcccagaggg gatttcatag tctcaacatt 360

aaaggcgagt ggtgctccag cacagataga tgccaccagt actgatagtg gaggcaaaaa 420

tggtcgaata actgttattc gaccattttt gcctccacta tcagtactgg tggcatctat 480

ctgtgctgga gcaccactcg cctttaatgt tgagactatg aaatcccctc tgggagttgc 540

tatcttgttg cttgttgttg cttttcattt gtcatctttt atagctggtt atatcctcag 600

tggatttgtc ttccgtgatt ctcttgatgt gaaggcactg caacgaacaa tttcctttga 660

gacaggtaca aagtttctcc attttcatac atcaaatgcc tgtaatagca tgttgtttca 720

ataacttcat tta 733

<210> 6

<211> 3668

<212> DNA

<213> Unknown (Unknown)

<400> 6

ggatccaaca atggccccgt ccgtgatggc ctcctccgcc accaccgtgg ccccgttcca 60

gggcctcaag tccaccgccg gcatgccggt ggcccgccgc tccggcaact cctccttcgg 120

caacgtgtcc aacggcggcc gcatccgctg catgccgcgc ggccagggca agcgcctcgc 180

ccagctcctc ggcgcccagc tcaagcagta cgccgccgag gtgcgcggca tctccaccgc 240

cggcggcgcc tcccgcggcg gcgcccgcgg cccggcctcc ccgtcctccc tcgagcagca 300

gacccgccag gtggcccagg tggccgtgca gcagtccacc cagcaggccg tgaaggtggt 360

ggtgccggcc atcaaggtgg acctcgtggg cgccgtgtcc tccgtgtccg agtccgacaa 420

ggtggagccg ggcgtgttca agaacgtgga cggccaccgc ttcgaggacg gccgctacgc 480

cgccttcgtg gaggagatca ccaagttcat cccgaaggag cgccagtact ccgacccggt 540

gcgcaccttc gcctacggca ccgacgcctc cttctaccgc ctcaacccga agctcgtggt 600

gaaggtgcac aacgaggacg aggtgcgccg catcatgccg atcgccgagc gcctccaggt 660

gccgatcacc ttccgcgccg ccggcacctc cctctccggc caggccatca ccgactccgt 720

gctcatcaag ctctcccaca ccggcaagaa cttccgcaac ttcaccgtgc acggcgacgg 780

ctccgtgatc accgtggagc cgggcctcat cggcggcgag gtgaaccgca tcctcgccgc 840

ccaccagaag aagaacaagc tcccgatcca gtacaagatc ggcccggacc cgtcctccat 900

cgactcctgc atgatcggcg gcatcgtgtc caacaactcc tccggcatgt gctgcggcgt 960

gtcccagaac acctaccaca ccctcaagga catgcgcgtg gtgttcgtgg acggcaccgt 1020

gctcgacacc gccgacccga actcctgcac cgccttcatg aagtcccacc gctccctcgt 1080

ggacggcgtg gtgtccctcg cccgccgcgt gcaggccgac aaggagctca ccgccctcat 1140

ccgccgcaag ttcgccatca agtgcaccac cggctactcc ctcaacgccc tcgtggactt 1200

cccggtggac aacccgatcg agatcatcaa gcacctcatc atcggctccg agggcaccct 1260

cggcttcgtg tcccgcgcca cctacaacac cgtgccggag tggccgaaca aggcctccgc 1320

cttcatcgtg ttcccggacg tgcgcgccgc ctgcaccggc gcctccgtgc tccgcaacga 1380

gacctccgtg gacgccgtgg agctcttcga ccgcgcctcc ctccgcgagt gcgagaacaa 1440

cgaggacatg atgcgcctcg tgccggacat caagggctgc gacccgatgg ccgccgccct 1500

cctcatcgag tgccgcggcc aggacgaggc cgccctccag tcccgcatcg aggaggtggt 1560

gcgcgtgctc accgccgccg gcctcccgtt cggcgccaag gccgcccagc cgatggccat 1620

cgacgcctac ccgttccacc acgaccagaa gaacgccaag gtgttctggg acgtgcgccg 1680

cggcctcatc ccgatcgtgg gcgccgcccg cgagccgggc acctccatgc tcatcgagga 1740

cgtggcctgc ccggtggaca agctcgccga catgatgatc gacctcatcg acatgttcca 1800

gcgccacggc taccacgacg cctcctgctt cggccacgcc ctcgagggca acctccacct 1860

cgtgttctcc cagggcttcc gcaacaagga ggaggtgcag cgcttctccg acatgatgga 1920

ggagatgtgc cacctcgtgg ccaccaagca ctccggctcc ctcaagggcg agcacggcac 1980

cggccgcaac gtggccccgt tcgtggagat ggagtggggc aacaaggcct acgagctcat 2040

gtgggagctc aaggccctct tcgacccgtc ccacaccctc aacccgggcg tgatcctcaa 2100

ccgcgaccag gacgcccaca tcaagttcct caagccgtcc ccggccgcct ccccgatcgt 2160

gaaccgctgc atcgagtgcg gcttctgcga gtccaactgc ccgtcccgcg acatcaccct 2220

caccccgcgc cagcgcatct ccgtgtaccg cgagatgtac cgcctcaagc agctcggccc 2280

gggcgcctcc gaggaggaga agaagcagct cgccgccatg tcctcctcct acgcctacga 2340

cggcgagcag acctgcgccg ccgacggcat gtgccaggag aagtgcccgg tgaagatcaa 2400

caccggcgac ctcatcaagt ccatgcgcgc cgagcacatg aaggaggaga agaccgcctc 2460

cggcatggcc gactggctcg ccgccaactt cggcgtgatc aactccaacg tgccgcgctt 2520

cctcaacatc gtgaacgcca tgcactccgt ggtgggctcc gccccgctct ccgccatctc 2580

ccgcgccctc aacgccgcca ccaaccactt cgtgccggtg tggaacccgt acatgccgaa 2640

gggcgccgcc ccgctcaagg tgccggcccc gccggccccg gccgccgccg aggcctccgg 2700

catcccgcgc aaggtggtgt acatgccgtc ctgcgtgacc cgcatgatgg gcccggccgc 2760

ctccgacacc gagaccgccg ccgtgcacga gaaggtgatg tccctcttcg gcaaggccgg 2820

ctacgaggtg atcatcccgg agggcgtggc ctcccagtgc tgcggcatga tgttcaactc 2880

ccgcggcttc aaggacgccg ccgcctccaa gggcgccgag ctcgaggccg ccctcctcaa 2940

ggcctccgac aacggcaaga tcccgatcgt gatcgacacc tccccgtgcc tcgcccaggt 3000

gaagtcccaa atctccgagc cgtccctccg cttcgccctc tacgagccgg tggagttcat 3060

ccgccacttc ctcgtggaca agctcgagtg gaagaaggtg cgcgaccagg tggccatcca 3120

cgtgccgtgc tcctccaaga agatgggcat cgaggagtcc ttcgccaagc tcgccggcct 3180

ctgcgccaac gaggtggtgc cgtccggcat cccgtgctgc ggcatggccg gcgaccgcgg 3240

catgcgcttc ccggagctca ccggcgcctc cctccagcac ctcaacctcc cgaagacctg 3300

caaggacggc tactccacct cccgcacctg cgagatgtcc ctctccaacc acgccggcat 3360

caacttccgc ggcctcgtgt acctcgtgga cgaggccacc gccccgaaga agcaggccgc 3420

cgccgccaag accgcctaag tagatgccga ccggatctgt cgatcgacaa gctcgagttt 3480

ctccataata atgtgtgagt agttcccaga taagggaatt agggttccta tagggtttcg 3540

ctcatgtgtt gagcatataa gaaaccctta gtatgtattt gtatttgtaa aatacttcta 3600

tcaataaaat ttctaattcc taaaaccaaa atccagtact aaaatccaga tcccccgaat 3660

taaagctt 3668

<210> 7

<211> 1136

<212> PRT

<213> Unknown (Unknown)

<400> 7

Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala

1 5 10 15

Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Ala Thr Arg Lys Ala Asn Gly Gly Pro Arg Gly Gln Gly Lys

35 40 45

Arg Leu Ala Gln Leu Leu Gly Ala Gln Leu Lys Gln Tyr Ala Ala Glu

50 55 60

Val Arg Gly Ile Ser Thr Ala Gly Gly Ala Ser Arg Gly Gly Ala Arg

65 70 75 80

Gly Pro Ala Ser Pro Ser Ser Leu Glu Gln Gln Thr Arg Gln Val Ala

85 90 95

Gln Val Ala Val Gln Gln Ser Thr Gln Gln Ala Val Lys Val Val Val

100 105 110

Pro Ala Ile Lys Val Asp Leu Val Gly Ala Val Ser Ser Val Ser Glu

115 120 125

Ser Asp Lys Val Glu Pro Gly Val Phe Lys Asn Val Asp Gly His Arg

130 135 140

Phe Glu Asp Gly Arg Tyr Ala Ala Phe Val Glu Glu Ile Thr Lys Phe

145 150 155 160

Ile Pro Lys Glu Arg Gln Tyr Ser Asp Pro Val Arg Thr Phe Ala Tyr

165 170 175

Gly Thr Asp Ala Ser Phe Tyr Arg Leu Asn Pro Lys Leu Val Val Lys

180 185 190

Val His Asn Glu Asp Glu Val Arg Arg Ile Met Pro Ile Ala Glu Arg

195 200 205

Leu Gln Val Pro Ile Thr Phe Arg Ala Ala Gly Thr Ser Leu Ser Gly

210 215 220

Gln Ala Ile Thr Asp Ser Val Leu Ile Lys Leu Ser His Thr Gly Lys

225 230 235 240

Asn Phe Arg Asn Phe Thr Val His Gly Asp Gly Ser Val Ile Thr Val

245 250 255

Glu Pro Gly Leu Ile Gly Gly Glu Val Asn Arg Ile Leu Ala Ala His

260 265 270

Gln Lys Lys Asn Lys Leu Pro Ile Gln Tyr Lys Ile Gly Pro Asp Pro

275 280 285

Ser Ser Ile Asp Ser Cys Met Ile Gly Gly Ile Val Ser Asn Asn Ser

290 295 300

Ser Gly Met Cys Cys Gly Val Ser Gln Asn Thr Tyr His Thr Leu Lys

305 310 315 320

Asp Met Arg Val Val Phe Val Asp Gly Thr Val Leu Asp Thr Ala Asp

325 330 335

Pro Asn Ser Cys Thr Ala Phe Met Lys Ser His Arg Ser Leu Val Asp

340 345 350

Gly Val Val Ser Leu Ala Arg Arg Val Gln Ala Asp Lys Glu Leu Thr

355 360 365

Ala Leu Ile Arg Arg Lys Phe Ala Ile Lys Cys Thr Thr Gly Tyr Ser

370 375 380

Leu Asn Ala Leu Val Asp Phe Pro Val Asp Asn Pro Ile Glu Ile Ile

385 390 395 400

Lys His Leu Ile Ile Gly Ser Glu Gly Thr Leu Gly Phe Val Ser Arg

405 410 415

Ala Thr Tyr Asn Thr Val Pro Glu Trp Pro Asn Lys Ala Ser Ala Phe

420 425 430

Ile Val Phe Pro Asp Val Arg Ala Ala Cys Thr Gly Ala Ser Val Leu

435 440 445

Arg Asn Glu Thr Ser Val Asp Ala Val Glu Leu Phe Asp Arg Ala Ser

450 455 460

Leu Arg Glu Cys Glu Asn Asn Glu Asp Met Met Arg Leu Val Pro Asp

465 470 475 480

Ile Lys Gly Cys Asp Pro Met Ala Ala Ala Leu Leu Ile Glu Cys Arg

485 490 495

Gly Gln Asp Glu Ala Ala Leu Gln Ser Arg Ile Glu Glu Val Val Arg

500 505 510

Val Leu Thr Ala Ala Gly Leu Pro Phe Gly Ala Lys Ala Ala Gln Pro

515 520 525

Met Ala Ile Asp Ala Tyr Pro Phe His His Asp Gln Lys Asn Ala Lys

530 535 540

Val Phe Trp Asp Val Arg Arg Gly Leu Ile Pro Ile Val Gly Ala Ala

545 550 555 560

Arg Glu Pro Gly Thr Ser Met Leu Ile Glu Asp Val Ala Cys Pro Val

565 570 575

Asp Lys Leu Ala Asp Met Met Ile Asp Leu Ile Asp Met Phe Gln Arg

580 585 590

His Gly Tyr His Asp Ala Ser Cys Phe Gly His Ala Leu Glu Gly Asn

595 600 605

Leu His Leu Val Phe Ser Gln Gly Phe Arg Asn Lys Glu Glu Val Gln

610 615 620

Arg Phe Ser Asp Met Met Glu Glu Met Cys His Leu Val Ala Thr Lys

625 630 635 640

His Ser Gly Ser Leu Lys Gly Glu His Gly Thr Gly Arg Asn Val Ala

645 650 655

Pro Phe Val Glu Met Glu Trp Gly Asn Lys Ala Tyr Glu Leu Met Trp

660 665 670

Glu Leu Lys Ala Leu Phe Asp Pro Ser His Thr Leu Asn Pro Gly Val

675 680 685

Ile Leu Asn Arg Asp Gln Asp Ala His Ile Lys Phe Leu Lys Pro Ser

690 695 700

Pro Ala Ala Ser Pro Ile Val Asn Arg Cys Ile Glu Cys Gly Phe Cys

705 710 715 720

Glu Ser Asn Cys Pro Ser Arg Asp Ile Thr Leu Thr Pro Arg Gln Arg

725 730 735

Ile Ser Val Tyr Arg Glu Met Tyr Arg Leu Lys Gln Leu Gly Pro Gly

740 745 750

Ala Ser Glu Glu Glu Lys Lys Gln Leu Ala Ala Met Ser Ser Ser Tyr

755 760 765

Ala Tyr Asp Gly Glu Gln Thr Cys Ala Ala Asp Gly Met Cys Gln Glu

770 775 780

Lys Cys Pro Val Lys Ile Asn Thr Gly Asp Leu Ile Lys Ser Met Arg

785 790 795 800

Ala Glu His Met Lys Glu Glu Lys Thr Ala Ser Gly Met Ala Asp Trp

805 810 815

Leu Ala Ala Asn Phe Gly Val Ile Asn Ser Asn Val Pro Arg Phe Leu

820 825 830

Asn Ile Val Asn Ala Met His Ser Val Val Gly Ser Ala Pro Leu Ser

835 840 845

Ala Ile Ser Arg Ala Leu Asn Ala Ala Thr Asn His Phe Val Pro Val

850 855 860

Trp Asn Pro Tyr Met Pro Lys Gly Ala Ala Pro Leu Lys Val Pro Ala

865 870 875 880

Pro Pro Ala Pro Ala Ala Ala Glu Ala Ser Gly Ile Pro Arg Lys Val

885 890 895

Val Tyr Met Pro Ser Cys Val Thr Arg Met Met Gly Pro Ala Ala Ser

900 905 910

Asp Thr Glu Thr Ala Ala Val His Glu Lys Val Met Ser Leu Phe Gly

915 920 925

Lys Ala Gly Tyr Glu Val Ile Ile Pro Glu Gly Val Ala Ser Gln Cys

930 935 940

Cys Gly Met Met Phe Asn Ser Arg Gly Phe Lys Asp Ala Ala Ala Ser

945 950 955 960

Lys Gly Ala Glu Leu Glu Ala Ala Leu Leu Lys Ala Ser Asp Asn Gly

965 970 975

Lys Ile Pro Ile Val Ile Asp Thr Ser Pro Cys Leu Ala Gln Val Lys

980 985 990

Ser Gln Ile Ser Glu Pro Ser Leu Arg Phe Ala Leu Tyr Glu Pro Val

995 1000 1005

Glu Phe Ile Arg His Phe Leu Val Asp Lys Leu Glu Trp Lys Lys Val

1010 1015 1020

Arg Asp Gln Val Ala Ile His Val Pro Cys Ser Ser Lys Lys Met Gly

1025 1030 1035 1040

Ile Glu Glu Ser Phe Ala Lys Leu Ala Gly Leu Cys Ala Asn Glu Val

1045 1050 1055

Val Pro Ser Gly Ile Pro Cys Cys Gly Met Ala Gly Asp Arg Gly Met

1060 1065 1070

Arg Phe Pro Glu Leu Thr Gly Ala Ser Leu Gln His Leu Asn Leu Pro

1075 1080 1085

Lys Thr Cys Lys Asp Gly Tyr Ser Thr Ser Arg Thr Cys Glu Met Ser

1090 1095 1100

Leu Ser Asn His Ala Gly Ile Asn Phe Arg Gly Leu Val Tyr Leu Val

1105 1110 1115 1120

Asp Glu Ala Thr Ala Pro Lys Lys Gln Ala Ala Ala Ala Lys Thr Ala

1125 1130 1135

<210> 8

<211> 2057

<212> DNA

<213> Unknown (Unknown)

<400> 8

ggatccaaca atggccccgt ccgtgatggc ctcctccgcc accaccgtgg ccccgttcca 60

gggcctcaag tccaccgccg gcatgccggt ggcccgccgc tccggcaact cctccttcgg 120

caacgtgtcc aacggcggcc gcatccgctg catggccacc aacgccgccg ccccgccgtg 180

cccgtgctac gacaccccgg agggcgtgga catcctcggc cgctacgacc cggagttcgc 240

cgccatcctc acccgcgacg ccctcgcctt cgtggccggc ctccagcgcg agttccgcgg 300

cgccgtgcgc tacgccatgg agcgccgccg cgaggcccag cgccgctacg acgccggcga 360

gctcccgcgc ttcgacccgg ccacccgccc ggtgcgcgag gccggcggct gggcctgcgc 420

cccggtgccg ccggccatcg ccgaccgcac cgtggagatc accggcccgg ccgagccgcg 480

caagatggtg atcaacgccc tcaactccgg cgccaaggtg ttcatggccg acttcgagga 540

cgccctctcc ccgacctggg agaacctcat gcgcggccag gtgaacctcc gcgacgccgt 600

ggccggcacc atcacctacc gcgacgccgc ccgcggccgc gagtaccgcc tcggcgaccg 660

cccggccacc ctcttcgtgc gcccgcgcgg ctggcacctc ccggaggccc acgtgctcgt 720

ggacggcgag ccggccatcg gctgcctcgt ggacttcggc ctctacttct tccactccca 780

cgccgccttc cgctccggcc agggcgccgg cttcggcccg ttcttctacc tcccgaagat 840

ggagcactcc cgcgaggccc gcatctggaa gggcgtgttc gagcgcgccg agaaggaggc 900

cggcatcggc cgcggctcca tccgcgccac cgtgctcgtg gagaccctcc cggccgtgtt 960

ccagatggag gagatcctcc acgagctccg cgaccactcc gccggcctca actgcggccg 1020

ctgggactac atcttctcct acgtgaagac cttccgcgcc cgcccggacc gcctcctccc 1080

ggaccgcgcc ctcgtgggca tggcccagca cttcatgcgc tcctactccc acctcctcat 1140

ccagacctgc caccgccgcg gcgtgcacgc catgggcggc atggccgccc agatcccgat 1200

caaggacgac gccgccgcca acgaggccgc cctcgagctc gtgcgcaagg acaagctccg 1260

cgaggtgcgc gccggccacg acggcacctg ggccgcccac ccgggcctca tcccggccat 1320

ccgcgaggtg ttcgagggcc acctcggcgg ccgcccgaac cagatcgacg ccgccgccgg 1380

cgacgccgcc cgcgccggcg tggccgtgac cgaggaggac ctcctccagc cgccgcgcgg 1440

cgcccgcacc gtggagggcc tccgccacaa cacccgcgtg ggcgtgcagt acgtggccgc 1500

ctggctctcc ggctccggct ccgtgccgct ctacaacctc atggaggacg ccgccaccgc 1560

cgagatttcc cgcgtgcaga actggcagtg gctccgccac ggcgccgtgc tcgacgccgg 1620

cggcgtggag gtgcgcgcca ccccggagct cctcgcccgc gtggtggagg aggagatggc 1680

ccgcgtggag gccgaggtgg gcgccgagcg cttccgccgc ggccgctacg ccgaggccgg 1740

ccgcatcttc tcccgccagt gcaccgcccc ggagctcgac gacttcctca ccctcgacgc 1800

ctacaacctc atcgtggtgc accacccggg cgcctcctcc ccgtgcaagc tctaagagct 1860

ctagatcgtt ctgcacaaag tggagtagtc agtcatcgat caggaaccag acaccagact 1920

tttattcata cagtgaagtg aagtgaagtg cagtgcagtg agttgctggt ttttgtacaa 1980

cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac 2040

caaaatccag gggatcc 2057

<210> 9

<211> 567

<212> PRT

<213> Unknown (Unknown)

<400> 9

Met Ala Thr Asn Ala Ala Ala Pro Pro Cys Pro Cys Tyr Asp Thr Pro

1 5 10 15

Glu Gly Val Asp Ile Leu Gly Arg Tyr Asp Pro Glu Phe Ala Ala Ile

20 25 30

Leu Thr Arg Asp Ala Leu Ala Phe Val Ala Gly Leu Gln Arg Glu Phe

35 40 45

Arg Gly Ala Val Arg Tyr Ala Met Glu Arg Arg Arg Glu Ala Gln Arg

50 55 60

Arg Tyr Asp Ala Gly Glu Leu Pro Arg Phe Asp Pro Ala Thr Arg Pro

65 70 75 80

Val Arg Glu Ala Gly Gly Trp Ala Cys Ala Pro Val Pro Pro Ala Ile

85 90 95

Ala Asp Arg Thr Val Glu Ile Thr Gly Pro Ala Glu Pro Arg Lys Met

100 105 110

Val Ile Asn Ala Leu Asn Ser Gly Ala Lys Val Phe Met Ala Asp Phe

115 120 125

Glu Asp Ala Leu Ser Pro Thr Trp Glu Asn Leu Met Arg Gly Gln Val

130 135 140

Asn Leu Arg Asp Ala Val Ala Gly Thr Ile Thr Tyr Arg Asp Ala Ala

145 150 155 160

Arg Gly Arg Glu Tyr Arg Leu Gly Asp Arg Pro Ala Thr Leu Phe Val

165 170 175

Arg Pro Arg Gly Trp His Leu Pro Glu Ala His Val Leu Val Asp Gly

180 185 190

Glu Pro Ala Ile Gly Cys Leu Val Asp Phe Gly Leu Tyr Phe Phe His

195 200 205

Ser His Ala Ala Phe Arg Ser Gly Gln Gly Ala Gly Phe Gly Pro Phe

210 215 220

Phe Tyr Leu Pro Lys Met Glu His Ser Arg Glu Ala Arg Ile Trp Lys

225 230 235 240

Gly Val Phe Glu Arg Ala Glu Lys Glu Ala Gly Ile Gly Arg Gly Ser

245 250 255

Ile Arg Ala Thr Val Leu Val Glu Thr Leu Pro Ala Val Phe Gln Met

260 265 270

Glu Glu Ile Leu His Glu Leu Arg Asp His Ser Ala Gly Leu Asn Cys

275 280 285

Gly Arg Trp Asp Tyr Ile Phe Ser Tyr Val Lys Thr Phe Arg Ala Arg

290 295 300

Pro Asp Arg Leu Leu Pro Asp Arg Ala Leu Val Gly Met Ala Gln His

305 310 315 320

Phe Met Arg Ser Tyr Ser His Leu Leu Ile Gln Thr Cys His Arg Arg

325 330 335

Gly Val His Ala Met Gly Gly Met Ala Ala Gln Ile Pro Ile Lys Asp

340 345 350

Asp Ala Ala Ala Asn Glu Ala Ala Leu Glu Leu Val Arg Lys Asp Lys

355 360 365

Leu Arg Glu Val Arg Ala Gly His Asp Gly Thr Trp Ala Ala His Pro

370 375 380

Gly Leu Ile Pro Ala Ile Arg Glu Val Phe Glu Gly His Leu Gly Gly

385 390 395 400

Arg Pro Asn Gln Ile Asp Ala Ala Ala Gly Asp Ala Ala Arg Ala Gly

405 410 415

Val Ala Val Thr Glu Glu Asp Leu Leu Gln Pro Pro Arg Gly Ala Arg

420 425 430

Thr Val Glu Gly Leu Arg His Asn Thr Arg Val Gly Val Gln Tyr Val

435 440 445

Ala Ala Trp Leu Ser Gly Ser Gly Ser Val Pro Leu Tyr Asn Leu Met

450 455 460

Glu Asp Ala Ala Thr Ala Glu Ile Ser Arg Val Gln Asn Trp Gln Trp

465 470 475 480

Leu Arg His Gly Ala Val Leu Asp Ala Gly Gly Val Glu Val Arg Ala

485 490 495

Thr Pro Glu Leu Leu Ala Arg Val Val Glu Glu Glu Met Ala Arg Val

500 505 510

Glu Ala Glu Val Gly Ala Glu Arg Phe Arg Arg Gly Arg Tyr Ala Glu

515 520 525

Ala Gly Arg Ile Phe Ser Arg Gln Cys Thr Ala Pro Glu Leu Asp Asp

530 535 540

Phe Leu Thr Leu Asp Ala Tyr Asn Leu Ile Val Val His His Pro Gly

545 550 555 560

Ala Ser Ser Pro Cys Lys Leu

565

<210> 10

<211> 47

<212> PRT

<213> Unknown (Unknown)

<400> 10

Met Ala Pro Ser Val Met Ala Ser Ser Ala Thr Thr Val Ala Pro Phe

1 5 10 15

Gln Gly Leu Lys Ser Thr Ala Gly Met Pro Val Ala Arg Arg Ser Gly

20 25 30

Asn Ser Ser Phe Gly Asn Val Ser Asn Gly Gly Arg Ile Arg Cys

35 40 45

<210> 11

<211> 42

<212> PRT

<213> Unknown (Unknown)

<400> 11

Met Ala Ser Ser Met Leu Ser Ser Ala Thr Met Val Ala Ser Pro Ala

1 5 10 15

Gln Ala Thr Met Val Ala Pro Phe Asn Gly Leu Lys Ser Ser Ala Ala

20 25 30

Phe Pro Ala Thr Arg Lys Ala Asn Gly Gly

35 40

<210> 12

<211> 838

<212> DNA

<213> Unknown (Unknown)

<400> 12

cgatcggcag gtcatgcgaa atcgcgacga ggctgcgtgc attttgactg attgacgact 60

cacgctgggt aggcccgagg gagaggtggc gcccgcgcct cctcctcctc ctccggcggc 120

ggcggcggcg tggatcgggg cgatgaggcg gtggcgagga ttggcgggag ccatggagat 180

tgccgccgct tggcgggagg aggaggagga ggaggagggg ttgcgtcggc atcggcgggg 240

aggaagcgtg cggaggcggg gcggcgacgt ggcggtggcg gagggcgaaa ggcggcagcg 300

atggctgctg cgtagcgagg caatcatgca gggggaggat gatgatgagg tcgctgccat 360

ctcttctctt ctcttctctt cttctccttc tcctttggcc agcgagagag cagtggcagt 420

gacagtggat gagaagggag ctgggagcag tggcagaggc caggtggaag agaggagatg 480

gcagcgacct catcatcatc ctccccctgc atgattgcct cgctacgcag cagccatcgc 540

tgccgccttt cgccctccgc caccgccacg tcgccgcccc gcctccgcac gcttcctccc 600

cgccgatgcc gacgcaaccc ctcctcctcc tcctcctcct cccgccaagc ggcggcaatc 660

tccatggctc ccgccaatcc tcgccaccgc ctcatcgccc cgatccacgc cgccgccgcc 720

gccggaggag gaggaggagg cgcgggcgcc acctctccct cgggcctacc cagcgtgagt 780

cgtcaatcag tcaaaatgca cgcagcctcg tcgcgatttc gcatgacctg ccgatcgg 838

<210> 13

<211> 491

<212> DNA

<213> Unknown (Unknown)

<400> 13

gccgtgacat aaatgacagt ctcagccaac aattgcaagc aaactttgtc taacagcagg 60

aatcgagcaa agaatggatc caaatatacc attcagagca taagcaatgg cacaaaatgg 120

gagagcctcg ggttccttgg cagacaaagc tgctgttcca agcccgtggg cactgaaaaa 180

taaaatttag gtcaagagag taagtaaata accatgcacg cacaaagaaa atgtcattca 240

actattattg catgtttggt atagattaat ttttgagaaa taatcactta ttttacagaa 300

actaatttta tttttcagtg cccacgggct tggaacagca gctttgtctg ccaaggaacc 360

cgaggctctc ccattttgtg ccattgctta tgctctgaat ggtatatttg gatccattct 420

ttgctcgatt cctgctgtta gacaaagttt gcttgcaatt gttggctgag actgtcattt 480

atgtcacggc c 491

Claims

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 glycollic acid 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 1, 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.