CN116987716B - Expression vector, application of OsSUT1 gene and transgenic rice - Google Patents

Abstract

The application belongs to the field of biology, and discloses an expression vector which comprises a vector, a functional gene connected to the vector and a promoter connected to the vector, wherein the functional gene has a nucleotide sequence shown as SEQ ID NO.1OsSUT1A gene; the promoter gene is used for promoting under drought stressOsSUT1Gene expression. After the expression vector is transformed into agrobacterium and infects rice, the drought resistance of the rice under drought stress can be obviously improved. Meanwhile, the application also discloses a deviceOsSUT1The application of the gene and transgenic rice.

Description

An expression vector,OsSUT1Gene use and transgenic rice

Technical Field

The application relates to the field of biology, in particular to an expression vector,OsSUT1The application of the gene and transgenic rice.

Background

The inventor previously puts forward an application patent CN114703221A to disclose a method for improving the sugar metabolism level and drought resistance of ricepOsHAK1:OsFLN2Expression vector and application thereof. Gene with up-regulated expression induced by drought/osmotic stressOsHAK1Is driven by a promoter encoding a fructokinase-like protein geneOsFLN2Expressed in rice, under drought stress,pOsHAK1:OsFLN2promote the synthesis and transportation of sugar in the body and obviously improve the drought resistance of plants. The saidpOsHAK1:OsFLN2The transgenic lines grew normally under control conditions without an adverse phenotype, whereas after PEG treatment, the glycometabolism index Pn, SPS, SER was significantly higher than that of WT, which was comparable to that of WTOsFLN2Inducible expression under drought stress is closely related and at T ₁ Substitution and T ₂ And (5) generation stable inheritance.

In this case, the number of the elements to be processed is,OsFLN2is a fructokinase-like protein gene,OsHAK1genes that are up-regulated in expression induced by drought/osmotic stress;

to those skilled in the art, verifying the correlation of plants in response to drought stress appears to be:

1. drought stress affects rice sugar metabolism, including but not limited to: net photosynthetic rate of leaf, SPS activity, and sucrose export rate (transport of sucrose from source to pool);

2. the drought inhibits the growth of the overground parts and root systems of plants;

3. influence of drought on the activity of sugar synthesis key enzyme SPS;

4. the influence of drought on the water holding capacity and lipid peroxidation degree of rice;

5. effects of drought on senescence, stress response gene expression;

and other factors not mentioned.

Regarding the transgenic coping mode of rice drought, the following scheme can be also referred to:

prior art 1: CN116121266A discloses rice geneqSS7The application in drought resistance illustrates the drought resistance application of the vascular related protein qSS 7;

prior art 2: CN116121293A disclosesNRT2.1The application of the gene in improving drought resistance and/or high temperature resistance of crops illustrates drought resistance application of nitrate transporter NRT 2.1;

prior art 3: CN115851660A discloses application of protein OsPIS and coding gene in improving stress resistance of plants, and describes rice phosphatidylinositol synthase geneOsPISDrought-resistant application of (2);

prior art 4: CN116218897A disclosesOsGolS2The application of the gene in improving the activity of rice seeds and resisting drought stress illustrates that the gene of the inositolgalactoside synthase is knocked outOsGolS2To improve drought resistance;

prior art 5: CN116064570A disclosesTORThe application of the gene in improving drought resistance and nitrogen utilization efficiency of crops illustrates rapamycin target kinase geneTORDrought-resistant application of (2);

prior art 6: CN115927445A disclosesOsPIL15The application of the gene in regulating water saving and drought resistance of rice illustrates the photosensitive pigment interaction factor geneOsPIL15Coping with drought conditions in the case of knockdown and overexpression;

prior art 7: CN114752573A discloses the application of rice OsGA20ox2 protein and a coding gene thereof in improving the abiotic stress resistance of plants, and illustrates the drought resistance application of OsGA20ox2 (gibberellin 20 oxidase 2);

prior art 8: CN114736890B discloses the use of rice chitinase and its encoding gene in enhancing plant resistance to abiotic stress, illustrating the drought-resistant use of chitinase OsGH 18;

prior art 9: CN114703199A discloses a gene related to drought resistance of plantsTaCML46And application thereof, describes the calmodulin geneTaCML46Drought-resistant application of (2);

prior art 10: CN114438103A discloses transcription factors for regulating drought and salt stress tolerance of riceOsNAC15Gene and application thereof, illustrating NAC transcription factor geneOsNAC15Drought-resistant application of (2);

prior art 11: CN114456248A discloses synergistic regulation of drought and rice blast resistance of riceSex abscisic acid receptorOsPYL2The gene and the application thereof illustrate the drought-resistant application of the abscisic acid receptor OsPYL 2;

prior art 12: CN114085854A discloses a drought-resistant and salt-resistant gene of riceOsSKL2And the application thereof, illustrate shikimate kinase geneOsSKL2Drought-resistant application of (2);

prior art 13: CN113913441A discloses a rice new-born polypeptide binding complex alpha subunitNACAThe application of the gene in the osmotic stress resistance of plants illustrates the drought resistance application of the nascent polypeptide combined complex protein NACA;

prior art 14: CN114015666A disclosesOsPARP3The application of the gene in regulating plant drought tolerance describes the poly ADP-ribose polymerase geneOsPARP3Drought-resistant application of (2);

prior art 15: CN113637682a discloses the application of OsMYB26 or mutants thereof in improving drought stress tolerance of plants, illustrating drought-resistant use of MYB transcription factor OsMYB 26;

prior art 16: CN113337521A disclosesOsNAC78The application of the gene in improving the drought tolerance of rice illustrates the drought resistance application of the NAC transcription factor OsNAC 78;

prior art 17: CN113046368A discloses rice geneOsPM1The application of the gene and the promoter in improving the high temperature stress resistance of rice illustrates the rice membrane protein geneOsPM1Drought-resistant application;

prior art 18: CN112608938A disclosesOsAO2The application of the gene in controlling the drought resistance of rice illustrates the knockoutOsAO2Drought-resistant application after gene;

prior art 19: CN114644690A discloses a rice geneKT572The application of the WRKY transcription factor KT572 in improving the stress tolerance of plants is described;

in addition, CN 114525302A%OsCRKD1Gene), CN 112210567A%OsPPCK2Gene), CN 114507672A%OsSLT1Gene), CN 112143744A%OsPLDδ3Gene), CN 114381467A%OsCRKS2Gene), CN 112322627A%OsZFP1Gene), CN112322598A (dependent glutamate dehydrogenaseGene AcGDH), CN 111909937B%OsUGT55Gene), CN 111961668A%OsSalTGene), CN 111394365A%OsDUF6Gene), CN 111423500A%SiMYB56Protein gene), CN 111334515A%OsSAPK7Gene), CN110577938A (abscisic acid, ABA) 8' -hydroxylase geneOsABA8ox2) CN110643618A (Jatropha curcas MYB transcription factor)JcMYB16Gene), CN110592137A (Arabidopsis thaliana)AT5G10290Gene), CN110656113A (AP 2/ERF transcription factor)OsERF65Gene), CN110760522A (drought-resistant QTL segmentAK209Gene), CN110408605A (GA 20 oxidase OsGA2ox8 protein), CN109400689A (rice transcription factor)OsHRS1Mutant), CN109456982A (MYB protein)OsMYB6Gene), CN111285927B (stress-tolerance-related protein SiWRKY 78), CN109112142A (rice drought-tolerance response gene)OsNMCP1) CN109055390A (AP 2/EREBP transcription factor OsERF 101), CN108314716A (Arabidopsis thaliana)AtXIW1Gene), CN108341858A (nitrate transporter gene)OsNAR2.1) CN108359674A (Rice serine/threonine protein kinase gene)OsSAPK8) CN107828805A (epoxy carotenoid dioxygenase)OsNCED3Gene), CN107881179A (indoleacetic acid amino synthase gene)OsGH3.6) CN108004255A (cytokinin oxidase/dehydrogenase gene)OsCKX4) CN107827963A (Arabidopsis thaliana)IDD14Gene), CN 107557370A%rel1Mutant), CN107326033A (HD-ZIP IV family transcription factor)OsROC4Gene), CN107354163A (RING finger family E3 ubiquitin ligaseOsDHSGene), CN109112147A (mitogen-activated protein kinase)OsMPKK10-2Gene), CN107033230A (basic leucine zipper transcription factor)OsbZIP86Gene), CN106754957A (Carrier protein)OsSCAMP13Gene), CN105543237A (WRKY transcription factor)KT572Gene), CN105255941A (double B-box zinc finger protein)OsBBX14Gene), CN105018522A (Gene AK287958 encoding bHLH domain), CN106318952A (Membrane protein Gene)OsAPM1) Etc.;

therefore, in the prior art, the method for coping with rice drought stress is mostly realized by over-expression or knocking out of characteristic genes; there are many kinds of over-expressed or knocked-out target genes, and there is no report on the response of sucrose transporters (SUTs) to drought stress.

Related prior art regarding SUTs is as follows:

prior art 20: CN103194458A discloses a method for improving phosphorus absorption efficiency of wheat plants by utilizing sucrose transporter gene, and proposes that the phosphorus absorption efficiency is improved by over-expressionTaSUT2The gene makes transgenic wheat have the phenotype of raising phosphorus absorbing efficiency and raising low phosphorus response.

Prior art 21: CN102775480A discloses a sucrose transporter derived from plantsSbSUT5The gene and the application regulate and control carbohydrate metabolism of the sweet sorghum, and finally improve the ethanol production index and economic benefit of the sweet sorghum.

Prior art 22: CN115043919A discloses a cotton sucrose transporter geneGhSUT6The application in improving the salt tolerance of plants; overexpression ofGhSUT6Salt tolerance enhancement and overexpression of Arabidopsis thalianaGhSUT6The arabidopsis thaliana can promote the absorption of sucrose, increase the accumulation of sucrose, has stronger tolerance to NaCl stress and improves the salt tolerance of plants.

Prior art 23: CN103880935a discloses the application of sucrose transporter AtSUT2 in cultivating high-yield transgenic plants, and the transgenic plants are obtained by introducing arabidopsis sucrose transporter 2 (AtSUT 2) into wild plants through genetic means, wherein the rice grain size, biomass and single plant yield of the transgenic plants are higher than those of the wild plants, so that the protein can be proved to be capable of improving the rice grain size, biomass and single plant yield of plants.

Prior art 24: CN101851629B discloses a gene for transforming rice sucrose transporterOsSUT5ZThe method for improving the crop yield and the application thereof, the encoding gene of the OsSUT5Z protein is introduced into a target plant to obtain a transgenic plant, and the method provides available data for other crops and production practices, thereby having great significance in theory and practice.

Prior art 25: CN102250227a discloses plant sucrose transporter, and coding gene and application thereof, and the specification thereof describes:

sucrose transporter (sucrose transporter, abbreviated as SUT or SUC) is a 12-time transmembrane binding protein, widely exists in various tissues and organs of higher plants, is responsible for transmembrane transport of sucrose, and plays an important role in various links of sucrose transport from a source to a warehouse and the like. Knowing the mechanism of action of the regulation of the plant sucrose transporter, revealing the relationship between the sucrose transporter and the adversity stress can help to reveal the regulating function of the sucrose transporter on the growth and development of plants and the adversity resistance, and provide genetic basis for genetic engineering research of improving crop yield and improving quality.

The protein and the coding gene thereof provided by the application can regulate plant growth and development, are induced by various stresses, and participate in response of the leymus chinensis to various stresses. The stress resistance of the plant can be improved by introducing the gene into the plant. In particular, the mowing (cut) can induce the expression of the protein, improve the transportation efficiency of sugar in plants, improve the expression quantity of the sucrose transport protein of crops through a transgenic means, improve the super-compensation growth and grazing tolerance of grasses after mowing, and be beneficial to realizing the healthy and sustainable development of a grassland ecological system. The protein and the coding gene thereof have important practical value for cultivating leymus chinensis and other new plant varieties with improved stress resistance. The method is helpful for understanding the action mechanism of potential internal and external factors on the regulation and control of the sucrose transport protein in the growth and development process of plants, and provides genetic basis for revealing the regulation function of the sucrose transport mechanism on the growth and development and stress resistance of plants and for genetic engineering research of improving crop yield and quality.

As can be seen from an analysis of prior art 20 to prior art 25, the method aims atSUTFor this general class of genes, a certain effect was shown in salt stress (prior art 22) and mowing stress (prior art 25), and further analysis was done for the above two documents as follows:

in prior art 22, the manner of dealing with salt stress is by over-expressionGhSUT6Promoting the sucrose transport capacity to increase sucrose accumulation at roots, increasing cell membrane osmotic pressure and enhancing the plant ability to respond to salt stress; the specific implementation mechanism can be seen in 163 to 17 of the specification of the prior art 22Paragraph 1 describes:

1. overexpression ofGhSUT6DPost-activating the TCA (tricarboxylic acid) cycle pathway, which directly affects the metabolism and transport of sugars in plants;

2. overexpression ofGhSUT6DPost-activation of a phenylpropane signal pathway closely related to up-regulation of three genes involved in cellular redox homeostasis, intracellular homeostasis and cellular homeostasis processes and down-regulated expression of 6 genes involved in translation processes, peptide biosynthesis, peptide metabolism, cellular amide metabolism, cellular nitrogen biosynthesis and amide biosynthesis;

3. overexpression of cotton induces the expression of salt tolerance gene, and reduces Na ⁺ /K ⁺ Ratio of; in particular with Na ⁺ And K ⁺ Up-regulation of expression levels of transport-related genes, includingGhNHX2、GhCHX15、GhCHX20、GhCIPK11、GhNCL、GhCML27Etc.;

it can be seen that in the prior art 22, the factors that cope with the saline-alkali stress are mainly: sucrose transport, phenylpropane signaling pathway, and expression of salt tolerance genes.

In the prior art 25, the number of steps taken in the prior art,LcSUT1the transcription level is obviously induced by stress, under the induction condition,LcSUT1the relative expression quantity of the gene is rapidly increased, and especially the increase of the expression quantity of ABA and mowing (cut) treatment is obvious;LcSUT1can code functional LCST 1 protein, and can complement the yeast mutant strain with the sucrose transporter with the function deleted so as to enable the yeast mutant strain to grow normally. It is clear that when it is against mowing stress, it is possible to cope with stress by promoting sucrose transport.

Furthermore, it can be seen from the analysis of prior art 22 and prior art 25 that the manner in which stress is handled is by means of overexpression.

Finally, it can be seen from the above document thatSUTGene families have multiple members in different species, as is well known in the art, when dealing with environmental stresses,SUTmost genes of the gene family do not play any role, and no document reports are availableSUTGene families have any response to drought stressStudy.

By riceSUTBy way of example, a gene family having 5 members, osSUT1-OsSUT5, each of which can obtain multiple different transcripts at the mRNA level by variable cleavage; in practical experiments we found that not every transcript in large numbers can function, especially well against drought stress.

What needs to be further explained is: in the inventor's prior application (CN 114703221 a), it has been demonstrated that the mere dependence on the transport of sucrose from source to pool is not the only factor in response to drought stress, as is well known in the art, a number of factors must be fully considered in the drought stress response of test plants (5 general factors as shown in prior application CN114703221a, 6 general factors as shown in CN115851660a, 6 general factors as shown in CN116218897aCATAGenes (gene),CATBGenes (gene),CATCGenes (gene),APXGenes (gene),SOD1AndSOD2gene expression these 6 major factors);

the technical problem that the present case solves is: how to improve the capacity of rice to cope with drought stress.

Disclosure of Invention

The first object of the present application is to provide an expression vector which can significantly improve drought resistance of rice under drought stress after being transformed into agrobacterium and infecting the rice.

Meanwhile, the application also discloses a deviceOsSUT1The application of the gene and transgenic rice.

In order to achieve the first object, the present application provides the following technical solutions:

an expression carrier is composed of carrier, functional gene linked to carrier and promoter gene linked to carrier, and said functional gene has nucleotide sequence shown in SEQ ID NO.1OsSUT1A gene; the promoter gene is used for induction under drought stressOsSUT1Gene expression.

In the above expression vector, the promoter isOsHAK1Genes (gene),SNAC1Genes (gene),OsMYB2Genes and the like are genes with obviously up-regulated expression induced by drought stress.

In the above expression vector, the vector is one of pTCK303 vector and pCAMBIA1300 vector.

At the same time, the application also discloses the method for expressing the polypeptide by inductionOsSUT1A method for improving the drought stress response capability of rice by using the gene.

Further, by inducing expressionOsSUT1The gene can improve the transportation capability of sucrose from source to reservoir when rice is subjected to drought stress.

Further, by inducing expressionOsSUT1The gene can improve the drought stress coping capability of the rice and reduce the growth inhibition capability of the overground part and the root system of the rice.

Further, by inducing expressionOsSUT1Gene for improving rice water retention capacity and/or reducing H when rice is subjected to drought stress ₂ O ₂ And the ability of MDA to accumulate.

Further, by inducing expressionOsSUT1Gene for reducing senescence indicator when rice is raised against drought stressSGRAbility to express, reduce stress response genesOsNAC2Capacity of expression, enhancement of stress response genesOsbZIP23And antioxidant genesOsCATBOne or more of the ability to express.

Finally, the application also discloses transgenic rice, the over-expression nucleotide sequence of which is shown as SEQ ID NO.1OsSUT1A gene;

further, the transgenic rice is obtained by transforming the expression vector as described in any one of the above into a bacterial cell, and infecting rice.

In the transgenic rice described above, the bacterial cells are either agrobacterium EHA105 or EHA 101.

Compared with the prior art, the application has the outstanding characteristics that:

1.OsSUT1when the gene is transcribed, different transcripts can be obtained due to different intron cutting mechanisms and RNA editing, and after repeated researches, the unique and effective transcripts can be determined, so that creative labor is paid.

2. For the followingThe management of drought stress is not determined by the factors sucrose from source to pool, as is known to those skilled in the art; it has surprisingly been found that,OsSUT1gene can reduce H in rice water retention capacity ₂ O ₂ And MDA accumulation ability, and aging-reducing indicator geneSGRAbility to express, reduce stress response genesOsNAC2Capacity of expression, enhancement of stress response genesOsbZIP23And antioxidant genesOsCATBThe ability to express is a significant advantage over WT samples; this is a difficult to expect result.

The improvement of the capacity is a key factor for improving drought resistance of transgenic rice during drought stress.

3. Another meaning of the application is that, compared withpHAK1:FLN2The application is based on the optimization of the basic functions of sucrose from source to library, combines with the improvement of the functions of other non-sucrose aspects, and can further facilitate the research on the further optimization of strategies (measures) for improving the drought resistance of rice after excluding the factors of sucrose production, consumption and the like.

The application utilizes drought-induced genesOsHAK1Promoter drive of (2)OsSUT1The expression of the sugar can improve the long-distance transportation of sugar under stress and the synchronous optimization of other functions, and the sugar can reach the aim ofpHAK1:FLN2Similar drought resistance improving effect of rice.

Drawings

FIG. 1 shows the result of drought stress on the upper part of wild riceOsSUT1An influence map of expression;

FIG. 2A is T ₁ Overground part of seedling stage of riceOsSUT1qRT-PCR analysis of (C);

FIG. 2B is T ₁ Sucrose export rate graph of rice, the object represented by each column in fig. 2B refers to the label of the column of fig. 2A;

FIG. 3A shows the growth of seedling stage plants under normal conditions;

FIG. 3B shows the growth of plants at seedling stage under 20% PEG treatment;

FIG. 3C is a graph of the state of overground growth of seedling stage plants;

FIG. 3D is a root growth state diagram of a seedling stage plant, and objects represented by the columns in FIG. 3D refer to the labels of the columns in FIG. 3C;

FIG. 4A is a graph of net photosynthetic rate of rice;

FIG. 4B is a graph of SPS activity of rice, with each column in FIG. 4B representing an object referenced by the column label of FIG. 4A;

FIG. 5A is a graph showing sucrose content in rice leaves;

FIG. 5B is a graph of sucrose content in the roots of rice, and the individual columns in FIG. 5B represent objects referenced to the labels of the columns in FIG. 5A;

FIG. 5C is a SER plot of rice leaves, with each column in FIG. 5C representing an object referenced to the column label of FIG. 5A;

FIG. 6A is a graph of total root length of rice;

FIG. 6B is a plot of root surface area of rice, with each column in FIG. 6B representing an object referenced by the column of FIG. 6A;

FIG. 7A is a graph of relative water content of rice;

FIG. 7B is a graph showing the leaf loss rate of rice;

FIG. 7C is rice H ₂ O ₂ Content map, the object represented by each column in fig. 7C refers to the label of the column of fig. 7A;

FIG. 7D is a graph of MDA content of rice, and the objects represented by the columns in FIG. 7D are referenced by the labels of the columns in FIG. 7A;

FIG. 8A is a rice plantSGRA gene expression level map;

FIG. 8B is a view of riceOsNAC2Gene expression level map, the object represented by each column in fig. 8B refers to the label of the column in fig. 8A;

FIG. 8C is a rice plantOsbZIP23Gene expression level map, the object represented by each column in fig. 8C refers to the label of the column of fig. 8A;

FIG. 8D is a view of riceOsCATBGene expression level map, the object represented by each column in fig. 8D refers to the label of the column in fig. 8A;

FIG. 9A shows WT and example [. About.pHAK1:SUT1-CDS1Abbreviated asHAK1-CDS1）、pHAK1: SUT1-CDS2(abbreviated asHAK1-CDS2）、HAK1-CDS3、HAK1-CDS4Is a phenotype map of (2);

FIG. 9B shows the conditions of WT, example, drought stress simulation,HAK1-CDS2、HAK1-CDS3、HAK1-CDS4Is a phenotype of (a).

Detailed Description

The following description of the embodiments of the present application will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.

The experimental raw materials and equipment used in the application are as follows:

pEASY-Blunt cloning vector: beijing full gold biotechnology Co., ltd;

GBclonart seamless cloning kit: suzhou Shenzhou Gene Co.

Example 1

Preparation of transgenic Rice

Amplification Using Nipponbare (Nipponbare) cDNA as templateOsSUT1The full-length coding sequence CDS1 (1836 bp) of the gene is connected to pEASY-Blunt cloning vector after purification and amplifiedOsHAK1Is ligated to BamHI-cut promoters (3037bp,SEQ ID NO.5 upstream of the initiation codon) using GBclonart seamless cloning kitOsSUT1The intermediate vector pHAK1-SUT1 (SEQ ID NO. 6) was obtained on the linear cloning vector, the pHAK1-SUT1 fragment was amplified using the vector as a template, and after purification, the vector was ligated to the HindIII and SpeI double digested pTCK303 linear vector by the same method to obtain the final vector pTCK303-pHAK1-SUT1. The vector was electrotransformed into Agrobacterium EHA105 and infected with Nippon callus, the genetic transformation method is described in Chen et al (cf. Chen G, hu J, lian J, et al Functional characterization of)OsHAK1promoter in response to osmotic/drought stress by deletion analysis inTransgenic force Plant Growth Regulation, 2019, 88 (3): 241-251), createdpHAK1: SUT1Transgenic rice.

pHAK1:FLN2Transgenic line refers to CN114703221A, and is pOsHAK1:OsFLN2 expression vector for improving sugar metabolism level and drought resistance of rice and application thereof.

T ₀ -T ₂ The transgenic material is planted in a transgenic nursery of Guangdong agricultural sciences institute agricultural quality standard and monitoring technology institute Guangzhou Dafeng test base. Adding 20% (w/v) polyethylene glycol (PEG) 6000 into the water culture IRRI nutrient solution to simulate drought stress (refer to Chen G, feng H, hu Q, et al Improving rice tolerance to potassium deficiency by enhancing)

OsHAK16p:WOX11

Controlled root development Plant Biotechnology Journal, 2015, 13 (6): 833-848.) 3 week old rice was treated for 7 days, phenotypes were recorded, various physiological and biochemical indices were measured, and gene expression was quantitatively analyzed. Experiments were performed in a climatic chamber with a 14h light (30 ℃) to 10h dark (25 ℃) photoperiod and a relative humidity of about 70%, with all treatments changing nutrient solutions every 2 days.

Performance testing

The test method is as follows:

1. determination of the net photosynthetic Rate (Pn) is described in Chen et al (Chen G, liu C, gao Z, et al Variation in the abundance of)OsHAK1transcript underlies the differential salinity tolerance of anindicaand ajaponicarice custivar, frontiers in Plant Science, 2018, 8:2216.), 9 am was measured using a Li-COR6400 portable photosynthesis apparatus (Li-COR, usa): 00 to 11: net photosynthetic rate of 00 rice leaves.

2. Sucrose Phosphate Synthase (SPS) activity measuring leaves were ground into powder by liquid nitrogen, extracted by SPS kit (Suzhou Ming Biotechnology Co., ltd.), and measured by the method of reference Chen et al (reference Chen G, zhang Y, ruan B, et al OsHAK1 controls the vegetative growth and panicle fertility of rice by its effect on potassium-mediated Sugar meta-stability, plant Science, 2018, 274:261-270.).

3. Determination of Sucrose Export Rate (SER) the phloem permeate was collected from the leaves by EDTA method of Chen et al (see Chen G, zhang Y, ruan B, et al, osHAK1 controls the vegetative growth and panicle fertility of rice by its effect on potassium-treated sugam meta solution Science, 2018, 274: 261-270.) and the sucrose concentration in the harvest was determined using a sucrose kit (Suzhou Kogyo Biotech Co.).

4. Determination of sucrose content leaves and root System were ground to powder with liquid nitrogen, extracted with sucrose kit, reference to Chen et al (reference to Chen G, hu J, dong L, et al The tolerance of salinity in rice requires the presence of a functional copy of)FLN2Biomacromolecules, 2020, 10 (1): 17.).

5. Real-time fluorescent quantitative PCR (qRT-PCR) was described in Chen et al (see Chen G, hu Q, luo L, et al Rice potassium transporter OsHAK1 is essential for maintaining potassium-mediated growth and functions in salt tolerance over low and high potassium concentration ranges. Plant, cell)&Environmental, 2015, 38 (12): 2747-2765.) to extract RNA from the corresponding parts of WT and each transgenic line under normal and drought treatment. Rice UBQ5 (LOC_Os01g 22490) is used as an internal reference gene, and is described in Chen et al (see Chen G, wu C, he L, et al Knocking out the gene)RLS1induces hypersensitivity to oxidative stress and premature leaf senescence in Rice International Journal of Molecular Sciences, 2018, 19 (10): 2853.) the algorithm determines relative expression abundance and quantitative primer sequences are shown in Table 1.

TABLE 1 primers for fluorescent quantitative PCR

Gene name	Forward primer (5 '-3')	Reverse primer (5 '-3')
			UBQ5	CTCGCCGACTACAACATCCA	TCTTGGGCTTGGTGTACGTCTT
OsSUT1	CGGTGACCCAAAGGGAACT	TGCCCTGACACCCTGGTT
			SGR	GCAATGTCGCCAAATGACG	GCTCACCACACTCATTCCCTAAAG
OsNAC2	AAAAACAACCGCATTGGCAG	AGTCCTCATCTCCTCTGTCTAATCC
			OsbZIP23	GGAGCAGCAAAAGAATGAGG	GGTCTTCAGCTTCACCATCC
OsCATB	GGTGGGTTGATGCTCTCTCA	ATTCCTCCTGGCCGATCTAC

6. Determination of total root length and root surface area is described in Chen et al (see Chen G, liu C, gao Z, et al Driving the expression of)RAA1with a dry-responsive promoter enhances root growth in rice, its accumulation of potassium and its tolerance to moisture stress, environmental and Experimental Botany, 2018, 147:147-156.), root systems were scanned using a root system analyzer (winrhizov 4.0b, regent Instrument, canada) and total root length and root surface area were recorded. Each treatment measures 5 individual plants per strain.

7. Measurement of relative moisture content and loss of water is described in Chen et al (see Chen G, liu C, gao Z, et al Driving the expression of)RAA1with a dry-responsive promoter enhances root growth in rice, its accumulation of potassium and its tolerance to moisture stress, environmental and Experimental Botany, 2018, 147:147-156.) to determine relative water content and in vitro leaf loss rate.

8. Hydrogen peroxide (H) ₂ O ₂ ) And Malondialdehyde (MDA) content measuring blade was ground into powder by liquid nitrogen, using H ₂ O ₂ And MDA kit (Suzhou Kogyo Biotechnology Co., ltd.) were separately extracted and measured by the method of Chen et al (refer to Chen G, liu C, gao Z, et al OsHAK1, a high-affinity potassium transporter, positively regulates responses to drought stress in price Frontiers in Plant Science, 2017, 8:1885.).

9. Statistical analysis the significance of differences between different strains and treatments was analyzed by Tukey method comparison of SPSS10 software, with different letters and asterisks indicated inPThere was a significant difference at the < 0.05 level, ns indicating no significant difference.

Test results

1.OsSUT1Response of genes to drought stress

qRT-PCR results show that drought stress significantly inhibits overground partsOsSUT1Is shown in FIG. 1. 20The transcript levels were reduced after%peg treatment 1 h, minimized after 6 h, and then the expression levels were maintained substantially 45% -55% under normal culture conditions (fig. 1).

Description of the drawings:OsHAK1transient expression in the aerial parts of drought-treated wild rice (Nippon-Qing). Seedlings of rice were grown in normal IRRI solution for 14 days and then transferred to nutrient solution containing 20% PEG for various periods of time (0, 1, 3, 6, 12 and 24 h). The expression level of treatment 0h was set to 1. The values show the mean ± SE (n=3).

2. ConstructionpHAK1:SUT1Transgenic rice

By passing throughpHAK1:SUT1And (3) carrying out differential analysis on drought response between the transgenic strain and the WT, and verifying whether the drought resistance of the rice can be improved by promoting long-distance transportation of sugar. Identification of positive plants by GUS staining, T ₀ A total of 21 independent transgenic lines were obtained. Select 5T ₁ Analysis of the Generation Positive lines and their corresponding segregant-free isolates (NS)SUT1The expression level and sucrose export rate of (2) show that under normal and drought conditions, there is no significant difference between NS and WT; drought inhibitionSUT1Expression in the aerial parts of WT and NS, but in transgenic plants,SUT1up-regulated by 2-fold (fig. 2A); at the same time, the rate of sucrose export was significantly lower in magnitude than both WT and NS (FIG. 2B), so subsequent experiments selected two homozygous T's that were all positive for GUS staining ₂ Substitution ofpHAK1:SUT1Strain and WT as the sole negative control.

Description of the drawings: fig. 2A: overground part of rice seedling stageOsSUT1qRT-PCR analysis of (C). Fig. 2B: sucrose export rate. 5 isolates without the fragment of interest were pooled as control 2 and 5 transgenic positive lines were pooledpHAK1:SUT1. The values show the mean ± SE, the different letters indicating significant differences at the P < 0.05 level.

3.pHAK1:SUT1Influence on the growth of rice in seedling stage

Culturing WT, T in normal and 20% PEG in nutrient solution ₂ Substitution ofpHAK1:SUT1AndpHAK1:FLN2homozygous transgenic lines, evaluationpHAK1:SUT1Is of (2)The effect on the growth and drought response of rice is achieved. Under normal culture conditions, transgenic plants were not significantly different from the growth of WT, and the aerial parts and root biomass were similar (fig. 3A and 3B). Under drought stress, WT leaves seriously wilt, and the water loss phenotype of transgenic plants is obviously reduced, and drought is opposite to that of plantspHAK1:SUT1AndpHAK1:FLN2the extent of inhibition of growth of both the transgenic lines was similar, significantly lower than WT, resulting in PEG treated aerial parts with dry weights higher than WT 7-12% and 13-14%, respectively (fig. 3C), and root systems with dry weights higher than WT 11-13% and 14-17%, respectively (fig. 3D).

Description of the drawings: fig. 3A-3B: growth of seedling stage plants under normal and 20% PEG treatment, scale = 5cm. C-D: biomass (dry weight) of the aerial parts (C) and root systems (D). The values show the mean ± SE (n=5), with and without significant differences between WT and transgenic lines at P < 0.05 level, respectively.

Further additional description: in the following several figures, the icon labels appear:pHAK1:SUT1-L1、pHAK1: SUT1-L2、pHAK1:FLN2-L1、pHAK1:FLN2-L2；

wherein L1 and L2 represent two strains obtained by the same method, the same expression vector and the same bacterial strain for the rice.

For example,pHAK1:SUT1-L1、pHAK1:SUT1-l2 represents two strains obtained by the same method and bacterial infection using pTCK303-pHAK1-SUT1 expression vector; same reasonpHAK1:FLN2-L1、pHAK1:FLN2-L2 is also of similar meaning.

4.pHAK1:SUT1Influence on sugar synthesis in Rice

First, for WT,pHAK1:SUT1AndpHAK1:FLN2transgenic plant leaf sugar synthesis level is analyzed and compared to verifypHAK1:SUT1Whether the expression of (a) improves drought resistance of rice mainly by promoting sugar transport, but not other sugar metabolism processes.

Consistent with the previous study results, under PEG treatment,pHAK1:FLN2the net photosynthetic rate (Pn) and SPS activity of the transgenic plants were significantly higher than that of WT (FIG. 4A), whereaspHAK1:SUT1The transgenic lines were not significantly different from the WT (fig. 4B), indicating that they were compared to WTpHAK1:FLN2In a different manner, the processing time is different,pHAK1:SUT1the expression of (2) does not alter sugar synthesis under drought stress.

Description of the drawings: fig. 4A: net photosynthetic rate. Fig. 4B: SPS activity. The values show the mean ± SE (n=5), with and without significant differences between WT and transgenic lines at P < 0.05 level, respectively. FW: fresh weight.

5.pHAK1:SUT1Influence on sugar transport in Rice

To WT,pHAK1:SUT1AndpHAK1:FLN2the sugar transport level of the transgenic plants is analyzed and compared, and the sugar transport level of the transgenic plants is foundpHAK1:SUT1AndpHAK1:FLN2the expression of the sugar can obviously improve the long-distance transportation of sugar under drought stress, and is particularly expressed aspHAK1:SUT1AndpHAK1:FLN2SER of leaves of transgenic lines was 31-35% and 26-36% higher than WT, respectively (FIG. 5C), whereaspHAK1:SUT1The sucrose content in the leaves and roots of the plants was 8-17% lower and 10-18% higher than WT, respectively (fig. 5A, fig. 5B).

Sugar is used as an osmotic substance, participates in abiotic stress response, helps plants resist adverse conditions,pHAK1:SUT1andpHAK1:FLN2the SER and root sucrose contents in the transgenic plants are similar and significantly higher than those of WT, which is beneficial to improving tolerance.

Description of the drawings: fig. 5A-5B: sucrose content in leaf (a) and root (B). C: sucrose Export Rate (SER). The values show the mean ± SE (n=5), with and without significant differences between WT and transgenic lines at P < 0.05 level, respectively. FW: fresh weight.

6.pHAK1:SUT1Influence on the root system configuration of rice

Definite and clearpHAK1:SUT1AndpHAK1:FLN2whether the transgenic plants and the WT root systems respond to drought or not are different.

PEG treatment significantly reduced total root length and root surface area for all plants (FIGS. 6A and 6B), but the effect on WT roots was relatively strong, whereaspHAK1:SUT1AndpHAK1:FLN2the root systems of the transgenic lines are similarly inhibited to a degree which is obviously lower than that of WT, and the transgenic lines are stressedpHAK1:SUT1AndpHAK1:FLN2the total root length of the plants was 16-19% and 11-27% higher than WT (fig. 6A), respectively, while the root surface area was 13-20% and 9-14% higher than WT (fig. 6B), respectively.

The biomass, total root length and root surface area of the root system under drought stress are all significantly higher than WT, which may be related to a sufficient supply of sucrose as a nutrient to the root system. Biomass and morphology of the root system are key factors for determining the acquisition capacity of water and nutrient of plants, so that optimization of the root system configuration is in positive correlation with drought resistance, and PEG treatment is performedpHAK1:SUT1AndpHAK1: FLN2the transgenic strain maintains a more luxuriant root system and contributes to the improvement of stress resistance at the morphological level.

Description of the drawings: fig. 6A: total root length. Fig. 6B: root surface area. The values show the mean ± SE (n=5), with and without significant differences between WT and transgenic lines at P < 0.05 level, respectively.

7.pHAK1:SUT1Influence on the water holding capacity and lipid peroxidation degree of rice

Drought stressed WT,pHAK1:SUT1AndpHAK1:FLN2the RWC of the transgenic lines were reduced,pHAK1:SUT1andpHAK1:FLN2no significant difference in RWC, but all significantly higher than WT (fig. 7A), the difference in RWC may be due to the difference in water loss rates of the plants: the WT in vitro leaf loss rate was higher than that of the transgenic strain (FIG. 7B), and these results indicatepHAK1:SUT1Expression of (2)pHAK1:FLN2Similarly, the water retention capacity of the rice is remarkably improved.

Under normal conditions, WT and transgenic rice plants have similar H ₂ O ₂ And MDA content, and after PEG treatment,pHAK1: SUT1andpHAK1:FLN2h of transgenic lines ₂ O ₂ The levels were below WT 8-15% and 15-18%, respectively (fig. 7C) and the MDA levels were below WT 13-20% and 17-19%, respectively (fig. 7D). In conclusion, the method comprises the steps of,pHAK1:SUT1andpHAK1:FLN2the drought resistance of transgenic lines is similar and significantly higher than that of WT, probably due to the stronger water retention capacity and less H of the plants ₂ O ₂ And MDA accumulation.

As drought stressIs unfavorable for plants to cope with drought stress, drought stress causes accumulation of ROS in rice, and excessive generation of ROS leads to membrane lipid structure damage. The main product of lipid peroxidation of plant cell membranes is MDA, and under drought stress, the accumulation amount of MDA is continuously increased. After PEG treatmentpHAK1:SUT1AndpHAK1: FLN2transgenic line H ₂ O ₂ And MDA content is significantly lower than WT, indicating that drought-induced cell membrane damage is lower in transgenic plants than in WT.

Description of the drawings: fig. 7A: relative water content. Fig. 7B: the rate of water loss from the blade. Fig. 7C: h ₂ O ₂ The content is as follows. Fig. 7D: MDA content. The values show the mean ± SE (n=5), with and without significant differences between WT and transgenic lines at P < 0.05 level, respectively. FW: fresh weight.

8.pHAK1:SUT1Influence on senescence, stress response and antioxidant-associated Gene expression

To further clarifypHAK1:SUT1The mechanism for improving drought resistance of rice for normal and drought stress WT,pHAK1:SUT1AndpHAK1:FLN2the transgenic lines were subjected to differential analysis of gene expression.

The genes selected are classified into three categories:SGRbelongs to the aging indicator gene, and belongs to the field of aging indicator genes,OsNAC2andOsbZIP23is a stress response gene, which is a stress response gene,OsCATBis an antioxidant related gene.

Under normal conditions, the differences between the selected genes in WT and transgenic plants were not apparent (FIGS. 8A-8D).

After drought treatment, the expression level of the gene is obviously increased compared with that of a control plant, and inpHAK1:SUT1AndpHAK1: FLN2up-regulation in strains is similar in magnitude and different from WT, resulting inSGRAt the position ofpHAK1:SUT1AndpHAK1:FLN2only 43-70% and 41-50% of WT in the plants (figure 8A),OsNAC2only 64-82% and 62-67% of the WT, respectively (FIG. 8B), andOsbZIP23at the position ofpHAK1:SUT1AndpHAK1:FLN2plants were 1.36-1.58 and 1.46-1.83 times WT (figure 8C),OsCATBare 1.39-1.43 and 1.53-1.92 times WT, respectively (fig. 8D).

Senescence-inducedSGRGenes play an important role in regulating chlorophyll degradation and are overexpressedSGRResulting in oxidative stress and plaque-like cell death in rice seedlings, and is used as a marker gene for leaf senescence.SGRAt the position ofpHAK1:SUT1AndpHAK1: FLN2the trend of the transgenic lines under drought induction and enhanced expression is similar and is obviously lower than that of WT, which indicates thatpHAK1:SUT1Expression of (2)pHAK1:FLN2Can slow down the aging of the leaves caused by PEG treatment.

Many studies have shown that differential expression of stress/ABA response genes enhances tolerance of plants to various stresses. OsNAC2 is passed through andOsAP37andOsCOX11is combined with the promoter to influence the accumulation of ROS and negatively regulate the drought tolerance of rice.OsbZIP23Is induced by drought, through positive regulationOsPP2C49AndOsNCED4affects ABA signal transduction and biosynthesis, and participates in drought stress response of rice. In the present application, as compared with WT after drought treatment,pHAK1:SUT1in transgenic plantsOsNAC2AndOsbZIP23the degree of induction is lower and higher, respectively, and is equal topHAK1:FLN2The expression trend of these genes is consistentpHAK1:SUT1Another important reason for the improvement of drought resistance of transgenic lines.

Catalase (CAT, EC.1.11.1.6) is an important antioxidant enzyme and plant CATs are usually encoded by three isozyme genes, in rice, there are also 3 isozyme genesOsCATA、OsCATBAndOsCATCwater stress inhibitionOsCATAAndOsCATCbut significantly improves the expression ofOsCATBIs a factor (B) of the expression level of (C). Drought inductionOsCATBUp-regulated expression, andpHAK1: SUT1andpHAK1:FLN2the transgenic lines were significantly amplified above WT.OsCATBMay be one of the important ways of alleviating oxidative damage in PEG-treated transgenic plants;

these results indicate that the inhibition of the upregulated expression of drought-induced senescence-indicating genes, negative-regulatory stress-responsive genes, and the promotion of the upregulated expression of drought-induced positive-regulatory stress-responsive genes, antioxidant-associated genes, arepHAK1:SUT1Drought resistance of transgenic linesOne of the important reasons for the improvement of the sex.

Description of the drawings: the genes quantitatively detected areSGR（A），OsNAC2（B），OsbZIP23(C) AndOsCATB(D) A. The application relates to a method for producing a fibre-reinforced plastic composite The values show mean ± SE (n=3), sum representing significant differences between WT and transgenic lines at levels P < 0.05 and P < 0.01, respectively, ns representing no significant differences.

9.OsSUT1Preparation of transgenic Rice from multiple transcripts of (A)

The application is characterized in thatOsSUT1In the transcription of the gene into mRNA, 4 transcripts were obtained by amplification, the transcripts used in example 1 beingSUT1-CDS1The method comprises the steps of carrying out a first treatment on the surface of the In addition, there are three transcriptsSUT1-CDS2（SEQ ID NO.2）、SUT1-CDS3（SEQ ID NO.3）、SUT1-CDS4（SEQ ID NO.4）。

Comparative examples 1 to 3

Using the procedure of example 1, transcripts were usedSUT1-CDS2、SUT1-CDS3、SUT1-CDS4Three transgenic rice plants are respectively prepared;

reference may be made to fig. 9A and 9B;

description of the drawings: FIG. 9A shows the WT, examples, and conditions under normal culture,HAK1-CDS2、HAK1-CDS3、HAK1-CDS4Is a phenotype of (2);

FIG. 9B shows the conditions of WT, example, drought stress simulation,HAK1-CDS2、HAK1-CDS3、HAK1-CDS4Is a phenotype of (2);

as can be seen from fig. 9A, under normal culture conditions, there was no significant difference between the plants;

as can be seen from FIG. 9B, the transgenic rice of the example was significantly better than WT,HAK1-CDS2、HAK1-CDS3、HAK1-CDS4Phenotype of (3) evenHAK1-CDS2、HAK1-CDS3、HAK1-CDS4Less than the WT samples;

rice plantSUTThe gene family hasOsSUT1～OsSUT5These 5 members, each of which can obtain multiple different transcripts at the mRNA level by variable cleavage; from the point of view of gene screening, this would result in tens of transcripts based on different splice formations;

further findings in a more advanced study,OsSUT1different transcripts formed by different splicing of gene introns are greatly different in practical application, and only OsSUT1-CDS1 used on the embodiment carrier can achieve the effect of improving drought resistance of rice, so that the variety of an intron cutting mechanism and an RNA editing mode can be proved to bring completely different results more definitely, and the finally used gene screened by the application has unpredictable creativity.

Summary

The innovative research and discovery of the present application is as follows:

1. rice plantOsSUT1When the gene is transcribed, different transcripts can be obtained due to different intron cutting mechanisms and RNA editing, and after repeated researches, the unique and effective transcripts can be determined, so that creative labor is paid.

2. For those skilled in the art, the response to drought stress is not determined by the factor sucrose from source to pool; it has surprisingly been found that,OsSUT1gene can reduce H in rice water retention capacity ₂ O ₂ And MDA accumulation ability, and aging-reducing indicator geneSGRAbility to express, reduce stress response genesOsNAC2Capacity of expression, enhancement of stress response genesOsbZIP23And antioxidant genesOsCATBThe ability to express is a significant advantage over WT samples; this is a difficult to expect result.

The improvement of the capacity is a key factor for improving drought resistance of transgenic rice during drought stress.

3. Another meaning of the application is that, compared withpHAK1:FLN2The application is based on the optimization of the basic functions of sucrose from source to warehouse, combines with the improvement of the functions of other non-sucrose aspects, and can further facilitate the research on the further optimization of the strategy (measure) for improving the drought resistance of rice after excluding the factors of sucrose production, consumption and the like.

The application utilizes drought-induced genesOsHAK1Promoter drive of (2)OsSUT1The expression of the sugar can improve the long-distance transportation of sugar under stress and the synchronous optimization of other functions, and the sugar can reach the aim ofpHAK1:FLN2Similar drought resistance improving effect of rice.

The foregoing examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application.

Claims (1)

1. Inducible expression of nucleotide sequence shown in SEQ ID NO.1OsSUT1The application of the gene in improving the drought stress response capability of rice is characterized by utilizing the drought induction type geneOsHAK1Promoter drive of (2)OsSUT1Expression of the genes.