CN117165626A - Method for improving salt tolerance of rice through sucrose transporter - Google Patents
Method for improving salt tolerance of rice through sucrose transporter Download PDFInfo
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- CN117165626A CN117165626A CN202311128952.7A CN202311128952A CN117165626A CN 117165626 A CN117165626 A CN 117165626A CN 202311128952 A CN202311128952 A CN 202311128952A CN 117165626 A CN117165626 A CN 117165626A
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Abstract
The application discloses a method for improving salt tolerance of rice by sucrose transport gene, and application of a gene OsSUT5, a vector containing the gene OsSUT5 or a host cell containing the gene OsSUT5 in improving salt tolerance of rice or preparing transgenic rice or in rice breeding; a method for improving salt tolerance of rice by using the gene OsSUT 5; the method utilizes the transgenic technology to over-express the key sucrose transporter OsSUT5 of the rice to promote the transportation and secretion of photosynthetic carbon into root systems and rhizosphere soil, improve the salt tolerance of the rice and promote the growth and grain yield of the rice under salt stress.
Description
Technical Field
The application belongs to the technical field of plant genetic engineering, and relates to a technology and application for improving salt tolerance of rice by utilizing a rice sucrose transporter gene OsSUT5.
Background
China has abundant backup farmland resources such as beach and saline-alkali soil, but is unfavorable for crop growth due to high soil salinity content (Ambastha, V., sopory, S.K., tiwari, B.S., tripathy, B.C.,2017.Photo-modulation of programmed cell death in rice leaves triggered by salinity. Apoptosis 22, 41-56.). Besides the engineering measures of salt reduction and salt elimination with high cost, the utilization of the saline-alkali soil also needs to combine biological and molecular biological technologies such as salt-tolerant crop breeding, salt-tolerant crop molecular breeding and the like.
In recent years, research and demonstration on 'sea rice' are carried out, and the technology is mainly implemented by selecting a salt-tolerant rice variety and combining desalination and seawater irrigation, has high cost and cannot be implemented in a large area at present. In improved tidal flats and saline-alkali lands, rice growth still faces the threat of high salt stress, which results in poor root growth, blocked nutrient absorption and utilization, and various metabolic disorders in the body, and lower yield. Wherein due to a large amount of Na + The accumulation in plants causes oxidative damage to photosynthetic organs, thereby weakening the synthesis of photosynthetic carbohydrates and the distribution and turnover of the photosynthetic carbohydrates in bodies, leading to insufficient carbon sources and carbon libraries in the plants, and being a key factor affecting the rice yield in saline-alkali soil (Abdullah, z., khan, m.a., flowers, T.J.,2001.Causes of Sterility in Seed Set of Rice under Salinity Stress.J.Agron.Crop Sci.187,25-32.). The oxidative damage of photosynthetic organs caused by environmental stress of plants such as rice can be alleviated by exogenously adding (root application or foliar spray) sucrose, and there have been numerous reports (Yang, S.Y., hao, D.L., jin, M, li, Y., su, Y.H.2020, international ammonium excess induces ros-mediated reaction and causes carbon scarcity in, etc., LI, Y, zhou, J.Y., hao, D.L., yang, S.Y., su, Y.H.,2020.Arabidopsis under ammonium over-supply: characteristics of ammonium toxicity in relation to the activity of ammonium transmitters, pedosphere,30 (3), 314-325). However, the continuous application of exogenous sucrose can cause the breeding of germs and the death of root systems, so the method cannot be popularized and applied in production.
Sucrose is the main transportation form of photosynthetic carbon in plants, so that the promotion of the transportation and distribution capacity of the sucrose of rice per se is a new effective idea for absorbing high salt stress and improving salt tolerance and yield. Meanwhile, the reinforcement of the sucrose transmission link can promote the efficient synthesis of photosynthetic carbon through feedback action. The transport and partitioning of sucrose in plants is mediated by specific sucrose transporters (SUTs). SUT is a class of transmembrane transporters that are capable of transporting sucrose from source tissue (e.g., leaves) to sink tissue (e.g., root system, stalk, grain, etc.) (Dhungana, s.r., braun, D.M.,2021.Sugar transporters in grasses:Function and modulation in source and storage tissues.J.Plant Physiol.266,153541.). There are 5 SUT genes in the rice genome, designated OsSUT1-OsSUT5, respectively. Wherein OsSUT5 is a SUT gene specifically expressed in phloem and root system, and is related to sucrose transport function (Sun, Y., reindex, A., lafleur, K.R., mori, T., ward, J.M.,2009.Transport activity of rice sucrose transporters OsSUT1 and OsSUT5.Plant and Cell Physiology,51 (1), 114-122.). At present, the correlation between the gene and the salt tolerance of rice has not been reported.
Disclosure of Invention
Aiming at the problems, the application provides a transgenic technology for improving the salt tolerance of rice based on the sucrose transporter OsSUT5 of the rice, which is easy to popularize and apply in the field.
Specifically, the application is realized by the following technical scheme:
firstly, the application provides the application of the gene OsSUT5 in improving the salt tolerance of rice or preparing transgenic rice or breeding rice, and the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11.
Secondly, the application provides the application of the vector containing the gene OsSUT5 in improving the salt tolerance of rice or preparing transgenic rice or in rice breeding, and the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11. Preferably, the vector containing the gene OsSUT5 comprises a ubiquitin promoter and a terminator NOS. Further preferably, the vector is a pCAMBIA1301 vector comprising a ubiquitin promoter and terminator NOS.
Third, the present application provides the use of a host cell for improving salt tolerance of rice or for preparing transgenic rice or for breeding rice, the host cell comprising the gene OsSUT5 or comprising a vector comprising the gene OsSUT 5; the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11. Further preferred, the host cell is Agrobacterium tumefaciens.
Fourth, the present application provides a method for improving salt tolerance of rice by sucrose transporter, comprising: a) Transforming the gene OsSUT5 and a vector containing the gene OsSUT5 into a rice plant; alternatively, b) transforming the gene OsSUT5 or a vector containing the gene OsSUT5 into a rice plant; alternatively, c) infecting rice plants with the host cell; the host cell contains a gene OsSUT5 or contains a vector containing the gene OsSUT 5; the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11.
Preferably, the above-mentioned transformation of the gene OsSUT5 and/or the vector containing the gene OsSUT5 into a plant means that the gene OsSUT5 and/or the vector containing the gene OsSUT5 is transformed into a plant by an Agrobacterium tumefaciens transformation method.
The full-length coding sequence of the OsSUT5 gene (the nucleotide sequence of which is shown as SEQ ID NO.11 and the protein sequence of which is shown as SEQ ID NO. 12) is obtained from a rice cDNA library through amplification by a high-fidelity PCR technology, cloned into a pUN1301 vector and placed under the drive of a monocot specific strong promoter ubiquitin. The expression vector is transferred into an agrobacterium tumefaciens strain GV3101, and bacterial liquid is cultured and amplified and then is transformed into rice (Japanese sunny) callus for over-expression, so as to construct an OsSUT5 over-expression rice plant material. The regenerated rice plants are subjected to hygromycin resistance screening and PCR verification, the obtained transgenic lines are subjected to propagation, and single copy plants with stable expression are obtained through homozygous line screening, namely the rice with improved salt tolerance (such as sodium ions) is obtained. In one embodiment of the application, the single copy plants obtained are further subjected to OsSUT5 gene abundance detection, and the result shows that compared with the reference gene, the OsSUT5 gene abundance is up-regulated by 13 times.
The application discovers the physiological function of the OsSUT5 gene (http:// rice. Uga. Edu/, LOC_Os02g 36700) in the in-vivo sucrose transportation of rice for the first time and applies the physiological function to the improvement of the salt tolerance of the rice. The mutant and the over-expression material of the OsSUT5 gene are obtained by using a transgenic technology, so that the dominant effect of the OsSUT5 in the transportation of sucrose in phloem of rice is clarified, and the practical application of the OsSUT5 transgenic technology in improving the salt tolerance of rice, promoting the growth and increasing the yield is verified by a full-growth period cultivation test. Compared with the prior art, the application provides a novel method for improving the salt tolerance of rice from the viewpoint of enhancing in-vivo sucrose transportation for the first time, which has the following advantages:
1) Repairing photosynthetic injury: the rice plant over-expressing the OsSUT5 gene can absorb oxidative damage caused by salt stress to photosynthetic organs of rice leaves through strengthening in-vivo sucrose transportation and distribution process, and improves the generation and distribution efficiency of photosynthetic products.
2) Promoting rice growth and yield formation: the rice plant over-expressing the OsSUT5 gene can improve the growth of rice under salt stress, increase the seed setting rate and grain weight, and has better salt-resistant yield-increasing application prospect.
3) Improving the rhizosphere soil environment: the rice plant over-expressing the OsSUT5 gene can promote the active carbon reservoir capacity of the rhizosphere of the rice through root carbon secretion and residue, and is helpful for promoting the recruitment of beneficial microorganisms of the rhizosphere and inhibiting pathogenic microorganisms.
Drawings
FIG. 1 is a schematic diagram showing the cloning result of a Osk-1 gene of rice; wherein M1 is 250bp DNA ladder marker,lane 21 is OsSUT5 (BamHI/KpnI PCR result) of rice.
FIG. 2 is a map of an overexpression vector (pUN 1301-OsSUT 5) used in OsSUT5 transgenic rice material.
FIG. 3 shows the comparison result of OsSUT5 transgenic enhanced expression homozygous lines.
FIG. 4 shows the mutation site results of Ossut5 mutant homozygous rice material.
FIG. 5 shows the evaluation results of growth conditions and yield indexes of rice materials under salt stress conditions.
FIG. 6 shows in vivo sucrose transport, photosynthetic carbon synthesis and Na synthesis of rice plants under salt stress conditions + Accumulation and oxidative damage index measurement results.
FIG. 7 shows the results of rhizosphere activated carbon and microbial copy number analysis of rice material under salt stress conditions.
Detailed Description
The raw materials, reagents involved in the examples: the materials and reagents used in the following examples were purchased from commercial sources unless otherwise specified.
pUN1301 vector (containing the ubiquitin strong promoter): the pCAMBIA1301 vector containing the ubiquitin promoter and terminator NOS is constructed by the applicant laboratory, and the vector construction method is a conventional technology in the field, and is carried out according to the method disclosed by the patent 'corn stomata open type potassium ion absorption channel Zm-1 molecule and application thereof' (CN 108440657A). In practice, any conventional vector comprising a Ubiquitin and a NOS terminator may be used to achieve the objects of the application.
Agrobacterium tumefaciens strain GV3101: purchased from south Beijing, department of biotechnology, inc.
Rice (japan): purchased from Beijing, unknown Kaitou agricultural biotechnology Co.
Cloning primer nucleotide sequence of OsSUT5 gene:
P1 CAGGTCGACTCTAGAGGATCCATGGAGGAAGGCCGCCGT(SEQ ID NO.1)；
P2 CTAGATCGGGAGCTCGGTACCCTAGTGACCGCCGGCGAC(SEQ ID NO.2)；
identification primer nucleotide sequence of OsSUT5 transgene expression abundance:
P3 AGCATCAAGGCTGTCTGCCTCG(SEQ ID NO.3)；
P4 GCGCGATGACCACCTGAGGGATGACGAT(SEQ ID NO.4)。
selection medium: n6 in a large amount of 25mL, N6 in a trace amount of 2.5mL, vitamin in a 2.5mL, fe 2 + EDTA stock solution 2.5mL,2,4-D (2, 4-dichlorophenoxyacetic acid) stock solution 0.625mL (Sigma, USA), enzymatic hydrolysis of casein (also called enzymatic hydrolysis of casein) 0.15g, sucrose 7.5g, agarose 1.75g, pH adjustment to 6.0, dH 2 O constant volume to 250mL. Wherein N6 is a large number (KNO) 3 28.3g,(NH 4 ) 2 SO 4 4.63g,KH 2 PO 4 4g,MgSO 4 ·7H 2 O 1.85g,CaCl 2 1.25g,dH 2 0 to 1000 ml), N6 trace (KI 0.08g, H 3 BO 3 0.16g,ZnSO 4 ·7H 2 O 0.15g,MnSO 4 ·4H 2 O 0.44g,dH 2 Constant volume O to 1000 ml); vitamin (Vitamin B30.1g, vitamin B10.1g, vitamin B60.1g, inositol 10g, glycine 0.2g, dH) 2 O constant volume to 1000 mL); fe (Fe) 2 + EDTA stock solution (FeSO) 4 ·7H 2 O2.78 g in 300ml dH 2 O, another 300ml of dH 2 O was heated to 70℃and dissolved 3.73g Na 2 EDTA·2H 2 O and cooling to room temperature, mixing the two solutions and using dH 2 Constant volume O to 1000 ml); 2,4-D (100 mg of 2,4-D, dH was dissolved with 1ml of 1N KOH shaking 2 O constant volume to 100 mL); 1N KOH (100 mL dH) 2 O dissolves 5.6g KOH). The above reagents were purchased from national pharmaceutical group chemical reagent limited except for individual labeling.
Differentiation medium: MS major amount 100mL, MS trace amount 10mL, vitamin 10mL, fe 2 + 10mL of EDTA stock solution, 2mL of 6-BA (6-benzylaminopurine), 2mL of kinetin (Sigma, USA), 0.2mL of IAA (indoleacetic acid), 0.2mL of NAA (naphthaleneacetic acid), 30g of sucrose, 1g of casein enzymatically hydrolyzed (Beijing Soy Corp., ltd.), 3g of Phytagel (plant gel), pH adjustment to 6.0, dH 2 O is fixed to 1000mL. Wherein MS is a plurality of (NH) 4 Cl 16.5g,NaNO 3 16.5g,KH 2 PO 4 1.7g,KNO 3 19g,MgSO 4 ·7H 2 O3.7g,CaCl 2 3.32g,dH 2 Constant volume O to 1000 ml); MS trace (MnSO) 4 ·4H 2 O 2.23g,ZnSO 4 ·7H 2 O0.86g,KI 0.083g,H 3 BO 3 0.62g,Na 2 MoO 4 ·2H 2 O 0.025g,CoCl 2 ·6H 2 O 0.0025g,CuSO 4 ·5H 2 O 0.0025g,dH 2 Constant volume O to 1000 ml); 6-BA (100 mg 6-BA, dH was dissolved by shaking with 1ml 1N KOH) 2 O constant volume to 100 mL); kinetin (100 mg kinetin, dH dissolved by 1ml 1N KOH concussion) 2 O constant volume to 100 mL); IAA (100 mg IAA, dH dissolved by shaking with 1ml of 1N KOH 2 O constant volume to 100 mL); NAA (100 mg NAA, dH dissolved with 1ml 1N KOH shaking) 2 O constant volume to 100 mL). The above reagents were purchased from national pharmaceutical group chemical reagent limited except for individual labeling.
EXAMPLE 1 cloning of Rice OsSUT5 Gene
1. Cloning of Rice OsSUT5 Gene
Total RNA of Nippon Rice grown in water for 10 days was extracted using TRIZOL reagent (Nanjinopran) and used as a template, and 1. Mu.g of RNA sample was subjected to reverse transcription using HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wind) kit (Nanjinopran) to obtain a reverse transcription product cDNA. According to the full-length coding sequence of the rice OsSUT5, designing primers for amplifying a complete coding reading frame, designing the primers according to requirements, and introducing different enzyme cutting sites on each primer to obtain an upstream primer P1 (SEQ ID NO. 1) and a downstream primer P2 (SEQ ID NO. 2).
PCR amplification was performed using high fidelity enzyme (50. Mu.L, 2X Phanta Max Master Mix was 25. Mu.L, 1. Mu.L each for the upstream and downstream primers (10. Mu. Mol/L concentration), 1. Mu.L for the total cDNA template, and ddH was added 2 O was made up to 50. Mu.L.
PCR reaction procedure: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30sec, annealing at 65℃for 30sec, extension at 72℃for 2min,30 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
The result of the electrophoresis detection gave a single product of about 1.6kb (FIG. 1). The PCR product was subjected to 1% agarose gel electrophoresis, and the gel block containing the target band was excised under ultraviolet irradiation, and the target fragment was recovered using a gel recovery kit (MACHEREY-NAGEL, T40609.250).
2. Construction of the overexpression vector
The target band recovered above was constructed into pUN1301 vector via cleavage site BamHI/KpnI by homologous recombination method using kit ClonExpress IIOne Step Cloning Kit (purchased from Nanjinouzan, see kit description for specific procedures), and after transformation of E.coli competent cell DH5 alpha (purchased from Optimum of the family Pratiaceae, cat# TSC-C14, see kit description for specific procedures), the plasmid was extracted and submitted for sequencing identification by Nanjinoqing biological sciences Co. The size of the target gene sequencing result is 1608bp, which is completely consistent with the reported OsSUT5 (http:// rice. Uga. Edu/, LOC_Os02g 36700) sequence, and shows that the recombinant plasmid pUN1301-OsSUT5 is successfully obtained, and the plasmid map is shown in figure 2.
3. Preparation of transgenic OsSUT5 rice material
The recombinant plasmid pUN1301-OsSUT5 obtained by the above construction was transformed into the rice Japanese variety by the Agrobacterium tumefaciens-mediated method (conventional method in the art, see the literature "Valvekens, D., van Montagu, M.and Van Lijsebettens, M. (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl Acad. Sci. USA,85,5536-5540." method disclosed).
Inducing rice to mature, picking out the callus growing to a proper size, placing the callus into agrobacterium suspension to infect for 5-10 minutes, taking out the callus, placing the callus on sterile filter paper to drain for 30-40 minutes, placing the callus on a co-culture medium, culturing the callus in dark at 28 ℃ for 3 days, transferring the callus on a selection medium containing 50mg/L hygromycin for screening, picking out the resistance callus with bright yellow color, transferring the resistance callus into a culture dish filled with a differentiation medium, and placing the culture dish into a constant temperature incubator to differentiate into seedlings. And then placing the strong seedlings in a rooting culture medium for one to two weeks. OsSUT5 overexpressing rice strain OsSUT5-OE was obtained by identification (gene abundance identification of OsSUT5 overexpressing material, FIG. 3).
Gene abundance identification of OsSUT5 overexpressing material
The total RNA of Nippon Rice and transgenic material rice grown in water culture for 10 days was extracted using TRIZOL and reverse transcribed using 1ug as a template to obtain cDNA (cloning of rice OsSUT5 gene using reagent and kit step 1).
The expression level of the OsSUT5 gene of the transgenic material is analyzed by real-time quantitative PCR, and an upstream primer P3 and a downstream primer P4 are designed by taking OsActin as an internal reference gene.
The real-time quantitative PCR reaction system was 20. Mu.L: 2X ChamQ Universal SYBR qPCR Master Mix is 10. Mu.L, the upstream and downstream primers (10. Mu. Mol/L) are each 0.4. Mu.L, the cDNA template is 1. Mu.L, and ddH is added 2 O was made up to 20. Mu.L.
Real-time quantitative PCR reaction procedure: pre-denaturation at 95 ℃ for 30sec; denaturation at 95℃for 10sec, annealing at 60℃for 30sec, elongation at 72℃for 30sec,40 cycles; dissolution profile was obtained by treating 15sec at 95℃for 60sec, and treating 15sec at 60℃for 95 ℃.
The analysis result is shown in figure 3, which shows that the OsSUT5 over-expression transgenic enhanced expression homozygous strain with 13 times background gene abundance is obtained, namely the OsSUT5 over-expression Japanese sunny OsSUT5-OE.
As a countercheck material, the embodiment simultaneously utilizes CRISPR/cas9 targeted gene editing technology to create a mutant material (OsSUT 5) of the OsSUT5 gene, which entrusts the generation of Wohan Bober remote biotechnology Co. Successful knockdown of the OsSUT5 gene product (protein) was demonstrated by sequencing the resulting osSUT5 mutant homozygous rice material (FIG. 4), which inserted a T base at 319bp (third exon) of the OsSUT5 gene coding sequence, resulting in premature termination of protein translation (111 aa).
EXAMPLE 2 salted soil pot test
1. Potted plant test material
A small plastic barrel with the same size is used as a container for soil filling test, and 3kg of soil is filled in each barrel (typical rice soil for common maturing in Jiangsu, black grid soil). To simulate the salt content of 1/3 of seawater, a mild saline soil experiment is carried out, and the chemical reagent grade NaCl is mixed into Na + The final concentration was 0.1% (i.e. the salt content was 0.1%). The application amount of the nitrogen, phosphorus and potassium elements refers to the fertilizer application standard of the high-yield rice in southwest: 0.916g NH is applied to 3kg soil 4 Cl，2.26g NaH 2 PO 4 ·2H 2 O and 1gK 2 SO 4 . Rhizosphere characteristics of transgenic rice were studied using root bag methods (Nie, s., li, h., yang, x., zhang, z., weng, b., huang, f., zhu, g.b., zhu, Y.G.,2015.Nitrogen loss by anaerobic oxidation of ammoniumin rice rhizosphere.ISME J.9,2059-2067.). The root bags were nylon nets of 30 μm with a diameter of 7 cm and a height of 12 cm, placed in barrels and filled with soil (the soil volume is included in 3kg per barrel), and each root bag was planted with 1 plant of rice. When the root system is full of the root bag, the soil in the root bag and the soil far away from the root bag correspond to rhizosphere soil and non-rhizosphere soil respectively for subsequent analysis and determination.
The rice seeds filled with WT (wild Nippon Temminck), osSUT5 (OsSUT 5 gene mutant Nippon Temminck obtained in example 1) and OsSUT5-OE (OsSUT 5 overexpressing Nippon Temminck obtained in example 1) of uniform size were sterilized under sterile conditions, germinated in clear water, and transplanted after 20 days of seedling age by culture using a rice nutrient solution formula (all reagents were purchased from national institute of medicine chemical Co., ltd.) published by International Rice institute (IRRI) for pot culture.
2. Transplanting and management of test rice materials
And taking out seedlings 20 days after germination, and planting 3 seedlings in each barrel of each plant line, wherein 1 seedling is planted in the root bag. 3 replicates were set. And respectively dressing 0.687g NH for each barrel on 10 th day and 40 th day after transplanting 4 Cl. The whole growth period keeps the same watering volume and frequency of each barrel. And if necessary, unified pest control measures are adopted.
3. Test results
The OsSUT5 transgene promotes the growth and yield of rice under the condition of salt stress as shown in FIG. 5, and the representative photographs (A) and (B) in FIG. 5 show the growth and single spike phenotype of rice plants in the grouting period respectively. It can be seen that under salt stress conditions, transgenic rice (OsSUT 5-OE) has greater biomass and a single-spike seed setting rate than wild-type rice; while mutant ossut5, which served as a negative control, was significantly weaker in growth and ear yield than wild-type and transgenic rice. This demonstrates that OsSUT5 transgenes are effective in overcoming the inhibition of rice plant growth and yield formation by salt stress, whereas mutant materials are more susceptible to salt stress and exhibit a greater degree of growth and yield-limiting characteristics than wild-type. This also demonstrates from the other side the close relationship between OsSUT5 sucrose transporter activity and rice salt tolerance.
The results of statistical analysis of the data at harvest time are shown in FIG. 5 (C), which shows that under salt stress, osSUT5-OE rice has a 27% increase in biomass over WT, while OsSUT5 mutant rice has a 50% decrease in biomass over WT; as shown in (D) of FIG. 5, the individual yield OsSUT5-OE and OsSUT5 mutants were increased by 38% and decreased by 75% respectively compared with WT rice, confirming the great contribution of the gene to rice yield under salt stress conditions.
Further, the yield was determined by the setting rate among the yield factors, and the result is shown in FIG. 5 (E); as shown in (F) of FIG. 5, it was found that the promotion effect of OsSUT5-OE on grain yield increase was mainly represented by an increase in seed setting rate (+35%) and grain weight (+14%; in contrast, the fruiting rate and hundred grain weight of ossut5 mutant were reduced by 54% and 39% respectively compared to WT rice. Therefore, the OsSUT5 transgene promotes the yield increase of the rice under the condition of salt stress mainly by increasing the biomass of the rice and improving the fruiting rate and the grain weight.
Example 3 analysis of mechanisms of OsSUT5 transgenesis to improve salt tolerance in Rice
1. Identification of transgenic material sucrose partitioning, photosynthetic carbon assimilation and salt tolerance physiology under salt stress condition
The photosynthetic products of plants are mainly delivered to various parts and organs of the plants through phloem in the form of sucrose, so that the sucrose level in phloem wound fluid reflects the strength of sucrose transport capacity in vivo. The phloem wound fluid of 20-day-old rice in example 2 was collected, and the sucrose content in the wound fluid was measured using a sucrose content measuring kit (purchased from nanjing) and the results are shown in fig. 6 (a): compared with WT, the phloem sucrose content of OsSUT5-OE is improved by 30%, while OsSUT5 is reduced by 35%, so that the dominant position of OsSUT5 in transportation and distribution of rice phloem sucrose is verified, the activity of the gene is enhanced by a transgenic technology, and transportation of phloem sucrose can be effectively promoted.
The net photosynthetic rate (Pn) of the leaf of rice was measured during the grouted period using a portable photosynthetic apparatus (LI-6400 XT) and the photosynthetic carbon fixation rate of rice was evaluated. The specific operation is as follows: light synthesis analyzer of LI-6400 portable blue light and red light source is used for measuring the light concentration of 100 mmol.m 2 s -1 PPFD and 500 mmol.s -1 Photosynthesis was measured at the flow rate. 9:00-11:00AM, under outdoor condition (30-35 ℃, relative humidity 67% -79%, illumination intensity 1200 mmol.m) 2 s -1 ，CO 2 Concentration of 400 mmol.mol –1 ) The net photosynthetic rate of the sword leaf of rice was measured, and then the CO2 fixation rate was calculated from the net photosynthetic rate, and the result was shown in FIG. 6 (B): compared with WT, the photosynthetic carbon fixation rate of OsSUT5-OE is improved by 30%, which indicates that the OsSUT5-OE can fix more CO in unit time 2 Further sucrose is generated for transport and distribution; whereas the photosynthetic carbon assimilation rate of ossut5 was drastically reduced by about 60% compared with WT, it is apparent that the lack of production and distribution of carbon source in vivo was caused.
In order to further clarify the transportation and distribution of sucrose in plants and the relation between the sucrose and OsSUT5 gene activity, samples of the Sword leaves (source), the three leaves (stock), the stem nodes (stock) and the root system (stock) of the rice subjected to salt treatment are collected in the rice grouting period in the embodiment 2, fresh samples are mixed with liquid nitrogen and ground, the sucrose synthase activity of the Sword leaves is measured by using a sucrose synthase kit (Nanjing built), and the sucrose content of each part is measured by using a sucrose content measuring kit (Nanjing built). The results are shown in fig. 6 (C): under the salt treatment condition, the sucrose content of the OsSUT5-OE rice sword leaf is obviously lower than that of WT (-17%), and the sucrose content of the inverted three leaves (+38%), the stems (+33%) and the root system (+30%) is obviously higher than that of WT, so that the OsSUT5 transgene has the function of promoting the strong transmission and distribution of sucrose from source tissues to organs in a body under the salt treatment condition.
In contrast, the ossut5 mutant rice had a content of Sword She Zhetang (source tissue) 16% higher than that of WT, whereas sucrose in the pool organs, the three leaves, the nodes and the roots were much lower than that of WT 56%, 60% and 55%, respectively, indicating that the sucrose transport and distribution process in rice was strongly inhibited.
2. Na of rice plant under salt stress + Accumulation and oxidative damage identification
Collecting rice plant samples in the grouting period, deactivating enzyme and grinding the samples, and then passing through H 2 SO 4 -H 2 O 2 After the method is performed for decoking, a flame photometer is used for analyzing the Na+ content of the plant, and the result is shown in (D) in fig. 6, compared with WT, the Na+ content of OsSUT5-OE is reduced by 21%, and the Na+ content of OsSUT5 is increased by about 50%, which indicates that the Na+ accumulation amount of the rice plant can be reduced by the OsSUT5 transgene, the Na+ toxicity is reduced, and the Na+ accumulation and the toxic effect can be aggravated by the gene mutation. This phenomenon can be further confirmed by accumulation of cytoplasmic membrane peroxo Malondialdehyde (MDA). The sword-leaved leaves of each rice plant were collected during the grouting period, ground after adding liquid nitrogen, and the MDA content of the sword-leaved leaves was measured using an MDA content measuring kit (built by Nanjing). As shown in FIG. 6 (E), the MDA content of OsSUT5-OE was reduced by 43% and the MDA content of OsSUT5 was increased by 38% as compared to WT. The reduction of MDA content shows that OsSUT5-OE can effectively relieve peroxidation injury of rice caused by salt stress. Therefore, osSUT5 can improve the salt tolerance of rice by reducing the Na+ accumulation of plants and reducing the peroxidation damage of cells.
Example 4 OsSUT5 transgenic improvement of active carbon library of rhizosphere of Rice under salt stress, recruitment of rhizosphere-beneficial microorganism
In example 2, at the late tillering stage of rice, collecting rhizosphere soil in WT, osSUT5-OE and OsSUT5 root bags and non-rhizosphere soil at a position distant from the root bags, and measuring the soluble carbon (DOC) content of rhizosphere and non-rhizosphere soil by using a low-temperature external thermal potassium dichromate oxidation-colorimetry method, and the results are shown in (A) of FIG. 7: the DOC content of the soil in the non-rhizosphere area is not significantly different; in rhizosphere soil, compared with WT, the DOC content of OsSUT5-OE is improved by 25%, the DOC content of OsSUT5 is reduced by 36%, which shows that the OsSUT5 transgene remarkably improves the storage capacity of rhizosphere active carbon sources, and the effect is possibly related to the enhancement of root system carbon secretion and activation capability.
Using FastDNA TM SPIN Kit for Soil kit (MP Biomedicals, U.S.A., see kit description for specific procedures), extracting DNA from rhizosphere soil and non-rhizosphere soil samples of WT, osSUT5-OE and OsSUT5, performing fluorescent quantitative analysis by using quantitative instrument Quantum studio3, respectively using Bacillus specific upstream and downstream primers P5/P6 (nucleotide sequences of which are shown as SEQ ID NO.5 and SEQ ID NO. 6), burkholderia specific upstream and downstream primers P7/P8 (nucleotide sequences of which are shown as SEQ ID NO.7 and SEQ ID NO. 8), and Pythium specific upstream and downstream primers P9/P10 (nucleotide sequences of which are shown as SEQ ID NO.9 and SEQ ID NO. 10), respectively analyzing copy numbers of the soil bacteria genus and pathogenic bacteria genus, respectively, to indicate changes of soil microorganisms. The method comprises the following specific steps:
taking 1ug of total DNA of each sample obtained above as a template, and carrying out quantitative PCR: the conditions are that the mixture is pre-denatured at 95 ℃ for 30 seconds, then denatured at 95 ℃ for 10 seconds, renatured at 60 ℃ for 30 seconds, and then 40 cycles are carried out, and finally a dissolution curve is obtained at 95 ℃ for 15 seconds, 60 seconds and 95 ℃ for 15 seconds; the quantitative results were plotted after analysis. Screening analysis shows that the copy numbers of two beneficial microorganisms of Bacillus (shown in (B) in FIG. 7) and Burkholderia (shown in (C) in FIG. 7) in rhizosphere bacteria of OsSUT5-OE are increased by 35% and 33% respectively compared with the WT rhizosphere, and have obvious rhizosphere recruitment characteristics.
The copy number of the rhizosphere pathogenic bacteria of OsSUT5-OE, namely the genus Pythium, is reduced by 22% compared with the WT rhizosphere. In contrast to this, ossut5 exhibited 44% and 25% lower copy numbers of Bacillus rhizosphere and Burkholderia, respectively, while the copy numbers of Pythium were increased by 80% above WT (shown as B-D in FIG. 7).
These results demonstrate that the OsSUT5 transgene can promote recruitment of beneficial microorganisms in the rhizosphere of rice under salt stress and inhibit the abundance of pathogenic bacteria, which may be related to the lifting effect of the active carbon library in the rhizosphere soil.
The above examples prove that the transgenic technology of the sucrose transporter OsSUT5 can obviously improve the salt tolerance of rice, has the effects of promoting growth and increasing yield of seeds with value, and has better effects of improving the active carbon reservoir capacity of the rhizosphere of the rice and recruiting beneficial microorganisms. The gene OsSUT5 can be used for preparing transgenic plants or for plant breeding.
Claims (9)
1. The gene OsSUT5 is used for improving the salt tolerance of rice or preparing transgenic rice or rice breeding, and the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11.
2. The carrier containing the gene OsSUT5 is used for improving the salt tolerance of rice or preparing transgenic rice or rice breeding, and the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11.
3. The host cell is used for improving the salt tolerance of rice or preparing transgenic rice or used for rice breeding, and contains a gene OsSUT5 or contains a vector containing the gene OsSUT5, and the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11.
4. The use according to claim 2, wherein the vector containing the gene OsSUT5 comprises a ubiquitin promoter and terminator.
5. The use according to claim 3, wherein the host cell is agrobacterium tumefaciens.
6. The use according to claim 4, wherein the terminator is terminator NOS.
7. A method for improving salt tolerance of rice by sucrose transporter gene, which comprises transforming gene OsSUT5 and/or a vector containing gene OsSUT5 into rice or infecting rice with a host cell containing gene OsSUT5 or a vector containing gene OsSUT 5; the nucleotide sequence of the gene OsSUT5 is shown as SEQ ID NO. 11.
8. The method according to claim 7, wherein the transformation of the gene OsSUT5 and/or the vector containing the gene OsSUT5 into a plant means transforming the gene OsSUT5 and/or the vector containing the gene OsSUT5 into rice by using Agrobacterium tumefaciens transformation method.
9. The method of claim 7, wherein the host cell is agrobacterium tumefaciens.
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