CN103710356B - A kind of method improving rice phosphorus specific absorption - Google Patents
A kind of method improving rice phosphorus specific absorption Download PDFInfo
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Abstract
The present invention discloses a kind of method improving rice phosphorus specific absorption.The present invention utilizes the full-length cDNA of pcr clone OsSUT2 gene, builds by the plant expression vector of corn Ubi promoters driven, obtains process LAN OsSUT2 trans-genetic hybrid rice by agrobacterium-mediated transformation.Transgenic homozygous paddy rice plant phosphorus absorbed dose under low-phosphorous growth conditions significantly improves, and plant height, tiller number, the number of blade and spike number significantly increase, and fringe examples explain, setting percentage also have increase trend.The present invention has excavated the novelty teabag of OsSUT2 gene.
Description
Technical field
The present invention relates to plant genetic engineering, be specially a rice sucrose transporter gene OsSUT2 and improving the application in rice phosphorus absorption.
Background technology
Phosphorus is one of most important element of growth and development of plants.The unavailable phosphorus mode being difficult to absorb with plant due to the phosphorus major part in soil exists, and the available phosphorus in the arable land of 30-40% can not meet the needs that plant normal growth is grown in the world.Crop lacks phosphorus phenomenon ubiquity.In addition due to the physicochemical property of soil, the utilization ratio of phosphate fertilizer this season also only has 10% ~ 25%, and the excessive phosphate fertilizer used brings the problem such as the wasting of resources, environmental pollution.Therefore the crop varieties cultivating efficiency utilization phosphate fertilizer is the basic of the above-mentioned series of problems of solution.
Plant is a complicated process to the absorption of phosphorus and utilization, long-term with soil Interaction, the a series of adaptation mechanism of plant evolution, as the quantity by increasing root and surface-area, increase the absorption to Soil Phosphorus, utilize mycorhiza to form symbiotic relationship and obtain phosphoric, and the expression changing phosphorus metabolism genes involved improves the mechanism such as the recycle of phosphorus.
Sucrose is the substrate of growth and development of plants and energy metabolism, and research in recent years finds: sucrose is important semiochemicals, excites and take part in the pathways metabolism of a lot of plant, increases sucrose and can improve plant to environment stress adaptability.
Sucrose transporter is a class high hydrophobicity albumen, be responsible for transhipment and the distribution of sucrose in plant materials, it is positioned on plant cell membrane, containing 12 membrane spaning domains, N-end and C-end are all positioned at cytoplasmic one side, medium surface has a large ring to the part of kytoplasm, and be divided into by albumen respectively containing two portions of 6 membrane spaning domains, N-terminal may play a decisive role to the affinity of substrate.
The major function of Sucrose Transporters in Plants is the transmembrane transport of sucrose in mediated plant body, and sucrose is preserved at the handling of phloem, the sucrose of storehouse tissue and supplied, the response of abiotic and biotic, interaction etc. between plant-microorganism have important relationship.Discovered in recent years, the conduction of sucrose transporter and invertase signal is closely related, the likely adaptation reaction that lacks phosphorus of involved in plant.The present invention extends the function of sucrose transporter.
Summary of the invention
The object of the present invention is to provide a kind of method improving rice phosphorus specific absorption, construct p1300UbiOsSUT2 expression vector, overexpression OsSUT2 gene in paddy rice, transgenic paddy rice shows low-phosphorous physiological compatibility in low-phosphorus stress environment, improve assimilated efficiency and the utilising efficiency of phosphorus, result of study utilizes paddy rice significant to cultivation phosphorus efficiency.
For achieving the above object, the present invention adopts following technical scheme:
Improve a method for rice phosphorus specific absorption, realized by overexpression encoding sucrose transporter gene OsSUT2 in paddy rice.
Described overexpression OsSUT2 gene, gene is numbered AB091672, the cDNA total length 1505bp of gene, and 501 amino acid of encoding, gene order is as shown in SQE ID NO:1.
Described overexpression utilizes PCR method homologous clone to go out paddy rice OsSUT2 gene, adopts F-primer:SQE ID NO:2 and R-primer:SQE ID NO:3 to amplify OsSUT2 gene cDNA; Build overexpression plant vector p1300UbiOsSUT2; Obtain by agrobacterium-mediated transformation and turn OsSUT2 trans-genetic hybrid rice; The homozygous lines of overexpression OsSUT2 gene is obtained by selfing separation screening.
Intermediate carrier pUbiOsSUT2 enzyme cuts back to close and is connected with p1300 and obtains by described plant expression vector p1300UbiOsSUT2; The rice sucrose translocator OsSUT2 cDNA be connected on T-easy sequencing vector is cut restructuring to plasmid pBluescriptIIKS through EcoRI enzyme, obtain intermediate carrier pBlueOsSUT2, then BamHI/KpnI double digestion intermediate carrier is used, OsSUT2 forward in pBlueOsSUT2 is connected to before terminator T-nos, obtains intermediate carrier pUbiOsSUT2; PUbiOsSUT2 enzyme being cut back to close UbiOsSUT2 fragment is connected on plasmid P1300, obtains conversion carrier P1300UbiOsSUT2, and conversion carrier P1300UbiOsSUT2 is imported agrobacterium strains LBA4404, obtains p1300UbiOsSUT2/LBA4404 engineering bacteria.
Conversion comprises the following steps:
1) induction of embryo callus subculture;
2) genetic transformation of embryo callus subculture;
3) screening of kanamycin-resistant callus tissue;
4) differentiation of resistant plant, root culture.
Improve a method for rice phosphorus specific absorption, its concrete steps comprise following:
1) using OsSUT2 gene fragment as applying gene, design primer, amplifies coding region, using strong promoter Ubi as driving, builds monocotyledon expression vector;
2) by agrobcterium-mediated transformation, overexpression transgenic paddy rice is obtained.
Described rice varieties is bright extensive 86, is transformed by Agrobacterium p1300UbiOsSUT2/LBA4404, obtains overexpression OsSUT2 trans-genetic hybrid rice plant.Described genetic transforming method specifically comprises the following steps:
1) seed disinfection and callus of induce
2) prepared by Agrobacterium p1300UbiOsSUT2/LBA4404
3) to infect and Dual culture
4) kanamycin-resistant callus tissue screening
5) kanamycin-resistant callus tissue differentiation, seedling are taken root.
Transgenic plant is transplanted, detect, filter out the transfer-gen plant of overexpression, is recombinated by selfing, obtains transgenic homozygous strain.Transgenosis high-generation homozygosis paddy rice is in low-phosphorus stress environment, and total root length, tip of a root number and root surface area, root/shoot ratio significantly improve, and plant total phosphorous significantly improves.
The present invention constructs p1300UbiOsSUT2 Overexpression vector, overexpression OsSUT2 gene in paddy rice, transgenic paddy rice shows low-phosphorous physiological compatibility in low-phosphorus stress environment, improve assimilated efficiency and the utilising efficiency of phosphorus, result of study can be used for cultivating phosphorus efficiency and utilizes rice varieties, reduce phosphate fertilizer in Rice Production process, the pollution reducing soil, water body and environment has expected using value, extends the function of OsSUT2 gene in theory.
Beneficial effect of the present invention is as follows:
1. the invention provides a kind of new opplication method of rice sucrose transporter gene.The present invention, by genetic transfoumation, obtains overexpression sucrose transporter gene OsSUT2 paddy rice; In low-phosphorus stress environment, the biomass of transgenic paddy rice increases by 43.74%, and root/shoot ratio improves 18.56%, and individual plant phosphorus content improves 24.24% than non-transgenic paddy rice.
2. the present invention is by overexpression sucrose transporter gene OsSUT2 in paddy rice, changes rice root configuration, and root total length adds 20.20%, root total surface area adds 21.92%, total radical adds 21.79%, and rice phosphorus receptivity improves.The present invention is for phosphate fertilizer in minimizing Rice Production process, and the pollution reducing soil, water body and environment has expected using value.
3. the present invention is by carbon distribution mode in transgenic regulation paddy rice body, reaches Crop Improvement nutrien utilization object, for providing new thinking by molecule manipulation Crop Improvement kind.
Accompanying drawing explanation
Fig. 1 .p1300UbiOsSUT2 carrier schematic diagram.
Fig. 2. the plant phenotype in seedling stage of transgenic paddy rice under low-phosphorus stress.Wherein A is transgenic paddy rice, and B is non-transgenic paddy rice; Transgenic paddy rice root system is more compared with non-transgenic paddy rice, blade is wider, leaf look dark green.
Fig. 3. transgenic paddy rice productive phase plant phenotype under low-phosphorus stress.Wherein A is transgenic paddy rice, and B is non-transgenic paddy rice; Transgenic paddy rice tiller number is more compared with non-transgenic paddy rice, spike number is more.
Fig. 4. transgenic paddy rice single-strain blade number under low-phosphorus stress.Wherein D86 represents contrast; DM4 represents transgenic paddy rice.* significant difference is represented; Compared with non-transgenic paddy rice transgenic paddy rice whole vegetative period plant the number of blade all remarkable in contrast.
Fig. 5. transgenic paddy rice individual plant tiller number under low-phosphorus stress.Wherein D86 represents contrast; DM4 represents transgenic paddy rice.* significant difference is represented; Transgenic paddy rice tillers digital display work more than contrast when heading stage compared with non-transgenic paddy rice.
Fig. 6. the root/shoot ratio of transgenic paddy rice different growing stages under low-phosphorus stress.Wherein D86 represents contrast; DM4 represents transgenic paddy rice.* significant difference is represented; Compared with non-transgenic paddy rice transgenic paddy rice seedling stage and tillering phase root/shoot ratio be significantly higher than contrast, but the ripening stage is significantly lower than contrast.
Fig. 7. the total grain number of transgenic paddy rice individual plant under low-phosphorus stress.Wherein D86 represents contrast; DM4 represents transgenic paddy rice.* significant difference is represented; Transgenic paddy rice total grain digital display work is more than contrast compared with non-transgenic paddy rice
Fig. 8. the phosphorus content of transgenic paddy rice different sites harvesting time and complete stool under low-phosphorus stress.Wherein D86 represents contrast; DM4 represents transgenic paddy rice.* significant difference is represented; The content of tatal phosphorus of the fringe of transgenic paddy rice, leaf, root and whole strain is significantly higher than contrast compared with non-transgenic paddy rice.
Embodiment
implement a rice sucrose translocator OsSUT2 full length cDNA clone
1. the clone of rice sucrose translocator OsSUT2
According to (Aoki N, Hirose T, waits The sucrose transporter gene family in rice .Plant Cell Physiol. 2003 Mar; 44 (3): 223-32.) gene number of registering in literary composition, OsSUT2 gene (GenBank:AB091672.1) total length CDS is checked in, design of amplification primers F primer:5'-ATGCCGCGGCGGCCTAGCGGCGGCG-3' and R primer:5'-TTATCGGTGACCTCTCCTCCTTGAT-3' by NCBI.Amplify OsSUT2 coding region, amplification condition is: (1) 94 DEG C of 5min; (2) 94 DEG C of 3min, 55 DEG C of 3min, 72 DEG C of 3min, react 35 circulations; (3) 72 DEG C extend 10min and reclaim fragment, be connected on T-easy sequencing vector, order-checking.
2. Overexpression vector builds
Rice sucrose translocator OsSUT2cDNA warp on T-easy sequencing vector will be connected to
ecorI enzyme cuts restructuring to plasmid pBluescriptIIKSm, obtains intermediate carrier pBlueOsSUT2, then warp
bamhI/
kpni double digestion intermediate carrier pUSCK, is connected to OsSUT2 fragment forward before terminator T-nos, obtains intermediate carrier pUbiOsSUT2.PUbiOsSUT2 enzyme being cut back to close UbiOsSUT2 fragment is connected on plasmid P1300, obtains conversion carrier P1300UbiOsSUT2.Carrier is shown in accompanying drawing 1.
implement two turns of OsSUT2 overexpression rice materials to obtain
Adopt agrobacterium-mediated transformation to obtain and turn OsSUT2 trans-genetic hybrid rice, concrete steps are as follows:
1. embryonic callus induction and subculture
The prematurity fringe of 20d after the pollination of water intaking rice, with 75% alcohol immersion 1min after peeling off, proceed in the chlorine bleach liquor of 25% the sterilizing 25min that vibrates again, aseptic water washing 4 times in Bechtop, chooses the rataria of seed after sterilization and is inoculated in inducing culture, about 30, every ware, in 28 DEG C of light culture 7d, excision radicle, continues cultivation 7 days d, after callus is grown up, carries out succeeding transfer culture.Suitable embryo callus can be selected from the third generation for Agrobacterium-mediated Transformation.
2. the preculture of callus
Choose nature dispersion, particulate state callus that color cadmium yellow, diameter are about 3mm, be transferred in precultivation medium, be placed in 27 DEG C of light culture 4d.
3. Agrobacterium is cultivated and bacterium solution preparation
From low temperature (-85 DEG C) cryopreservation tube, scrape the bacterium liquid that takes a morsel be inoculated in additional 50mg.L
-1kantlex and 50mg.L
-1on the YEB solid medium of Rifampin, carry out bacterial strain activation in 28 DEG C of light culture 72h; Bacterium is drawn in single bacterium colony switching of getting on activation flat board, after 28 DEG C of cultivation 2h, with being added with 100 μMs of .L
-1the AAM liquid nutrient medium of Syringylethanone, by bacterium wash-out, is used containing 100 μMs of .L
-1the AAM liquid nutrient medium constant volume of Syringylethanone is to about 20ml, and after violent shake 1min, adjustment bacterial concentration, to OD600nm=2.0, leaves standstill 1h, for subsequent use.
4. agroinfection and Dual culture
The callus of preculture 2d is transferred in aseptic triangular flask, add above-mentioned Agrobacterium bacterium liquid, slightly leave standstill 30min after shake, go Agrobacterium, the callus infected is placed in and is inoculated on Dual culture base after aseptic filter paper dries 60min, 25 DEG C of light culture 3d.
5. remove Agrobacterium
Callus after picking Dual culture, in aseptic triangular flask, with aseptic water washing 4 times, shakes for several times, until lose thread thalline in water at every turn.Last use is containing 100mg.L
-1the sterilized water of carboxylic Bian penicillin soaks 60min, then outwells liquid, is placed on aseptic filter paper dries removing the callus after bacterium.
6. resistant calli screening
Callus is transferred to containing 30mg.L
-1cultivate in the Selective agar medium of Totomycin, switching in every two weeks 1 time, after 3 weeks, resistant calli grows.
7. transformed plant regeneration
Be transferred to by resistant calli on division culture medium, after 2 weeks, callus starts to turn green, and can put out new shoots after 3 weeks, root also grows subsequently.Seedling is moved on root media, 1, every culturing bottle clone.After seedling takes root and grows up to, shift out culturing bottle, after cleaning the substratum on root, move to greenhouse pot culture.
embodiment three. transgenic paddy rice Tolerant to low P is tested
1. the low-phosphorous cultivation of transgenic paddy rice
Isozygoty paddy rice for material, with non-transgenic reference bright extensive 86 for contrast with transgenosis T4 generation.Get full rice paddy seed, room temperature is soaked seed, and after 2d, seed is placed in 30 DEG C of incubator vernalization 2d, and after seed germination, seedling moves into cultivates 5d containing in 1/2MS substratum.Being moved into by seedling high for about 5cm is that matrix contains in the cultivated box without soil of the liquid nutrient medium of different phosphate concentration and cultivates with quartz sand, and incubator is placed in rainproof, and surrounding is ventilated, in sun-drenched plastic greenhouse.Quartz sand soaks 5d with dilute hydrochloric acid in advance, then uses deionized water shower number time, cleans hydrochloric acid, measures pH value.Between incubation period, 1 week seedling stage mended one time of nutrition liquid, within after tillering phase 3 days, add one time of nutrition liquid, the nutritive medium that displacement in every 2 weeks is once new, deionized water rinsing quartz sand is used before replacing new nutrition, growing period is looked rising situation and is mended deionized water, ensures that in each incubator, moisturizing volume is identical during moisturizing.The formula (IRRI discussion paper series NO 22 " Screening rice for salinity tolerance ") that paddy rice soilless culture nutrient fluid is researched and developed with reference to International Rice, in formula, phosphorus content is set to 2 phosphorus process: low-phosphorous formula phosphorus amount is reduced to 1mgL
-1, be 1/10 in formula, all the other constituent contents are constant; Normal phosphorus formula is then complete in recipe configuration.In table 1.
table 1. International Rice institute rice nutrition liquid formula
2. the metamorphosis of transgenic paddy rice in low-phosphorous environment
After seedling cultivates the 35th day, carry out examining seedling.Often 4 strain materials are got in process, repeat for 3 times.After clear water is cleaned, statistics tiller number, the number of blade and height of seedling.The plant underground part investigated and overground part are separately loaded in little paper bag, indicate numbering, the baking oven putting into 105 DEG C completes 30 min, then dries to constant weight at 80 DEG C of temperature, weighs.Root structure is analyzed: often 3 strains are got in process, after cleaning substratum, are cut by root, with EPSON scanner scanning (pixel is dpi300), and image ten thousand dark root system analysis software.
3. the receptivity of the phosphorus of transgenic paddy rice in low-phosphorous environment
Paddy rice full phosphorus flow measurement: paddy rice to be measured tap water wash clean, then rinse several times with deionization.After sample 105 DEG C of 30min that complete, then baking 48 is little of constant weight in 80 DEG C of baking ovens, and 80 mesh sieves are crossed in grinding.Accurately take paddy rice dry sample, material is through pre-treatment H
2sO
4-H
2o
2disappear after boiling, adopt the full phosphorus amount of the yellow Their Determination by Spectrophotometry rice plant of vanadium molybdenum.
Content of inorganic phosphorus measures: accurately take in different phosphate concentration the fresh blade 1g grown after 20 days, add the perchloric acid 4ml of 10%, use mortar homogenate, 15000rpm is centrifugal, gets 400 μ l supernatants, adds 4ml containing 15mM ammonium molybdate and 100mM zinc acetate (pH5.0), add the fresh xitix of 1ml (10% again, pH5.0), after 30 DEG C of water-bath 15min, the absorbance value of 850 nm is measured.Do typical curve with the light absorption value of gradient concentration standard phosphorus solution, calculate phosphorus content.
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCE LISTING
<110> Fujian Province Agriculture Science Academy, Institute of Biotechnology
<120> mono-kind improves the method for rice phosphorus specific absorption
<130>3
<160>3
<170>PatentIn version 3.3
<210>1
<211>1869
<212>DNA
<213>OsSUT2 gene
<400>3
ggcaagagac aacccactcc tcttctgaac taacccaaag atcaggtcgg aggcggaggc 60
aaacacgaga agaagatgcc gcggcggcct agcggcggcg gcggcggcgc tggtcccgcg 120
gcggccgcgg tgcgcaaggt ccccctgcgg aagcttctgc gtgcggcgtc ggtggcgtgc 180
ggcgtgcagt tcgggtgggc gccgcagctc tccctgctga ccccctacgt ccaggagctc 240
ggcatcccgc acgccttcgc cagcctcgtc tggctctgcg gccccctctc cggcctcctc 300
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acacagggac cctgcagggc cttccccgcc gacctcaccg agaatgaccc aaagaggact 600
cggatagcta atgcttactt ctcattgttc atggccctgg gaaacatact tggatatgcc 660
actggggcat acagtggctg gtacaagata ttcccgttca ccgttactcc atcatgtagc 720
atcagctgtg ccaacctcaa gtcggccttt ctacttgata ttatcatttt ggtggtcact 780
acatgcatca ctgtagcatc agtgcaagag cctcaatcct ttggaagtga tgaagcagat 840
caccctagca cagaacagga agctttcctc tgggaacttt ttggatcatt ccggtacttt 900
acattaccgg tttggatggt tttgattgtt actgccctca catggattgg atggtttcca 960
tttatcctct ttgataccga ttggatgggt cgagagatct atcgtggaag tccagatgat 1020
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aactcggtcc ttcttggatt cacttctatt gtactagaga agttatgtcg gaagtgggga 1140
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attgtcattg cttccctggt agtttttaca attttaggag cgcccctggc gatcacgtac 1320
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gggccctggg accaactgtt tggtggtggc aatgcaccag cctttgcagt ggctgctgct 1500
gcatctttta tcggtgggct ggtggctatt ctgggccttc cacgagcccg cattgcatca 1560
aggaggagag gtcaccgata agaatattgc tacatataaa ttgtcggcca ttctttgcaa 1620
ttcgactcat aagaggcact cggaacgcta tgcagtgcat gggggaattg tatattatct 1680
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aaaaaaaaa 1869
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Claims (3)
1. improve a method for rice phosphorus specific absorption, it is characterized in that: realized by overexpression encoding sucrose transporter gene OsSUT2 in paddy rice, described overexpression OsSUT2 gene, gene order is as shown in SQE ID NO:1; Described overexpression utilizes PCR method homologous clone to go out paddy rice OsSUT2 gene, adopts F-primer:SQE ID NO:2 and R-primer:SQE ID NO:3 to amplify OsSUT2 gene cDNA; Build overexpression plant vector p1300UbiOsSUT2; Obtain by agrobacterium-mediated transformation and turn OsSUT2 trans-genetic hybrid rice; The homozygous lines of overexpression OsSUT2 gene is obtained by selfing separation screening.
2. a kind of method improving rice phosphorus specific absorption according to claim 1, is characterized in that: intermediate carrier pUbiOsSUT2 enzyme cuts back to close and is connected with p1300 and obtains by described plant expression vector p1300UbiOsSUT2; The rice sucrose translocator OsSUT2 cDNA be connected on T-easy sequencing vector is cut restructuring to plasmid pBluescriptIIKS through EcoRI enzyme, obtain intermediate carrier pBlueOsSUT2, then BamHI/KpnI double digestion intermediate carrier is used, OsSUT2 forward in pBlueOsSUT2 is connected to before terminator T-nos, obtains intermediate carrier pUbiOsSUT2; PUbiOsSUT2 enzyme being cut back to close UbiOsSUT2 fragment is connected on plasmid P1300, obtains conversion carrier P1300UbiOsSUT2.
3. a kind of method improving rice phosphorus specific absorption according to claim 2, is characterized in that: transform and comprise the following steps:
1) induction of embryo callus subculture;
2) genetic transformation of embryo callus subculture;
3) screening of kanamycin-resistant callus tissue;
4) differentiation of resistant plant, root culture.
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| CN107771660A (en) * | 2017-11-24 | 2018-03-09 | 扬州大学 | A kind of method that Tolerant to low P rice varieties are screened from multi items |
| CN115976042A (en) * | 2022-09-28 | 2023-04-18 | 上海市农业科学院 | Phosphorus efficient gene applied to rice and germplasm cultivation of rice |
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| CN101402958A (en) * | 2008-11-21 | 2009-04-08 | 南京农业大学 | Genetic engineering uses of rice phosphate transfer protein gene OsPht1;6 |
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