CN104140971A - Application of paddy rice genes OsMADS27 to promoting tap root system plant lateral root growth - Google Patents

Application of paddy rice genes OsMADS27 to promoting tap root system plant lateral root growth Download PDF

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CN104140971A
CN104140971A CN201410352797.1A CN201410352797A CN104140971A CN 104140971 A CN104140971 A CN 104140971A CN 201410352797 A CN201410352797 A CN 201410352797A CN 104140971 A CN104140971 A CN 104140971A
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plant
osmads27
expression vector
growth
application
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CN104140971B (en
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甘银波
于春燕
苏莎
刘一华
刘伯涵
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Zhejiang University ZJU
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Zhejiang University ZJU
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Abstract

The invention discloses application of paddy rice genes OsMADS27 to promoting tap root system plant lateral root growth. The base sequence of the genes OsMADS27 is shown in SEQ ID No.1. The application includes the steps that the paddy rice genes OsMADS27 are connected into a plant expression vector, and a recombinant expression vector is obtained through establishment; the recombinant expression vector is transformed into a receiver plant. According to the application, by cloning the paddy rice genes OsMADS27 and conducting genetic transformation on tap root system plants such as arabidopsis thaliana through the genes, external source paddy rice genes OsMADS27 are over-expressed through the plants, and therefore generation and growth of plant lateral roots can be promoted. The test result shows that an external source NO3- and the genes OsMADS27 are cooperative in promoting growth of the receiver plant. By the adoption of the application, nitrogen key controlling genes relevant to paddy rice lateral root growth can be researched, and the application has significance in screening and breeding plant varieties capable of efficiently absorbing nutrients.

Description

Rice Os MADS27 gene is in the application promoting in the growth of taproot system plant lateral roots
Technical field
The invention belongs to technical field of bioengineering, relate in particular to rice Os MADS27 gene in the application promoting in the growth of taproot system plant lateral roots.
Background technology
Paddy rice is one of most important food crop in the world, and China is maximum in the world Rice Production and country of consumption.Heterotic successfully utilization is greatly improved rice yield, has made huge contribution for solving worldwide crisis in food.But since the eighties in 20th century, the output of hybrid rice is the situation in fluctuating just.The input of nitrogenous fertilizer in Rice Production just becoming one of effective measure of volume increase, but the input of a large amount of nitrogenous fertilizer has not only reduced nitrogen utilization efficiency, also brought the series of environmental problems such as the waste of resource and the eutrophication of water body simultaneously.People regulate to improve nitrogen utilization efficiency aspect two at present, and the one, improve root plasma membrane to NH 4 +receptivity; The 2nd, improve the root structure high to nitrogen susceptibility, expand the absorption area of root.Therefore, the molecular mechanism of Study On Rice root system response nitrogen nutrition, the potentiality that fully excavation paddy rice self absorbs nitrogen are to improve Nitrogen Use Efficiency in Rice, not only there is important theory significance for the plasticity-of explaining plant root growth, and there is important production practice value for the output that improves paddy rice unit surface.
Root system, as one of plant important absorption, synthetic, fixing and support organ, plays an important role in the growth and development process of crop, and healthy and strong root system can provide sufficient nutrient and moisture for plant-growth, is the basis of high-yield plant.At present, oneself has the basic scientific research task of potentiality using root system research as further of improving farm crop productivity in the world.Rice root is the vitals that absorb nutrient and moisture, is also a lot of material assimilations, conversion or synthetic place, still carries out the metabolism organ of material exchange with overground part.Its growing state and vigor be the growing of the whole paddy rice of impact, trophic level and yield level directly.And, under the condition of resource enrichment, resource in the increase of root system branches and raising environment accurately absorbs closely related, so depend on very much the understanding that how to affect root system branches for inherent and external nutritional factor for the research of Root Absorption nutrition mechanism.
Although above-mentioned result of study has accumulated very valuable starting material for studying morphogenesis and the root system of nitrogen to rice root to absorbing of nitrogen, but these research great majority all concentrate on the observation of phenomenon and the change detection aspect of substances content, and there is no the deep internal cause changing of explaining, therefore also there is very large distance from the essence of the relation between understanding nitrogen and the growth of rice root.Therefore, thinking and the technique means of nutrition absorption utilization research in reference mode plant, separate and the key gene relevant to Nitrogen Absorption in qualification paddy rice, for illustrating the Nitrogen Absorption utilization of paddy rice and the molecular mechanism of adjusting has important theory significance, simultaneously also to thering is potential more practical value with the new rice variety that utilizes modern biotechnology means to cultivate the utilization of nitrogen efficient absorption.
Summary of the invention
The invention provides rice Os MADS27 gene in the application promoting in the growth of taproot system plant lateral roots, in the taproot system plants such as Arabidopis thaliana, heterogenous expression rice Os MADS27 gene can promote generation and the growth of lateral root.
Rice Os MADS27 gene is in the application promoting in the growth of taproot system plant lateral roots, and the nucleotide sequence of described rice Os MADS27 gene is as shown in SEQ ID No.1.
Described application comprises:
(1) described rice Os MADS27 gene is connected in plant expression vector, builds and obtain recombinant expression vector;
(2) by described recombinant expression vector transformation receptor plant.
The structure of recombinant expression vector can adopt ordinary method, as adopted Gateway system (Invitrogen company) that rice Os MADS27 gene is connected in plant expression vector.
Described plant expression vector can be selected pH2GW7 or pK2GW7.
When transformation receptor plant, can adopt the method for agrobacterium mediation converted, described Agrobacterium is specifically as follows Agrobacterium GV3101, EHA105 etc.
In the present invention, the taproot system plant such as described recipient plant is Arabidopis thaliana; Described Arabidopis thaliana is Columbia type.
Utilize the taproot system plants such as described rice Os MADS27 gene pairs Arabidopis thaliana to carry out genetic transformation, find after tested, the time of origin of transfer-gen plant the first lateral root has shifted to an earlier date compared with wild-type, the speed of growth of taproot and lateral root is faster than wild-type, thereby can promote the early growth and development of taproot system plant seedlings.
The present invention also provides a kind of recombinant expression vector, comprises the goal gene of initial carrier and the described initial carrier of insertion, and the base sequence of described goal gene is as shown in SEQ ID No.1.The construction process of described recombinant expression vector is the same.
In described recombinant expression vector, the promotor that starts described rice Os MADS27 genetic expression is strong promoter.As preferably, described strong promoter is 35S.The expression paddy rice that strong promoter can strong promoter can effectively improve outer rim gene, starts rice Os MADS27 gene overexpression, can promote generation and the growth of lateral root.
Described initial carrier is above-mentioned plant expression vector, can select pH2GW7 or pK2GW7.
The present invention also provides a kind of transformant that comprises described plant expression vector.
The Host Strains of described transformant can be Agrobacterium, is specifically as follows Agrobacterium GV3101 or EHA105.
Utilize described transformant to infect recipient plant, can realize the conversion of recombinant expression vector to recipient plant.
Compared with prior art, beneficial effect of the present invention is:
The present invention is by cloning rice OsMADS27 gene, and utilize the taproot system plants such as this gene pairs Arabidopis thaliana to carry out genetic transformation, made it expression external source paddy rice OsMADS27 gene, thereby can promote generation and the growth of taproot system plant lateral roots, promote the early growth and development of taproot system plant seedlings, in exogenous NO gas 3 -in situation about existing, growth-promoting effect is more remarkable, and test-results shows, exogenous NO gas 3 -work in coordination with the growth-promoting effect of OsMADS27 gene pairs recipient plant lateral root.
The present invention contributes to the nitrogen key controlling gene of research and paddy rice lateral root related to development, screening and seed selection to plant variety that can efficient absorption nutrient are significant, also can be and explain that the physiological mechanism of Nitrogen in Rice utilization, nitrogen utilization efficiency and the kind genetic improvement thereof of raising paddy rice provide theoretical foundation and technical support, have important theory significance and potential using value simultaneously.
Brief description of the drawings
Fig. 1 is different concns KNO 3with the growth-promoting effect design sketch of OsMADS27 gene overexpression to transgenic arabidopsis;
Wherein, first LRs represents the first lateral root, and PR represents main root, and PR >=2cm represents that main root length is more than or equal to 2cm, and Advancement (%) represents growth promotion rate (%); " * * " represents significant difference compared with wild-type Arabidopis thaliana, and " * * * " represents that difference is very remarkable compared with wild-type Arabidopis thaliana.
Embodiment
Below in conjunction with specific embodiments and the drawings, the present invention is further explained.
Embodiment 1 prepares transgenic arabidopsis
(1) get the root of rice seedling, liquid nitrogen grinding, adds TRIzol reagent (Invitrogen company), extracts RNA.Getting 0.5 μ g RNA carries out reverse transcription reaction and obtains cDNA.
(2) according to the OsMADS27 gene order design special primer on Genbank, add Sal I and Not I restriction enzyme site respectively at primer 5 ' end, sequence is as follows:
Upstream primer: 5 '-GCGTCGACATGGGGAGGGGGAAGATTGT-3 ' (SEQ ID No.2);
Downstream primer: 5 '-TTGCGGCCGCTCATGGATTCAACTGTAACC-3 ' (SEQ ID No.3).
(3) carry out pcr amplification taking full-length cDNA as template.
Adopt high-fidelity enzyme hS DNA Polymerase (TaKaRa Code:DR010A) system:
5×PrimeSTAR?Buffer(Mg 2+plus)10μl，
DNTP Mixture (each 2.5mM) 4 μ l,
Upstream primer (10 μ M) 1 μ l,
Downstream primer (10 μ M) 1 μ l,
Template cDNA1 μ l,
HS?DNA?Polymerase0.5μl，
Sterile purified water adds to cumulative volume 50 μ l.
PCR cycling condition is: 98 DEG C of denaturations 5 minutes, and 98 DEG C of sex change 10 seconds, 55 DEG C of annealing 15 seconds, 72 DEG C are extended 90 seconds, 30 circulations of increase, last 72 DEG C are extended 10 minutes, finish to react.
(4) by being cut and be connected to Gateway cloning system entry vector pENTR1A by enzyme after the recovery of PCR product, transform bacillus coli DH 5 alpha, check order and analyze, OsMADS27 gene order is as shown in SEQ ID NO.1.
(5) correct order-checking pENTR1A plasmid and Overexpression vector pH2GW7 containing OsMADS27 total length CDS are carried out to LR permutoid reaction.
Use Gateway LR Clonase TM Enzyme Mix (Invitrogen company, Cat.No.11791-019), system is as follows:
Entry clone (entry vector) 50-150ng,
Destination vector (object carrier) 150ng,
5×LR?Clonase?Reaction?Buffer2μl，
ddH 2O?up?to8μl，
Add 2 μ l LR Clonase enzyme mix, of short duration vortex mixes for twice, slightly centrifugal, in 25 DEG C of water-bath 8h, then adds 1 μ l Proteinase K solution (Proteinase K solution) to mix, and places for 37 DEG C and within 10 minutes, finishes reaction.
Get 5 μ l reaction product and transform bacillus coli DH 5 alpha, picking list bacterium colony is verified positive colony through PCR, and positive colony extracts plasmid after cultivating and carries out double digestion checking, obtains recombinant expression plasmid.
(6) transform Agrobacterium
From-80 DEG C of refrigerators, take out EHA105 competent cell, be placed on ice and thaw, the recombinant expression plasmid of getting 2 μ l left and right is added in competent cell, mixes and is placed on 30min on ice, again all mixtures in centrifuge tube are added in the electric shock cup of precooling, place on ice.Open Bole's electroporation, program is adjusted to Agr, draw the LB liquid nutritional base of 1ml, electric shock cup salient point is pushed to electroporation inwardly, click plus key, hear buzzer, take out rapidly electric shock cup, and to the LB liquid nutritional base that adds 1ml in electric shock cup, re-suspended cell, be transferred in the centrifuge tube of 1.5ml, be placed on interior 28 DEG C of eppendorf mixed instrument, more than 700rpm shakes 2.5h, coat (Rif (Rifampin) 50mg/L on resistance YEP flat board, Spec (spectinomycin hydrochloride) 100mg/L), be inverted for 28 DEG C and cultivate 2 evenings so that grow single bacterium colony.
(7) the Agrobacterium mono-clonal that picking grows is to containing in corresponding antibiotic 5mL YEP liquid nutrient medium (corresponding microbiotic is Rif50mg/L spec100mg/L), 28 DEG C of 150rpm shaking culture 48 hours.Fresh bacterium liquid is seeded in corresponding antibiotic YEP liquid medium of containing of 200ml, and 28 DEG C, 160rpm expansion is cultured to OD 600for 1.2-1.6, to collect bacterium piece after the centrifugal 15min of 4000rpm, bacterium piece is resuspended in to (MgCl in infiltration damping fluid at 4 DEG C 210mM, sucrose 5%, Silwet0.05%), make the OD of bacterium liquid 600for 0.6-0.8, be placed on ice.
(8) soak method arabidopsis thaliana transformation with thaliana flower:
1) the whole strain of Arabidopis thaliana of fruit pod being removed to clean, remaining inflorescence coil with cave together with back-off in the bacterium liquid of Agrobacterium, soak seedling 5min, constantly rock during this time bacterium liquid;
2) after having infected, take out plant, be sidelong in pallet, cover the black plastic cloth of lucifuge, be placed in growth room, after 24h, open;
3) Arabidopis thaliana plant is placed under the condition of natural lighting and cultivates, water weekly water 1-2 time.After seed maturity, results T 0for seed;
4) by the T of results 0after seed sterilization and disinfection, be planted in the solid medium of MS+50mg/L Hygromycin, after 4 DEG C of dark vernalization 3d, culture dish is placed in growth cabinet and is cultivated, observe the upgrowth situation of Arabidopis thaliana plant.The transgenic seed with hygromycin resistance will be grown in screening culture medium, and dies with regard to albefaction soon after not genetically modified seed germination.
The lateral root growth of embodiment 2 transgenic arabidopsis is observed statistics
Arabidopis thaliana is axial root system plant, and first long main root is sent out lateral root after main root growth for some time again.To the transgenic arabidopsis (T of embodiment 1 obtaining 0for the T obtaining after seed plantation 3for homozygote) containing different concns KNO 3b5 improved culture medium on root growth situation add up.
Point has the plank of transgenic arabidopsis to be first put in 4 DEG C of refrigerators two days, then vertically put into growth room, check root system situation every day, the time that mark the first lateral root occurs and main root arrive the time of 2cm, calculate first lateral root and the main root growth promotion rate of transgenic arabidopsis with respect to wild-type Arabidopis thaliana.
The calculation formula of Advancement (growth promotion rate, %) is:
Advancement (growth promotion rate, %)=(m control-d n)/m control× 100%;
Wherein, m controlrepresent the time (number of days) of the first lateral root time of origin (number of days) or the main root arrival 2cm of wild-type Arabidopis thaliana, d nrepresent the time (number of days) of the first lateral root time of origin (number of days) or the main root arrival 2cm of transgenic arabidopsis.
Statistics is in table 1 and Fig. 1.
Table 1
From Fig. 1 and table 1, do not containing KNO 3b5 improved culture medium on, with respect to wild-type Arabidopis thaliana, the first lateral root time of origin of transgenic arabidopsis has shifted to an earlier date approximately 0.51 day, the first lateral root growth promotion rate reaches 4.72%; Main root arrive 2cm time advance approximately 0.30 day, main root growth promotion rate reaches 2.72%.
And, along with KNO in B5 improved culture medium 3the continuous rising of concentration, also more and more early, the time of main root arrival 2cm is shorter and shorter for the first lateral root time of origin of transgenic arabidopsis.Wherein, at the KNO of 2mM 3under concentration, the first lateral root growth promotion rate reaches 7.68%, at the KNO of 10mM 3under concentration, main root growth promotion rate reaches 4.52%, at the each KNO for examination 3under concentration for the highest.At the KNO of 10mM 3under concentration, the first lateral root growth promotion rate declines relatively, and the amplification of main root growth promotion rate also declines, indication KNO 3excessive concentration is unfavorable for root growth.
And, each KNO 3concentration to the growth promotion rate of transgenic arabidopsis higher than KNO 3the summation of independent role and OsMADS25 gene independent role.As do not contained KNO 3b5 improved culture medium on, transgenic arabidopsis is 4.72% with respect to the first lateral root growth promotion rate of wild-type Arabidopis thaliana; With not containing KNO 3b5 improved culture medium on the wild-type Arabidopis thaliana of growing compare, containing 0.1mMKNO 3b5 improved culture medium on the first lateral root growth promotion rate of the wild-type Arabidopis thaliana of growing be about 10.13%; And with not containing KNO 3b5 improved culture medium on the wild-type Arabidopis thaliana of growing compare, at 0.1mMKNO 3b5 improved culture medium on the growth promotion rate of the transgenic arabidopsis of growing reach 15.09%.
These results suggest that, OsMADS25 gene overexpression can significantly improve growing of transgenic arabidopsis the first lateral root and main root, at certain density external source KNO 3exist lower this growth to promote that phenomenon is more remarkable, show external source KNO 3work in coordination with the growth-promoting effect of OsMADS25 gene.

Claims (9)

1. rice Os MADS27 gene, in the application promoting in the growth of taproot system plant lateral roots, is characterized in that, the nucleotide sequence of described rice Os MADS27 gene is as shown in SEQ ID No.1.
2. application as claimed in claim 1, is characterized in that, comprising:
(1) described rice Os MADS27 gene is connected in plant expression vector, builds and obtain recombinant expression vector;
(2) by described recombinant expression vector transformation receptor plant.
3. application as claimed in claim 2, is characterized in that, described recipient plant is Arabidopis thaliana.
4. a recombinant expression vector, comprises initial carrier and inserts the goal gene of described initial carrier, it is characterized in that, the nucleotide sequence of described goal gene is as shown in SEQ ID No.1.
5. recombinant expression vector as claimed in claim 4, is characterized in that, the promotor that starts described paddy gene OsMADS27 expression is strong promoter.
6. recombinant expression vector as claimed in claim 5, is characterized in that, described promotor is 35S.
7. recombinant expression vector as claimed in claim 4, is characterized in that, described initial carrier is pH2GW7 or pK2GW7.
One kind comprise as arbitrary in claim 4～7 as described in the transformant of recombinant expression vector.
9. transformant as claimed in claim 8, is characterized in that, Host Strains is Agrobacterium GV3101 or EHA105.
CN201410352797.1A 2014-07-23 2014-07-23 Rice Os MADS27 gene is in the application promoting in the growth of system of taproot plant lateral roots Expired - Fee Related CN104140971B (en)

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CN109355305A (en) * 2018-11-19 2019-02-19 浙江大学 Rice Os MADS27 gene and albumen are improving rice to the application in utilization rate of nitrogen fertilizer
CN110028563A (en) * 2018-01-11 2019-07-19 中国科学院植物研究所 Application of the protein OsMADS27 in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds
CN114686488A (en) * 2020-12-25 2022-07-01 中国科学技术大学 Rice salt-tolerant stress gene OsAGL16 and application of encoding protein thereof

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CN107779471B (en) * 2017-10-17 2020-10-13 中国计量大学 Application of methanobacterium thermoautotrophicum MTH1745 gene in improving stress tolerance of plants

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CN110028563A (en) * 2018-01-11 2019-07-19 中国科学院植物研究所 Application of the protein OsMADS27 in the suspend mode of the sprout time and regulation vegetable seeds of regulation vegetable seeds
CN109355305A (en) * 2018-11-19 2019-02-19 浙江大学 Rice Os MADS27 gene and albumen are improving rice to the application in utilization rate of nitrogen fertilizer
CN114686488A (en) * 2020-12-25 2022-07-01 中国科学技术大学 Rice salt-tolerant stress gene OsAGL16 and application of encoding protein thereof

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