CN107012153A - Applications of the nitrogen nutrition transporter gene OsNPF8.1 in rice tillering number is improved - Google Patents

Abstract

The invention discloses nitrogen nutrition transporter geneOsNPF8.1Application in rice tillering number is improved, belongs to plant genetic engineering field.OsNPF8.1The amino acid sequence of DNA encoding the protein is as shown in SEQ ID NO.1, and cDNA sequence is as shown in SEQ ID NO.2.The present invention is by building paddy riceOsNPF8.1Gene overexpression plant,OsNPF8.1Gene disturbs plant, finds by improvingOsNPF8.1Gene expression, can make normal rice tillering number and panicle number per hill increase, thereforeOsNPF8.1Gene can be used in paddy rice seed selection improving rice yield.OsNPF8.1Gene has important application value in terms of Nitrogen effect plant growth and growth course is illustrated and in terms of plant type of rice improvement.

Description

Nitrogen nutrition transporter geneOsNPF8.1Application in rice tillering number is improved

Technical field

The invention belongs to plant genetic engineering field, and in particular to nitrogen nutrition transporter geneOsNPF8.1Improving paddy rice point Application in tiller number.

Background technology

Rice in China cultivated area accounts for the 20% of the total cultivated area of world crop, but amount of application of nitrogen fertilizer accounts for the world and always applied The 37% of consumption；Nineteen ninety-five China's nitrogen fertilizer production amount and usage amount reach No. 1 in the world, but nitrogenous fertilizer service efficiency is relatively low, nitrogenous fertilizer Amount of application compared with 50 years before increase by 20 times, by this trend, it is contemplated that to the year two thousand fifty, it will turn over 3 times again.The excessive meeting of nitrogen application Cause the ecological pollution problem such as body eutrophication（Xu Guohua, model dawn honor paddy rice nitre transporter genesOsNRT1.1aWithOsNRT1.1bFunctional study Agricultural University Of Nanjing, 2011：4-6）.More nitrogen nutrition pass through denitrification, water and soil It is lost in, volatilizees naturally, microorganism such as utilizes to be wasted at the approach.

If the absorption efficiency of nitrogen is improved into 1%, it is equivalent to save more than ten hundred million dollars of spending every year.From China National conditions are analyzed, and the potentiality that expansion cultivated area is increased the total yield are very limited, and the only way out is raw on limited soil The more paddy of output, that is, increase the yield per unit area.In traditions of the past farming, by selecting nitrogen use efficiency higher Crop, to improve the utilization ratio of nitrogen；But compared with the breeding on molecular level, this process seems slow and poorly efficient（Zhang Hong Journey, wears genotypic difference and physiological Mechanism research Yangzhou Universitys, 2008 that its root Nitrogen in Rice is utilized：10-13）.Improve Nitrogen use efficiency, it is necessary to find breach from the molecule absorption mechanism of nitrogen.Nitrate anion transporter gene family is divided into low-affinity nitre Acid group transporter gene and the class of high-affinity nitrate anion transporter gene two（Zhou Shiyi carbohydrates and amino acid are to the high parent of paddy rice induction type With the influence Central China University of Science and Technology of power nitrate transport system, 2009：15-16）.By nitrogen assimilation, by nitrate nitrogen and ammonium State nitrogen absorbs and is converted into amino acid, and referred to as the first kind of nitrogen absorbs.The increase of seed nutritional material is made by the transport to nitrogen, increased Plus plumpness, referred to as nitrogen Equations of The Second Kind absorb, that is, nitrogen recycling（Kant S, Bi Y, Steven J, et al. Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency . Journal of Experimental, 2011, 62(4): 1499- 1509）.Increase nitrogen absorption and accumulation amount or N transformation amount, can increase production.Therefore, in agricultural modernization construction, molecule is passed through Breeding technique improves utilization ratio of the paddy rice to nitrogenous fertilizer, it is possible to reduce nitrogenous fertilizer pollutes, moreover it is possible to increase yield.

NRT1/PTR families（NRT1/PTR family, NPF）It is the small molecule for referring to mediate 2-3 amino acid residue The material such as peptide and nitrate anion carries out the albumen of transdermal delivery（Rentsch D, Schmidt S, Tegeder M. Transporters for uptake and allocation of organic nitrogen compounds in plants. FEBS Let, 2007, 581: 2281-2289）.NRT1/PTR family members take part in during Seed Development Micromolecule polypeptide form is transported after protein degradation in the accumulation and sprouting of protein（Martre P, Porter J R, Jamieson P D, et al. Modeling grain nitrogen accumulation and protein composition to understand the sink/source regulations of nitrogen remobilization for wheat. Plant Physiol, 2003, 133: 1959-1967）.At present on NPF family The report of race member research is seldom, of the present inventionOsNPF8.1Gene is a gene of paddy rice NPF gene families.This hair It is bright to findOsNPF8.1Gene pairs rice tiller regulating has extremely important effect, can be applied to the improvement of plant plant type so that water Rice is increased production.

The content of the invention

It is an object of the invention to solve problems of the prior art, there is provided paddy rice NPF gene family membersOsNPF8.1Application of the gene in rice tillering number is improved.

The purpose of the present invention is achieved through the following technical solutions：

The present invention is with the NPF gene family members of paddy riceOsNPF8.1Gene is object, is cloned in spending 11 from paddy riceOsNPF8.1CDNA sequence.By buildingOsNPF8.1Gene overexpression vector, using AgrobacteriumEHA105The heredity of mediation Method for transformation, overexpression vector is imported and spent in normal japonica rice variety in 11, is obtainedOsNPF8.1Gene overexpresses plant, its point Tiller number is significantly improved compared with spending 11 in control wild type.Built by RNAi technologyOsNPF8.1Gene interference expression vector, Spend in 11, obtain during interference expression vector is importedOsNPF8.1The interference plant that gene expression amount declines, point of interference plant Tiller number is significantly reduced compared with spending 11 in.These results indicate that by improvingOsNPF8.1The expression of gene, can make normal Rice tillering number increase, so as to improve spike number and rice yield.

Based on present invention discover thatOsNPF8.1The function of gene,OsNPF8.1Gene can be used in paddy rice seed selection.It is described Paddy rice seed selection for improve rice tillering number, so as to improve spike number and rice yield.Specifically can be by improvingOsNPF8.1Gene Expression make rice tillering number and panicle number per hill increase, reach improve rice yield purpose.

OsNPF8.1Gene can also be used for improving the yield of other plant, such as be made by transgenosisOsNPF8.1Gene is being planted In thing（Excess）Expression, to improve the branch quantity of plant, and then is improved the yield of plant.Described plant refers to list Cotyledon plant or dicotyledon；Such as：Wheat, tomato, turfgrass or clover etc..

DescribedOsNPF8.1The amino acid sequence of the OsNPF8.1 albumen of gene code is as shown in SEQ ID NO.1；Institute StateOsNPF8.1The cDNA sequence of gene is preferably as shown in SEQ ID NO.2.

It is construed as, on the premise of OsNPF8.1 protein actives are not influenceed（I.e. not in the activated centre of albumen）, this Art personnel can carry out various substitutions, additions and/or deletions one or several to the amino acid sequence shown in SEQ ID NO.1 Individual amino acid obtains the amino acid sequence with equal function.Therefore, OsNPF8.1 albumen also includes ammonia shown in SEQ ID NO.1 Base acid sequence is substituted, replaces and/or increased the protein having with isoreactivity that one or several amino acid are obtained.In addition, It should be understood that the preferences of the degeneracy and different plant species codon in view of codon, those skilled in the art can basis Need to use the codon of suitable particular species expression.

Advantages of the present invention and effect：

（1）What the present invention was clonedOsNPF8.1Strengthen Tillering Ability in Rice after gene overexpression, explanationOsNPF8.1Gene pairs Improve rice yield more apparent, therefore, improved by technique for gene engineeringOsNPF8.1The expression of gene can improve plant production Amount.It is not only does this facilitate and cultivates high-yield rice under the conditions of nitrogen by normally applying, the product of plant can also be carried out by molecular breeding Plant improvement.

（2）OsNPF8.1The successful clone of gene, further demonstrate important work of the NPF families in nitrogen absorption process With, there is important meaning to the biological function for illustrating NPF families, in addition to further appreciating that plant nitrogen metabolism approach, raising Nitrogen absorption efficiency has great impetus.

（3）Although being cloned into some genes for improving plant products at present, to the molecular mechanism of plant yield-increasing still It is unclear.And what the present invention was clonedOsNPF8.1Gene can improve the yield of paddy rice, the key factor of pair determination plant yield-increasing There is great impetus.

Brief description of the drawings

Fig. 1 be spend 11 in control,OsNPF8.1Gene overexpress 2 strains of plant andOsNPF8.1Gene disturbs plant 2 The whole strain phenotypic map of individual strain.

Fig. 2 be spend 11 in control,OsNPF8.1Gene overexpress 2 strains of plant andOsNPF8.1Gene disturbs plant 2 The statistics block diagram of individual strain tiller number, data carry out variable analysis using SPSS softwares（ANOVA）, use Duncan ' s to exist Significance difference analysis, different group lowercases are carried out in 0.05 level（a、b、c）Represent significant difference.

Fig. 3 be spend 11 in control,OsNPF8.1Gene overexpress 2 strains of plant andOsNPF8.1Gene disturbs plant 2 In individual strainOsNPF8.1The statistics block diagram of gene relative expression quantity, data carry out variable analysis using SPSS softwares （ANOVA）, use Duncan ' s to carry out significance difference analysis, different group lowercases in 0.05 level（a、b、c）Table Show significant difference.

Embodiment

With reference to embodiment, the present invention will be further described in detail, but the implementation of the present invention is not limited to this. Unless otherwise specified, the conventional meanses that the technological means used in following embodiments is well known to those skilled in the art；Used Experimental method is conventional method, and can be according to the recombinant technique described（Referring to molecular cloning, laboratory manual, second edition, CSH Press, Cold SpringHarbor, New York）Complete；Material, reagent used etc., are commercially obtained.

Embodiment 1OsNPF8.1Gene overexpresses the structure of plant

Extract in paddy rice and spend 11 RNA, and its reverse transcription is utilized into primer pair into cDNA：

F1：5'-GGTACCATGGGAGAGGTTGCAGAAGACATC-3'（kpnI）,

R1：5'-TCTAGAGTTTGCTCCGGCGTGCTCGGCCTT-3'（XbaI）；

Expanded by PCROsNPF8.1After the cDNA of gene, then pass throughkpnI、XbaPCAMBIA-1306 carriers are connected into after I digestions （PCAMBIA-1306 carriers are purchased from Cambia companies）, constructOsNPF8.1The overexpression vector of geneOsNPF8.1- p1306.Using AgrobacteriumEHA105The genetic transforming method of mediation, overexpression vector is imported in normal rice varieties and spends 11 In.

Obtained all transgenic plants are transplanted in the basket with soil, periodically watered, applies fertilizer, treats that seedling grows tall about During 10cm, plant in big Tanaka, after seedling is grown up, extract genomic DNA and detect that detection is drawn to transfer-gen plant by PCR Thing to for：

F2：5'-GATGTTGGCGACCTCGTATT-3',

R2：5'-TCGTTATGTTTATCGGCACTTT-3'.

If amplifying 517bp fragment, it is positive plant to illustrate transfer-gen plant.Positive plant individual plant sowing is simultaneously planted Plant, until T2 generations identify the transfer-gen plant of homozygosis, that is, obtainOsNPF8.1Gene overexpresses plant.OsNPF8.1Gene surpasses The tiller number of plant is expressed far more than spending 11 plant in control, significant difference, as shown in Figure 1, 2.

TakeOsNPF8.1Gene overexpresses plant leaf, extracts RNA and by its reverse transcription into cDNA, fixed by real-time fluorescence Measure PCR detectionsOsNPF8.1The expression quantity of gene, as a result shows（Fig. 3）Overexpress plantOsNPF8.1The expression quantity of gene with it is right 11 are spent according in compared to significantly rise.Real-time fluorescence quantitative PCR the primer to for：

F3：5'-GCTGCACGAGACGCTCGACA-3',

R3：5'-AGCAGCCGCACCACGCTCTT-3'.

Embodiment 2OsNPF8.1Gene disturbs the structure of plant

Extract in paddy rice and spend 11 RNA, and its reverse transcription is utilized into primer pair into cDNA：

F4：5'-GGTACCGGGGTGGTGTTCTTGGCGCTGTAC-3'（KpnI）,

R4：5'-GGATCCACGAGCACCTGCGCGATCCT-3'（BamHI）；

F5：5'-ACTAGTGGGGTGGTGTTCTTGGCGCTGTAC-3'（SpeI）,

R5：5'-GAGCTCACGAGCACCTGCGCGATCCT-3'（Sac I）；

Respective PCR is amplifiedOsNPF8.1After the cDNA fragments of gene, by being connected into after corresponding digestion with restriction enzyme PTCK303 carriers, are constructedOsNPF8.1The interference expression vector of geneOsNPF8.1-pTCK303.Using AgrobacteriumEHA105 The genetic transforming method of mediation, interference expression vector is imported and spent in normal japonica rice variety in 11.

Obtained all transgenic plants are transplanted in the basket with soil, periodically watered, applies fertilizer, treats that seedling grows tall about During 10cm, plant in big Tanaka, after seedling is grown up, extract genomic DNA and detect that detection is drawn to transfer-gen plant by PCR Thing to for：

F2：5'-GATGTTGGCGACCTCGTATT-3',

R2：5'-TCGTTATGTTTATCGGCACTTT-3'.

If amplifying 517bp fragment, it is positive plant to illustrate transfer-gen plant.Positive plant individual plant sowing is simultaneously planted Plant, until T2 generations identify the transfer-gen plant of homozygosis, that is, obtainOsNPF8.1Gene disturbs plant.OsNPF8.1Gene is disturbed The tiller number of plant is far fewer than spending 11 plant in control, significant difference, as shown in Figure 1, 2.

TakeOsNPF8.1Gene disturbs plant leaf, extracts RNA and by its reverse transcription into cDNA, passes through real time fluorescent quantitative PCR is detected OsNPF8.1The expression quantity of gene, as a result shows（Fig. 3）Disturb plantOsNPF8.1The expression quantity of gene is with compareing In spend 11 compared to significantly reducing, as shown in Figure 3.Real-time fluorescence quantitative PCR the primer be the same as Example 1.

The above results show, by improvingOsNPF8.1The expression of gene, can increase the tiller number of paddy rice, and then improve Spike number and rice yield.

Above-described embodiment is preferably embodiment, but embodiments of the present invention are not by above-described embodiment of the invention Limitation, other any Spirit Essences without departing from the present invention and the change made under principle, modification, replacement, combine, simplification, Equivalent substitute mode is should be, is included within protection scope of the present invention.

SEQUENCE LISTING

<110>Wuhan Bioengineering Institute

<120>Applications of the nitrogen nutrition transporter gene OsNPF8.1 in rice tillering number is improved

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<170> PatentIn version 3.3

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Claims (8)

1.OsNPF8.1Application of the gene in rice tillering number is improved, it is characterised in that：By improvingOsNPF8.1Gene Expression makes the increase of rice tillering number.

2.OsNPF8.1Application of the gene in rice yield is improved, it is characterised in that：By improvingOsNPF8.1The table of gene Up to increasing rice yield.

3.OsNPF8.1Application of the gene in paddy rice seed selection.

4.OsNPF8.1Application of the gene in plant branching number and yield is improved.

5. application according to claim 4, it is characterised in that：Described plant refers to monocotyledon or dicotyledon.

6. the application according to claim 4 or 5, it is characterised in that：Described plant include wheat, tomato, turfgrass or Clover.

7. the application according to claim any one of 1-4, it is characterised in that：DescribedOsNPF8.1Gene code The amino acid sequence of OsNPF8.1 albumen is as shown in SEQ ID NO.1；Or OsNPF8.1 albumen is amino shown in SEQ ID NO.1 Acid sequence is substituted, replaces and/or increased the protein having with isoreactivity that one or several amino acid are obtained.

8. application according to claim 7, it is characterised in that：DescribedOsNPF8.1The cDNA sequence of gene such as SEQ ID Shown in NO.2.