CN107022563A - Genetically modified plants - Google Patents

Abstract

The genetically modified plants with increased nitrogen metabolism of the nucleic acid construct of the albumen worked as movable signal are encoded the present invention relates to expression.

Description

Genetically modified plants

Invention field

The present invention relates to improved characteristic, for example, grow, and the expression movable signal HY5 of improved nitrogen metabolism turns Gene plant, prepares the method for such plant and the method for improving nitrogen metabolism and growth.

Background technology

Nitrogen (N) is basic to Crop development, because it forms the basic ingredient of many organic molecules, nucleic acid and albumen.N Nutrition influences the plant function of all levels, from metabolism to resource allocation, growth and development.Due to the organic and fertilizer N of administration Strong nitrification, be the nitrate anion (NO3 in natural aerobic soil for the N of the plant roots most abundant sources obtained^-)。

Between past five decade, world crops production capacity product write increase, and this makees article mainly due to improvement Plant and chemical fertilizer, a large amount of inputs for being particularly nitrogen.However, for many crops, fertilizer N utilization ratios are only about 30-50%, big portion Point it is lost in environment, causes various adverse effects, the deterioration of such as air and water quality and the forfeiture of bio-diversity (Ju, Et al. X.T. Proc Natl AcadSci USA.106,3041-3046 (2009)).In addition, nitrogen is the battalion of the most expensive of supply Support one of thing and commercially available fertilizer represents prime cost in plant production.It is estimated that N unnecessary in environment is spent every year at present Take 70,000,000,000 Euros to 320,000,000,000 Euros of European Union (Sutton et al., Nature 472,159-61 (2011).In China, past 30 The increase of grain yield is drastically reduced to the fertilizer N that 20kg cereal/kg is applied along with N utilization ratios (NUE) from 55 between year (Guo, et al. Science 327,1008-1010 (2010)).Increase NUE and limitation nitrogen Fertilizer application are all environmental protections and changed Kind sustainable and voluminous agriculture significant challenge.The challenge is especially with needing a large amount of N fertilizer to obtain maximum production and estimate Cereal crops of the NUE much smaller than 50% it is related (Hirel et al., Journal ofExperimental Botany, Vol.58, No.9, pp.2369-2387 (2007)).

It is therefore important that differentiating the committed step of control plant N metabolism (including N utilization ratios (NUE) and N intakes).It is right In most plants species, how NUE depends primarily on plant from soil extract inorganic nitrogen, absorbs nitrate and ammonium, and follow Ring organic nitrogen.NUE can be defined as obtainable N (including remaining N present in soil and fertilizer) in the soil of per unit Cereal yield, i.e. output (total plant N, cereal N, Biomass yield, cereal yield) and input (total N, the soil N or N- of administration Fertilizer) ratio.Therefore, NUE can be divided into two processes：Ingestion efficiency (NupE；Plant is gone from soil with nitrate anion and ammonium ion Except N ability) and utilization ratio (NutE；The ability of grain yield is produced using N).

Utilization of the plant to nitrogen is related to multiple steps, including intake, absorb, migration, and when plant senescence, recycling and Reactivation.In order to control the nitrate concentration changed in soil, plant roots develop at least three kinds nitrate anion intake systems, two High-affinity transporter system (HATS) and a low-affinity delivery system (LATS), are responsible for the acquisition (Crawford of nitrate anion and Glass Trends Plant Sci3：389-395(1998)).Composing type HATS (cHATS) and nitrate-inducible HATS (iHATS) operations with low nitrate concentration in external agency so that absorb nitrate anion, and saturation range is 0.2- 0.5mM.Compared to before, LATS is worked in nitrate anion acquisition with higher outside nitrate concentration.Pass through LATS's and HATS Absorb and mediated by the nitrate anion transport protein for being belonging respectively to NRT1 and NRT2 families.Via the intake of root by negative-feedback regu- lation, The expression and activity that nitrate anion is absorbed are associated with the N-state of plant.

Arabidopsis (At) albumen HY5, is a kind of basic leucine zipper (bZIP) transcription factor, it is known that it integrates plant more (for example, auxin and the abscisic acid) of thing hormone and (for example, low temperature) signal of environment, and in the photosensitive form hair of control plant Played a role (18,28-30) in raw development (photomorphogenic development).It it is known that lateral root formation in funeral Losing in the mutant of hy5 functions increases (18,28).

It has been established that, the long range signals conduction induction root growth of tender shoots to root and N intakes (26,27).The present inventor is first The secondary molecular basis that authenticated the signal and show that HY5 is N is metabolized in mediated plant tender shoots to root movable signal.This is provided It is used to improve the alternate strategies of nutritional utilization efficiency in crop.

The a large amount of expensive formulation fertilizer containing nitrogens of the agricultural demand of fecund.The NUE for improving crop plants is therefore most important.Need for Crop plants provide more nutrition effective gene type and, to ensure the sustainable crop production for global food safety, and subtracted The cost and negative environment effect of few chemical fertilizer input, such as to air and the negative environment effect of water quality, and bio-diversity Lose.Present invention seek to address that the demand.

Summary of the invention

Present inventors have surprisingly discovered that, HY5 participates in N metabolism.Show in example as described herein, turning base Cause the increase that nitrate anion is absorbed in root because expressing AtHY5 in plant.In addition, inventors have shown that, HY5 is as response light from tender The movable signal that bud is moved to root works and mediates root growth and nitrate anion to absorb.Therefore, although HY5 is produced in root, Only when the HY5 albumen produced due to light sensation in tender shoots/leaf is moved to root, it builds up to level of significance, and induces root to send out Educate and NO3^-Intake.

HY5 is to adjust the carbon and nitrogen metabolism of photoresponse, enable plant in the whole biological response changed to ambient light Having now surprisingly been found that for the middle tender shoots-root floating preteins for coordinating tender shoots and root growth, opens enhancing crop nutritional utilization, life The method of the biotechnology approach of long and productivity ratio, is used to have especially with tissue-specific promoter to lift HY5 levels The effect of profit.

In the first aspect, the present invention relates to for increasing the method for nitrogen metabolism in plant, it is included in introducing in plant And express the nucleic acid construct for including HY5 nucleotide sequences.

On the other hand, the present invention relates to genetically modified plants, it is included is adjusted containing HY5 nucleotide sequences and tissue specificity The nucleic acid construct of sequence.

On the other hand, the present invention relates to genetically modified plants, it includes the nucleic acid construct containing HY5 nucleotide sequences, its Described in plant be not arabidopsis.

On the other hand, the present invention relates to the nucleic acid construct for including HY5 nucleotide sequences and tissue-specific promoter.

Further, the present invention relates to the carrier for including nucleic acid construct as described herein.

On the other hand, the present invention relates to host cell, it is comprising nucleic acid construct as described herein or as herein Described in carrier.

Further, the present invention relates to the method for producing the plant absorbed with increased nitrogen, it includes Introduced in plant and express the nucleic acid construct comprising HY5 nucleotide sequences and tissue-specific promoter.

On the other hand, the present invention relates to for increasing the method for the presence of HY5 albumen in plant roots, it is included in plant It is middle to introduce and express the nucleic acid construct for including the HY5 nucleotide sequences being operatively connected with chlorenchyma specificity promoter.

On the other hand, the present invention relates to the method for adjusting C and N metabolic balances in plant, it is included in plant Introduce and express the nucleic acid construct for including HY5 nucleotide sequences.

On the other hand, the present invention relates to for increasing the method for the presence of HY5 albumen in plant roots, it is included in plant Chlorenchyma in introduce and express the nucleic acid construct for including HY5 nucleotide sequences.

Last in terms of, the present invention relates to the plant of hereditary change, wherein the plant is in endogenous HY5 nucleotide sequences Or mutation is carried in endogenous HY5 promoters, or wherein described be mutated introduces at least one additional copy into Plant Genome HY5 nucleic acid.

In summary, the present invention provides following technical proposals：

1. for increasing the method for nitrogen metabolism in plant, methods described, which is included in plant, to be introduced and expresses comprising HY5 cores The nucleic acid construct of acid sequence.

2. the method according to the 1st, wherein the nucleic acid construct includes coding SEQ ID NO：3 are limited The SEQ ID NO of AtHY5 albumen or its functional variety：1 or 2 or coding SEQ IDNO：The nucleotide sequence of 3 homologue.

3. the method according to the 2nd, wherein the homologue and SEQ ID NO：3 have at least 30% sequence same One property.

4. the method according to the 2nd or 3, wherein the homologue is selected from SEQ ID NOs：4 to 29.

5. the method according to foregoing items, wherein the plant is selected from corn, paddy rice, wheat, colea/plus taken Big rape, sorghum, soybean, sunflower, clover, potato, tomato, tobacco, grape, barley, pea, beans, broad bean, lettuce, cotton Flower, sugarcane, sugar beet, broccoli or other brassica vegetables or willow.

6. the method according to foregoing items, wherein the nucleic acid construct includes regulatory sequence.

7. the method according to the 6th, wherein the regulatory sequence is selected from constitutive promoter or tissue specificity is opened Mover.

8. the method according to the 7th, wherein the tissue-specific promoter is chlorenchyma specificity promoter.

9. genetically modified plants, it includes the nucleic acid construct containing HY5 nucleotide sequences and tissue specificity regulatory sequence.

10. the genetically modified plants according to the 9th, wherein the tissue-specific promoter is chlorenchyma specificity Promoter.

11. the genetically modified plants according to the 10th, wherein the nucleic acid construct includes coding SEQ ID NO：3 limits The SEQ ID NO of fixed AtHY5 albumen：1 or 2 or coding SEQ ID NO：The nucleotide sequence of 3 homologue.

12. the genetically modified plants according to the 11st, wherein the homologue and SEQ ID NO：3 have at least 30% Sequence identity.

13. the genetically modified plants according to the 11st or 12, wherein the homologue comes from SEQID NOs：4 to 29.

14. the genetically modified plants according to any one of 9-13, wherein the plant be selected from corn, it is paddy rice, small Wheat, colea/canola, sorghum, soybean, sunflower, clover, potato, tomato, tobacco, grape, barley, pea, Beans, broad bean, lettuce, cotton, sugarcane, sugar beet, broccoli or other brassica vegetables or willow.

15. genetically modified plants, it includes the nucleic acid construct containing HY5 nucleotide sequences, wherein the plant is not to intend south Mustard.

16. nucleic acid construct, it includes HY5 nucleotide sequences and tissue-specific promoter.

17. the nucleic acid construct according to the 16th, wherein the tissue-specific promoter is that chlorenchyma is special Property promoter.

18. the nucleic acid construct according to the 16th or 17, wherein the nucleic acid construct includes coding SEQ ID NO：The SEQ ID NO of the 3 AtHY5 albumen limited：1 or 2 or coding SEQID NO：The nucleotide sequence of 3 homologue.

19. the nucleic acid construct according to the 18th, wherein the homologue and SEQ ID NO：3 have at least 30% Sequence identity.

20. the nucleic acid construct according to the 18th, wherein the homologue is selected from SEQ IDNOs：4 to 29.

21. the nucleic acid construct according to any one of the 16th to 20, wherein the plant be selected from corn, paddy rice, Wheat, colea/canola, sorghum, soybean, sunflower, clover, potato, tomato, tobacco, grape, barley, pea Beans, beans, broad bean, lettuce, cotton, sugarcane, sugar beet, broccoli or other brassica vegetables or willow.

22. carrier, it includes the nucleic acid construct according to any one of the 16th to 21.

23. host cell, it is comprising the nucleic acid construct according to any one of the 16th to 21 or according to the 22nd Described carrier.

24. the method for producing the plant absorbed with increased nitrogen, it includes introducing and expressing into plant including HY5 nucleotide sequences and the nucleic acid construct of tissue-specific promoter.

25. for increasing the method for the presence of HY5 albumen in plant roots, its be included in plant introduce and express comprising with The nucleic acid construct for the HY5 nucleotide sequences that chlorenchyma specificity promoter is operatively connected.

26. the method for adjusting the balance in plant between C and N metabolism, it, which is included in plant, introduces and expresses bag The nucleic acid construct of the nucleotide sequence containing HY5.

27. for increasing the method for the presence of HY5 albumen in plant roots, it, which is included in the chlorenchyma of plant, introduces simultaneously Expression includes the nucleic acid construct of HY5 nucleotide sequences.

28. the plant of hereditary change, wherein the plant is taken in endogenous HY5 nucleotide sequences or endogenous HY5 promoters With mutation or wherein described mutation is by the HY5 nucleic acid into plant genomes of at least one additional copy.

The present invention is further described in following non-limitative drawings.

Accompanying drawing

Fig. 1 .HY5 regulate and control root growth and the NO3 of NRT2.1- dependences^-The tender shoots of intake-irradiation promotes.(A) distinctiveness is tender Bud/root irradiates the diagram of condition.By 5- ages in days seedling exposed to 3 days distinctiveness light processing (100 μm of ol.s^-1.m^-2)：Irradiation (S (L)) or secretly raw (S (D)) tender shoots, (R (L)) of irradiation or secretly raw (R (D)) root.(B) to WT and hy5-526 primary roots Distinctiveness tender shoots/root illumination effect of growth.Arrow shows tip of a root position when experiment starts.Engineer's scale, 1cm.(C) to WT, The distinctiveness tender shoots of hy5-526, hy5 and cop1-4 seedling primary root development length/root irradiation influence.(D) to seedling root¹⁵NO3^-The distinctiveness tender shoots of intake/root irradiation influence.(E) to the difference of the primary root development length of the plants of 10- age in days graftings Property the irradiation influence of tender shoots/root.Grafting is expressed as scion/stock (for example, HY5/hy5-526 has HY5 scion and hy5-526 Stock).(F) to such as the root of the plant of grafting in (E)¹⁵NO3^-The distinctiveness tender shoots of intake/root irradiation influence.(C-F) data It is expressed as average value ± s.e.m. (n=30).Inapparent difference (P ＜ between identical lowercase letter average value 0.05)。

Fig. 2 .HY5 migrate regulation root growth and NO3 from tender shoots to root^-Intake.

(A) distinctiveness tender shoots/root irradiation on the nascent root extension of genetically modified plants influences.(B) HY5-GFP exists pHY5；Distribution in HY5-GFP hy5 roots.(C) in pCAB3：Detectable HY5-GFP distribution in HY5-GFP hy5 roots. (D) the relative myc-HY5 transcript abundances in 14- ages in days plant, relative to pCAB3：Turn of the level of myc-HY5hy5 plants Record is set to 1.Data display is average value ± s.e.m. (n=3).(E) in tender shoots (S) and root (R) myc-HY5 immunology inspection Survey, HSP90 loading controls.(F) pCAB3 of tender shoots-irradiation：HY5-GFP point in the root of HY5-GFP hy5/hy5 graftings Cloth.(G) pCAB3 of the expression TEV protease of tender shoots-irradiation：2×GUS TEV^reHY5- in the root of-HY5-GFP hy5 plants GFP detection.(H) distinctiveness tender shoots/root irradiation on the nascent root extension for the plant for expressing TEV protease influences.(I) To the root for the plant for expressing TEV protease¹⁵NO3^-The distinctiveness tender shoots of intake/root irradiation influence.(J) to pHY5 in root：GFP turns The distinctiveness tender shoots of the expression of gene/root irradiation influence.(K) in seedling root (grafting as shown) HY5-GFP distribution.(B, C, F, G, J, K) engineer's scale, 50 μm.(A, D, H, I) data are expressed as average value ± s.e.m. (n=30).Identical lowercase Represent inapparent difference (P ＜ 0.05) between average value.

Fig. 3 .HY5 coordinate N and C metabolism.(A) in nascent seedling root (genotype/graft chimaera as shown) NRT2.1 transcript levels, the transcription relative to the level of WT roots is set to 1.Data display is average value ± s.e.m. (n=3). (B) the root NO3 of graft chimaera^-Intake.Data display is average value ± s.e.m. (n=30).(C) show for coming from 14- Age in days pHY5：The NRT2.1 promoter fragments of the ChIP analyses of the extract of myc-HY5hy5 plants.Data display is average value ± s.e.m (n=3).Arrow represents C/G box sequence motifs.(D) the fragment 3 and MBP-HY5 from (C) is incubated.With 10,20, 50 or 100- times of excessive unlabelled probe be at war with.(E) PSY, TPS1, SWEET11 and SWEET12 in 7- ages in days seedling The relative abundance of transcript.Value represents that data are average value ± s.e.m. (n=3) relative to WT levels.(F) ChIP is determined. Fragment containing G- box motifs in TPS1, SWEET11 and SWEET12 promoter is used to come from 14- ages in days pHY5：myc-HY5hy5 The ChIP analyses of the extract of plant.Data display is average value ± s.e.m (n=3).(G) sucrose influence NRT2.1 transcripts Abundance.Transcription relative to the level of WT S (L)/R (D) seedling is set to 1.Data display is average value ± s.e.m. (n=3). (H) sucrose influence root¹⁵NO3^-Intake.Data display is average value ± s.e.m. (n=30).(I) HY5 is to NRT2.1 promoters Binding in vivo.Use 10- ages in days pHY5：Myc-HY5hy5 plants carry out ChIP-PCR analyses.Data display be average value ± S.e.m (n=3).Inapparent difference (P ＜ 0.05) between identical lowercase letter average value.

The luminous environment plant growth coordinating of Fig. 4 .HY5 response fluctuations and nutrition.(A) with fluence rate (fluence as shown Rate) the primary root development length of the seedling of growth.Data display is average value ± s.e.m. (n=30).(B) with as shown The seedling of fluence rate growth¹⁵NO3^-Intake.Data display is the children of the different fluence rates of average value ± s.e.m. (n=30) (C) Seedling tender shoots biomass.Data display is average value ± s.e.m. (n=30).(D) with fluence rate of not sharing the same light in soil-grown 21 days The whole phytomass of the plant of (16h photoperiods).(E) C content of plant shown in D.(F) N of plant shown in D contains Amount.(G) with the C/N contents ratio of the 21- age in days plants of fluence rate growth as shown.(D-G) data display be average value ± S.e.m. (n=16).Inapparent difference (P ＜ 0.05) between identical lowercase letter average value.

Expression and OsHY5 expression in arabidopsis of Fig. 5 .PpHY5 in arabidopsis.From arabidopsis, paddy rice The conservative function of the HY5 homologues of (Oryza sativa) and small liwan moss (Physcomitrella patens).(A-C) light The hypocotyl length of the 6- ages in days arabidopsis thaliana (as indicated, WT, hy5 and hy5 of the transgenosis containing expression HY5-GFP) of growth Spend (A), primary root meristematic cell number (B) and root NRT2.1 transcriptional levels (C).Data display is average value ± s.e.m (n =30).(D) GFP merged in the stock part (WT roots) of the plant of grafting with AtHY5, OsHY5 or PpHY5 distribution.

Fig. 6 .a) AtHY5 and homologue comparison；B) AtHY5 and the comparison of AtHYH sequences, c) homologue tree.

Fig. 7 distinctivenesses tender shoots/root irradiates the influence to lateral root development.(A) distinctiveness tender shoots/root irradiation is to lateral root development Influence.3- age in days WT seedling is transferred to new plate, 10d distinctivenesses light processing (100 μm of ol.s are then exposed to^-1.m^-2)。 Engineer's scale, 1cm.(B) lateral root in different disposal is produced.Data display is average value ± s.e.m. (n=30).Identical small letter Letter represents inapparent difference (P ＜ 0.05) between average value.

The allelic variation of Fig. 8 .HY5 sequences.(A) the splice site mutation in hy5-526.Dark grey frame represents outer aobvious Son, black line represents introne, and numeral represents extron size (bp).(B) albumen between HY5 and the hy5-526 of mutation Sequence compares.The numeral on right side represents the resi-dues in holoprotein.Identical residue is represented by Dark grey frame, and variant is residual Base is represented by light grey frame.

The 6- ages in days seedling of Fig. 9 photoproduction length containing expression HY5-GFP or myc-HY5 (as indicated, WT, hy5 and turn base The hy5 of cause) hypocotyl length.Data display is average value ± s.e.m. (n=30).Identical lowercase letter average value Between inapparent difference (P ＜ 0.05).

Plant pCAB3s of Figure 10 in grafting：Detectable HY5-GFP distribution in HY5-GFPhy5 scion.(A) test Use hypocotyl graft chimaera.(B) the HY5-GFP distributions in the scion leaf of the plant of 10- ages in days grafting.

HY5-GFP distribution in the root of the plant of Figure 11 .10- age in days graftings.(A) grafting plant pHY5：HY5- Detectable HY5-GFP distribution in GFPhy5 stock roots.Engineer's scale, 50 μm.(B) phase in the tender shoots and root of the plant of grafting To HY5-GFP transcript abundances, as expressed in (A), relative to the pHY5 of the plant of grafting：The water of myc-HY5hy5 scion leaves Flat transcription is set to 1.Data display is average value ± s.e.m. (n=3).(C) HY5-GFP in scion (tender shoots) and stock (root) Immunology detection, utilize HSP90 loading controls.

Figure 12 distinctivenesses tender shoots/influence of the root irradiation to the distribution of the HY5-GFP in leaf.(A) tender shoots-irradiation pCAB3：2×GUS-TEV^reHY5-GFP distribution in the leaf of-HY5-GFP hy5 plants.(B) expression TEV protease tender shoots- The pCAB3 of irradiation：2×GUS-TEV^reHY5-GFP distribution in the leaf of-HY5-GFP hy5 plants.

Figure 13 .HYH are not the tender shoots for the light regulation for regulating and controlling root growth to root movable signal.(A) tender shoots-irradiation is to 6- ages in days The influence of the primary root growth of Ws, hyh-1 and hy5hyh-1 seedling.(B) Ws, hyh-1 and hy5hyh-1 seedling primary root are prolonged The distinctiveness tender shoots of elongation/root irradiation influence.Data display is average value ± s.e.m. (n=30).Identical lowercase Represent inapparent difference (P ＜ 0.05) between average value.(C) HYH-GFP is moved to root from tender shoots undetectablely.Will be by pSUC2：The scion (leaf) of the plant for the 10- age in days graftings that HYH-GFP hyh-1 scion and HYH (Ws) stock are constituted and stock HYH-GFP in (root) is distributed and by pSUC2：The 10- age in days graftings that HY5-GFP hy5 scion and HY5 (Col) stock are constituted The scion (leaf) of plant compares with the HY5-GFP distributions in stock (root).

Figure 14 .HY5 combination HY5 promoters.(A) ChIP is determined.The HY5 for being used for ChIP analyses of chart drawing presumption is opened Mover and fragment (1-7).Use 14- ages in days pHY5：Myc-HY5hy5 plants carry out ChIP-PCR.Data display be average value ± S.e.m (n=3).(B) EMSA is determined.By the HY5 promoter fragments of the box containing T/G- shown in A-motif and shown MBP- HY5 is incubated.For the HY5 competitions combined be utilized respectively containing T/G- box motifs 10 ×, 20 ×, 50 × with 100 × unmarked Probe carry out.

Figure 15 glucose to root NRT2.1 transcript abundances and¹⁵NO3^-The influence of intake.(A) in the root of WT and hy5 seedling NRT2.1 transcript abundances.Transcript level is represented with respect to the abundance of arabidopsis actin 2.Data display be average value ± S.e.m. (n=3).(B) 7- ages in days WT and hy5 seedling roots¹⁵NO3^-Intake.Data display is average value ± s.e.m. (n= 10)。

Influence of Figure 16 sucrose to HY5 promoter activities, HY5 transcripts and HY5 to the binding affinity of HY5 promoters. (A) sucrose level is transcribed (such as by by pHY5 to HY5 in root：The GFP expression of GFP transgenosis driving can show) and HY5 it is steady It is qualitative (from pHY5：HY5-GFP expression HY5-GFP) influence.Engineer's scale, 50 μm.(B) sucrose level is to root HY5 transcripts The influence of abundance.Transcript level is represented with respect to the abundance of arabidopsis actin 2.Data display is average value ± s.e.m. (n =3).(C) using the 10- ages in days pHY5 grown on the 1/2MS culture mediums containing 1% or 3% sucrose：Myc-HY5hy5 plants The ChIP-PCR analyses of progress.Data display is average value ± s.e.m. (n=3).Utilize Student ' s t- to examine and produce P Value.

Figure 17 .myc-HY5 expression recovers the hy5 sucrose sensitiveness to WT levels.(A) hy5 of sucrose-processing and pHY5：The level of NRT2.1 transcripts in myc-HY5hy5 roots.Abundance table of the transcript level relative to arabidopsis actin 2 Show.Data display is average value ± s.e.m. (n=3).(B) hy5 and pHY5 of sucrose-processing：In myc-HY5hy5 roots¹⁵NO3^- Uptake ratio.Data display is average value ± s.e.m (n=30).The presence of identical lowercase represents not show between average value The difference (P ＜ 0.05) of work.

Figure 18 increase influence of the light fluence rate to plant growth.Not share the same light, fluence rate grows 21 days (16h in soil Photoperiod) WT plants tender shoots (above) and root (figure below) system.Engineer's scale, 1cm.

It is described in detail

Present invention will be further described now.In paragraphs below, the different aspect of the present invention is limited in more detail.Can be by this The various aspects that sample is limited are combined with any other one side or many aspects, are indicated unless there are clearly opposite.Especially, refer to It is bright can be with being indicated as being preferred or favourable any other feature or multiple features for preferred or favourable any feature Combination.

Unless otherwise directed, practice of the invention will be trained using the botany in art technology, microbiology, tissue Foster, molecular biology, chemistry, biochemistry and recombinant DNA technology, the routine techniques of bioinformatics.Such technology is in text Fully explained in offering.

As used in this article, term " nucleic acid ", " nucleotide sequence ", " nucleotides ", " nucleic acid molecules " or " polynucleotides " It is intended to include DNA molecular (for example, cDNA or genomic DNA), RNA molecule (for example, mRNA), naturally occurring, mutation, conjunction Into DNA or RNA molecule and using nucleotide analog produce DNA or RNA analog.It can be single-stranded or double-stranded 's.Such nucleic acid or polynucleotides include, but not limited to the coded sequence of structural gene, antisense sequences, and do not encode MRNAs or protein product non-coding regulatory sequence.These terms also include gene.Term " gene " or " gene order " extensively Ground is used to refer to the DNA nucleic acid related to biological function.Therefore, gene can include such as the introne in genome sequence and outer aobvious Son, or can only include such as the coded sequence in cDNA, and/or the eDNA that is combined with regulatory sequence can be included.

Term " peptide ", " polypeptide " and " albumen " are used interchangeably herein, and refer to what is linked together by peptide bond With the amino acid of the Multimeric forms of any length.

As used in this article, term " hereditary change " includes, but not limited to genetically modified plants and mutant plant.

For the purposes of the present invention, " transgenosis ", " transgenosis " or " restructuring " is meant on such as nucleotide sequence, expression Box, gene construct or carrier comprising nucleotide sequence or conversion have nucleotide sequence of the present invention, expression cassette or carrier Organism, all that construct is produced by recombination method, wherein

(a) nucleotide sequence of the albumen for method of the invention is encoded, or

(b) genetic control sequences being operatively connected with nucleotide sequence of the present invention, such as promoter, or

(c) a) and b) it is not at their natural genetic environment or is modified by recombination method, for modification, example Such as, the form of substitution, addition, deletion, inversion or the insertion of one or more nucleotide residues may be taken.Natural genetic environment Presence in natural gene group or the chromogene seat or genomic library that are interpreted as meaning in former plant.In genomic library In the case of, it is preferable that at least partly retain the natural genetic environment of nucleotide sequence.Environment connects simultaneously in nucleotide sequence at least side side And with least 50bp, preferably at least 500bp, particularly preferably at least 1000bp, most preferably at least 5000bp sequence length.When The expression cassette by non-natural, synthesis (" artificial ") method such as, for example, mutagenic treatment modify when, naturally occurring expression cassette-example Such as corresponding nucleic sequence of polypeptide of the natural promoter of nucleotide sequence as defined above with encoding the method for the present invention Naturally occurring combination-be changed into transgene expression cassette.Suitable method, for example, describing in US 5,565,350 or WO 00/ In 15815, it is by reference to combining.

In certain embodiments, for purposes of the present invention genetically modified plants are therefore understands that to mean, such as institute above State,, can for nucleic acid for nucleic acid not their natural gene seat in the genome of the plant of the method for the present invention Can homologous or heterogenous expression.Therefore, plant express transgenic.However, as mentioned, in certain embodiments, transgenosis is also Mean, when nucleic acid its natural place in the genome of plant described in the different embodiments of the present invention, with regard to native sequences For, the sequence has been modified, and/or the regulatory sequence of native sequences is modified, for example, modified by mutagenesis.

Transgenosis is preferably interpreted as meaning, nucleic acid of the present invention the non-native gene seat of genome expression, i.e., Occur the homologous expression of nucleic acid or preferably occur the heterogenous expression of nucleic acid.According to the present invention, transgenosis stable integration enters plant, And plant is homozygosis preferably for transgenosis.

The aspect of the present invention is related to recombinant DNA technology, and exclusion is based only upon by routine in preferred embodiments The embodiment that breeding method produces plant.

For the purposes of the present invention, " mutant " plant is the hereditary change compared with naturally occurring wild type (WT) plant Plant.In one embodiment, mutant plant is compared with naturally occurring wild type (WT) plant, to use mutagenesis side Method, the plant that method of mutagenesis as described herein changes.In one embodiment, method of mutagenesis targeted genomic modification or Genome editor.In one embodiment, compared with wild-type sequence, using method of mutagenesis change endogenous HY5 nucleic acid or HY5 promoter sequences.Compared with wild-type plant, these mutation can cause activation or in addition enhancing HY5 promoters or its work( Can homologue or variant activity or the expression of HY5 nucleic acid or function homologue or its variant can be strengthened.With wild type Plant is compared, and such plant has the phenotype changed as described herein, such as increased nitrogen metabolism.Therefore, in the example In, the endogenous HY5 genes or HY5 promoter sequences that there is mutation in Plant Genome assign increased nitrogen metabolism.Preferred Embodiment in, using the selectively targeted endogenous HY5 genes of targeted genomic modification or HY5 promoter sequences, also, deposit The presence of the HY5 genes or HY5 promoter sequences of mutation is not assigned in the transgenosis expressed in plant.In alternative embodiment party There is provided expression SEQ ID NO in case：The mutant plant of the nucleic acid limited in 1,2 or 3 or its function homologue or variant.This Outside, compared with wild-type plant, such plant has the phenotype changed and shows increased nitrogen metabolism.In addition, again, In one embodiment, the phenotype of such plant is not assigned by the presence of one or more transgenosis.

The inventors discovered that, HY5 is the coupling for adjusting the light regulation that tender shoots growth and C absorptions are absorbed with root growth and N Tender shoots is to root movable signal.HY5 is transferred to root and then activates root HY5 by itself regulation feedback loop and expresses from tender shoots.This Outside, as shown in arabidopsis, AtHY5 directly adjusts nitrate anion transport protein AtNRT2.1 expression.Therefore, HY5 tender shoots- Extremely-root migration is that the AtNRT2.1 transcriptions of photoactivation are absorbed (with NO3 with N in root^-Form) required for.In other words, to the greatest extent Pipe HY5 is produced in root, still, only when the HY5 albumen produced due to light sensation in tender shoots/leaf is moved to root, and it is accumulated Effective level and root development and N- is induced to absorb.

Therefore, in one aspect, the present invention relates to for increasing the method for the metabolism of the nitrate anion in plant, it is included in plant The nucleic acid construct for including plant HY5 nucleotide sequences is introduced and expressed in thing.

HY5 is highly conserved in terrestrial plant.Conservative function is by specifically described herein it is demonstrated experimentally that and the present inventor Show, when paddy rice HY5, OsHY5 are expressed in transgenic arabidopsis, AtNRT2.1 can be induced, and from liver moss, small vertical bowl The HY5 of moss (Physcomitrella) can also induce AtNRT2.1 (Fig. 5).

According to various aspects of the invention, including the above method, the plant HY5 protein specificities of plant HY5 nucleic acid coding exist In COP1 interaction domains and basic leucine zipper domain presence (Holme et al., EMBO journal, Vol.20, the 118-127 pages, 2001).Sequence for AtHY5 these domains is shown below.However, these sequences It is highly conserved (see, for example, Fig. 6) in other HY5 albumen, and technical staff therefore be possible to easily identify except arabidopsis Outside plant HY5 albumen.

Therefore, the conservative of one or more following discriminatings is included according to the plant HY5 albumen of various aspects of the invention Domain has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77,78% with the motif that is shown below, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, The domain of 94%, 95%, 96%, 97%, 98% or at least 99% complete sequence homogeneity.

AtHY5 albumen includes following COP1 interaction domains：ESDEEIRRVPEF(SEQID NO：30, referring to figure 6).This includes CKII sites (the ESDEE SEQID NO for phosphorylation of presumption：31).The domain (including COP1 is mutual Acting domains) core V-P-E/D-X-G motifs (SEQ ID NO：32, X be hydrophobic residue) it is highly conserved structure base Sequence, it is required for the interaction with COP1.

AtHY5 albumen also includes basic leucine zipper domain shown below：

Alkaline motif：KRLKRLLRNRVSAQQARERKK(SEQ ID NO：33)

Leucine zipper：LENRVKDLENKNSELEERLSTLQNENQML(SEQ IDNO：34)

In one embodiment, HY5 nucleic acid constructs include coding SEQ ID NO：The SEQ of the 3 AtHY5 albumen limited ID NO：1 or 2 or its functional variety or homologue.

Term " functional variety of nucleotide sequence " as used in this article, with regard to SEQ ID NO：It is for 1 or another sequence Refer to and retain the variant gene sequence of the biological function of non-variant sequence thereof or the part of gene order completely, for example, planted when in transgenosis Increased growth or yield are assigned when being expressed in thing.Functional variety also includes the variant for grinding target gene, and it, which has, does not influence work( The sequence of energy changes, such as change in non-conservative residue.It is also included to be and shown wild-type sequence phase herein Than, it is substantially the same, i.e., only there is the mutually change of some sequences, such as change in non-conservative residue, and be to have biology The variant of activity.

Therefore, as understood by a person skilled in the art, it is understood that aspect of the invention, including method and purposes, not only wrap Include and include or by SEQ ID NO：The nucleotide sequence of 1 composition, but also including do not influence the bioactivity of resulting albumen with The SEQ ID NO of function：1 functional variety or part.Cause do not influenceing what is encoded to anchor point generation different aminoacids The change of the nucleotide sequence of the functional characteristic of polypeptide is as known in the art.For example, (a kind of hydrophobic for amino acid alanine Amino acid) codon can be encoded another less hydrophobic residue, such as glycine, or more hydrophobic residue, such as valine, bright ammonia The codon substitution of acid or isoleucine.Similarly, a kind of electronegative residue is caused to substitute another, as aspartic acid is replaced Another, such as change of lysine replacement asparagine, it is also contemplated that production is substituted for the residue of glutamic acid, or a kind of positively charged The product that raw function phase is worked as.The nucleotides of the N- ends of peptide molecule and the change of C- end sections is caused to change and will not be expected Change the activity of polypeptide.The modification of every kind of suggestion is fully in the routine techniques of this area, and this is that coded product retains bioactivity Determinant.

Functional variety and non-Variant amino acid sequences have at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%th, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%th, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%th, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%th, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%th, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% complete sequence homogeneity.

Technical staff will be understood that the invention is not restricted to for the use of AtHY5.Therefore, one in terms of the present invention In embodiment, nucleic acid sequence encoding SEQ ID NO：3 homologue.

Term homologue as used in this article also means the AtHY5 homologues from other plant species.AtHY5 is more The homologue of peptide or AtHY5 nucleotide sequences is respectively with the increased order of priority and SEQ ID NO：3 represent amino acid or with SEQ ID NO：Nucleotide sequence shown in 1 or 2 has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%th, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%th, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%th, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%th, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%th, 93%, 94%, 95%, 96%, 97%, 98% or at least 99% complete sequence homogeneity.In one embodiment, Complete sequence homogeneity is at least 37%.In one embodiment, complete sequence homogeneity be at least 70%, 71%, 72%, 73%th, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%th, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%, most preferably 90%, 91%th, 92%, 93%, 94%, 95%, 96%, 97%, 98% or at least 99%.

The functional variety of HY5 homologues is also within the scope of the invention.

If the nucleotides or amino acid residue sequence in two sequences are entering to most homogeneous as described below respectively Row is identical when comparing, and two nucleotide sequences or polypeptide are referred to as " same ".In the feelings of one or two nucleic acid or peptide sequence Under condition, term " same " or percentage " homogeneity " refer to when such as using one of following sequence comparison algorithm or by manually comparing Pair and visual inspection measurement, when most homogeneous being compared and compared through comparison window, identical or specified with identical Amino acid residue or two or more sequences of nucleotides percentage or subsequence.When same using sequence for albumen or peptide During the percentage of one property, it is believed that usually difference is that conserved amino acid replaces to the resi-dues differed, wherein amino acid residue Substituting other has the amino acid residue of similar chemical character (for example, electric charge or hydrophobicity), and does not therefore change molecule Functional characteristic.When sequence difference is conservative replacement, percent sequence identities can raise to correct substituted conservative Matter.Carry out this regulation mode be well known to a person skilled in the art.Compare for sequence, a usual sequence is used as referring to sequence Row, cycle tests is compared with it.When using sequence comparison algorithm, test and reference sequences are inputted into computer, specify subsequent Coordinate, and if desired, specified sequence algorithm routine parameter.The program parameter of acquiescence can be used, or can specify and replace The parameter in generation.Program parameter is then based on, sequence comparison algorithm calculates percent sequence of the cycle tests relative to reference sequences Homogeneity.The non-limiting examples for being adapted to determine that the algorithm of sequence identity and sequence similarity are that BLAST and BLAST 2.0 are calculated Method.

The example of homologue is in Fig. 6, table 1 and SEQ ID NOs：Shown in 4-29.Inventors have shown that, to found bowl from childhood Moss (Physcomitrella patens) has the homologue of 38.6% sequence identity with AtHY5 and had with AtHY5 The OsHY5 of 65.5% sequence identity induces AtNRT2.1 (Fig. 5) when being expressed in arabidopsis, and saves function mutation The hy5 losses of body.

The identification of the domain that suitable homologue can be compared and be guarded by sequence differentiates.Existing in this area can For differentiating the fallout predictor of such sequence.The function of homologue can differentiate as described herein, and for example plant In thing be overexpressed when or when being expressed by saving mutation type surface in hy5 afunction mutant, technical staff thus, it is possible to Enough confirm the function.

Therefore, HY5 nucleotide sequences of the invention and specifically described herein can be used for from other biological, be particularly other Plant, such as crop plants separate corresponding sequence.By this way, such as PCR, hybridization method can be used for based on them and this The sequence homology of sequence described in text differentiates such sequence.When differentiating and separating homologue, it is also contemplated that sequence Topological structure and feature structure domain structure.With whole sequence or with the sequence identity of its fragment sequence can be separated based on them Row.In hybridization technique, it is known that all or part of nucleotide sequence is used as probe, and it is with being present in the plant from selection Other corresponding nucleotide sequences in the genomic DNA fragment of clone or colony's (that is, genome or cDNA library) of cDNA fragments Column selective hybridizes.Hybridization probe can be genomic DNA fragment, cDNA fragments, RNA fragments or other oligonucleotides, and Detectable group, or any other detectable mark substance markers can be used.For hybridizing and for construction cDNA and gene The preparation method of the probe in group library it is general it is known in the art that and in Sambrook, et al., (1989) Molecular Cloning：A Library Manual (second edition, Cold Spring Harbor Laboratory Press, Plainview, New York) described in.

The hybridization of such sequence can be carried out under strict conditions." stringent condition " or " stringent hybridization condition " means this The condition of sample, under this condition, the detectable degree that probe hybridizes with its target sequence is higher than the degree of hybridization (example with other sequences Such as, higher than at least 2- times of background).Stringent condition is sequence dependent and in varied situations by difference.It is miscellaneous by controlling The stringency and/or wash conditions of friendship, it is possible to authenticate the complementary target sequence (same to source detection) with probe 100%.It is alternatively possible to Stringency is adjusted to allow some mispairing in sequence, so as to detect the similitude (heterologous detection) of lower degree.Generally, Probe length is less than about 1000 nucleotides, and preferred length is less than 500 nucleotides.

Generally, stringent condition will be such condition：Wherein it is less than about 1.5M Na ions in the salinity of pH 7.0 to 8.3, Normally about 0.01 to 1.0M Na ion concentrations (or other salt), and for short probe (for example, 10 to 50 nucleotides), temperature Degree is at least about 30 DEG C, and for long probe (for example, more than 50 nucleotides), temperature is at least about 60 DEG C.Hybridization continues Time is generally less than about 24 hours, normally about 4 to 12 hours.It can also obtain strict by adding destabilizing agent such as formamide Condition.

, according to the invention it is preferred to AtHY5 homologues be selected from corn, paddy rice, wheat, colea/canola, height Fine strain of millet, soybean, sunflower, clover, potato, tomato, tobacco, grape, barley, pea, beans, broad bean, lettuce, cotton, sugarcane, sugar Garden beet, broccoli or other brassica vegetables or willow.

According to method specifically described herein, plant expression is " external source " polynucleotides, " external source for bion " polynucleotides are the polynucleotides by any mode introduced plant in addition to sexual hybridization.It is described below by it The example of the mode of this point can be realized.In an embodiment of method, the nucleic acid of external source is the table in genetically modified plants Reach, it is not endogenous for the plant, and is derived from the plant HY5 nucleotide sequences of another plant species.For example, AtHY5 can not be expression in another plant of arabidopsis (Arabidopsis).It is interior in an embodiment of method Property nucleic acid in source is expressed in genetically modified plants, and it is the nucleic acid construct comprising endogenous HY5 nucleic acid or the sequence from it.Example Such as, OsHY5 can be expressed in paddy rice.

According to various aspects of the invention, method described herein and the plant of application, including genetically modified plants, Ke Yishi Unifacial leaf or dicotyledon.

Dicotyledon can the section selected from including but not limited to the following：Composite family (Asteraceae), Cruciferae (Brassicaceae) (such as colea (Brassica napus)), Chenopodiaceae (Chenopodiaceae), Curcurbitaceae (Cucurbitaceae), pulse family (Leguminosae) (Caesalpiniaceae (Caesalpiniaceae), Aesalpiniaceae, bashfully Careless section (Mimosaceae), Papilionaceae or pulse family (Fabaceae)), Malvaceae (Malvaceae), the rose family Or Solanaceae (Solanaceae) (Rosaceae).For example, plant can selected from lettuce, sunflower, arabidopsis, broccoli, spinach, It is watermelon, winter squash, wild cabbage, tomato, potato, Chinese yam, capsicum, tobacco, cotton, gumbo, apple, rose, strawberry, clover, beans, big Beans, broad bean, pea, French beans, peanut, chick-pea, apricot, pears, peach, grapevine, pimento, capsicum or citrus species.

Monocotyledon can be, for example, selected from section's Arecaceae (Arecaceae), Amaryllidaceae (Amaryllidaceae) Or grass family (Poaceae).For example, plant can be cereal crops, such as corn, wheat, paddy rice, barley, oat, sorghum, black Wheat, grain, buckwheat or forage crop such as Lolium (Lolium species) or Festuca (Festuca species), or make Thing such as sugarcane, onion, leek, Chinese yam or banana.

Also included is bio-fuel and bio-energy crop in China such as rape/canola, sugarcane, sugar grass, switchgrass (Panicum virgatum) (switchgrass (switchgrass)), linseed, lupin and willow, willow, willow cenospecies, Chinese silvergrass (Miscanthus) or gymnosperm, such as torch pine.Also included is to be used for ensilage (corn), herd or grass Expect (grass, clover, sanfoin, clover), fiber (such as cotton, flax), construction material (such as pine tree, Oak Tree), slurrying (such as willow), the charging deposit (such as high erucic acid Oilseed rape, linseed) for chemical industry and for comfortable mesh (such as the sod grass for golf course), ornamental plant (such as toad's-mouth, petunia, the rose in both privately and publicly owned garden Rare, fish pelargonium, Nicotiana) and the plant of family expenses and cut-flower (African violet (African violets), begonia (Begonias), Chrysanthemum (chrysanthemums), fish pelargonium, hair spider plant (Coleus spider plants), dragon tree (Dracaena), rubber plant) crop.

It is preferred that, plant is crop plants.Crop plants mean with commercial-scale plantation be used for human or animal consumption or Any plant used.In preferred embodiments, plant is cereal.

Most preferred plant is corn, paddy rice, wheat, colea/canola, sorghum, soybean, sunflower, lucerne Mu, potato, tomato, tobacco, grape, barley, pea, beans, broad bean, lettuce, cotton, sugarcane, sugar beet, broccoli or its His brassica vegetable or willow.

Term " plant " as used in this article includes complete plant, the ancestors of plant and offspring and plant part, bag Include seed, fruit, tender shoots, stem, leaf, root (including stem tuber), flower, tissue and organ, wherein it is foregoing each include as herein Described nucleic acid construct.Term " plant " also includes plant cell, suspension culture, callus, embryo, meristematic zone, spouse Body, sporinite, pollen and microspore, similarly, wherein it is foregoing each include nucleic acid construct as described herein.

Methods described can also include filtering out comprising polynucleotide constructs as described herein and/or having from plant Plant any phenotype (such as increased nitrogen metabolism) specifically described herein, and select have the phenotype (such as increased Nitrogen metabolism) plant.In another embodiment, further step includes measuring the nitrogen in described plant generations or part thereof Metabolism, and compared nitrogen metabolism with check plant.Preferably, by progeny plants stable conversion, and included in plant cell Heredity keep external source polynucleotides, and methods described can include checking construct stable integration the step of.The side Method can also include the other step that seed is collected from the progeny plants of selection.

The aspect of the present invention also extends to from the plant and harvests partly derivative, preferably directly derivative product, such as Dry particl or powder, oil, fat and aliphatic acid, starch or albumen.The invention further relates to plant comprising the present invention or part thereof Food and food supplement.

According to various aspects of the invention, compared with check plant, the nitrogen metabolism increase in genetically modified plants.As herein The check plant according to all aspects of the invention used is the plant of method modification not according to the present invention.Therefore, will not Check plant genetic modification encodes HY5 nucleic acid to express.In one embodiment, check plant is wild-type plant.It is right It is typically identical plant species according to plant, preferably there is identical genetic background with the plant of modification.

Included obtaining (NO3 according to increased NUE and nitrogen of the invention according to the nitrogen metabolism of the present invention^-Intake).According to this The term " increase ", " improvement " or " enhancing " that the various aspects of invention are used is used interchangeably.NUE can be defined as in soil often The obtainable N (including remaining N present in soil and fertilizer) of unit grain yield.The overall N service efficiencies bag of plant The efficiency containing uptake and utilization and it may be calculated UpE.In one embodiment, compared with check plant, NUE increases 5%- More than 50%, for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.Implement another In scheme, compared with check plant, nitrogen, which is absorbed, increases more than 5%-50%, and for example, at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

Increased nitrogen metabolism causes increased yield.Therefore, method of the invention can be used for increased yield.Term " yield " includes the one or more in the non-limiting list of following characteristics：Early flowering time, the biomass (biology of plant Measure (root and/or tender shoots biomass) or seed/cereal biomass), seed/cereal yield, seed/cereal viability and rudiment effect What rate, seed/cereal size, cereal starch content, early stage vigor, greenness index, increased growth rate, chlorenchyma postponed declines Always.Term " yield " generally means that the measurable generation of economic value typically related with period to specified crop, region.Individual Plant part directly facilitates yield based on its quantity, size and/or weight.Actual yield is the production of annual every square metre of crop year Rate, it is by using total output (including harvest and yield of assessment) divided by square metre determination of plantation.

Therefore, according to the present invention, yield includes the one or more of the following, and can be by evaluating the following In one or more measurements：The increased seed yield of each plant, the increased full rate of seed, the number of increased full seed Amount, increased harvest index, increased viability/germination efficiency, the number of increased seed/capsule (capsule)/pod/grain Amount or size, increased growth or increased branch, such as inflorescence with more multi-branched, increased biomass or cereal are filled out Fill.Preferably, increased yield include increased grain/seed/capsule/pod quantity, increased biomass, increased growth, The quantity of the flower of increased floral organ and/or increase branch.Relative to the increase of check plant yield.For example, with check plant phase Than, yield increase by 2%, 3%, 4%, more than 5%-50%, for example, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50%.

The method for being used to increase nitrogen metabolism in plant as described above (is included in plant and introduces and express comprising plant The nucleic acid construct of HY5 nucleotide sequences) further step can be included, the step includes assessing the table of genetically modified plants Type, measurement NUE and/or NO3^-Absorb, compared NUE and/or NO3 with check plant^-Intake, measurement yield and with check plant ratio Compared with one or more of yield.

The method for transformation of genetically modified plants for producing the present invention is as known in the art.Therefore, according to the present invention Various aspects, the nucleic acid of HY5 nucleic acid, such as SEQ D NO.1 will be included, its functional variety or homologue introduced plant simultaneously make For transgene expression.Nucleotide sequence is introduced into the plant by being referred to as the process of conversion.Term " introducing " as referred to herein Or " conversion " includes the polynucleotides of external source being transferred to host cell, regardless of whether the method for transfer.No matter passing through organ Generation or embryo occur that the plant tissue of follow-up clonal propagation, can be converted with the genetic constructs of the present invention, and From the overall plant of its generation.The particular organization of selection is by according to for obtained by the particular species of conversion and optimal with it The clonal propagation systems change of adaptation.Exemplary target tissue site include leaf dish, pollen, embryo, cotyledon, hypocotyl, egagametophyte, Callus, existing separate living tissue (for example, apical meristem, axillary bud and root separate living tissue), and mitogenetic group induced Knit (for example, cotyledon meristem and hypocotyl separate living tissue).Polynucleotides can instantaneously or stably introduce host cell, and It can keep not integrating, for example, as plasmid existing.Alternatively, it can be integrated into host genome.It will then can obtain The plant cell of conversion be used for regenerating transformed plant in the manner known to persons skilled in the art.

Foreign gene is transferred in the genome of plant and is referred to as converting.In many species, the conversion of plant is now Routine techniques.Advantageously, any of a variety of method for transformation can be used for target gene introducing suitable progenitor cells.For It can be used for instantaneous or stable conversion from the method described by plant tissue or transforming plant cells and aftergrowth.Method for transformation Including the use of liposome, DNA is injected directly into plant by electroporation, the chemical agent of increase dissociative DNA intake, Gun Bombardment, Converted using virus or pollen and microprojection.Method can be selected from the calcium/polyethylene glycol method for being used for protoplast, electroporation Protoplast, microinjection enter vegetable material, DNA or the coated particle bombardments of RNA-, with (circles) virus infection etc..Turn Gene plant, including transgenic crop plant, are preferably situated between by Agrobacterium tumefaciens (Agrobacterium tumefaciens) The conversion led is produced.

In order to select the plant of conversion, the vegetable material obtained in conversion is subjected to selective conditions, so as to turn The plant of change is mutually distinguished with unconverted plant.For example, the seed obtained in the above described manner can be planted, also, in starting life After long-term, suitably selected by spraying.Other possibilities are if appropriate, to use suitable choosing after sterilization Select and seed is planted on the agar plate of agent, so that the seed of only conversion can be grown to plant.Alternatively, the plant of conversion is sieved Select the presence of selective key thing.After DNA is shifted and regenerated, the plant for the conversion that for example can also be estimated using DNA analysis evaluation The presence of target gene, copy number and/or genome composition in thing.Alternately, or additionally, can use RNA traces and/or The expression for the DNA that western blot analysis monitoring is newly introduced, both technologies are all known in those of ordinary skill in the art 's.

The conversion plant of generation can breed in several ways, such as pass through clonal propagation or classical breeding technique.For example, The plant of the first generation (or T1) conversion with selfing and can select the second generation (or T2) transformant of homozygosis, and T2 plants can be with Further bred afterwards by classical breeding technique.The biology of the conversion of generation can be diversified forms.For example, they can be turned The chimera of the cell of change and unconverted cell；Clonal transformants (for example, converting all cells to contain expression cassette)；Turn The grafting (for example, in plant, being grafted onto the conversion rhizome of unconverted scion) of tissue change and unconverted.

The method that the present invention is used to increase nitrogen metabolism in plant includes introducing and expressing the core for including plant HY5 nucleotide sequences Acid con-struct.The nucleic acid construct preferably comprises the regulating element being operatively connected with plant HY5 nucleotide sequences.

According to all aspects of the invention, including the above method and including plant as described herein, method and use On the way, term " regulating element " is used alternatingly with " control sequence " and " promoter " herein, and all terms are with wide model Consideration is enclosed, refers to influence the regulation nucleotide sequence of the expression of sequence in connection.

Term " promoter " typically refers to the nucleic acid control sequence of the upstream of the transcription initiation positioned at gene, and it participates in RNA The combination of polymerase and other albumen, so as to instruct the transcription of nucleic acid being operatively connected.What above-mentioned term included be derived from through Allusion quotation eukaryotic genomic gene (including TATA boxes needed for accurate transcription starting, be with or without CCAAT boxes sequence) and other tune Save element (i.e. upstream activating sequence, enhancer and silencer) transcriptional regulatory sequences, its respond develop and/or outside stimulus or Change gene expression in the way of organizing specific.The transcriptional regulatory sequences of the also included prokaryotic gene for being classics in the term, In this case, it can include -35 box sequences and/or -10 box transcriptional regulatory sequences.

Term " regulating element " also includes imparting, activation or the expression of enhancing cell, tissue or organ nucleic acid molecule The fusion molecule or derivative of synthesis.

" plant promoter " includes the regulating element of the expression of coding sequence fragment in regulation and control plant cell.Therefore, plant Promoter is necessarily plant origin, but can be derived from virus or microorganism, such as virus from attack plant cell.″ Plant promoter " may originate from plant cell, for example originating from being expressed in the method with the present invention and specifically described herein The plant of nucleotide sequence conversion.This is also applied for other " plant " Regulate signals, such as " plant " terminator.Side for the present invention The promoter of the nucleotide sequence upstream of method can be modified by the substitution of one or more nucleotides, insertion and/or deletion, without dry Disturb promoter, ORFs (ORF) or 3 '-regulatory region such as terminator or other the 3 ' regulatory regions work(of any one away from ORF Energy or activity.It is still possible that the activity of promoter is increased by modifying their sequences, or they are opened by more active completely Mover is substituted, or even is substituted by the promoter from heterologous organisms.For the expression in plant, nucleic acid point as described above Son, is preferably operatively connected with suitable promoter or comprising suitable promoter, the promoter correct time point simultaneously And with required spatial expression pattern expressing said gene.

For identification function promoter of equal value, the promoter intensity and/or expression pattern of alternate promoters can be such as By the way that promoter and reporter gene are operatively connected and expression of the reporter gene in the different tissues of plant is determined Analyzed with pattern.Suitable known reporter gene is known to technical staff, and including such as beta-glucuronidase or β- Galactosidase.

Term " being operatively connected " as used in this article refers to that the function between promoter sequence and target gene connects Connect, so that the promoter sequence can originate the transcription of target gene.

For example, nucleotide sequence can use the promoter expression that driving is overexpressed.Expressed the meaning to turn base according to the mistake of the present invention Because with the horizontal expression of the expression higher than the endogenous homologue driven by its internal promoter.For example, being overexpressed to make With strong promoter, such as constitutive promoter is carried out." constitutive promoter " refers in the most of but unnecessary of growth and development During all stages and under most environmental conditions, there is the promoter of transcriptional activity at least one cell, tissue or organ. The example of constitutive promoter include cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoters, Maize ubiquitin promoter, ribulose diphosphate hydroxylase (rubisco) small subunit, corn or clover H3 histones, OCS, SAD1 Or 2, GOS2 or any promoter for producing enhanced expression.Alternatively, enhanced or increased expression can be by using turning Record or translational enhancer or activator are realized, and enhancer can be incorporated into gene with further increase expression.

In one embodiment, promoter is composing type or strong promoter.In preferred embodiments, regulatory sequence It is inducible promoter or stress-induced type promoter.Stress-induced type promoter is selected from following non-limiting list：HaHB1 is opened Mover, RD29A (it drives DREB1A drought-inducible expression), (it drives by corn rabl7 drought-inducible promoters, P5CS1 Dynamic Proline synthesis enzyme P5CS1 drought-inducible expression), arabidopsis clade A PP2C ABA- and drought-inducible Promoter (ABI1, ABI2, HAB1, PP2CA, HAI1, HAI2 and HAI3) or their corresponding crop homologues.

Promoter can also be organizing specific.The promoter of these types is described in the prior art, but non-limiting Example is listed below.Other suitable promoters are also known to technical staff.

Tissue-specific promoter is special time during development of plants only in specific cells or organized (such as in nutrition Tissue or germinal tissue) in active transcriptional control element.The example bag of tissue-specific promoter under development control Include only (or main only) particular organization (such as nutritive issue, for example, root or leaf, or germinal tissue, such as fruit, ovule, seed, Pollen, gynoecium (pistols), flower) or any embryo tissue or epidermis or mesophyll in originate the promoter of transcription.Germinal tissue is special Promoter can be, for example, ovule-special, embryo-special, endosperm-special, integument-special, seed and plant skin- Special, pollen-special, petal-special, sepal-special, or their some combinations.In some embodiments, Promoter is that cell type is special, for example, guard cell-special.

In one embodiment, the promoter of chlorenchyma-special can be used.Chlorenchyma includes leaf and tender shoots. For example, chlorenchyma-special promoter can be selected from corn orthophosphoric acid kinase promoter, corn phosphoenolpyruvate carboxylate carboxylic Change enzyme promoters, paddy rice phosphoenolpyruvate carboxylate carboxylase promoter, paddy rice small subunit ribulose diphosphate hydroxylase (rubisco) promoter, paddy rice β expansin EXBP9 promoters, pigeonpea small subunit ribulose diphosphate hydroxylase (rubisco) promoter, mesophyll specificity promoter CAB3 (Warnasooriya, S.N., and Montgomery, B.L. (2009), Plant Physiol 149,424-433) or pea RBS3A promoters.

The example of the promoter of leaf-special also includes diphosphoribulose carboxylase (RBCS) promoter.For example, tomato RBCS1, RBCS2 and RBCS3A gene are expressed in the seedling of leaf and photoproduction length, and only RBCS1 and RBCS2 are in developmental tomato (Meier FEBS Lett.415 are expressed in fruit：91-95,1997).It can use by Matsuoka Plant J.6：311- The diphosphoribulose carboxylase of high level expression in the mesophyll cell almost only in blade and leaf sheath of 319,1194 descriptions Promoter.Another leaf-special promoter is light harvesting chlorophyll a/b binding-protein gene promoters, see, e.g., Shiina Plant Physiol.115：477-483,1997；Casal Plant Physiol.116：1533-1538,1998.Li FEBS Lett.379：1 17-121, the arabidopsis myb- related gene promoters (Atmyb5) of 1996 descriptions are also leaf-special. Developmental leaf epidermal hair (leaf trichomes), stipule and table of the Atmyb5 promoters on young flower ring and stem leaf edge Expressed in chrotoplast, and immature seed.Atmyb5mR A occur in 16 cell stages of fertilization and embryonic development, and retain super Spend embryonic stage (heart stage).

Another type of useful nutritive issue-special promoter is the promoter of separate living tissue, and it drives tender shoots point Expression in end.It is, for example, possible to use " SHOOTMERISTEMLESS " and " SCARECROW " promoter, they are developmental It is active in tender shoots or roots and tops separate living tissue, by Di Laurenzio, Cell 86：423-433,1996；And Long, Nature 379：66-69,1996 descriptions.Another useful promoter is such promoter, and it controls 3- hydroxy-3-methyls The expression of glutaryl CoA reductase HMG2 genes, its expression is limited to separate living tissue and flower (the secretion area of column cap, mature pollen Grain, the ovule of gynoecium vascular tissue and fertilization) tissue is (see, e.g., Enjuto Plant Cell.7：517-527, 1995).Also available is the knl- related genes from corn He other species, and it shows the expression of separate living tissue-special, ginseng See, for example, Granger Plant Mol.Biol.31：373-378,1996；Kerstetter Plant Cell 6：1877- 1887,1994；Hake Philos.Trans.R.Soc.Lond.B.Biol.Sci.350：45-51,1995.

The invention further relates to the plant of hereditary change, it includes HY5 nucleotide sequences and tissue specificity regulatory sequence.

In one aspect, included the present invention relates to genetically modified plants containing HY5 nucleotide sequences and tissue specificity regulation sequence The nucleic acid construct of row.

Term HY5 nucleotide sequences and tissue specificity regulatory sequence are described elsewhere herein.Preferably, organize special Specific regulatory sequences are leaf or the specific promoter of tender shoots.In one embodiment, the plant is not arabidopsis.

The present invention also extends to genetically modified plants, and it includes the nucleic acid construct containing HY5 nucleotide sequences, wherein described plant Thing is not arabidopsis.

The invention further relates to nucleic acid construct, it includes the plant HY5 cores being operatively connected with tissue-specific promoter Acid sequence.Tissue-specific promoter can be selected from chlorenchyma specificity promoter as described above.The invention further relates to Expression vector, it includes the nucleic acid construct containing the plant HY5 nucleotide sequences being operatively connected with tissue-specific promoter.

On the other hand, the present invention relates to the host cell of separation, its convert have nucleic acid construct as described above or Carrier.Host cell can be bacterial cell, such as Agrobacterium tumefaciens, or separation plant cell.The invention further relates to cultivate Base or kit, the host cell of separation of the kit comprising culture medium and as described above.

Nucleic acid construct or carrier described above is used for method for transformation generation as known in the art and turns base Because of plant.Therefore, on the other hand, the present invention relates to above-mentioned nucleic acid construct or carrier in terms of nitrogen metabolism in increasing plant Purposes.On the other hand, the present invention relates to the nucleic acid construct comprising HY5 nucleotide sequences and tissue-specific promoter or carrier Purposes in increase plant roots in terms of HY5 levels.

The invention further relates to produce the method for the plant with increased nitrogen metabolism, it, which is included in plant, introduces and expresses Include the nucleic acid construct for the HY5 nucleotide sequences being operatively connected with chlorenchyma specificity promoter.

On the other hand, the present invention relates to for increasing the method for the presence of HY5 albumen in plant roots, it is included in plant Introduce and express the nucleic acid construct for including the HY5 nucleotide sequences being operatively connected with chlorenchyma specificity promoter.

Inventors have also demonstrated that the balance between HY5 regulation C and N metabolism.The profit of coding and the fixed carbon of sucrose form The phase of the genetic transcription thing of transporter (such as SWEET11 and 12) is arranged outside the related albumen (such as TPS1) of expenditure or encoding sucrose Abundance is reduced strongly in hy5 seedling (lacking HY5), the two in terms of the migration of HY5 regulation Sucrose synthesis and C metabolism is pointed out. They also found that sucrose or glucose substantially strengthen AtNRT2.1 transcript abundances and the NO3 in WT plants^-Intake, but In hy5 mutant, these effects are substantially reduced.The HY5- dependences that high-affinity nitrate anion transhipment sublist is reached and N- is absorbed Sucrose induction promotes the homeostasis of the coordination of C and N metabolism.Therefore, the invention further relates to adjust C and N in plant to be metabolized it Between balance method, it is included in the nucleic acid construct for introducing and expressing in plant and include HY5 nucleotide sequences.In an implementation In scheme, HY5 nucleotide sequences are operatively connected with chlorenchyma specificity promoter.

The invention further relates to increase the method for HY5 levels in plant roots, its be included in plant introduce and express comprising with The nucleic acid construct for the HY5 nucleotide sequences that chlorenchyma specificity promoter is operatively connected.

The present invention is also extended to by method as described herein, and the method for such as increase nitrate anion metabolism obtains or can The plant of acquisition.

On the other hand, the present invention relates to mutant plant, i.e. the plant with increased endogenous HY5 expression of nucleic acid, Wherein endogenous HY5 promoters carry the mutation introduced by the genome editor of mutagenesis or targeting, and the mutation causes Increased endogenous HY5 expression of nucleic acid.In this embodiment, the increase of expression is relative in control or wild-type plant For level, as described elsewhere herein.In one embodiment, the genome editor of targeting is used to modify (example Such as, insert or change) at least one of the nucleotide sequence of HY5 promoters or its function homologue or variant nucleic acid.Preferred Embodiment in, the mutation strengthen internal promoter activity.For example, the genomic modification of targeting can be used for being inserted into Few an enhancer or promoter site, such as TATA boxes (TATAAA), GC boxes (GGGCGG) or CAAT (GGCCAATCT) box, or Its functional variety.On the other hand, the present invention relates to the plant with increased endogenous HY5 expression of nucleic acid, wherein endogenous HY5 nucleic acid carries the mutation introduced by the genome editor of mutagenesis or targeting, and it causes the increased expression of the nucleic acid. On the other hand, the present invention relates to the plant with increased HY5 expression of nucleic acid, wherein HY5 is expressed by the way that more than one is extra The HY5 nucleic acid of copy is incorporated into Plant Genome and increased.In preferred embodiments, the combination genome editor is real It is existing.

In one embodiment, HY5 nucleic acid is SEQ ID NO：1 or 2 or its function homologue or variant.In a reality Apply in scheme, internal promoter is SEQ ID NO：35 or its function homologue or variant.

In another aspect of the present invention there is provided the method for nitrogen metabolism in increase plant, methods described includes will at least one Mutation is planted to be introduced into endogenous HY5 sequences or its regulatory sequence to strengthen the expression of endogenous HY5 nucleic acid.There is also provided producing C and N generations in the method for the presence of HY5 albumen, regulation plant in the method for the plant absorbed with increased nitrogen, increase plant roots The method of the presence of HY5 albumen in the method and increase plant roots that are balanced between thanking, wherein methods described is included at least one Mutation introduces endogenous (i.e. natural or innately) HY5 sequences or its regulatory sequence to strengthen the expression of endogenous HY5 nucleic acid.

In one embodiment, at least one described mutation is introduced using the genome editor of targeting.In an implementation In scheme, the genome editor of targeting is used to modify (for example, insertion or change) HY5 promoters or its function homologue or change At least one nucleic acid in the nucleotide sequence of body.In preferred embodiments, the mutation strengthens the activity of internal promoter. For example, the genomic modification of targeting can be used for inserting at least one enhancer or promoter site, such as TATA boxes (TATAAA), GC boxes (GGGCGG) or CAAT (GGCCAATCT) box, or its functional variety.In another embodiment, the genome of targeting is compiled Collecting is used to more than one HY5 nucleic acid copied in addition being incorporated into Plant Genome.

In one embodiment, HY5 nucleic acid is SEQ ID NO：1 or 2 or its function homologue or variant.In a reality Apply in scheme, internal promoter is SEQ ID NO：35 or its function homologue or variant.

In the above-described embodiment, ' endogenous ' nucleic acid can refer to born or natural sequence in Plant Genome.

The genomic modification of targeting or the genome editor of targeting are made by the recombination event of homologous recombination (HR)-mediation The genome project technology of (DSBs) stimulated gene group editor is broken with the DNA double chain of targeting.In order to by introducing site-special DNADSB realize efficient gene group editor, four kinds of major type of customizable DBPs can be used：From micro- The meganuclease (meganucleases) of the mobile genetic elements of biology, the ZF ribozymes based on the eukaryotic transcription factor, from Huang The transcription activation factor sample effector (TALEs) of zygosaccharomyces bacterium (Xanthomonas bacteria), and from II type bacteriums The DNA endonucleases Cas9 of adaptive immune system CRISPR (the short palindrome in rule cluster interval is repeated) RNA- guiding.Greatly Meganuclease, ZF and TALE albumen recognize specific DNA sequence dna by protein-dna interaction.Although a wide range of nucleic acid Enzyme integrates nuclease and DNA- binding structural domains, ZF and TALE albumen is respectively by for 3 or 1 nucleotides (nt) for targetting DNA Module is constituted.The nuclease domain that ZF and TALE can assemble and be connected to FokI with required combining is directed to guiding The nucleolytic activity of specific gene group locus.

When being delivered to by bacterial type III secretion system in host cell, TAL effectors enter core, with reference to host's base Because of the sequence and activated transcription of the effector in promoter-special.Their targeting specific is by 33-35 amino acid connecting The central domain repeated is determined.The repetition of this then 20 amino acid of single truncation.It is most of check it is naturally occurring There are TAL effectors 12 and 27 to repeat completely.

These repeat only to be different from two adjacent amino acid, their repetition-variable two residue (RVD) each other.Determine TAL effectors will recognize the RVD of which single nucleotide acid：One RVD corresponds to a nucleotides, and four kinds of most common RVD are each From preferably associated with one of four kinds of bases.Naturally occurring recognition site as one man the T needed for TAL effector activities it Afterwards.TAL effectors can be blended in the catalyst structure domain of FokI nucleases to produce TAL effectors nuclease (TALEN), its Producing the DNA double chain fracture (DSBs) of targeting in vivo is used for genome editor.Use of the technology in genome editor is at this Sufficiently described in field, such as in US 8,440,431, US 8,440,432 and US 8, described in 450,471.With reference to Document 38 describes the plasmid of one group of customization, and it can be used together to assemble multiple DNA pieces with Golden Gate cloning process Section.As described therein, Golden Gate methods use IIS type restriction endonuclease, its lateral incision outside its recognition site Cut and produce unique 4bp protrusions.Clone is accelerated by digesting and connecting in same reactant mixture, and reason is correct group Dress eliminates enzyme recognition site.The assembling of the TALEN or TAL effector constructs of customization is related to two steps：(i) mould will be repeated Block is assembled into the 1-10 intermediate arrays repeated, and intermediate arrays are connected as skeleton to prepare final construct by (ii).

Another genome edit methods that can be used according to various aspects of the invention are CRISPR.The technology is in gene Use in group editor is fully described in the art, such as in US 8,697,359 and references cited herein Fully describe.In short, CRISPR is bacteriophage and the microbial nucleic acids enzyme system of plasmid for participating in resisting invasion.Microorganism place CRISPR locus in master contains related (Cas) genes of CRISPR- and can program the nucleic acid cutting of CRISPR- mediations (sgRNA) combination of specific non-coding RNA element.(the I- of three types is identified in the bacterial host of wide scope III) CRISPR systems.One key feature of each CRISPR locus is between short non repetitive sequence section (spacerarm) Every repetitive sequence (repeat in the same direction (direct repeats)) array presence.Non-coding CRISPR arrays are transcribed, and The short crRNA containing individual spacer sequence is segmented into repeating inscribe in the same direction, it guides Cas nucleases to target site (between former Every (protospacer)).II types CRISPR is one of system for most fully characterizing, and carries target in four sequential steps To DNA double chain fracture.First, two non-coding RNAs, i.e., before-crRNA arrays and tracrRNA, turn from CRISPR locus Record.Second, the tracrRNA-crRNA with before duplicate block hybridize, and-crRNA is processed as containing individual spacerarm sequence before mediation The ripe crRNA of row.3rd, ripe crRNA：TracrRNA complexs by the spacerarm on crRNA and with original by being spaced The Watson Crick base pairing between former interval on the adjacent target DNA of adjacent motif (PAM) guides Cas9 to target DNA, This is required in addition for target identification.Finally, Cas9 regulates and controls the cutting of target DNA to produce double-strand break in former interval.

Therefore, Cas9 is the marker protein of II type CRISPR-Cas systems, and is big monomer DNA nucleases, its quilt The complex of two non-coding RNAs (CRISPR RNA (crRNA) and de-activated crRNA (tracrRNA)) is directed to and PAM (the adjacent motif in original interval) adjacent DNA target sequence of sequence motifs.Cas9 albumen contain two it is homologous with RuvC and HNH nucleases Nuclease domain.HNH nuclease domains cut complementary dna chain, and incomplementarity chain is cut in RuvC- spline structures domain, and Thus passivity cutting is introduced in target DNA.Cas9 can be by the double-strand break in site-special together with sgRNA heterogenous expression (DSBs) genomic DNA from various biological living cells is introduced.In order to applied to eucaryote, use Cas9 codon Optimize version, it initially comes from bacterium streptococcus pyogenes (Streptococcus pyogenes).

Single guiding RNA (sgRNA) is the second component of CRISPR/Cas systems, and it forms compound with Cas9 nucleases Body.SgRNA is the prna chimera body by the way that crRNA to be merged to the synthesis produced with tracrRNA.SgRNA positioned at its 5 ' end Homing sequence assigns DNA target mark specificity.Therefore, by modifying homing sequence, it is possible to create with different target specificities sgRNA.The typical length of homing sequence is 20bp.In plant, using plant RNA polymerase III promoters (such as U6 and U3) sgRNA is expressed.

The Cas9 expression plasmids used in the method for the present invention can be built as described in the art.

Therefore, aspect of the invention is related to the method for mutagenesis of targeting, particularly genome editor, and in preferred implementation In scheme, the embodiment for being based only upon and plant being produced by traditional breeding way is excluded.

In another aspect of the present invention there is provided detection with increased or high HY5 expressions and/or increased or high The screening technique of the plant variety of nitrogen metabolism level, methods described is included in detection HY5 expressions at least one plant, and And selection is with one or more plants described in highest or higher HY5 expressions.In preferred embodiments, the plant of selection Thing also in several ways, breed by as described above those.

Term HY5 and plant are defined above.

Foregoing disclosure provides the general description for the theme being included within the scope of the invention, including prepares and use this The method of invention, and its best mode there is provided following instance with further such that those skilled in the art practice the present invention, and Its complete written description is provided.It will be understood by those skilled in the art, however, that the particular content of these examples should not be construed The limitation present invention, its scope should be from the claim appended by the disclosure and its physical solution of equal value.According to present disclosure, this hair Bright various other aspects and embodiment will be apparent for those skilled in the art.

" and/or " it is used herein in the case of to be considered two specific characteristics or composition each with or without Another specific disclosure.For example " A and/or B " is understood to (i) A, each specific public affairs in (ii) B and (iii) A and B Open, individually listed herein just as each.

Unless the context indicates otherwise, the definition of the description of features described above is not limited to any particular aspects or reality of the present invention Scheme is applied, and is equally applicable to all aspects and embodiment of description.

Aforementioned application, and all documents and Sequence accession number (" application wherein or in their checking process quoted are quoted Document ") and and application quote document in quote or reference all documents, and it is cited herein or reference all texts Offer (" the document cited herein "), and all documents quoted or referred in the document cited herein, together with this article or Herein by the operation instruction of any manufacturer of any product with reference to described in any document combined, specification, production Product specification and product brochure, herein by reference to being incorporated into herein, and can be used for the practice of the present invention.More specifically, The document of all references is by reference to being bound to seeming to indicate that each individual document is specific and identical by reference to combination respectively Degree.

Embodiment

Material and method

Vegetable material and growth conditions.By arabidopsis seed in 4 DEG C of imbibitions three days, it is subsequently placed on 1/2MS culture mediums.Will The seedling spilt is exposed to the 16h photoperiods at 22 DEG C.The experiment for being related to sapling grafting carries out (17) as described above.

Plasmid Constructs.Amplification HY5cDNA is simultaneously subcloned into pCaMV35S：Nos carriers (32).By HY5, SUC2 and CAB3 The sequence amplification of promoter is simultaneously subcloned into pCaMV35S：Both no carriers, HY5 promoters and GFP coded sequences are also so To produce pHY5：GFP expression cassettes.HY5 coded sequences are cloned into pSK-N-Tagged-myc carriers (33), and then sub- gram It is grand enter pCaMV35S：Nos carriers.In order to prepare pCAB3：2 × GUS-TEVrs-HY5-GFP fusion constructs, will by PCR TEV recognition sites (TEV^rs) 3 '-end of 2 × GUS coded sequences is blended in, and PCR primer is introduced into pCAB3：HY5-GFP Carrier.TEV protease is expanded and is cloned into pCaMV35S：Nos carriers (32).In order to build pHY5：HY5-GFP transgenosis, HY5 coded sequences are cloned into pHY5：GFP constructs.It is given in Table 2 for the PCR primer sequences expanded.

Transcript is analyzed.Total serum IgE is extracted using TRIzol reagents (Invitrogen, New York, USA), and is used M-MLV Reverse Transcriptase Reagents kits (Promega, Wisconsin, USA) reverse transcription.QRT-PCR analyses (34) are carried out as previously described. Repeated to represent each experiment by three secondary pollutants, each biology repeats at least three times technologies and repeated.Arabidopsis actin 2 is used Make reference gene.Relevant primer is given in Table 3.

Immunoblotting assay.The preparation of Crude protein passes through in 50mM Tris-HCl (pH 7.5), 150mM KCl, 10mM Extract and obtain in MgCl2,1mM EDTA, 10% glycerine, 0.1%NP-40,1 × adequate proteins enzyme inhibitor (Roche).Will etc. Point sample is through 10% (w/v) SDS- polyacrylamide gel electrophoresises and is transferred on Hybond ECL nitrocellulose filters.Then Film is operated according to the scheme (35) set up.Anti- myc antibody (Santa Cruz Biotechnology, Santa are used respectively Cruz, USA) and anti-GFP antibody (Roche Diagnostics GmbH, Germany) detection myc-HY5 and HY5-GFP fusion eggs In vain, and using uperSignal West Pico chemical luminous substrates (Thermo Fisher Scientific, Waltham, USA signal) is shown.

The measurement of total carbon and nitrogen content.The plant tissue of dehydration is ground, and uses ElementarVario PYRO Cube analyzers (Elementar Analysensysteme GmbH, Frankfurt, Germany), use high-temp combustion process (36) to determine the total carbon and nitrogen content of powdered material.

¹⁵NO3- picked-up activities are determined.(3) determine root as described in elsewhere¹⁵NO3^-Flow into.Root is dried at 80 DEG C Night, and use ANCA-MS systems (PDZ Europa Ltd) measurement¹⁵N content.

ChIP-PCR is determined.By hy5 and pHY5：Myc-HY5hy5 seedling grows 14 days on 1/2MS plates.Then by plant The 2g aliquots of tissue are fixed by formaldehyde crosslinking.As described above (37), ChIP, which is determined, uses anti-myc antibody (Santa Cruz Biotechnology, Santa Cruz, USA).The enrichment of DNA fragmentation is analyzed by qRT-PCR and determined.Carry out three times Independent biology is repeated.Relevant primer sequence is given in Table 4.

EMSA is determined.HY5cDNA is expanded and pMAL is cloned into^TM- c2X carriers (New England Biolabs, Ipswich, USA).According to the explanation of manufacturer, MBP and MBP-HY5 fusion proteins are purified.DNA amplification probe simultaneously uses biology Plain labelling kit (Invitrogen, New York, USA) is with biotin in its 3- end mark.Use LightShiftization Learn luminous EMSA kits (Thermo Fisher Scientific, Waltham, USA) and carry out DNA gel migration measure.Make Primer sequence is given in Table 5.

As a result

Although plant tender shoots and root coordinate its biology with excellent in the environment of fluctuation according to different development tracks Change overall plant performance.The coordination of metabolic adsorption is most important：Carbon (the C that tender shoots is fixed in air；CO₂), root obtain soil from Sub- nitrogen (N；Mainly nitrate anion (NO3^-))(1).Because N is required, soil NO3^-Level usually limits natural and agricultural environment In productivity ratio (2,3).The regulation of known N and C acquisitions rate is tight association (4,5), and the two processes are adjusted using light (6).However, potentially the molecular mechanism of regulation long range tender shoots-root communication is still unknown.

As obvious report of regulation tender shoots-root communication, and because root is generally not exposed to light, we it is thus determined that Separated tender shoots or root irradiate the influence (Figure 1A) to root growth.It was found that wild type (WT) arabidopsis (Col-0 tests strain) Seedling (its tender shoots (only) exposed to light (S (L)/R (D)) primary root and be completely exposed to light (S (L)/R (L) WT seedling Primary root has similar-length (Figure 1A-C).By contrast, the root of WT S (D)/R (L) seedling is shorter than S (L)/R (D) seedling, And there is similar-length (Figure 1B, C) with S (D)/R (D) seedling.In addition, the lateral root propagation of S (L)/R (D) seedling is similar to S (L)/R's (L), and the lateral root of S (D)/R (L) seedling propagation much less, similar to S (D)/R (D) lateral root propagation (Fig. 7).Cause This, tender shoots irradiates most probable and promotes root growth by tender shoots-extremely-root signal transduction.

We are then screened by the Arabidopsis Mutants of specificity destruction in terms of the root growth that tender shoots-irradiation promotes, and And find, new HY5 afunction allele (hy5-526 is contained in one of they；Figure 1B；Fig. 8).Further analysis shows, Hy5 amorphs (7；Fig. 8) also eliminate the root growth (Fig. 1 C) that tender shoots-irradiation promotes.HY5 codings are photosensitive morphogenetic BZIP transcription factors HY5 (8).HY5 is by COP1 ubiquitinbond enzyme adjustments, and the ubiquitin ligase targets HY5, in the dark Protein degradation (9,10).Therefore, it has been found that root growth is tender in the dark raw seedling of afunction copl-4 mutant simulation Bud-irradiation promotes (Fig. 1 C).These results are pointed out, and tender shoots-irradiation of HY5 regulation and control root growths promotes.

We then have found, WT roots NO3^-Absorb and promoted by the irradiation of tender shoots, but (Fig. 1 D) is not promoted by the irradiation of root. NO3^-Taken the photograph by plant via dual low-affinity/high-affinity CHL1/NRT1.1/NPF6.3 and high-affinity NRT2.1 transhipment Take (11-15).The NO3 that tender shoots-irradiation promotes^-Absorb and largely reduced in the nrt2.1-2 mutant for lacking NRT2.1, but (Fig. 1 D) is relatively unaffected in the chl1-5 mutant for lacking CHL1/NRT1.1/NPF6.3, shows root NO3^-That absorbs is tender It is notable NRT2.1- dependences that bud-irradiation, which promotes,.In addition, the root NO3 that tender shoots-irradiation promotes^-Intake is in hy5 and hy5-526 Largely eliminate (Fig. 1 D) in the two, this prompting HY5 regulation NRT2.1- dependences to root NO3^-The tender shoots of intake-irradiation promotes.

Subsequent experiment uses hypocotyl Transplantation chimera, and it was found that HY5 scion allows root growth and NO3^-Intake Tender shoots-irradiation promotes (relative to hy5-526 scion；Fig. 1 E, F), tender shoots-derivative Signal Regulation of this prompting HY5- dependence Root NO3^-Intake.In order to determine whether HY5 is the signal (part), we then use non-tissue specificity pHY5 (8), light It is charge-coupled knit-special pCAB3 (16,17) or bast companion cell-special pSUC2 promoters (18) express in hy5 and encode The transgenosis of HY5-GFP or myc-HY5 fusion proteins, finds both HY5-GFP and myc-HY5 supplement hy5 phenotypes (Fig. 9), and And therefore retain HY5 activity.It has been found that pHY5：HY5-GFP、pCAB3：HY5-GFP and pSUC2：HY5-GFP full recoveries The tender shoots irradiation regulation (Fig. 2A) of hy5 primary root growth.Because the expression of pCAB3- drivings is photosynthetic tissue-specific, These observations result prompting HY5 transcripts, the signal of HY5 or HY5- dependences are moved to root from tender shoots.

We then have found that HY5-GFP can be in S (L)/R (D) pHY5：Detected in the whole root of HY5-GFP hy5 seedling, but (Fig. 2 B) is not detected in S (D)/R (D), this prompting HY5 stablizes relatively in secretly taking root, and particularly root HY5 abundance Irradiated and adjusted by tender shoots.We are in addition in S (L)/R (D) pCAB3：HY5-GFP is detected in the root of HY5-GFP hy5 seedling, but (Fig. 2 C) is not detected in S (D)/R (D) control, this implies that HY5 transcripts or HY5 (or the two) are moved to from tender shoots Root.We then compare pCAB3：Myc-HY5 transcripts and myc-HY5 distribution (Fig. 2 D, E) in myc-HY5hy5 seedling.To the greatest extent Pipe myc-HY3 transcripts can only be detected (Fig. 2 D) in S (L)/R (D) tender shoots, and myc-HY5 is in S (L)/R (D) tender shoots and root Detect (Fig. 2 E).The result is by detecting the pCAB3 of S (L)/R (D)-growth：HY5-GFP hy5/hy5 graft chimaeras HY5-GFP in root confirms (Fig. 2 F；Figure 10).Utilize pHY3：Plant of the further experiment of HY5-GFP hy5 seedling in grafting S (L)/R (D) stock root in detection HY5-GFP (Figure 11), this prompting in bast vascular HY5 be moved to root from tender shoots.

We then express HY5-GFP, TEVrs (TEV protease recognition site) and double β-glucosides of fusion from pCAB3 Sour enzyme (2 × GUS) (2 × GUS-TEVrs-HY5-GFP) albumen.The albumen is detected in tender shoots, but in S (L)/R (D) plant Root in do not detect, probably due to its relatively large size prevents tender shoots-root movement (Fig. 2 G；Figure 12).However, 35S： TEVP (expression TEV protease) coexpression makes it possible in TEV^rsCut (19), this causes to detect in tender shoots and root HY5-GFP (Fig. 2 G；Figure 12).These ambulant changes of HY5-GFP abreast reflect the primary root promoted tender shoots-irradiation Grow (Fig. 2 H) and NO3^-Absorb the influence of (Fig. 2 I).These results indicate that HY5 tender shoots-root movement is that tender shoots-irradiation promotes Root growth and NO3^-Necessary to intake.It is interesting that HYH, i.e., a kind of and closely related HY5 transcriptional regulatory agent (20), It is not tender shoots-root mobile model (Figure 13).Therefore, HY5 (but not being HY5mRNA) is regulation and control root growth and NO3^-The light of intake is adjusted The tender shoots of section-root movable signal.

By pHY5：HY5-GFP (Fig. 2 B), pCAB3：HY5-GFP (Fig. 2 C) and pHY5：HY5-GFP hy5/hy5 graftings are embedding The HY5-GFP distributions that fit (Figure 11) assigns hy5 roots are different.pHY5：HY5-GFP causes HY5-GFP to be detected in whole root, And pCAB3：HY5-GFP and pHY5：HY5-GFP hy5/hy5 seedling assigns and the more closely related HY5-GFP of root vascular system Position.The difference is probably because HY5 activation root HY5 expression.First, in hy5, pHY5：(report pHY5's GFP transgenosis lives Property) root expression tender shoots-radiation-induced reduction (Fig. 2 J), the HY5 of this prompting from tender shoots generally activates root pHY5.Second, HY5-GFP can tender shoots-irradiation pCAB3：myc-HY5hy5/pHY5：Detected in the root of HY5-GFP hy5 graftings, but In the hy5/pHY5 of tender shoots-irradiation：It can not be detected in the root of HY5-GFP hy5 graftings (Fig. 2 K).3rd, use pHY5： The EMSA of the root of myc-HY5hy5 seedling is determined and ChIP is analyzed to identify combinations (21 of the HY5 to pHY5；Figure 14).These observation knots Fruit shows that the HY5 of tender shoots-root transfer activates root HY5 by the feedback control loop that is self-regulated.

We then have found that tender shoots-irradiation of root NRT2.1 transcript levels promotes largely to eliminate (Fig. 3 A) in hy5. Further experiment shows, HY5 scion/pHY5：HY5-GFP hy5 stocks or pCAB3：HY5-GFP hy5 scion/HY5 stocks are permitted Perhaps tender shoots-irradiation-dependence of root NR72.1 transcript levels is increased to the similar degree to being observed in HY5 plants, But pCAB3：HY5-GFP hy5 scion/hy5 stocks horizontally relative in HY5 plants observe reduction (Fig. 3 A).These The parallel reflection of difference effect is to tender shoots-irradiation and the NO3 of NRT2.1- dependences^-The influence (Fig. 3 B) of intake, and be related in root The need for feature HY5.ChIP and EMSA determine show HY5 to NRT2.1 promoters it is internal and external combine (Fig. 3 C, D), this shows that HY5 directly adjusts NRT2.1 expression.To sum up, these results prompting ambulant HY5 of tender shoots-root passes through by root HY5 Self-activation amplification mechanism activation root NRT2.1, thus increase NO3^-Intake.

Recent studies have indicated that HY5 parts are by controlling the chlcrophyll biosynthesis gene related to light compositing (such as eight Hydrogen lycopene synthase (PHYTOENE SYNTHASE) (PSY, Fig. 3 E)) expression adjustment light compositing ability (22,23).It is fixed C sagging tissue (sink tissues) (for example, root) (24), and marine alga are mainly transported to through bast in the form of sucrose Sugar precursor trehalose-6-phosphate salt (T6P) is used as the sub (25) of photosynthetic carbohydrate state.It was also found that HY5 is by promoting TPS1 (gene (25) of encoding trehalose-6-phosphate salt synthase) and SWEET11 and SWEET12 (coding sugarcanes The gene of sugar outflow transporter) expression (24) influence Sucrose Metabolism and tender shoots-root transhipment (Fig. 3 E).Further ChIP Combinations (Fig. 3 F) of the experimental verification HY5 to TPS1, SWEET11 and SWEET12 promoter.Therefore, HY5 adjusts C and fixes and input Phloem cell, for tender shoots-root migration.

Because C- status adjustment N- states (4,5), we then determine whether sugar level influences root NRT2.1 transcript water The NO3 of the NRT2.1- dependences of gentle light-regulation^-Intake.We have found that sugared (sucrose or glucose) enhancing NRT2.1 expression and NO3^-The tender shoots of intake-irradiation promotes (effect largely eliminated in hy5) (Fig. 3 G, H；Figure 15).In addition, ChIP determines table Bright, sucrose promotes the myc-HY5 of photoactivation to the Binding in vivo of NRT2.1 promoters, and the effect is mutated in sugared non-sensitive type (Fig. 3 I) is eliminated in body sis4.Because HY5 promoter activities and HY5-GFP abundance are not increased (Figure 16) by sucrose, increased HY5 levels can not possibly promote increases of the myc-HY5 of sucrose induction to NRT2.1 promoter binding affinities.No matter which kind of machine System, NRT2.1 expression and NO3^-The sucrose induction of intake depends on HY5 (Figure 17).To sum up, the movement of HY5 albumen promotes C and N metabolism Coordination homeostasis.

Because the natural influence of fluctuations C- of incident optical energy metric density (incident light fluence) fix (26) and Both HY5 abundance (27), we are it is thus determined that increase energy density (5-100 μm of ol.s^-1.m^-2) to root growth and NO3^-Intake Influence.We have found that increase energy density promotes WT primary roots development length (Fig. 4 A) and NO3^-Absorb (Fig. 4 B), this causes life The increase (Fig. 4 C) of energy density-dependence of object amount accumulation.However, these energy densities-dependence effect is a large amount of in hy5 Eliminate (Fig. 4 A-C).We then determine whether HY5 continues harmonic growth, C and N metabolism in more ripe plant.First, I Find, lack the biomass of HY5 reductions 21- ages in days soil-growing plants, with the increased effect of increased energy density (Fig. 4 D, Figure 18).Then, it has been found that the C content of 21- age in days WT and hy5 full plants is relative with increased energy density Constant (Fig. 4 E).By contrast, although the N content of WT full plants is remained relatively unchanged over, hy5 N content is close with energy Degree increase is remarkably decreased (Fig. 4 F).As a result, lack HY5 cause C/N contents than the increase of energy density-dependence (it is in WT In remain relatively unchanged over；Fig. 4 G).These results are pointed out, the coordination that mobile HY5 regulation tender shoots and root growth, C and N are obtained.Especially It is that HY5 maintains the homeostasis of C and N metabolism in the optical energy density of change.

Although research before shows, the sucrose of bast-movement is as next during the early stage seedling development of arabidopsis The signal of cotyledon is come to control primary root to extend (28), tender shoots-root long range signals conduction of regulation lateral root growth and N intakes Molecular mechanism be still unclear (29).Here, we show that HY5 is that the growth of regulation and control tender shoots and C absorb and absorbed with root growth and N Light regulation association tender shoots-root movable signal.The HY5- that the association is fixed by C in tender shoots is adjusted and by root Sucrose-enhanced promotion of the N- intakes of HY5- dependences is realized.As a result, HY5 regulates and controls full plants C relative to full plants The steady-state adjustment of N-state.It is known in the control of plant growth and development, HY5 integrates various plants hormones (for example, de- Fall acid) and environment (for example, low temperature) signal transduction input (30,31).

We increase the knowledge other dimension on the discovery that HY5 is movable signal.Our moved on HY5 is adjusted Controlling the discovery of the stable state coordination of C and N metabolism strengthens the understanding to how to maintain plant C and N nutrient balance in the environment of fluctuation, And point out to improve the new strategy of Crop Species nutrition service efficiency.

The discriminating of HY5 homologues

Between two protein sequences the percentage of homogeneity by by coupling number divided by total position (including gap position) come Obtain.

Table 1：HY5 homologues homogeneity (is based on protein sequence)

At：Arabidopsis (Arabidopsis thaliana)；Bn：Colea (Brassica napus)；Gb：Sea island cotton (Gossypium barbadense)；Gm：Soybean (Glycine max)；Hv：Barley (Hordeum vulgare)；Ta：Commonly Wheat (Triticum astivum)；Os：Paddy rice (Oryza sativa)；So：Sugarcane (Saccharum officinarum)； Zm：Corn (Zea mays)；Sm：Selaginella tamariscina (Selaginella moellendo rffii)；Pp：Small liwan moss (Physcomitrella patens)。

OsHY5 expression (Fig. 5) in the expression of PpHY5 in arabidopsis and arabidopsis

In order to examine whether the evolutionary conservatism that there is HY5 functions, we then produce uses arabidopsis HY5 in hy5 The promoter of gene expresses the genetically modified plants of OsHY5-GFP and PpHY5-GFP fusion proteins.We have found that pAtHY5： OsHY5-GFP and pAtHY5：PpHY5-GFP supplement hy5 hypocotyl extension phenotypes (Fig. 5 a).It was also found that pAtHY3： OsHY5-GFP and pAtHY5：Both PpHY5-GFP recover hy5 root separate living tissue size and tender shoots-irradiation of NRT2.1 expression Adjust (Fig. 5 b, c).Then experiment uses hypocotyl graft chimaera, and it was found that, OsHY5-GFP and PpHY5-GFP point Cloth can detect that this prompting both PpHY5-GFP and OsHY5-GFP fusion protein is all to move in the stock root of the plant of grafting Dynamic (Fig. 5 d).

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Sequence information

Arabidopsis

SEQ ID NO：1 is used for the AtHY5CDS of construct：

atgcaggaacaagcgactagctctttagctgcaagctctttaccatcaagcagcgagaggtcatcaagctctgctcc acatttggagatcaaagaaggaattgaaagcgatgaggagatacggcgagtgccggagtttggaggagaagctgtcg gaaaagaaacttccggtagagaatctggatcggcgaccggtcaggagcggacacaggcgactgtcggagaaagtcaa aggaagcgagggaggacaccggcggagaaagagaacaagcggctgaagaggttgttgaggaacagagtttcagctca gcaagcaagagagaggaaaaaggcttacttgagcgagttggaaaacagagtgaaagacttggagaacaaaaactctg aacttgaagagcgactctctactcttcagaacgagaaccagatgcttagacatattctgaagaacacaacaggaaac aagagaggaggtggtggtggttctaatgctgatgcaagcctttga

SEQ ID NO：2AtHY5mRNA(NM_121164)

cagagatctgacggcggtagccagagtaatctattccttcccaaaatgtctcgcaattagattctttccaagttctt ctgtaaatcccaagtcccgctcttttcctctttatccttttcaccagcttcgctactaagacaacaaatctttccct ctctctctcgcctgatcgatcttcaaagagtaagaaaaatgcaggaacaagcgactagctctttagctgcaagctct ttaccatcaagcagcgagaggtcatcaagctctgctccacatttggagatcaaagaaggaattgaaagcgatgagga gatacggcgagtgccggagtttggaggagaagctgtcggaaaagaaacttccggtagagaatctggatcggcgaccg gtcaggagcggacacaggcgactgtcggagaaagtcaaaggaagcgagggaggacaccggcggagaaagagaacaag cggctgaagaggttgttgaggaacagagtttcagctcagcaagcaagagagaggaaaaaggcttacttgagcgagtt ggaaaacagagtgaaagacttggagaacaaaaactctgaacttgaagagcgactctctactcttcagaacgagaacc agatgcttagacatattctgaagaacacaacaggaaacaagagaggaggtggtggtggttctaatgctgatgcaagc ctttgatctccttcttcttcttgtgttatatttttgtggataaaatttacagagaattgtatcaataattatcatgt taaaattatatgggatgtgagagctaatattgcaattgtagaccaagttctcttattgtagtcttagatttctctta attgaaacataatgttgttttataacaaaaataagctaatttttgttctatgata

SEQ ID No：3AtHY5 albumen

mqeqatsslaasslpssserssssaphleikegiesdeeirrvpefggeavgketsgresgsatgqertqatvgesq rkrgrtpaekenkrlkrllrnrvsaqqarerkkaylselenrvkdlenknseleerlstlqnenqmlrhilknttgn krgggggsnadasl

Paddy rice (Oryza sativa)

SEQ ID NO：4 are used for the OsHY5CDS of construct：

atgcaggagcaggcgacgagctcgcggccgtccagctccgagaggtcgtccagctccggcggccaccacatggagat caaggaaggaatggagagcgacgaggagatagggagagtgccggagctggggctggagccgggcggcgcttcgacgt cggggagggcggccggcggcggcggcggcggggcggagcgcgcgcagtcgtcgacggcgcaggccagcgcgcgccgc cgcgggcgcagccccgcggataaggagcacaagcgcctcaaaaggttgctgaggaaccgggtatcagcgcagcaggc aagggagagaaagaaggcatacttgaatgatcttgaggtgaaggtgaaggacttggagaagaagaactcagagttgg aagaaagattctccaccctacagaatgagaaccagatgctcagacagatactgaagaatacaactgtgagcagaaga gggccaggtagcactgctagtggagagggtcaatag

SEQ ID NO：5OsHY5mRNA

atgcaggagcaggcgacgagctcgcggccgtccagctccgagaggtcgtccagctccggcggccaccacatggagat caaggaaggaatggagagcgacgaggagatagggagagtgccggagctggggctggagccgggcggcgcttcgacgt cggggagggcggccggcggcggcggcggcggggcggagcgcgcgcagtcgtcgacggcgcaggccagcgcgcgccgc cgcgggcgcagccccgcggataaggagcacaagcgcctcaaaaggttgctgaggaaccgggtatcagcgcagcaggc aagggagagaaagaaggcatacttgaatgatcttgaggtgaaggtgaaggacttggagaagaagaactcagagttgg aagaaagattctccaccctacagaatgagaaccagatgctcagacagatactgaagaatacaactgtgagcagaaga gggccaggtagcactgctagtggagagggtcaatag

SEQ ID NO：6OsHY5 albumen

mqeqatssrpssserssssgghhmeikegmesdeeigrvpelglepggastsgraaggggggaeraqsstaqasarr rgrspadkehkrlkrllrnrvsaqqarerkkaylndlevkvkdlekknseleerfstlqnenqmlrqilknttvsrr gpgstasgegq

Small liwan moss

SEQ ID NO：7 are used for the PpHY5CDS of construct：

atggcagacgcacaaaatggtaagggcttttcgcagttgacttcagttattggaaacatcaatagtgttgcaaatag tagcaggaggggtcccagagaagcggttgatattggccggaactggaaacctgtcaattttggtgagtaccaaggtt tgggcgaaatcttgcccatgcaggcctctactgtgggtcccgcttcttctccgccctcttcgaagcagcagacgggc actgatatatcagtatcacctcctttggcgactgcagctgttgacaaactcatgaaagacggcaatgaaagcgactc tgatgttaggagggttccagagctatctgcgaagactagtggaggggtttctgggtcgcatacacaagataaaggtc tcactggatcttctcatcggaaaagagggggagcttctgctgataaagaacacaagcgtctgaagaggctgcttagg aaccgtgtttcagctcagcaagccagagaacgaaaaaaagcatatctcggtgaattggaagttagatcaaaggagtt ggagcatcgaaatgcagaattagaagaaagagtgtccaccctacaaagggagaaccagatgttgcgtcaaatcgtca agaacactgctctgaagaagacttatagtggtggtaacgctgaggatggtgcgcaatga

SEQ ID NO：8：PpHY5mRNA

cagttttgttccattgtagtatgtactttgagccagtaatgcgttgtagttaaactgagagtcctgcactattattt agctaggtctgttcatgtttttcccattgcagtagggattgaagagtgttggaaacaacgacagtaaaactaaacgt tcatatgggatggcagacgcacaaaatggtaagggcttttcgcagttgacttcagttattggaaacatcaatagtgt tgcaaatagtagcaggaggggtcccagagaagcggtttgtgttggccggaactggaaacctgtcaattttggtgagt accaaggtttgggcgaaatcttgcccatgcaggcctctactgtgggtcccgcgtcttctccgccctcttcgaagcag cagacgggcactgatatatcagtatcacctcctttggcgactgcagctgttgacaaactcatgaaagacggcaatga aagcgactctgatgttaggagggttccagagctatctgcgaagactagtggaggggtttctgggtcgcatacacaag ataaaggtctcactggatcttctcatcggaaaagagggggagcttctgctgataaagaacacaagcgtctgaagagg ctgcttaggaaccgtgtttcagctcagcaagccagagaacgaaaaaaagcatatctcggtgaattggaagttagatc aaaggagttggagcatcgaaatgcagaattagaagaaagagtgtccaccctacaaagggagaaccagatgttgcgtc aaatcgtcaagaacactgctctgaagaagacttatagtggtggtaacgctgaggatggtgcgcaatgatggaatatt cgagagatgtgtggcctaccgttttttgttattcataatcaaccatttagtatgtaatgcaggaagtttttatttca agtatgccccgcttcacattaagttcaaaataagtttggtaatcgaaggtcagaattctctcagcgttgtccttcat tccatcaacttccgttgttttatggaatgaccgttggatgctttgttgctgttacaatcgcaagaactatatttcca ttgaagtcatatttttgaagcaattctcaataactgtatgaaggccatggttcattataaccaagcgattcttgata gcat

SEQ ID NO：9PpHY5 albumen

Madaqngkgfsqltsvigninsvanssrrgpreavcvgrnwkpvnfgeyqglgeilpmqastvgpassppsskqqtg tdisvspplataavdklmkdgnesdsdvrrvpelsaktsggvsgshtqdkgltgsshrkrggasadkehkrlkrllr nrvsaqqarerkkaylgelevrskelehrnaeleervstlqrenqmlrqivkntalkktysggnaedgaq

Selaginella tamariscina (Selaginella moellendorffii)

SEQ ID NO：10SmHY5A mRNA(XM_002970018)

atagtcacgtgaaatatctttctctcttggcggagaagctgaaagcggtccaagagctttcggtgtccggatcgagc tagtgcttgggggaggcggtgaattttggttccatcgcaatgcggtgactcgggggtgtgaggagtatctattcagt catgcgatcggcgtccgcggtgaagaggccggttgcgagtttcaatcccggcagggtggagaatttgtcgcggaaga aaggctcgattggcggtgaagcagcggagaatgccgaggtcgtggatgatttgagcagcctcaaagctgagtgtgat atcgagtctgttggatacgagagccaggcggcggccagcattgcgaatttcggctgcaagtcgtttgcaggggcttg gaagcccgtcatcgacggaaaaatccaacctttggagccaccaaacagcgccaccaacagcatcacctggaattgga cgagcgagtccaacaggacgaattcgtcatcgaggcatggatcagacaggccctctgtttcgaacatctccggctca tcggatatgaagaaagacgacgaaggcaacgatagcgactcggacatcagacgagtgcccgagctgccagagaagag cagcaaaggtcgctctcagaagcttgtcggtggaagctcgtcgtcgagaaggcgatccggaggctcttccaatgaca aggaaggcaagcgactaaagaggttgctgaggaatcgcgtgtcggcacaacaggcccgagagcgcaaaaaggcttac ttggtcgagctggagcaaaaggccaaggatctggaaactagaaacgctgagctggaggagaaaaacgcgacgcttca aagagaaaactacatgctccgacagattgtcaagaacactaccatccggggcggtggcgattgataatccaaatctt ccaagacgaaagaaacaggagtaaacaagatctttggccattttacaacatacattggctatataacaagagaagtt atggtgttacaacaaaaataatttagctatgactaagat

SEQ ID NO：11SmHY5A albumen

Mrsasavkrpvasfnpgrvenlsrkkgsiggeaaenaevvddlsslkaecdiesvgyesqaaasianfgcksfagaw kpvidgkiqpleppnsatnsitwnwtsesnrtnsssrhgsdrpsvsnisgssdmkkddegndsdsdirrvpelpeks skgrsqklvggssssrrrsggssndkegkrlkrllrnrvsaqqarerkkaylveleqkakdletrnaeleeknatlq renymlrqivknttirgggd

SEQ ID NO：12SmHY5BmRNA(XM_002973152)

Ggagttttcttggtgctggtgtttctcgcttccacagctcggcattgccccaatcgcgacgagaggacgcatccata gcaatcgacgcgatgattctgaggaacgattgctagaagttcctctctctctctctctgcctctccagccagctcca gatttgttccaggatgcaagcagcggcatcagcgtcgccgacaatgcacaagcaattcagcgatttgatggctcttc ccaacgccgagatgaaagtggacgaggattgggcaggaaatgagagtgactcggaagtaagaaaggtccccgactta cctggaggtaaaatcgtgactgcgttgccagagcaagacacggcagcatccaattctcgcaagaggggtgctgttcc tgctgacaaagaacacaagcgattgaaaagacttttacgcaatcgagtctcggcacaacaagccagggagagaaaaa aggcttacgttgttgagctcgaagcaaaagcccgggatttggagctcaggaatgcggagctagaggaacgggtaaac acgttacaaaaggaaacattcatgctgcgacagattttgaagaacataaagaacaatggctccactgctggactaga acaagcacagtaaaaaaccaaaaactttacttgagggggccaacgtagcaacgaaagtcggccaccactgtaagatt gtttctccacaatcagttttggtaaccatcttctcggc

SEQ ID NO：13SmHY5B albumen

mqaaasasptmhkqfsdlmalpnaemkvdedwagnesdsevrkvpdlpggkivtalpeqdtaasnsrkrgavpadke hkrlkrllrnrvsaqqarerkkayvveleakardlelrnaeleervntlqketfmlrqilkniknngstagleqaq

Barley (Hordeum vulgare)

SEQ ID NO：14HvHY5mRNA(AK365526)

Ggttttcctcgggattgggagggacgagagaggcggggagagaatgcaggagcagggggagagctcgcggccttcga gcagcgagaggtcgtccagctccggcaaccacatggagcacaaggaagggatggagagcgacgacgaaatagggacg gtgccggagctaggcctggggccaagcggcgcgtccacgtccggcaggagggaagccgacgggccggagcgtgccca gtcctccaacgcgcaaggcagcgcgcgccgccgcggacgcaccccggctgacaaggagcacaagcgcctcaagaggt tgctgaggaaccgggtatcagcccagcaggcaagggagaggaagaaagcttatttgggcgatctggaggtgaaggtg aaggacctagagaagaagaattcggagctggaagagaggcattccaccctacagaatgagaaccagatgctccgaca gatcctgaagaacaccactgtgagcagaagagggccaagtgagggtcaatagcacagaagttgtaagggtcgattcg cagaattttcacagcagaatcaaagaagccctaggatcgaatatagctgcgttgattgatcccaaaatacaccatgt ctcgaacttaaaatggttggaagctttctgaccaatggataacctcaaaaactggggtcaaaaacctgtgtagatct tcagagatgtccccatcatactctatgaagttcagcactacgtgttgcacttcagtaataatttcagaaaattagtt ttgggtggtttaacatatgatctgtactatcatttttatgtatctacaagtacaatccaaacattttattgttggat aatttactttctactattggaaaatgcgc

SEQ ID NO：15HvHY5 albumen (AK365526)

mqeqgessrpssserssssgnhmehkegmesddeigtvpelglgpsgastsgrreadgperaqssnaqgsarrrgrt padkehkrlkrllrnrvsaqqarerkkaylgdlevkvkdlekknseleerhstlqnenqmlrqilknttvsrrgpse gq

Common wheat (Triticum astivum)

SEQ ID NO：16TaHY5mRNA

Gagtaagtagcagctgggaggaggagccgaggaagaggagcagaagataggaggagaggagcagcggtagcctcgtc ttcctcgggattgggagggacgagagaggcggggggagaatgcaggagcagggggagagctcgcggccttcgagcag cgacaggtcgtccagctccggcaaccacatggggcacaaggaagggatggagagcgacgacgagatagggacggtgc cggagctgggcctggggccaagcggcgcgtccacgtccggcaggagggaagccgacgggccggagcgtgcccagtcc tccaccgcgcaaggcagcgcgcgccgccgcggacgcaccccggccgacaaggagcacaagcgcctcaagaggttgct gaggaaccgggtatcagcccagcaggcaagggagaggaagaaagcttatttgggcgatctggaggtgaaggtgaagg acctagagaagaagaattcggagctggaagagaggcattccaccctacagaatgagaaccagatgctccgacagatc ctgaagaacaccactgtgagcagaagagggccaagtgagggtcaatagcacagaggttgtcagggttgattcacaga attttcacagcagacagttcaattccagtgaatcaaagaggccctaggatcgaatatagccgcgttgatcgatccca aaacacaccatgtctggaacttaaactggttggaagctttctggccaaaggataacctcaaaaaccgggggcctaaa cctgtgtagatcttcacagatgtccccatcatactctatgaagttcagcaccacgtgctgtgcttcgttaataattt cacaaaattagttttgggtgatttaacatatgatctgtatcattttttatgtatctacaagcacaatccaaacattt tattgttgggcaatttactttgtactattggaaaatgcgcacgtccggtgtcggcaatgcagccctgtcatgttggc tcttgcagaccagactatttgttttgttgaacttgcttcaataaagtggccgcatgcatctttccgatgcagaggcc ggggaagtcctccttttcaaaaaaaaaaaaaaaacga

SEQ ID NO：17TaHY5 albumen

mqeqgessrpsssdrssssgnhmghkegmesddeigtvpelglgpsgastsgrreadgperaqsstaqgsarrrgrt padkehkrlkrllrnrvsaqqarerkkaylgdlevkvkdlekknseleerhstlqnenqmlrqilknttvsrrgpse gq

Corn (Zea mays)

SEQ ID NO：18ZmHY5/ZmbZIP61mRNA(KJ726945)

Atgcaggagcaggcggcgagctcgcggccttccagcagcgagaggtcgtccagctccgggcaccacgtggacatgga ggtcaaggaagggatggagagcgacgatgagataaggagagtgccggagctgggcctggagttgccgggagcctcca cgtcgggcagggaggctggccctggcgctgcgggcgcagaccgcgcccttgcccagtcgtccacggcgcaggccagc gcgcgccgccgcgtccgcagccacgccgacaaggagcacaagcgcctcaaaaggttactgaggaacagggtgtcagc tcaacaggctagagagaggaagaaggcttatttaactgatctggaggtgaaggtgagagatctggagaagaagaact cggagatggaagagaggctctccaccctccagaacgagaaccagatgctccgacagatactgaagaacaccgctgta aacagaagaggttcaggaagcactgctagtggagagggccacggccaa

SEQ ID NO：19ZmHY5/ZmbZIP61 albumen

mqeqaassrpssserssssghhvdmevkegmesddeirrvpelglelpgastsgreagpgaagadralaqsstaqas arrrvrshadkehkrlkrllrnrvsaqqarerkkayltdlevkvrdlekknsemeerlstlqnenqmlrqilkntav nrrgsgstasgeghgq

Sugarcane (Saccharum officinarumL.)

SEQ ID NO：20SoHY5mRNA(CA121289)

Agacaggaaggatcgcaggggaggaggagatagggaaggagaagcggagtgcgcgcgggcgactctgcagggcctca gtcggaggcggaggtggagagcgagccagaatgcaggagcaggcgacgagctcgcggccttccagcagcgagaggtc gtccagctccgcgcaccacatggacatggaggtcaaggaagggatggagagcgacgaggagataaggagagtgccgg agctgggcctggagctgccgggcgcttccacgtcgggcagggaggctggcccgggcgccgccggcgcagaccgcgcc ttggcccagtcgtccacggcgcaggccagcgagcgccgccgcgtccgcagccccgccgacaaggagcacaagcgcct caaaagattactgaggaaccgggtgtcagctcaacaggcaagagagaggaagaaggcttatttgactgatctggagg tgaaggtgaaacaccctgaagaagaagaacttcgaggttcgaagaagaggctctctacccttaaaaacgaagaacca gatgctccgggcagatacctgaagaatacccactgtaagcagaaagaggtttacggaagcactggttagtggaaaaa ggccaattagttcaaaatgacaggaaaaatggtaatggcctatgcttaaatatatgtttatgggga

SEQ ID NO：21SoHY5 albumen

mqeqatssrpssserssssahhmdmevkegmesdeeirrvpelglelpgastsgreagpgaagadralaqsstaqas errrvrspadkehkrlkrllrnrvsaqqarerkkayltdlevkvkhpeeeelrgskkrlstlkneepdapgrylknt hckqkevygstg

Colea

SEQ ID NO：22BnHY5mRNA(EV071015)

gaaagtcccgctcttttccatctctatcttcatcaccagcttctgtaaatcccaatccatcttcaaaggagattcaa agagtaaggaaaaaaaaatgcaggagcaaacgactagctctttacctgcaagctctctaccatcaagcagcgagaga tcctcaagctctgctcctcatttggagatcaaagaaggaattgaaagcgatgaagagatacggcgagttccggagtt tggaggagaagctaccggaaaggaaatctctggatcggcgaccggtcaggaccagacacaagcaacggtcggaggag agggtcaaaggaagagagggaggactccggctgagaaagagaccaagcggcttaagaggttgttgaggaacagagtt tcagcacagcaagcaagagagaggaagaaagcttacttgggtgagttggaaaacagagtgaaagacttggagaacag aaactctgaacttgaagagagactctctaccttgcagaacgagaaccagatgcttagacagattctgaagaacacaa caggaaacaagaggggaagcggtggttctaacgctgatgcaagcctatgatctccttgttcttgtattattatttac ctggataaactttacaaggaattgtattaaataaatattttt

SEQ ID NO：23BnHY5 albumen

mqeqttsslpasslpssserssssaphleikegiesdeeirrvpefggeatgkeisgsatgqdqtqatvggegqrkr grtpaeketkrlkrllrnrvsaqqarerkkaylgelenrvkdlenrnseleerlstlqnenqmlrqilknttgnkrg sggsnadasl

Sea island cotton (Gossypium barbadense)

SEQ ID NO：24GbHY5mRNA(JK803801)

Ccaaaaaattaattttattttgttttttctataacgaagaaatgcaagaacaaggaacgagttcaatagcagctagt tccttaccttcaagcagtgaaagatcttcaagctctgctcttcaagttgaagtcaaggaaggcatggaaagtgatga agagatccggagagtgcctgagataggaggtgaagcatcagcagctccggccgccggtcgtgaacccggttcactga cccgactggaccggcctcaaccatcgggtgaaggcggtcagagaaagagagggagaagcccaacggataaagaaaac aagcgcttaaagaggttgttgaggaacagagtatcagcgcaacaagcaagggaaaggaaaaaggcgtacttgaatga accggaaaccagagttagagacttggagaagaagaactctgaactagaagagaggctatccacgttgcacaatgaga atcagatgcttcgacaaatagtcaagaacacaactgctagcaggagaggtggaaatggcagttcaaatgcagctgat ggaaccctttaaaggaaatgcatgggtataaaaaattataaagggattttttttaaaaaaatattaattttatgctt tagggaaaaaaaaaccgctattagcaatgcaatgaagtagcaaaacatgacaattggaactttggttctctctaatc ttaatcataaaagtaaattatc

SEQ ID NO：25GbHY5 albumen

mqeqgtssiaasslpssserssssalqvevkegmesdeeirrvpeiggeasaapaagrepgsltrldrpqpsgeggq rkrgrsptdkenkrlkrllrnrvsaqqarerkkaylnepetrvrdlekknseleerlstlhnenqmlrqivknttas rrggngssnaadgtl

Soybean

SEQ ID NO：26GmHY5LmRNA(BT093548)

gggaagaaagagagagagagagagagagagaggtgtgaagttggtgaaggtttttgagaagaaagatggaacgaagt ggcggaatggtaactgggtcgcatgaaaggaacgaacttgttagagttagacacggctctgatagtaggtctaaacc cttgaagaatttgaatggtcagagttgtcaaatatgtggtgataccattggattaacggctactggtgatgtctttg tcgcttgtcatgagtgtggcttcccactttgtcattcttgttacgagtatgaactgaaacatatgagccagtcttgt ccccagtgcaagactgcattcacaagtcaccaagagggtgctgaagtggagggagatgatgatgatgaagacgatgc tgatgatctagataatgagatcaactatggccaaggaaacagttccaaggcggggatgctatgggaagaagatgctg acctctcttcatcttctggacatgatttctcaaataccaaacccccatctagcaaacgggcaaccgatgtctggtga gtttccatgtgctacttctgatgctcaatctatgcaaactacatctataggtcaatccgaaaaggttcactcacttt catatgctgatccaaagcaaccaggtcctgagagtgatgaagagataagaagagtgccagagattggaggtgaaagt gccggaacttcggcctctcagccagatgccggttcaaatgctggtacagagcgtgttcaggggacaggggagggtca gaagaagagagggagaagcccagctgataaagaaagtaaacggctaaagaggctactgaggaaccgagtttcagctc agcaagcaagggagaggaagaaggcatacttgattgatttggaaacaagagtcaaagacttagagaagaagaactca gagctcaaagaaagactttccactttgcagaatgagaaccaaatgcttagacaaatattgaagaacacaacagcaag caggagagggagcaataatggtaccaataatgctgagtgaacataatgtcaaaagatggcagagaaaacttatagat ggaatagatttagaaagagagaatacattagccagaaagagaaaaaaaaaattggacattagttgatgattctttct aggtgtgcgtttggaatacaatgaagtaaaggatgaaccttaagacatgctttatcctaaaatagtgtgatctgata ttccattgttaatgagtaatgtaattatcatacaaacaatttgtagtctcattttaattaataattattaaactact tgattaaaaaaaaaaaaaaaaa

SEQ ID NO：27GmHY5L albumen

mrstmaketvprrgcygkkmltslhlldmisqipnphlangqpmsgefpcatsdaqsmqttsigqsekvhslsyadp kqpgpesdeeirrvpeiggesagtsasqpdagsnagtervqgtgegqkkrgrspadkeskrlkrllrnrvsaqqare rkkaylidletrvkdlekknselkerlstlqnenqmlrqilknttasrrgsnngtnnae

SEQ ID NO：28GmbZIP36/STF1mRNA(NM_001250343.1)

actgaagtaagaaagagagagagagagagaaagagaagtgtgtagttggtgaagtttttgagaagaatatggaacga agtggcggaatggtaacggggtcgcatgaaaggaacgaacttgttagagttagacacggttctgacagtgggatttg tcaaatatgtggtgacaccattggattaacggctactggtgacctctttgttgcttgtcatgagtgtggcttcccac tttgtcattcttgttacgagtatgagctgaaaaatgtgagccaatcttgtccccagtgcaagactacattcacaagt cgccaagagggtgctgaagtggagggagatgatgatgacgaagacgatgctgatgatctagataatgggatcaacta tggccaaggaaacaattccaagtcggggatgctgtgggaagaagatgctgacctctcttcatcttctggacatgatt ctcatataccaaacccccatctagtaaacgggcaaccgatgtctggtgagtttccatgtgctacttctgatgctcaa tctatgcaaactacatcagatcctatgggtcaatccgaaaaggttcactcacttccatatgctgatccaaagcaacc aggtcctgagagtgatgaagagataagaagagtgccggagattggaggtgaaagcgctggaacttcagcctctcggc cagatgccggttcaaatgctggtacagaacgtgctcaggggacaggggacagccagaagaagagagggagaagccca gctgataaagaaagcaagcggctaaagaggctactgaggaatagagtttcggctcagcaagcaagggagaggaagaa ggcatatttgattgatttggaaacaagagtcaaagacttagagaagaagaactcagagctcaaagaaagactttcca ctttgcagaatgaaaaccaaatgcttagacaaatattgaagaacacaacagcaagcaggagagggagcaatagtggt accaataatgctgagtaaacttatagatggagtagatatagagagagagaaagagaaaaaaattaaacattagttga tgattctttctaggtgtgcgtttggaatacaatgaagtaaaggatgaaccttaagacatgctttgtcctaaaatagt gtgatctgatgtaccattgttgatgagtaatgtaattatcatacacagttttttacagtctcattttaattaataat tatcaaactacttgattacttatggttaa

SEQ ID NO：29GmbZIP36/STF1 albumen

Mersggmvtgshernelvrvrhgsdsgskplknlngqicqicgdtigltatgdlfvachecgfplchscyeyelknv sqscpqckttftsrqegaevegddddeddaddldnginygqgnnsksgmlweedadlssssghdshipnphlvngqp msgefpcatsdaqsmqttsdpmgqsekvhslpyadpkqpgpesdeeirrvpeiggesagtsasrpdagsnagteraq gtgdsqkkrgrspadkeskrlkrllrnrvsaqqarerkkaylidletrvkdlekknselkerlstlqnenqmlrqil knttasrrgsnsgtnnav

SEQ ID NO：35：HY5 promoters (2.2.kb)

cttcgtcgtcaggattatccgtcaacagagttctgtttcggaatcggaacgctagtccttgtggtctatctcttcca attctcaatccgtctcggtctgtacttgtttttgctcgtggcaagaatcgaaaaggattcgtgtcttcgtcttcttc gtctccgaagaaaaacaaaaaggtaagaaaatcagaatgagaatgatttatgttcgcaattgctcttacctgctgct ttcgccgaatttgactagaatttggatacactaactaagtgagctattatcggttagaattggtgaatcctcgaatt agactttggctctttttgattttgtatcagcctttgatttcatggtttgtggcagaaaagtttggatggagctgata atggaggaggtgaggaggaggaggatccatttgaggcattgtttaatcttttagaagaagatttgaagaatgataac tcggatgatgaggagataagtgaggaagagttagaggcattagccgatgagttagctcgagcattgggtgttggtga cgatgttgatgacatcgacttgtttggttcagtcactggtgatgttgatgttgatgttgataatgatgatgatgata atgatgatgatgataatgatgatgatgatgatgacagcgaagaagatgaaagaccgactaagctgaagaattggcag cttaaaagactggcttatgcattgaaagctgggcgtcgtaaaactagtgtaagttttagttcagtgttgaagtgatg ttgaatacattgtgtaaagcatagtgcttgagttagtgtctgctttggagattttgcttacgaattgtttaagagtt gacaaagaacaagtagttctctggttttcaaatgcataattgataacggatggttttgttgtgatctgtagattaaa aatctggcagctgaggtttgtcttgacagggcttatgttcttgaattgcttcgtgacccgcctccaaagctgctaat gctaagtgctacactaccagatgaaaaaccgccagtagcagcacctgagaactcctcacctgatccaagtcctgtgg aatcattatcagctgaagatgttgtggtcgaacctaaggaaaaagtaaaagatgaagcagttcatgtgatgcaacag agatggtctgctcagaaaagagtgaagaaagctcacattgaaacgctagagaaagtttacagaagatcaaaacgacc cactgtaaggattctccttttacatttgaatcaatttctatgttacttgaatgctctatctcacatatgatcatgtt tgatgatgctgtgaatagaatgctgtggttagcagcattgttcaagtgaccaatcttccaaggaagcgagttttgaa gtggttcgaagataaaagagcagaagacggagttccagataagcgagctccatatcaagctccggtttgatctaatg ttaacgttgagatggcaatgatttgtatacttgattctcagaaactcatcaacattgtcgtcaaggacaagtttttt tggtgatacgaggagtgtttatagtagtagattctgtccaatggtgtggctggatatgttggactatgaaattttag gatatcttgtattcagtttttagttatttccttgctgagattgtgtcttgtagaaaaccgtttcaactttgtttggt ttatggcggctataaagtttaattttaatgcatgacaaaaacaaatcaccaaaaataaaataaattactttcacgac acttttgaaagcactgccctaggcgtgggccatgtgacagaatgaaagaactcagaccaaacttttctgtccaagga caggaatggggcccacccaattagctcccctatccattattcaccgtaagatgctaaccagatctaacggctaaaat ccacccacgttccaatctcaattgcctttggatccttgtatttcctcaaggctcacctttctccacgattcactctc gatatccgttcgattcttcagagatctgacggcggtagccagagtaatctattccttcccaaaatgtctcgcaatta gattctttccaagttcttctgtaaatcccaagtcccgctcttttcctctttatccttttcaccagcttcgctactaa gacaacaaatctttccctctctctctcgcctgatcgatcttcaaagagtaagaaa

Table 2：Primer sequence for producing DNA construct

Table 3：The primer sequence analyzed for qRT-PCR

Primer	Primer sequence (5 ' to 3 ')	SEQ ID NO：
			Q actins 2-F	CTGGATCGGTGGTTCCATTC	57
Q actins 2-R	CCTGGACCTGCCTCATCATAC	58
			qHY5-F	GAACGAGAACCAGATGCTTAGAC	59
qHY5-R	TGCAATATTAGCTCTCACATCCC	60
			OCS-R	CATAGGCGTCTCGCATATCTC	61
qHY5-GFP-F	CAGAACGAGAACCAGATGCTTAG	62
			qHY5-GFP-R	CAGATGAACTTCAGGGTCAGC	63
qNRT1.1-F	TCTAAGACCGCTTCAACGGATCG	64
			qNRT1.1-R	ACTGTTGGACCATGAGCGTGTG	65
qNRT2.1-F	AACAAGGGCTAACGTGGATG	66
			qNRT2.1-R	CTGCTTCTCCTGCTCATTCC	67
qTPS1-F	GGTCATTTCTTGGGGAAGGA	68
			qTPS1-R	TCTCCTGATGATGACTTGGC	69
qSWEET11-F	GCGAACAAGTGTACCTGCGG	70
			qSWEET11-R	GGGTACACGTGGTGGTTGGT	71
qSWEET12-F	TCGTCCGATCGGTGAACACA	72
			qSWEET12-R	ACTAGTACACGTGGACAATGGTGA	73

Table 4：Primer sequence for ChIP-PCR

Table 5：The DNA probe determined with dry EMSA

Claims (17)

1. for increasing the method for nitrogen metabolism in plant, methods described, which is included in plant, to be introduced and expresses comprising HY5 nucleic acid sequences The nucleic acid construct of row.

2. according to the method described in claim 1, wherein the nucleic acid construct includes coding SEQ ID NO：3 are limited The SEQ ID NO of AtHY5 albumen or its functional variety：1 or 2 or coding SEQ ID NO：The nucleotide sequence of 3 homologue.

3. method according to claim 2, wherein the homologue is selected from SEQ ID NOs：4 to 29.

4. according in the method described in previous claims, wherein the plant is selected from corn, paddy rice, wheat, colea/plus taken Big rape, sorghum, soybean, sunflower, clover, potato, tomato, tobacco, grape, barley, pea, beans, broad bean, lettuce, cotton Flower, sugarcane, sugar beet, broccoli or other brassica vegetables or willow.

5. according in the method described in previous claims, wherein the nucleic acid construct includes regulatory sequence.

6. method according to claim 5, wherein the regulatory sequence is selected from constitutive promoter or tissue specificity is opened Mover.

7. method according to claim 6, wherein the tissue-specific promoter is chlorenchyma specificity promoter.

8. nucleic acid construct, it includes HY5 nucleotide sequences and tissue-specific promoter.

9. nucleic acid construct according to claim 8, wherein the tissue-specific promoter is chlorenchyma specificity Promoter.

10. nucleic acid construct according to claim 8 or claim 9, wherein the nucleic acid construct includes coding SEQ ID NO：3 The SEQ ID NO of the AtHY5 albumen limited：1 or 2 or coding SEQ ID NO：The nucleotide sequence of 3 homologue.

11. nucleic acid construct according to claim 10, wherein the homologue is selected from SEQ ID NOs：4 to 29.

12. recombinant vector, it includes the nucleic acid construct according to any one of claim 8 to 11.

13. host cell, it is comprising the nucleic acid construct according to any one of claim 8 to 11 or according to claim Recombinant vector described in 12.

14. the method for producing the plant absorbed with increased nitrogen, it includes introducing and expressing into plant comprising HY5 cores Acid sequence and the nucleic acid construct of tissue-specific promoter.

15. for increasing the method for the presence of HY5 albumen in plant roots, it, which is included in plant to introduce and express, includes and green The nucleic acid construct for the HY5 nucleotide sequences that tissue-specific promoter is operatively connected.

16. the method for adjusting the balance in plant between C and N metabolism, it, which is included in plant, introduces and expresses comprising HY5 The nucleic acid construct of nucleotide sequence.

17. for increasing the method for the presence of HY5 albumen in plant roots, it, which is included in the chlorenchyma of plant, introduces and expresses Include the nucleic acid construct of HY5 nucleotide sequences.