CN108707594B - 1 gene family of -1 related protein kinase of maize sucrose non-fermented and its application - Google Patents

Abstract

The present invention provides 1 gene family of -1 related protein kinase of maize sucrose non-fermented and its application, it includes following 3 members: ZmSnRK1.1 gene, ZmSnRK1.2 gene and ZmSnRK1.3 gene that the present invention reports maize sucrose non-fermented -1 related protein kinase 1 (SnRK1) gene family for the first time；And the function of 3 genes is studied from tissue expression, subcellular localization, the phenotypic characteristic for being overexpressed arabidopsis etc..Present invention discloses functional character of the corn SnRK1 member in terms of energy sensing signal, adjusting carbohydrate metabolism, Controlling the flowering period and plant type, this provides potential valuable properties and characteristics and genetic resources for high-yield breeding of crops.

Description

1 gene family of -1 related protein kinase of maize sucrose non-fermented and its application

Technical field

The invention belongs to technical field of molecular biology, and in particular to arrive -1 related protein kinase 1 of maize sucrose non-fermented Gene cloning, expression analysis, Function Identification and its application of gene family member.

Background technique

Sucrose non-fermented -1 (SNF1) related protein kinase 1 (SnRK1) is the important energy guarded in evolution Regulatory factor (Emanuelle et al., 2016).It is 1 albumen of yeast cane sugar non-fermented (Hedbacker and Carlson, 2008) and homologous base of the mammal AMP- activated protein kinase (Hardie et al., 2012) in plant Cause.SNF1/AMPK/SnRK1 protein kinase in regulating cell metabolism and maintains energy balance as key energy sensor Play a significant role in the process (Polge and Thomas, 2007；Hey et al.,2010).In plant, SnRK1 is regulating and controlling Played during growth and development and abiotic and biotic tolerance most important effect (Polge and Thomas, 2007；Baena-González and Sheen,2008；Halford and Hey,2009；Hulsmans et al., 2016).SnRK1 is swum across by the Direct Phosphorylation to key enzyme or to the extensive regulation of gene expression dose to play it under The adjustment effect (Broeckx et al., 2016) of journey.In structure, SnRK1 is by N-terminal catalyst structure domain and C-terminal Regulatory domain Composition, and functioned by forming heteromeric complexes with β and γ subunit (Ghillebert et al., 2011； Emanuelle et al.,2015)。

In plant first certified SnRK1 gene be from rye clone in come out (Alderson et al., 1991), hereafter, homologous gene of the gene in multiple plant species it is identified come out (Halford and Hardie, 1998；Halford et al., 2003), and its sugar perceptual signal transmitting and in terms of machine System is gradually revealed.Arabidopsis SnRK1 gene family include two functional members, SnRK1.1 (or AKIN10) and SnRK1.2 (or AKIN11).Arabidopsis kin10kin11 double-mutant fail to open the transcriptional switching of the hungry stress of response, thus It influences night starch to mobilize, leads to have accumulated a large amount of starch (Baena-Gonzalez et al., 2007) in blade.With this On the contrary, antisense expression SnRK1 gene in potato, causes the expression of its sucrose synthase gene to be lowered, forms sediment to reduce in stem tuber Powder content (Purcell et al., 1998)；And it is overexpressed SnRK1 gene and then has activated relevant 2 key enzymes of Starch synthesis Activity: sucrose synthase and ADP-glucose pyrophosphorylase, to increase (the McKibbin et of content of starch in stem tuber al.,2006).Antisense expression SnRK1 gene causes its pollen development to be obstructed in barley, contains only a small amount of in pollen or does not contain Starch (Zhang et al., 2001).And inhibit SnRK1 gene expression that its embryo is caused to be developed in later period maturation in pea It is obstructed and has accumulated less starch (Radchuk et al., 2010).In addition, being overexpressed MhSnRK1 gene in tomato makes The increase (Wang et al., 2012) of content of starch in tomato leaf and ripening fruits.The above result shows that SnRK1 is in source The resource of the progress starch distributed between the tissue of library is most important.

More and more researchs demonstrate SnRK1 and participate in plant growth and development and period regulation of blooming now, this sends out growth It educates, breeding time transformation is adjusted with metabolism and environmental factor connects.Turn SnRK1.1 (KIN10) gene Arabidopsis plant Show as florescence and Leaf Senescence delay (Baena-Gonzalez et al., 2007), while mesoderm growing early stage lotus throne leaf Blade becomes smaller.In contrast, be overexpressed SnRK1.2 (KIN11) arabidopsis show as blooming in advance, and lotus throne leaf blade compared with Greatly, it is opposite (Williams et al., 2014) to be overexpressed phenotype with SnRK1.1 for this.The reason of supposition causes this species diversity, can It can be that two members are different to the regulation for related gene of blooming or they act synergistically from different albumen and participate in different biological mistakes Journey (Williams et al., 2014).Nearest report shows, SnRK1s and bloom regulatory factor can be with direct interaction. FUSCA3 encodes B3 domain transcription factor, regulation plant organ development (such as embryo, cotyledon, silique and floral organ) and breeding time Transformation, SnRK1.1 (AKIN10) is by the stability of direct interaction and phosphorylation FUSCA3, to postpone nutrient growth and open Flower (Gazzarrini et al., 2004；Tsai and Gazzarrini,2012).IDD8 is one INDETERMINATE DOMAIN (IDD)-structural domain transcription factor, and by adjusting SUC (Sucrose synthesis) gene expression shadow Ring florescence.IDD8 defect mutant shows as blooming in advance, and is overexpressed and then blooms postponement (Seo et al., 2011). SnRK1.1 (AKIN10) can imply that SnRK1.1 passes through antagonism IDD8 function with phosphorylation IDD8 to reduce its transcriptional activation activity Flowering time (Jeong et al., 2015) can be controlled.In addition, bZIP63 mutant table in the aging course of dark induction Reveal delay, and SnRK1.1 can inhibit it to express (Mair with Direct Phosphorylation bZIP63 and in the aging course of starvation induction et al.,2015).Ethylene-EIN3 signal pathway plays a significant role in the adjusting of aging course, and SnRK1.1 passes through phosphoric acid Change and inhibit EIN3 activity, so as to cause leaf senile delay (Kim et al., 2017).

Corn is important industrial crops, and wherein silage corn, which is used directly for aquaculture, fermented can also be made feeding Material or the raw material of industry.Therefore, it is very necessary for increasing dynamics of the biotechnology in corn germplasm innovation and breed of variety.But It is, currently, there has been no relevant reports for -1 related protein kinase gene family of sucrose non-fermented in corn.Therefore, it clones, identify SnRK1 gene family in corn is studied the mechanism of action of the different SnRK1 albumen in corn growth and metabolism, is disclosed Functional character in terms of corn SnRK1 member's energy sensing signal, adjusting carbohydrate metabolism, Controlling the flowering period and plant type, It will be of great significance for corn with high yield breeding and raising maize economy benefit etc..

Summary of the invention

In view of the deficiencies of the prior art, the present invention provides maize sucrose non-fermented -1 related protein kinase 1 (SnRK1) gene Family and its application.The method that the present invention utilizes homologous clone, has cloned 3 SnRK1 gene families from Maize genome Member ZmSnRK1s, and from tissue expression, subcellular localization, the phenotype for being overexpressed arabidopsis phenotypic characteristic and transgenic corns Feature etc. studies the function of 3 genes, provides choosing for corn with high yield breeding and raising maize economy benefit etc. It selects.

Maize sucrose non-fermented -1 related protein kinase 1 (SnRK1) gene family of the present invention include following 3 at Member: ZmSnRK1.1 gene, ZmSnRK1.2 gene and ZmSnRK1.3 gene；Wherein, the cDNA sequence of the ZmSnRK1.1 gene Column are as shown in SEQ ID No.1, and encoding amino acid sequence is as shown in SEQ ID No.2；The cDNA of the ZmSnRK1.2 gene Sequence is as shown in SEQ ID No.3, and encoding amino acid sequence is as shown in SEQ ID No.4；The ZmSnRK1.3 gene CDNA sequence is as shown in SEQ ID No.5, and encoding amino acid sequence is as shown in SEQ ID No.6.

ZmSnRK1.1, ZmSnRK1.2 and ZmSnRK1.3 coding albumen respectively include 509,503 and 499 amino acid.With Arabidopsis AKIN10 is compared, and has 73%, 75% and 74% homology (Fig. 1) respectively.ZmSnRK1.1 and sorghum SbSnRK1b are same Source property highest, holoprotein sequence have 96% homology.And ZmSnRK1.2 and ZmSnRK1.3 is the most similar to rice OSK1, point Not Ju You 90% and 85% homology.

Expression pattern analysis and its subcellular localization of ZmSnRK1s the result shows that, ZmSnRK1s family member ZmSnRK1.1, ZmSnRK1.2 and the ZmSnRK1.3 wide expression in the tissue of all detections, and expressed most in the tender female fringe of children It is high.Three kinds of ZmSnRK1 albumen have positioning in nucleus and cytoplasm.

To ZmSnRK1s stress response and inducing expression pattern analysis, show in three members, only ZmSnRK1.1 is by famine It starves stress (dark) to induce and high expression, and adds the high table for the ZmSnRK1.1 that glucose can inhibit due to dark induction It reaches, illustrates that ZmSnRK1.1 is the important factor for perceiving and responding energy i (in vivo) level in corn.

3 ZmSnRK1s genes of heterologous overexpression show the hypersensitization to dextrose and saccharose, transgenosis in arabidopsis The more sucrose accumulated in Arabidopsis plant blade and mature seed, and glucose content reduces.Compared with wild type, table is crossed Shift to an earlier date up to ZmSnRK1.1 Arabidopsis plant florescence, and being overexpressed ZmSnRK1.2 and ZmSnRK1.3 postpones the plant blossom phase, And transgenic plant aging postpones, biomass and seed production increase.Further study show that ZmSnRK1 passes through regulation Some carbohydrate metabolisms, gene relevant with aging of blooming expression control arabidopsis growth and development.

Present invention discloses corn SnRK1 members in energy sensing signal, adjusts carbohydrate metabolism, Controlling the flowering period With the functional character in terms of plant type, this provides potential valuable properties and characteristics and genetic resources for high-yield breeding of crops.

Another aspect of the present invention also provides the recombinant expression carrier comprising the corn SnRK1 gene family；The recombination Expression vector separately includes ZmSnRK1.1 gene, the full length cDNA sequence of ZmSnRK1.2 gene and ZmSnRK1.3 gene.By institute It states gene order both ends and adds BamHI and KpnI connector respectively, be connected to support C UB, obtain CUB-pUBI::ZmSnRK1s- P35S::bar skeleton carrier.

Overexpression, Antisense Suppression, the RNA that recombinant expression carrier of the invention can be used for corn ZmSnRK1s gene are dry Disturb with CRISP/CAS9 gene editing etc..

The present invention also provides the transformant containing the recombinant expression carrier.The recombinant expression carrier is transformed into agriculture It is obtained in bacillus.The transformant is for obtaining transgenic plant.

Research shows that ZmSnRK1.2 and ZmSnRK1.3 postpone the plant blossom phase, and transgenic plant aging postpones, and plants Strain biomass and seed production increase.Wherein, ZmSnRK1.3 is weighed with axis respectively, axis is thick, grain is wide and plant height is significantly associated with, thus Illustrate that the natural variation of the gene can influence the growth and development of corn ear character.

Therefore, further aspect of the present invention also provides the corn SnRK1 gene family in delay plant senesecence, increases and plant Application in terms of strain biomass and seed production.

High-quality silage corn is obtained for example, the ZmSnRK1.2 gene and ZmSnRK1.3 gene are transferred in corn.

High-yield corn is obtained for example, the ZmSnRK1.3 gene is transferred in corn.

The invention has the following beneficial effects:

Present invention firstly discloses three members of 1 gene family of -1 related protein kinase of maize sucrose non-fermented and its cores Thuja acid, encoding amino acid sequence disclose its evolutionary relationship, the organ-tissue specificity of expression and inducing properties etc..And it uses Transgenic method had studied in arabidopsis gene family member to carbohydrate metabolism, bloom and the influence of aging.And Transgenic experiments are carried out in corn, obtain breeding time extended plant.

Present invention discloses corn SnRK1 members in energy sensing signal, adjusts carbohydrate metabolism, Controlling the flowering period With the functional character in terms of plant type, this provides potential valuable properties and characteristics and genetic resources for high-yield breeding of crops.

The present invention is to further understand corn SnRK1 gene regulating and controlling carbohydrate metabolism, blooming and the tune in aging Control mechanism provides theoretical basis, and has great importance to aging delay and high-yield transgenic new varieties is cultivated.

Detailed description of the invention

Fig. 1 is the chadogram of the SnRK1 protein sequence of higher plant.SnRK1 protein sequence is compared with ClustalX2 It is right, systematic evolution tree is constructed using the adjacent method in MEGA7.0.

Fig. 2 is corn ZmSnRK1s transcription analysis and subcellular localization.Wherein, Fig. 2 a indicates to pass through qRT-PCR pairs The tissue specific expression of ZmSnRK1.1, ZmSnRK1.2 and ZmSnRK1.3 gene is analyzed.Use FPGS transcript as inside Control, and the transcript abundance of ZmSnRK1.1 in young fringe is used as calibration.Numerical value is that there are three the duplicate standard deviations of biology for tool (SD) average value.Fig. 2 b indicates the subcellular localization of three kinds of ZmSnRK1 albumen, scale=10 μm.

Fig. 3 is sugar, ABA and dark processing to corn ZmSnRK1.1, the shadow of ZmSnRK1.2 and ZmSnRK1.3 expression It rings.

Fig. 4 is to influence after being overexpressed ZmSnRK1 gene in arabidopsis on glucose sensitive.The PCR of a transgenic plant is identified. OE1.1, OE1.2 and OE1.3 are respectively to turn ZmSnRK1.1, ZmSnRK1.2 and ZmSnRK1.3 gene Arabidopsis plant.M: DL200marker；1: positive plasmid control；2: wild type control；3-11: transgenic plant.B wild type and transgenic line Semi-quantitative RT-PCR analysis.AtUBQ10 is internal reference.C glucose (Glc), sucrose (Suc) and sorbierite (Sorb) to wild type and The influence of transgenic plant seedling stage phenotype and root long.Scale=1cm.

Fig. 5 is the influence that ZmSnRK1 gene pairs florescence, aging and carbohydrate metabolism are overexpressed in arabidopsis.A is wild Raw type and transgenic plant entirety phenotype.Scale=10cm.B, c be wild type and transgenic plant florescence, lotus throne number of sheets mesh, Chlorophyll content compares.D-f is glucose (d), sucrose (e) and starch (f) content in blade and seed.Data are as shown in the figure Test duplicate mean+SD 3 times.* it is respectively indicated with * * and tests significant difference (0.01 compared with wild type " ANOVA " < P < 0.05) and it is extremely significant (P < 0.01).

Fig. 6 is the qRT-PCR analysis of carbon metablism, aging and florescence related gene in arabidopsis.A carbon metablism dependency basis Cause.B aging related genes.C florescence related gene.AtUBQ10 is internal reference.Data as shown in the figure are that 3 experiments are duplicate flat Means standard deviation.

Fig. 7 is vegetative growth phase arabidopsis phenotype and potassium iodide coloration result.A vegetative growth phase wild type and transgenosis are planted Strain phenotype.Potassium iodide coloration result of the b Arabidopsis plant after dark and after illumination.Scale is 5 centimetres in figure.

Fig. 8 is maturity period wild type and transgenic Arabidopsis plants entirety phenotype.Scale is 10 centimetres in figure.

Fig. 9 is that the genotype of three genes in 368 parts of corn inbred lines and phenotype TASSEL software analyze result.

Figure 10 and Figure 11 is the PCR qualification result for turning ZmSnRK1s gene corn plant.

Figure 12 is the seedling stage phenotype for turning ZmSnRK1.3 gene corn plant.

Figure 13 is the Mature phase phenotype for turning ZmSnRK1.3 gene corn plant.

Specific embodiment

Technical solution of the present invention and technical effect are described further below with reference to specific experiment and attached drawing, following the description For explaining only the invention, it should not be construed as limiting the scope of the invention.In following experiments unless otherwise specified, used Method is conventional method in that art, the method that such as " Molecular Cloning:A Laboratory guide " is recorded.Agents useful for same in the present invention, such as without spy Different explanation, is this field conventional reagent, can obtain from commercial channels.Instrument of the present invention, equipment etc., such as such as without special Illustrate, is this field conventional instrument, equipment.

1. 1 (SnRK1) gene ZmSnRK1s of -1 related protein kinase of maize sucrose non-fermented clone and phylogenetic analysis

Using arabidopsis Akin10/Akin11 amino acid sequence, by NCBI (https: // Www.ncbi.nlm.nih.gov/ it) or in Maize GDB (https: //www.maizegdb.org/) database carries out homologous Search, identifies the homologous gene of SnRK1 from corn inbred line B73.Then, it clones and obtains from maize leaf cDNA library The overall length of three genes is respectively designated as ZmSnRK1.1 (GRMZM2G107867), ZmSnRK1.2 (GRMZM2G077278) and ZmSnRK1.3(GRMZM2G180704).Its cDNA sequence is respectively such as SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 It is shown.

ZmSnRK1.1, ZmSnRK1.2 and ZmSnRK1.3 coding albumen respectively include 509,503 and 499 amino acid.Sequence Column as shown in SEQ ID No.2, SEQ ID No.4, SEQ ID No.6, compared with arabidopsis AKIN10, have respectively respectively 73%, 75% and 74% homology (Fig. 1).N-terminal kinase catalytic domain is conservative in different eucaryotes, such as beautiful Rice SnRK1s, arabidopsis AKIN10, rice OSK1, sorghum SbSnRK1b and yeast SNF1.But C-terminal regulatory region is being planted Difference in height is shown in object protein.ZmSnRK1.1 and sorghum SbSnRK1b homology highest, holoprotein sequence have 96% it is same Source property.And ZmSnRK1.2 and ZmSnRK1.3 is the most similar to rice OSK1, is respectively provided with 90% and 85% homology.

The building of 2.ZmSnRK1s gene recombinant vectors

With the area the cDNA design primer of three gene orders, piece will be expanded plus BamHI and KpnI connector at both ends respectively Duan Huishou digestion is connected to support C UB, i.e. acquisition CUB-pUBI::ZmSnRK1s-p35S::bar skeleton carrier.Connector and expansion Increase primer sequence such as the following table 1:

Table 1

The expression pattern analysis and its subcellular localization of 3.ZmSnRK1s

It is (long in young fringe), boot leaf, silk, bract, grind in the various corn tissues such as prematurity tassel and mature stamen Tissue-specific transcription's abundance of ZmSnRK1s is studied carefully.Expression quantity of three ZmSnRK1s family member genes in young fringe is most Height, in boot leaf and bract expression quantity medium level.The transcriptional level of ZmSnRK1.1 and ZmSnRK1.2 is lower in stamen, and ZmSnRK1.3 transcriptional level in stamen and stamen is higher.As shown in Figure 2 a.

The subcellular localization of ZmSnRK1 albumen and its biological function are highly relevant.GFP is fused to three by us The C-terminal and the transient expression in Corn Protoplast of ZmSnRK1.Green florescent signal shows all three albumen in nucleus And have positioning in cytoplasm.As shown in Figure 2 b.

4.ZmSnRK1s is analyzed by the inducing expression of sugar, ABA and dark processing

Existing research shows that SnRK1 has played important function (Baena-Gonz á in Energy-aware and ABA signal pathway lez et al.,2007；Radchuk et al., 2010), therefore we have studied sugar, ABA and dark processings to corn The influence of 3 gene expression doses of ZmSnRK1s.By 2 weeks big corn seedlings be transferred to respectively containing 6% (w/v) glucose, 6% (w/v) sucrose, 6% (w/v) sorbierite, 10 μM of ABA 1/2MS culture medium in, separately take 2 groups (without glucose and containing 6% Glucose) dark processing is carried out, 0,1,2 and 4 hour in processing is drawn materials, and is extracted RNA and is detected using qRT-PCR method The expression of ZmSnRK1.1, ZmSnRK1.2 and ZmSnRK1.3 gene changes.

As a result as shown in figure 3,3 genes are by grape compared with untreated control and 6% sorbierite (osmotic stress control) Sugared inducing expression is not significant.ABA can inhibit the expression of 3 genes, but be not up to the level of signifiance.In dark processing condition Under, ZmSnRK1.1 expression is increased 30 times when handling 4 hours by the high inducing expression of dark processing (starvation coerce), and This high expression is significantly inhibited by glucose again.In contrast, ZmSnRK1.2 expression is inhibited by dark processing, And this inhibiting effect can be alleviated by glucose.

The above result shows that the high expression of dark processing induction ZmSnRK1.1, and this high expression is by the suppression of glucose System；And dark processing inhibits the expression of ZmSnRK1.2, and this inhibition can be alleviated by glucose.And glucose and ABA has no significant effect the expression of 3 genes.

5. ZmSnRK1s is overexpressed so that transgenic plant becomes sensitive to glucose in arabidopsis

The ZmSnRK1s gene recombinant vectors of building are transformed into Agrobacterium respectively, pass through agrobacterium mediation converted Method acquisition turns corn ZmSnRK1.1, the Arabidopsis plant of ZmSnRK1.2 or ZmSnRK1.3 gene, and utilizes PCR method to sun Property plant identified (Fig. 4 a), each transgenic experiments at least obtain 20 positive strains, respectively take a strain to carry out following Test and simultaneously the function of 3 genes researched and analysed, this 3 strains are respectively designated as OE1.1 respectively, OE1.2 and OE1.3.Semiquatitative RT-PCR assay the result shows that, express the ZmSnRK1.1 of higher level in 3 strains respectively, ZmSnRK1.2 or ZmSnRK1.3, and do not have expression (Fig. 4 b) in wild type.

In plant, SnRK1 can perceive energy i (in vivo) level and maintain plant growth and development and life by adjusting metabolism Grow necessary energy supply ((Baena-Gonzalez et al., 2007；Zhang et al.,2001).When by wild type With transgenic Arabidopsis plants respectively when being cultivated on the 1/2MS culture medium for containing or not contain 4% glucose, transgenic line Show to grow suppressed, root long shortens (Fig. 4 c).Root long is measured to turn SnRK1 gene strain compared with wild type and show The root long being substantially reduced increases glucose-sensitive.And when being grown on the culture medium containing 4% sorbierite, wild type It ties up in root long and is not significantly different with transgenic line, thus be excluded that as caused by the osmotic stress difference of root long.More than As a result the result one to high sugar processing sensitivity shown with overexpression OsSnRK1 in overexpression AKIN10 in arabidopsis and rice Cause (Jossier et al., 2009；Cho et al.,2012).

6. ZmSnRK1s, which is overexpressed, in arabidopsis influences carbohydrate metabolism

We have detected glucose, sucrose in the wild type of growth 45 days and the lotus throne leaf and mature seed of transgenic plant With the variation (Fig. 5) of content of starch.The result shows that compared with wild type, Portugal in transgenic plant OE1.1, OE1.2 and OE1.3 The content of grape sugar is remarkably decreased, and glucose content is reduced to the 67%-77% of wild type in blade, is reduced in mature seed The 53%-65% (Fig. 5 d) of wild type.On the contrary, cane sugar content dramatically increases in transgenic line, compared with wild type, in blade 134%, 151% and 167% has been increased separately, 74%, 106% and 133% (Fig. 5 e) has been increased separately in mature seed.Turn base Because the content of starch in plant leaf and seed increased, but compared with wild type, no significant difference (Fig. 5 f).It is raw to nutrition The coloration result of wild type and rotaring gene plant blade potassium iodide shows after the dark of a whole night for a long time, transgenic plant Blade dyeing is dark-brown, and wild type is light brown, illustrates still to accumulate by the consumption of starch of a whole night, transgenic plant More starch；And pass through after illumination in one day, wild type and transgenic plant uniform dyeing are dark-brown, and phenotype is without significant Difference (Fig. 7).7. ZmSnRK1s, which is overexpressed, in arabidopsis influences to bloom and aging

Existing research shows that being overexpressed AKIN10/SnRK1.1 Arabidopsis plant florescence postpones, and is overexpressed AKIN11/ SnRK1.2 then make arabidopsis florescence shift to an earlier date (Baena-Gonzalez et al., 2007；Williams et al.,2014). Currently, not clear to the influence for being overexpressed corn ZmSnRK1 gene pairs florescence.It is quasi- to ZmSnRK1 gene is turned in this research Southern mustard studies have shown that when under normal growing conditions (12 hours illumination/12 hour dark) cultivate when, compared with wild type, Turning ZmSnRK1.1 gene Arabidopsis plant florescence is advanced by 3 days, and turns the plant of ZmSnRK1.2 and ZmSnRK1.3 gene arabidopsis Strain florescence has postponed 5 days and 17 days (Fig. 5 a, b) respectively.It is aobvious to reach difference between different strains for the number of lotus throne leaf when blooming It writes, lotus throne number of sheets mesh when transfer ZmSnRK1.3 gene Arabidopsis plant is bloomed dramatically increases (Fig. 5 b).Chlorophyll content is One index of leaf senile degree, the detection to 45 days wild types and transgenic plant lotus throne leaf Determination of Chlorophyll contents of growth The result shows that comparing wild type, turns ZmSnRK1.1, had accumulated in ZmSnRK1.2 and ZmSnRK1.3 gene plant blade more Chlorophyll content, blade have it is longer hold the green phase, imply that the photosynthesis of long period.

8. ZmSnRK1s is overexpressed the influence to economical character in arabidopsis

2. wild type of table (Col) and turn ZmSnRK1.1, ZmSnRK1.2 and ZmSnRK1.3 gene plant maturity period agronomy Shape testing result

The above numerical value is to test duplicate mean+SD 6 times.* it is respectively indicated compared with wild type with * *, " ANOVA " tests significant difference (0.01 < P < 0.05) and extremely significant (P < 0.01).

Since transgenic plant aging postpones and maintains the photosynthesis of long period, maturity period transgenosis is planted Strain obtains higher biomass (table 2；Fig. 8).Although turning ZmSnRK1.1 plant arabidopsis florescence slightly to shift to an earlier date, entirety Plant type is bigger than wild type, therefore biomass and mass of 1000 kernel (increasing 11%) dramatically increase, in every plant of silique number and every silique Seed number has increase, but not up to significant difference is horizontal.Due to turning ZmSnRK1.2 and ZmSnRK1.3 gene plant with aobvious The florescence of work, plant type aging delay and increased, therefore, compared with wild type, OE1.2 and OE1.3 maturity period biomass point 75% and 95% are not increased.Every plant of silique number has increased separately 41% and 62%.In addition, the seed number in the every silique of OE1.3 Mesh increases 16% than wild type.Compared with wild type, the mass of 1000 kernel of OE1.2 and OE1.3 have increased separately 14% and 16% (table 2)。

9. ZmSnRK1 gene overexpression influences the expression of carbon metablism, aging and florescence related gene in arabidopsis

For a further understanding of the molecular mechanism for being overexpressed ZmSnRK1 gene pairs arabidopsis Phenotype, we are extracted The wild type of growth 45 days and the RNA of transgenic plant lotus throne leaf have simultaneously carried out RNA-seq analysis, to transcribe in full-length genome The ZmSnRK1 signal pathway that may regulate and control is parsed in this change level.We are by the RNA-seq data of transgenic plant It is compared respectively with wild type, and filters out differential expression multiple in the gene of 2 times or more.Compared with wild type, turn There are the gene of 42 expression up-regulations and the gene of 105 expression downwards in ZmSnRK1.1 gene strain respectively；In contrast, turn The gene number raised in ZmSnRK1.2 gene plant is 102, and the gene number lowered is 66；Turn in ZmSnRK1.3 gene plant The gene number of up-regulation is 210, and the gene number of downward is 101, and the number for raising gene is all larger than the number of down-regulated gene, shows It is different from OE1.1.In the gene of each strain all differences expression, 23 down-regulated genes and 5 up-regulation genes are to turn base at 3 Because being existed simultaneously in strain and having identical expression trend.These common difference expression genes are distinguished by biological function It is referred to that defence is corresponding, is metabolized, blooms and the processes such as aging, we have carried out further qRT-PCR to some of genes Verifying.

AMY1 (At4g25000) belongs to the gene that expression is lowered between each transgenic line.The gene encodes alphalise starch Enzyme may participate in the degradation of starch after cell death in aging and the inducing expression in biological, abiotic stress blade Journey (Doyle et al., 2007).Consistent with RNA-seq result, which is turning ZnSnRK1.1, ZnSnRK1.2 and Expression in ZnSnRK1.3 gene strain reduces 4 times, 7 times and 9 times respectively.(Fig. 6 a).Arabidopsis DIN6/ASN1 (AT3G47340) encoding asparagine rely on glutamine synthelase, the gene by various stress inducing expression and by sugar Inhibition.DIN6 by KIN10/KIN11 activation and can be used as the active indicator (Baena- of internal KIN10/KIN11 Gonzalez et al.,2007).Compared with wild type, the expression of DIN6 increases 2.7-4 in 3 transgenic lines Times, show SnRK1 activity with higher in transgenic Arabidopsis plants, and corn SnRK1 is in regulation DIN6 expression On, show the conservative functionally with arabidopsis KIN10/KIN11.

Consistent with the phenotype of aging delay that strain is shown is overexpressed, we have found one in RNA-seq data Significant change occurs for the expression of gene relevant to leaf senile a bit, such as SAG13 and WRKY53.SAG13 is in arabidopsis One of aging related genes (SAGs) member, the gene in ageing leaves special up-regulated expression (Weaver, et al., 1998； Chen et al.,2017).Compared with wild type, SAG13 expression is significantly lowered in transgenic plant, and in OE1.2 and Multiple is lowered in OE1.3 reaches 3 and 4.WRKY53 be first positive regulation leaf senile being accredited WRKY transcription because Son (Miao et al., 2004).In transgenic plant OE1.2 and OE1.3, which is remarkably decreased, with wild type Plant is compared, and has dropped 4.2 times and 2.5 times respectively, and the expression in the OE1.1 of the gene changes less than 2 times.

The process of blooming is process of the higher plant from nutrient growth to reproductive growth transformation and by finely regulating.Turn ZmSnRK1.2 and ZmSnRK1.3 arabidopsis shows different degrees of delay of blooming, and is consistent with this, our mileometer adjustments on OE1.3 In the gene reached, it is found that some significantly express to gene generation relevant with circadian rhythms of blooming changes (Fig. 6 c).FLC A MADS-box transcription factor is encoded, is the inhibiting factor (Michaels and Amasino, 1999) bloomed.Arabidopsis ATXR7/SDG25 encodes H3K4 methylase, modification is methylated by H3K4 to activate FLC to express, to inhibit process of blooming (Tamada etal.,2009；Berr et al.,2009).SUPPRESSOR OF FRIGIDA 4 (SUF4) encodes one and contains Zinc fingers transcription factor, it can inhibit the process of blooming (Kim et by binding directly FLC promoter and activating its expression al.,2006；Kim and Michaels 2006).In transgenic arabidopsis OE1.3 plant, ATXR7/SDG25 and SUF4's Expression significantly raises, and expression multiple variation respectively reaches 10 and 19, and in OE1.1 and OE1.2, the table of two genes Do not have significant change (Fig. 6 c) up to level.By detecting to FLC expression, the gene expression water in OE1.3 is found It is flat to rise 3 times, imply that the ATXR7/SDG25 and SUF4 of up-regulated expression have expressed activation to FLC, to inhibit out Flower, makes delay of blooming.In addition, in RNA-seq data, it has been found that some participation circadian rhythms and photoperiod rely on The expression of gene of process of blooming significant change, such as FIONA1 (FIO1) and NIGHT LIGHT-INDUCIBLE occurs AND CLOCK-REGULATED2(LNK2)(Kim et al.,2008；Rugnone et al.,2013).Two genes exist 2.5 times and 2.4 times of up-regulated expression is distinguished in OE1.3.These results suggest that participating in the gene in florescence and biocycle regulation Important function has been played in the florescence control that ZmSnRK1 is mediated.

10.ZmSnRK1s candidate gene association analyzes result

For the effect of the regulation of supposition ZmSnRK1s gene pairs corn growth, we utilize existing database 368 parts of three bases in corn inbred line all over the world in http://modem.hzau.edu.cn/maizego/ The genotype and phenotype of cause are downloaded and are analyzed using TASSEL software, as a result as shown in figure 9, as threshold value P < 0.01, ZmSnRK1.3 respectively with axis weight, axis is thick, grain is wide and plant height etc. is significantly associated with, thus illustrate that the natural variation of the gene being capable of shadow Ring the growth and development of corn ear character.

The molecular Evidence of 11.ZmSnRK1s overexpression corn

For the effect for further studying regulation of the ZmSnRK1s to corn growth, the present invention is by building ZmSnRK1.3 over-express vector is transformed into agrobacterium strains AGL1, is obtained by the rataria genetic transforming method of mediated by agriculture bacillus It must turn the plant of corn ZmSnRK1.3 gene, and positive plant is identified using PCR and Southern hybridizing method.Figure 10, the visible present invention of Figure 11 has successfully obtained transgenic corns positive plant, and the copy number of external source riddled basins is singly to copy Shellfish.

The phenotype of 12.ZmSnRK1s overexpression corn

Transgenic corns chamber planting material seedling stage and Mature phase observed result are shown in Figure 12 and 13 respectively, visible in figure It is transferred to the corn of ZmSnRK1.3 gene, is compared compared with control, breeding time extends, and biomass increases.This is found to be further reason Solve corn SnRK1 gene in regulation carbohydrate metabolism, blooming provides theoretical basis with the regulatory mechanism in aging, and right Cultivating aging delay and high-yield transgenic new varieties has great importance.

SEQUENCE LISTING

<110>Corn Institute, Shandong Academy of Agricultural Sciences's (Shandong Academy of Agricultural Sciences's corn Engineering Technical Research Centre)

<120>1 gene family of -1 related protein kinase of maize sucrose non-fermented and its application

<130>

<160> 12

<170> PatentIn version 3.3

<210> 1

<211> 1530

<212> DNA

<213> Zea mays L.

<400> 1

atggatggaa gtagtaaagg gagtgggcat tctgaagcat taaggaacta caacctggga 60

agaactttag gtatcggtac atttggaaaa gtgaagattg cagagcataa gcttactgga 120

catagggttg ctataaagat catcaactgc cgccaaatga gaaatatgga aatggaagag 180

aaagcaaaga gagaattcaa gatattgaag ttgttcattc acccccatat cattcggctt 240

tatgaggtca tatacacacc tacagatata tatgttgtga tggaatattg taagtatggc 300

gagttatttg attacattgt tgagaaaggc agattacagg aagatgaagc tcgtcgaatc 360

ttccagcaga tcatatctgg cgtcgaatac tgccatagaa acatggttgt ccaccgtgac 420

ctaaagccgg aaaacttgtt acttgattca aagtataatg taaaacttgc ggattttggt 480

ctgagcaatg tcatgcatga tggccatttt ctgaagacta gctgtgggag tccgaactat 540

gctgctccag aggtaatatc tggtaaacta tatgctggac ctgaggtcga tgtatggagt 600

tgtggggtga ttctttatgc tcttctttgt ggaactcttc catttgatga tgagaatatt 660

cccaatctgt tcaaaaaaat taagggaggt atctacacac ttccaagtca tttgtctgct 720

ttggccaggg atttgatccc acgaatgctt gttgttgagc ctatgaagag aatcacaatt 780

agggaaattc gggagcatca atggttccag attcgccttc cacgttactt ggcagtgcct 840

ccaccagata cgacacaaca agccaaaatg attgatgaag atacacttcg agatgttgtt 900

aatatgggat ttaacaagaa ccatgtgtgt gaatcactgt gcagcagact tcaaaatgag 960

gcaactgttg catattattt actattggac aatcggttta gagcaactag tggctatctt 1020

ggggcagatt atcaagaatc aatggacagg aatttaaatc agctggcgtc atctgaatca 1080

tctagttctg gtacgaggaa ttatgttcca ggaagcagtg atcctcatag cagtggtttg 1140

cggccatatt atcctgttga aagaaaatgg gcgcttggac ttcagtctcg ggcccaccct 1200

cgtgaaataa tggttgaggt cttaaaagca cttcaagaat taaacgtcag atggaagaag 1260

aatgggcact acaacgtgaa atgcagatgg tgcccagggt ttcctgaagt taatgacacg 1320

ttagatgcca gcaacagctt tcttggtgac tctaccatca tggataatga tgatgctaat 1380

gggaggctac ctactgtgat caagtttgaa ttccagcttt acaagacgaa ggacgacaag 1440

tacctcttag atatgcagag agttactgga cctcagctgc tcttccttga cttctgtgcg 1500

gccttcctta ccaagcttag ggttctatag 1530

<210> 2

<211> 509

<212> PRT

<213> Zea mays L.

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Met Asp Gly Ser Ser Lys Gly Ser Gly His Ser Glu Ala Leu Arg Asn

1 5 10 15

Tyr Asn Leu Gly Arg Thr Leu Gly Ile Gly Thr Phe Gly Lys Val Lys

20 25 30

Ile Ala Glu His Lys Leu Thr Gly His Arg Val Ala Ile Lys Ile Ile

35 40 45

Asn Cys Arg Gln Met Arg Asn Met Glu Met Glu Glu Lys Ala Lys Arg

50 55 60

Glu Phe Lys Ile Leu Lys Leu Phe Ile His Pro His Ile Ile Arg Leu

65 70 75 80

Tyr Glu Val Ile Tyr Thr Pro Thr Asp Ile Tyr Val Val Met Glu Tyr

85 90 95

Cys Lys Tyr Gly Glu Leu Phe Asp Tyr Ile Val Glu Lys Gly Arg Leu

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Gln Glu Asp Glu Ala Arg Arg Ile Phe Gln Gln Ile Ile Ser Gly Val

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Glu Tyr Cys His Arg Asn Met Val Val His Arg Asp Leu Lys Pro Glu

130 135 140

Asn Leu Leu Leu Asp Ser Lys Tyr Asn Val Lys Leu Ala Asp Phe Gly

145 150 155 160

Leu Ser Asn Val Met His Asp Gly His Phe Leu Lys Thr Ser Cys Gly

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Ser Pro Asn Tyr Ala Ala Pro Glu Val Ile Ser Gly Lys Leu Tyr Ala

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Leu Cys Gly Thr Leu Pro Phe Asp Asp Glu Asn Ile Pro Asn Leu Phe

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Lys Lys Ile Lys Gly Gly Ile Tyr Thr Leu Pro Ser His Leu Ser Ala

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Leu Ala Arg Asp Leu Ile Pro Arg Met Leu Val Val Glu Pro Met Lys

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Arg Ile Thr Ile Arg Glu Ile Arg Glu His Gln Trp Phe Gln Ile Arg

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275 280 285

Lys Met Ile Asp Glu Asp Thr Leu Arg Asp Val Val Asn Met Gly Phe

290 295 300

Asn Lys Asn His Val Cys Glu Ser Leu Cys Ser Arg Leu Gln Asn Glu

305 310 315 320

Ala Thr Val Ala Tyr Tyr Leu Leu Leu Asp Asn Arg Phe Arg Ala Thr

325 330 335

Ser Gly Tyr Leu Gly Ala Asp Tyr Gln Glu Ser Met Asp Arg Asn Leu

340 345 350

Asn Gln Leu Ala Ser Ser Glu Ser Ser Ser Ser Gly Thr Arg Asn Tyr

355 360 365

Val Pro Gly Ser Ser Asp Pro His Ser Ser Gly Leu Arg Pro Tyr Tyr

370 375 380

Pro Val Glu Arg Lys Trp Ala Leu Gly Leu Gln Ser Arg Ala His Pro

385 390 395 400

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Arg Trp Lys Lys Asn Gly His Tyr Asn Val Lys Cys Arg Trp Cys Pro

420 425 430

Gly Phe Pro Glu Val Asn Asp Thr Leu Asp Ala Ser Asn Ser Phe Leu

435 440 445

Gly Asp Ser Thr Ile Met Asp Asn Asp Asp Ala Asn Gly Arg Leu Pro

450 455 460

Thr Val Ile Lys Phe Glu Phe Gln Leu Tyr Lys Thr Lys Asp Asp Lys

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Tyr Leu Leu Asp Met Gln Arg Val Thr Gly Pro Gln Leu Leu Phe Leu

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Asp Phe Cys Ala Ala Phe Leu Thr Lys Leu Arg Val Leu

500 505

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<213> Zea mays L.

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atggagggag cgggaagaga tgccaaccct ttgagcggtt acagaattgg caaaaccctg 60

ggaattgggt cgttcggtaa agtgaagatc gcggaacata tattgactgg tcataaggtg 120

gcgatcaaga ttctcaatcg caagaagatc agaagcatgg atatggaaga gaaagttaag 180

agagaaatca agatactgag attatttatg catcctcata tcatacgcct ttatgaggtg 240

atagatacac ctgctgatat ctgtgttgtt atggagtatg ttaaatctgg agagttgttt 300

gattacatcg ttgagaaggg aaggctacac gaagaggaag cccgacactt ttttcagcag 360

atcatatctg gtgttgaata ttgccatagg aacatggttg ctcaccgtga tttaaagcca 420

gagaatcttc ttttggattc aaaatgcaat gttaagattg ccgattttgg cttaagtaat 480

attatgcgtg atggtcactt tcttaagacg agttgtggta gcccgaatta tgcagcacct 540

gaggtcatat ctggtaaact atatgctggt cctgaagttg acgtctggag ctgtggagtt 600

attctttatg ctcttctttg tggcactctc ccatttgacg atgagaatat tccaaacctt 660

ttcaagaaaa taaagggtgg aatatatacc cttcctagtc atttgtcacc ttcagcgagg 720

gacttgattc ccagaatgct ggttgttgat ccaatgaaaa ggattacaat acgtgaaatc 780

cgtgaacatg tgtggttcaa gatccgactt ccgcgctatt tggctgtgcc gcctccagac 840

actgctcaac aagttaaaaa ggtcgacgag gaaactctta atgatgttat taagatgggt 900

tttgacaaga atcagctaat tgaatctctg caaaacagat tgcagaatga ggcaacagtt 960

gcctattatt tactcttgga caataggctt cgtacaacca gtggttatct tggatctgag 1020

tttcaagaat ctatggactc atctttgtct caagtaatcg ctgaaacacc aacttcagca 1080

actgaacttc gtcagcatgg gttttcagaa tctccaggtt ctggcttgag gcagcatttt 1140

gcagctgaaa ggaaatgggc ccttggtctt cagtctcgag cacatccacg agaaataata 1200

agtgaagtgc ttaaagctct gcaagaactg aatgtttact ggaaaaagat tggacactac 1260

aacatgaaat gcagatggag tcctggctgc cttgagagta tgatgcataa cagtgatagc 1320

ttcagtgcgg agtctgctat aattgaaact gatgttttca tggagaaatc aaccccgaca 1380

gtgaagtttg agattcagct ttacaaaacg agggatgaga agtaccttct tgacctgcaa 1440

agggtcagtg gatcacatct tctctttctg gacttgtgtt ccgcctttct aactcagctg 1500

agagttcttt ga 1512

<210> 4

<211> 503

<212> PRT

<213> Zea mays L.

<400> 4

Met Glu Gly Ala Gly Arg Asp Ala Asn Pro Leu Ser Gly Tyr Arg Ile

1 5 10 15

Gly Lys Thr Leu Gly Ile Gly Ser Phe Gly Lys Val Lys Ile Ala Glu

20 25 30

His Ile Leu Thr Gly His Lys Val Ala Ile Lys Ile Leu Asn Arg Lys

35 40 45

Lys Ile Arg Ser Met Asp Met Glu Glu Lys Val Lys Arg Glu Ile Lys

50 55 60

Ile Leu Arg Leu Phe Met His Pro His Ile Ile Arg Leu Tyr Glu Val

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Ile Asp Thr Pro Ala Asp Ile Cys Val Val Met Glu Tyr Val Lys Ser

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Gly Glu Leu Phe Asp Tyr Ile Val Glu Lys Gly Arg Leu His Glu Glu

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Glu Ala Arg His Phe Phe Gln Gln Ile Ile Ser Gly Val Glu Tyr Cys

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His Arg Asn Met Val Ala His Arg Asp Leu Lys Pro Glu Asn Leu Leu

130 135 140

Leu Asp Ser Lys Cys Asn Val Lys Ile Ala Asp Phe Gly Leu Ser Asn

145 150 155 160

Ile Met Arg Asp Gly His Phe Leu Lys Thr Ser Cys Gly Ser Pro Asn

165 170 175

Tyr Ala Ala Pro Glu Val Ile Ser Gly Lys Leu Tyr Ala Gly Pro Glu

180 185 190

Val Asp Val Trp Ser Cys Gly Val Ile Leu Tyr Ala Leu Leu Cys Gly

195 200 205

Thr Leu Pro Phe Asp Asp Glu Asn Ile Pro Asn Leu Phe Lys Lys Ile

210 215 220

Lys Gly Gly Ile Tyr Thr Leu Pro Ser His Leu Ser Pro Ser Ala Arg

225 230 235 240

Asp Leu Ile Pro Arg Met Leu Val Val Asp Pro Met Lys Arg Ile Thr

245 250 255

Ile Arg Glu Ile Arg Glu His Val Trp Phe Lys Ile Arg Leu Pro Arg

260 265 270

Tyr Leu Ala Val Pro Pro Pro Asp Thr Ala Gln Gln Val Lys Lys Val

275 280 285

Asp Glu Glu Thr Leu Asn Asp Val Ile Lys Met Gly Phe Asp Lys Asn

290 295 300

Gln Leu Ile Glu Ser Leu Gln Asn Arg Leu Gln Asn Glu Ala Thr Val

305 310 315 320

Ala Tyr Tyr Leu Leu Leu Asp Asn Arg Leu Arg Thr Thr Ser Gly Tyr

325 330 335

Leu Gly Ser Glu Phe Gln Glu Ser Met Asp Ser Ser Leu Ser Gln Val

340 345 350

Ile Ala Glu Thr Pro Thr Ser Ala Thr Glu Leu Arg Gln His Gly Phe

355 360 365

Ser Glu Ser Pro Gly Ser Gly Leu Arg Gln His Phe Ala Ala Glu Arg

370 375 380

Lys Trp Ala Leu Gly Leu Gln Ser Arg Ala His Pro Arg Glu Ile Ile

385 390 395 400

Ser Glu Val Leu Lys Ala Leu Gln Glu Leu Asn Val Tyr Trp Lys Lys

405 410 415

Ile Gly His Tyr Asn Met Lys Cys Arg Trp Ser Pro Gly Cys Leu Glu

420 425 430

Ser Met Met His Asn Ser Asp Ser Phe Ser Ala Glu Ser Ala Ile Ile

435 440 445

Glu Thr Asp Val Phe Met Glu Lys Ser Thr Pro Thr Val Lys Phe Glu

450 455 460

Ile Gln Leu Tyr Lys Thr Arg Asp Glu Lys Tyr Leu Leu Asp Leu Gln

465 470 475 480

Arg Val Ser Gly Ser His Leu Leu Phe Leu Asp Leu Cys Ser Ala Phe

485 490 495

Leu Thr Gln Leu Arg Val Leu

500

<210> 5

<211> 1500

<212> DNA

<213> Zea mays L.

<400> 5

atggaggggg caggcaagga tggcaacccg ttgaggaatt atcggattgg caagactctc 60

ggaattggct cattcgggaa ggtgaaaatt gcggagcata tcagcactgg acacaaggtg 120

gcaatcaaga ttctcaaccg ccgtaaaatc agaggcatgg agatggaaga gaaagttaaa 180

agagagatta agatattgag gttatttatg catccacata ttatccgcct ctatgaggtt 240

atagacacac cggctgatat ttatgttgtt atggagtatg ttaagtgtgg ggaattattt 300

gattacattg ttgagaaagg taggctgcag gaggaagagg ctcgccgttt cttccaacag 360

attatatccg gtgttgaata ttgccataga aacatggtgg tgcatcgtga tctaaagcca 420

gaaaacctcc tattggattc aaaatgcaat gttaagattg cagattttgg cttaagtaat 480

gttatgcggg atggtcattt tctgaagaca agttgtggta gcccaaatta tgctgctcct 540

gaggtgatat ctggtaaact atatgctgga cctgaagttg atgtgtggag ctgtggggtt 600

attctttatg ctcttttatg tggtactctg ccatttgatg acgagaacat accaaacctt 660

tttaagaaaa taaagggtgg aatatatacc cttcccagcc atttgtctgg tgcagcaagg 720

gatttgattc caagaatgct agttgtcgat cctatgaagc ggatcaccat tcgtgaaatt 780

cgcgaacatg attggttcaa aattcttctc ccgcgctatt tgactgtgcc tcctccagat 840

agtgcgcaac aagtcaaaaa ggttgatgag gaaactctcc gtgaggtttt aggtatggga 900

tatgacaaga acctgttggt ggaatcaatc caaaaaaggc tgcaaaatga ggcaactgtt 960

gcatattact tactcttgga caataggctc cgtacaacca gtggctatct tggagctgaa 1020

tgtcaagaag ctatggactc ctcattctca aacatcgcat catatgaaac accaagttca 1080

gcacgtggga atagacagca aatatttatg gagtctccag ttggcttgag accacatctt 1140

ccagctgaga ggaaatgggc tcttggtctt cagtctcgag cacatccaaa agaaataatg 1200

tctgaagtgc tgaaagctct gcaagaatta aatgtttact ggaaaaagat aggtcactat 1260

aacatgaagt gcagatggag tcctggcttt cctgctcaaa ttcataacaa tcataacttc 1320

agtgcagggt ccattgaaac tgatagcctg agtgagaggt taagtttaat taagtttgaa 1380

attcagctgt acaaaacaag agacgagaaa tacctcctcg atttgcaaag agtcagtggg 1440

ccacagctcc tctttctgga cttgtgcgcg gcctttctaa ctcaactgag agttctttga 1500

<210> 6

<211> 499

<212> PRT

<213> Zea mays L.

<400> 6

Met Glu Gly Ala Gly Lys Asp Gly Asn Pro Leu Arg Asn Tyr Arg Ile

1 5 10 15

Gly Lys Thr Leu Gly Ile Gly Ser Phe Gly Lys Val Lys Ile Ala Glu

20 25 30

His Ile Ser Thr Gly His Lys Val Ala Ile Lys Ile Leu Asn Arg Arg

35 40 45

Lys Ile Arg Gly Met Glu Met Glu Glu Lys Val Lys Arg Glu Ile Lys

50 55 60

Ile Leu Arg Leu Phe Met His Pro His Ile Ile Arg Leu Tyr Glu Val

65 70 75 80

Ile Asp Thr Pro Ala Asp Ile Tyr Val Val Met Glu Tyr Val Lys Cys

85 90 95

Gly Glu Leu Phe Asp Tyr Ile Val Glu Lys Gly Arg Leu Gln Glu Glu

100 105 110

Glu Ala Arg Arg Phe Phe Gln Gln Ile Ile Ser Gly Val Glu Tyr Cys

115 120 125

His Arg Asn Met Val Val His Arg Asp Leu Lys Pro Glu Asn Leu Leu

130 135 140

Leu Asp Ser Lys Cys Asn Val Lys Ile Ala Asp Phe Gly Leu Ser Asn

145 150 155 160

Val Met Arg Asp Gly His Phe Leu Lys Thr Ser Cys Gly Ser Pro Asn

165 170 175

Tyr Ala Ala Pro Glu Val Ile Ser Gly Lys Leu Tyr Ala Gly Pro Glu

180 185 190

Val Asp Val Trp Ser Cys Gly Val Ile Leu Tyr Ala Leu Leu Cys Gly

195 200 205

Thr Leu Pro Phe Asp Asp Glu Asn Ile Pro Asn Leu Phe Lys Lys Ile

210 215 220

Lys Gly Gly Ile Tyr Thr Leu Pro Ser His Leu Ser Gly Ala Ala Arg

225 230 235 240

Asp Leu Ile Pro Arg Met Leu Val Val Asp Pro Met Lys Arg Ile Thr

245 250 255

Ile Arg Glu Ile Arg Glu His Asp Trp Phe Lys Ile Leu Leu Pro Arg

260 265 270

Tyr Leu Thr Val Pro Pro Pro Asp Ser Ala Gln Gln Val Lys Lys Val

275 280 285

Asp Glu Glu Thr Leu Arg Glu Val Leu Gly Met Gly Tyr Asp Lys Asn

290 295 300

Leu Leu Val Glu Ser Ile Gln Lys Arg Leu Gln Asn Glu Ala Thr Val

305 310 315 320

Ala Tyr Tyr Leu Leu Leu Asp Asn Arg Leu Arg Thr Thr Ser Gly Tyr

325 330 335

Leu Gly Ala Glu Cys Gln Glu Ala Met Asp Ser Ser Phe Ser Asn Ile

340 345 350

Ala Ser Tyr Glu Thr Pro Ser Ser Ala Arg Gly Asn Arg Gln Gln Ile

355 360 365

Phe Met Glu Ser Pro Val Gly Leu Arg Pro His Leu Pro Ala Glu Arg

370 375 380

Lys Trp Ala Leu Gly Leu Gln Ser Arg Ala His Pro Lys Glu Ile Met

385 390 395 400

Ser Glu Val Leu Lys Ala Leu Gln Glu Leu Asn Val Tyr Trp Lys Lys

405 410 415

Ile Gly His Tyr Asn Met Lys Cys Arg Trp Ser Pro Gly Phe Pro Ala

420 425 430

Gln Ile His Asn Asn His Asn Phe Ser Ala Gly Ser Ile Glu Thr Asp

435 440 445

Ser Leu Ser Glu Arg Leu Ser Leu Ile Lys Phe Glu Ile Gln Leu Tyr

450 455 460

Lys Thr Arg Asp Glu Lys Tyr Leu Leu Asp Leu Gln Arg Val Ser Gly

465 470 475 480

Pro Gln Leu Leu Phe Leu Asp Leu Cys Ala Ala Phe Leu Thr Gln Leu

485 490 495

Arg Val Leu

<210> 7

<211> 27

<212> DNA

<213>artificial sequence

<400> 7

cgggatccta ggtgttctcc ccgtctc 27

<210> 8

<211> 23

<212> DNA

<213>artificial sequence

<400> 8

cgggtacccc tatttacatg ctt 23

<210> 9

<211> 27

<212> DNA

<213>artificial sequence

<400> 9

cgggatccag gtacacgatg gagggag 27

<210> 10

<211> 25

<212> DNA

<213>artificial sequence

<400> 10

cgggtaccgg tatttcttag tttgg 25

<210> 11

<211> 28

<212> DNA

<213>artificial sequence

<400> 11

cgggatcccc tcctttctcc tccgacct 28

<210> 12

<211> 29

<212> DNA

<213>artificial sequence

<400> 12

cgggtaccac gcaaggaatg caactgatg 29

Claims (3)

1. 1 gene family of -1 related protein kinase of maize sucrose non-fermented increases biomass and kind in delay plant senesecence Application in terms of suboutput, 1 gene family of -1 related protein kinase of maize sucrose non-fermented include 3 members: ZmSnRK1.1 gene, ZmSnRK1.2 gene and ZmSnRK1.3 gene；The cDNA sequence such as SEQ of the ZmSnRK1.1 gene Shown in ID No.1；The cDNA sequence of the ZmSnRK1.2 gene is as shown in SEQ ID No.3；The ZmSnRK1.3 gene CDNA sequence as shown in SEQ ID No.5, the ZmSnRK1.2 gene and ZmSnRK1.3 gene are transferred in corn obtain it is excellent Matter silage corn.

2. application as described in claim 1, characterized in that the ZmSnRK1.3 gene is transferred in corn and obtains high yield jade Rice, specifically: building ZmSnRK1.3 over-express vector is transformed into agrobacterium strains AGL1, passes through the rataria of mediated by agriculture bacillus Genetic transforming method obtains the plant for turning corn ZmSnRK1.3 gene；

The ZmSnRK1.3 over-express vector is by ZmSnRK1.3 gene order both ends plus BamHI and KpnI connector, connection To support C UB, CUB-pUBI::ZmSnRK1s-p35S::bar skeleton carrier is obtained.

3. application as claimed in claim 2, characterized in that the ZmSnRK1.3 gene order is full length cDNA sequence.