CN104388447A - Application of arabidopsis cell cycle dependency protein kinase gene AtCDKG2 in regulating and controlling plant flowering phase switching - Google Patents

Abstract

The invention discloses application of an arabidopsis cell cycle dependency protein kinase gene AtCDKG2 in regulating and controlling plant flowering phase switching. The experiment shows that the flowering time of arabidopsis can be brought forward if the AtCDKG2 gene is subjected to deletion mutantion, after the AtCDKG2 gene is over-expressed in arabidopsis by using a transgenic technology, the flowering time of an over-expressed transgenic strain is remarkably delayed, and moreover the negative regulation and control function of AtCDKG2 to flowering phase switching of arabidopsis is not affected by the photoperiod. The flowering time of plants is closely related to the yield and the quality, due to the application of the AtCDKG2 gene in regulating and controlling the plant flowering phase switching, the nutrient growth time of plants can be shortened and prolonged by controlling the expression of the gene, the breeding is accelerated, the crop growth amount is increased, assistance and support can be provided to the improvement of the crop variety, increase of the crop yield and improvement of the crop quality, and great significance on agricultural production is achieved.

Description

The application of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 in the conversion of regulating plant florescence

Technical field

The present invention relates to the function and application of arabidopsis cell cyclin dependent protein kinase gene, particularly relate to the application of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 in the conversion of regulating plant florescence, belong to genetically engineered field.

Background technology

CDKG2 belongs to cyclin-depended kinase (cyclin-dependent kinase, CDKs) G class subfamily, G class subfamily has two members, CDKG1 and CDKG2, they contain conservative PLTSLRE motif (Menges M et al.Global analysis of the core cell cycle regulators of Arabidopsis identifies novel genes, revealsmultiple and highly specific profiles of expression and provides a coherent model for plant cellcycle control.The Plant Journal, 2005).There are some researches show, AtCDKG1 participates in the montage of the Pre-mRNA of CalS5 gene, and then participate in the regulation process of pollen wall early origin, the degradation (Huang et al.CYCLIN-DEPENDENT KINASE G1is Associated with theSpliceosome to Regulate CALLOSE SYNTHASE5 Splicing and Pollen Wall Formation inArabidopsis.The Plant Cell Preview, 2013) of male fertile can be caused after knocking out the CDKG1 gene of Arabidopis thaliana.

Chip data shows, AtCDKG2 the latter stage of the elementary dormancy of seed and the initial stage expression amount of secondary dormancy relatively high, infer that AtCDKG2 may work in regulation and control seed dormancy process.In blade, the expression amount of AtCDKG2 will apparently higher than mesophyll cell in guard cell, and in guard cell, the expression of AtCDKG2 is not induced by ABA, infers that this gene does not participate in the regulating and controlling effect of ABA to stomatal movement.Through retrieval, arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 changes the effect in regulation and control at the plant florescence and applies and has no report at present.

Summary of the invention

For the deficiencies in the prior art, the object of this invention is to provide the application of a kind of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 in the conversion of regulating plant florescence.

The application of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 of the present invention in the conversion of regulating plant florescence.

Wherein: the sequence of described arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 is announced (gene is numbered NW_001843868.1); Described plant refers to cress, preferred Arabidopis thaliana, rape or Chinese cabbage.

Applicant early stage, find no matter be under long day or short day condition, cdkg2-1 and cdkg2-2 mutant all showed early floral formation with cdkg2-1 and cdkg2-2 mutant for experimental subjects.Be cloned into arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 further by RT-PCR technology, build the plant over-express vector of this gene, and carry out plant transgene operation, obtain transgenic plant.The flowering time of plant has obviously been postponed after found that AtCDKG2 gene overexpression.

Based on the above results, applicant tentatively confirms the supressor AtCDKG2 of blooming of an Arabidopis thaliana, when promoting Arabidopis thaliana Blooming after AtCDKG2 gene generation deletion mutantion.Further experiment confirms: utilize transgenic technology high expression level arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 in Arabidopis thaliana, to be found by the flowering time observing process LAN plant, the significant prolongation flowering time of plant after AtCDKG2 process LAN.

Flowering time and the yield and quality of plant are closely related, the application of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 provided by the invention in the conversion of regulating plant florescence, be expected to offer help in variety of crops improvement and crop yield or quality-improving and support, significant to the agriculture production of China.

Accompanying drawing explanation

Fig. 1: the expression pattern analysis of cyclin-depended kinase gene C DKG2.

The result of GUS dyeing shows, the expression amount of CDKG2 in dry seeds very high (see Fig. 1-I), and after seed germination, expression amount reduces, and does not almost express (see Fig. 1-II, III, IV) in the cotyledon of seedling in early days.Along with the growth of seedling, in blade, start there is expression, at microtubule tissue, leaf primordium, the tip of a root and side root nidus expression amount higher (see Fig. 1-V, VI, VII, VIII).CDKG2 also has expression in flower tissue, and wherein the expression amount of column cap, anthocaulus and stamen shank is higher, and the expression amount in mellow fruit pod is higher than young tender fruit pod (see Fig. 1-Ⅸ, Ⅹ, Ⅺ, Ⅻ).Be different at the expression amount of the Different growth phases CDKG2 of Arabidopis thaliana, it works in florescence conversion plant of the higher CDKG2 of imply that of expression amount of flower tissue.

Fig. 2: cdkg2 mutant and the bloom situation of CDKG2 process LAN strain under short day condition.

Wherein, Col-0 is wild type control, cdkg2-1 (SALK_012428) and cdkg2-2 (SALK_090262) is two mutant strains, all purchased from ABRC (Arabidopsis Biological Resource Center), VC is empty carrier untransformed controls strain, OE1 and OE2 is the transgenosis process LAN strain of two CDKG2.Under short day condition, the bolting time of cdkg2-1 and cdkg2-2 mutant is obviously early than wild-type Col-0 (see Fig. 2-A).On the contrary, the bolting time of two CDKG2 process LAN strains is then later than empty vector control (see Fig. 2-B).This proterties illustrates, AtCDKG2 gene plays negative regulation effect in the florescence switching process of Arabidopis thaliana.

To bloom in Fig. 3: cdkg2 mutant the expression of genes involved.

Realtime-PCR result shows, to bloom in approach of independently blooming the upstream gene of suppressor gene FLC: FCA, FY, FPA, LD, FLD and FLK etc. are at wild-type Col-0 and mutant cdkg2-1, and the expression amount in cdkg2-2 does not obviously distinguish (see Fig. 3-A).The expression of suppressor gene FLC in mutant cdkg2-1 and cdkg2-2 of blooming reduces, and approach of blooming conformity gene FT and the expression amount of floral meristem specific gene AP1 and LFY in mutant cdkg2-1 and cdkg2-2 are higher than wild-type Col-0 (see Fig. 3-B).Therefore, we infer that arabidopsis cell cyclin dependent albumin A tCDKG2 is positioned at the upstream of suppressor gene FLC of blooming, by regulating and controlling the expression of FLC and then suppressing flowering of plant.

Embodiment

Following examples are used for illustrating the present invention, but are not used for limiting the scope of the invention.Unreceipted concrete experimental technique in following Examples, all can conventionally carry out, or according to the operation instruction of product manufacturing production firm.

The molecular cloning of embodiment 1 arabidopsis cell cyclin dependent protein kinase gene CDKG2

1. the clone of arabidopsis cell cyclin dependent protein kinase gene CDKG2

CDNA sequence (gene is numbered NW_001843868.1) according to the arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 announced designs primer, wherein:

Forward primer is CDKG2-F:5 '-ATTGTTCAATGGGAAAACGG-3 ';

Reverse primer is CDKG2-R:5 '-TTCACAAATCACCACGGACC-3 '.

TRIzol test kit (Roche) is utilized to extract wildtype Arabidopsis thaliana RNA, the total length CDS sequence of RT-PCR method amplification AtCDKG2 gene.RT-PCR amplification program is as follows: 94 DEG C of 4min; 94 DEG C of 30s, 57 DEG C of 30s, 72 DEG C of 90s, totally 35 circulations; Last 72 DEG C extend 10min.The AtCDKG2 gene cDNA of acquisition is cloned on pEASY-Blunt (Trans Gen) intermediate carrier through operations such as recovery, connections, then the full length gene PCR carrying out intermediate carrier verifies and double digestion checking, finally carry out sequencing, obtain the correct sequence of cDNA.

2. the sequence information of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 and specificity analysis

The coding region cDNA of AtCDKG2 gene is 2275bp.Database is predicted, CDKG2 has serine/threonine or tyrosine protein kinase and a similar Dehydrin functional domain.

The expression pattern analysis of embodiment 2 arabidopsis cell cyclin dependent protein kinase gene CDKG2

The structure of 1.35S::CDGK2::GFP transient expression vector

Obtain CDKG2 full length sequence with BamH I and Sal I double digestion intermediate carrier, be connected on pBI121-GFP carrier, obtain 35S::CDGK2::GFP transient expression vector.Use MN to measure plasmid extraction kit except in intracellular toxin, extract plasmid and transformed wild type Arabidopis thaliana mesophyll protoplast, incubated at room 16h, with observing CDKG2::GFP fusion rotein ubcellular situation under laser confocal microscope 488nm wavelength.Result shows, CDKG2 mainly expresses in nucleus.

2.GUS staining analysis

(1) promoter sequence clone

Design primer, use high-fidelity enzyme Fast PFU from leaf genomic dna, clone the promoter sequence (front 4 bases clone of ATG) of ATG upstream 2045bp, PCR primer is directly connected transformation of E. coli DH5 α with pEASY-Blunt carrier, after that antibiotic-screening of card, direct picking colony carries out bacterium colony PCR, positive colony carries out digestion verification after extracting plasmid, and picking enzyme is cut correct bacterium colony and sent company to check order.

(2) pCDKG2::GUS vector construction

Cut back to close by pCDKG2 sequence enzyme, the pCambia-Ubi ＇ Gus cut back to close with enzyme is connected transformation of E. coli DH5 α, and after that resistance screening of card, the positive colony that picking grows carries out bacterium colony PCR, and extracts plasmid and carry out digestion verification.

(3) transgenic arabidopsis is obtained

Extraction PCR and enzyme cut the correct pCDKG2::GUS plasmid of qualification, transformation Agrobacterium EHA105, and bacterium colony PCR detects positive bacterium colony, by bacterium colony enlarged culturing correct for qualification, and transform Col-0 wildtype Arabidopsis thaliana.Collect T1 for seed, sow on the MS culture medium flat plate containing 30 μ g/mL hygromycin resistances, cultivate observation in about 10 days, have hygromycin resistance, the positive plant of successful conversion foreign gene can grow true leaf and normal root, and the seedling of feminine gender can not be sprouted and maybe can only grow cotyledon and very short root.Move in Nutrition Soil by positive seedling, 4-6 leaf period to be grown to, a little blade of clip extracts DNA, PCR and detects transgenic line.

(4) GUS staining analysis

After results to the seed of CDKG2 promotor+GUS fusion gene conversion, respectively GUS dyeing is carried out to histoorgans such as seed, seedling, inflorescences, analyze CDKG2 promotor tissue specific expression situation.First, normal seed germination process is analyzed.Result can find, pCDKG2 is at dry seeds high expression level, once infiltrate, expression amount obviously declines.After seed starts sprouting, its expression is confined to radicle tip, and along with radicle extends, its expression amount declines to some extent (see Fig. 1-I, II, III, IV).Carry out GUS dyeing to seedling, coloration result, pCDKG2 is at root, shoot apical meristem, and microtubule tissue, side root generating area etc. are high expression level (see Fig. 1-V, VI, VII, VIII) locally.Flower tissue and fruit pod coloration result are learnt, pCDKG2 expression characteristic is: mature tissue's expression amount comparatively tender tissue expression amount is wanted high (as pollen, petal, anthocaulus and androphore etc.), at column cap, mature pollen, androphore, microtubule tissue local expression amount higher (see Fig. 1-Ⅸ, Ⅹ, Ⅺ, Ⅻ).

The transgenosis application of embodiment 3 arabidopsis cell cyclin dependent protein kinase gene CDKG2

The structure of 1.pSTART-CDKG2 expression vector

By AtCDKG2-pEASY-Blunt plasmid enzyme restriction correct for order-checking, pSTART carrier after the object fragment be recovered to being cut with enzyme is connected, transformation of E. coli DH5 α, that resistance culture base of card screens positive bacteria and falls, and drops into performing PCR and double digestion checking to positive bacteria.

2. obtain transfer-gen plant

Extract the pSTART-CDKG2 plasmid built, transformation Agrobacterium EHA105, PCR detect positive bacterium colony, by positive bacterium colony enlarged culturing, and transform Col-0 wildtype Arabidopsis thaliana.Collect T1 for seed, sow on the MS substratum containing 50 those resistances of μ g/mL card, cultivate observation in about ten days, the positive plant with resistance can grow green true leaves, and the not long true leaf of negative seedling of unsuccessful conversion or true leaf present yellow.Move in Nutrition Soil by positive seedling, after cultivating two weeks, a little blade of clip extracts DNA, utilizes PCR to detect.Screening is continued, until obtain transgenic homozygous strain after identifying positive seedling.

3. transfer-gen plant Molecular Identification

The Transgenic wheat line obtained is carried out to the detection of gene expression dose.First, extract the total serum IgE of transfer-gen plant and WT lines, utilize RT-PCR method, analyze the CDKG2 gene expression difference of process LAN transfer-gen plant and WT lines.Screening CDKG2 expression amount carries out apparently higher than the process LAN transgenic line of WT lines Physiological Traits analysis of blooming.

The functional verification of 4.CDKG2 gene

(1) cross table strain OE1 and OE2 to mutant cdkg2-1 (SALK_012428) and cdkg2-2 (SALK_090262) and transgenosis to bloom character analysis

First be taped against on 1/2MS substratum by the mutant of surface sterilization, process LAN and wild-type and empty vector control seed, 4 DEG C of dark vernalization are put into greenhouse and continue to cultivate after 3 days.Selecting the consistent seedling of size growing way after 2 weeks moves on in soil, in short day hot-house culture, observes lobe numbers during their flowering time and bolting.

Under short day condition, the flowering time of mutant cdkg2-1 and cdkg2-2 is obviously early than wild type control, and the bolting time of mutant is 22 days, and the bolting time of wild type control is about 27 days (see Fig. 2-A).Correspondingly, during bolting the lotus throne number of sheets order of mutant also than few about 3 of the lotus throne number of sheets order of wild-type.The flowering time of CDKG2 process LAN strain is more late than the flowering time of empty vector control (see Fig. 2-B).Under long-day conditions, mutant morning floral formation and process LAN late floral formation with show under short day condition consistent.This illustrates that arabidopsis cell cyclin dependent protein kinase gene CDKG2 participates in blooming suppression approach, and CDKG2 is not by Photoperiod to the restraining effect of blooming.

(2) analyze the expression of a series of genes involved of blooming, determine the effect of DCKG2 gene in the conversion of plant florescence further.

The approach of blooming of plant mainly contains four, Photoperiod pathway, vernalization approach, gibberellin pathway and autonomous pathway.We experimentally results presumption CDKG2 mainly carried out the flowering time of regulating plant by autonomous pathway.In order to further clear and definite CDKG2 suppresses the molecule mechanism of flowering of plant, we have studied the expression of autonomous pathway genes involved in mutant by real-time quantitative PCR.The over-ground part that we choose the wild-type after bolting and mutant Arabidopis thaliana is experiment material, utilizes TRIzol test kit to extract Arabidopis thaliana RNA, and with the cDNA of reverse transcription acquisition for template carries out Realtime-PCR.

Found that, the expression of suppressor gene FLC of blooming in mutant reduces, and is arranged in the gene of suppressor gene FLC upstream of blooming: FCA, FY, FPA, LD, FLD and FLK etc. then obviously do not distinguish (see Fig. 3-A) at the expression amount of wild-type and mutant in approach of independently blooming.Approach of blooming conformity gene FT and the expression amount of floral meristem specific gene AP1 and LFY in mutant are higher than wild-type (see Fig. 3-B).Real-time quantitative PCR result shows, cell cycle dependant PROTEIN C DKG2 participates in independently blooming approach also by the expression of regulation and control FLC really, have impact on the expression of bloom approach conformity gene and floral meristem Te Jiyin, and then suppresses flowering of plant.

Based on above-mentioned experiment, confirm that CDKG2 is really a supressor of blooming, GUS dyeing finds that this gene has expression in flower tissue and fruit pod, imply that CDKG2 may play certain effect in the process of blooming of plant.Further experiment finds when causing plant Blooming after CDKG2 gene generation deletion mutantion, and that is CDKG2 plays negative regulation effect in the florescence conversion of plant.Real-time quantitative PCR result shows, CDKG2 regulates and controls the expression of FLC by the approach that participates in independently blooming, and then have impact on the expression of bloom approach conformity gene and floral meristem Te Jiyin, suppresses flowering of plant.We utilize transgenic technology high expression level CDKG2 gene in plant subsequently, and the flowering time observing process LAN plant finds, CDKG2 process LAN pusher flowering of plant late.The flowering time of plant and its Yield and qualities closely related, application of the present invention is expected to the expression by this gene, shorten or increase the time of vegetation growth of plant, for accelerating breeding or improving crop growth amount, offer help with the object reaching its high yield and high quality and support.

Claims (3)

1. the application of arabidopsis cell cyclin dependent protein kinase gene AtCDKG2 in the conversion of regulating plant florescence.

2. application according to claim 1, is characterized in that: described plant is cress.

3. application according to claim 2, is characterized in that: described cress is Arabidopis thaliana, rape or Chinese cabbage.