CN108148821A - A kind of regulation and control cucumber roundleaf character-related protein and its encoding gene and application - Google Patents

Abstract

The invention discloses a kind of regulation and control cucumber roundleaf character-related protein and its encoding gene and application, the amino acid sequence such as sequence table SEQ ID NO of the albumen：Shown in 2, the protein kinase of PID gene code serine/threonines belongs to agc kinase family, the encoding gene such as sequence table SEQ ID NO of the albumen：Base sequence shown in 1, the base sequence is replaced in the guanine (G) of 1092bp by adenine (A), the Val (valine) of 346 of the amino acid residue sequence of coding sports Met (methionine), the albumen inhibits Polar Transport of Auxin, lead to Auxin transport polarity defect, specific plant phenotype is leaf edge-smoothing, and sawtooth protrudes unobvious, and blade is in subcircular；Main root is undeveloped, and main side root does not have significant difference；Male and female flowering is without calyx, female flower infertility etc..The discovery of the gene and albumen can be improved for cucumber leaf type, yield increases and ideotype is cultivated and the research of auxin signal path molecular regulation mechanism provides basis.

Description

A kind of regulation and control cucumber roundleaf character-related protein and its encoding gene and application

Technical field

The present invention relates to genetic engineerings and the albumen and gene of cucumber, belong to molecular genetic breeding field, and in particular to one Kind regulation and control cucumber roundleaf character-related protein and its encoding gene and application.

Background technology

Cucumber (Cucumis sativus are L.2n=2x=14) the category annual sprawling herbs plant of Curcurbitaceae Cucumis, one As blade split in palmate 3-5, sliver triangle simultaneously has sawtooth.With the development of genomics, mutant material becomes research base Because function is direct, one of effective approach.In the cucumber mutant library built with EMS, a list that can stablize heredity is found Gene controls cucumber roundleaf mutant.Mutant shows blade edge smoothly in subcircular, no calyx, female flower infertility, cotyledon Heterauxesis and root anormogenesis isophenous.Blade is the major organs and vegetal inspired of photosynthesis and respiration One of important indicator that state is built up, having on the growth and development of plant greatly influences.It is leaf greatly to affect plant type, light Area, yield and commodity property etc. are closed, leaf margin shape largely maintains leaf diversity, and the development of floral organ is not Can only influence plant breeding and it is also closely related with the size of cucumber yield.The leaf molecular regulation mechanism of cucumber is still at present Unknown, floral organ research is concentrated mainly on sex determining gene, and the research for forming controlling gene is less, therefore, the mutant It is the ideal material for studying leaf cucumber and development of floral organs controlling gene and its regulatory mechanism.With new-generation sequencing technology Development, using improved MutMap methods to roundleaf mutant and wild type F₂Mixed pond sequencing is carried out for group, in No.1 chromosome The upper section for just navigating to 19.1Mb a to 22.0Mb, by EMS mutagenesis principle (G → A and C → T) and Δ SNP indexes >= 0.8, it is identified to obtain SNP, a SNP-1G19389144, on second extron of gene C sa1M537400, draw The amino acid sequence for playing coding albumen conserved domain changes, the final gene for determining regulation and control cucumber roundleaf mutant Csa1M537400 is named as CsPID.

Blade is the first kind lateral organ that shoot apical meristem generates (including cotyledon), is had in development of plants important Status.A large amount of evidences show leaf development by internal genetic mechanism and the dual regulation of the vitro factor.Plant hormone, especially It is that auxin plays indispensable effect in inside and outside regulation mechanism is coordinated.The stable state regulation and control of auxin, polar translocation The overall process of leaf development is influenced with signal transduction.Auximone takes part in many lifes as a kind of important plant hormone Manage the adjusting and control of biochemical process.The formation of elongation growth and geotropism, adventitious root such as root, the differentiation of vascular tissue, top End advantage, cambial cell division, the elongation growth of cell, plant tropism etc..The polar translocation of auxin and being permitted for plant More growth and development processes are closely related, but still not comprehensive to the understanding of its regulatory mechanism.Research shows that auxin is to plant growth The adjusting of development depends on the auxin concentration gradient of polar translocation formation.The reversible phosphorylation activity of PIN albumen is exchanged Section Polar Transport of Auxin plays key effect, and this process need serine/threonine protein kitase PINOID (PID) and The common participation of PP2A phosphorylases.Evidence suggests PID is to be given birth to by adjusting the subcellular localization of PIN albumen come control polarity The transhipment of long element, CsPID abnormal expressions may change auxin steady-state level cause Polar Transport of Auxin major defect from And lead to the change of cucumber leaves shape.

The protein kinase (protein-serine/threonine kinase) of PID gene code serine/threonines, Belong to agc kinase family, be present in cotyledon former base, blade, flower tissue and the nearly cardinal extremity of organ or separate living tissue developed In micro-pipe tissue (Benjamins et al., 2001).((PID) plays the positive tune of Polar Transport of Auxin to protein kinase PINOID Control agent effect (Friml et al., 2004).AtPID, OsPID overexpression can generate and the increased correlation table of Auxin transport Type, it may be possible to the positive regulatory factor (Morita and Kyozuka, 2007) of Polar Transport of Auxin.PID participates in auxin Polar translocation and signal transduction, afunction mutant is combined with other multiple auxin related mutants leads to embryo and children The cotyledon missing of seedling.Arabidopsis insertion mutation body pid：：En197 and pid：：En310 show flower generally comprise it is little or no Sepal, gynoecium is small and does not generate or only generates seldom seed, about 50% cotyledon of mutant seedlings display exception etc. Phenotype, (Benjamins et al., 2001) similar to this research cucumber roundleaf mutation type surface.Auxin is sent out in blade at present Function during educating, enable we get a glimpse of auxin be how to regulate and control certain organs development and certain organs be again How the regulation and control of specific hormone are responded.With going deep into for research, one increasingly complex and clearly leaf development and morphogenesis Regulated and control network will be revealed.These results improve cucumber blade profile, yield increases and ideotype nurturing research also has weight The meaning wanted.

Invention content

The technical problems to be solved by the invention are to provide a kind of regulation and control cucumber roundleaf character-related protein and its coding base Cause and application.The protein kinase of PID gene code serine/threonines, belongs to agc kinase family, in the feelings of amino acid substitution Under condition, regulate and control Polar Transport of Auxin, mutant causes Polar Transport of Auxin defect, shows leaf edge-smoothing, sawtooth is not Apparent prominent, blade is in subcircular；Generally there is three pieces cotyledon in cotyledon heterauxesis；Main root is undeveloped, and main side root is not bright Aobvious difference；Male and female flowering is without calyx, female flower infertility isophenous.Can be the improvement of cucumber leaf type, yield increases and ideotype training It educates and creates conditions with the research of auxin signal path molecular regulation mechanism.

Technical solution is used by the present invention solves above-mentioned technical problem：Navigate to a cucumber roundleaf character correlation egg In vain, CsPID is named as, from cucumber, CsPID is following albumen a) or b)：

A) by SEQ ID NO in sequence table：The protein of amino acid sequence composition shown in 2；

B) in sequence table SEQ ID NO：In amino acid sequence shown in 2 by substitution one amino acid and with cucumber roundleaf It is mutated the relevant protein as derived from a).

Amino acid sequence is made of 474 amino acid residues, belongs to agc kinase family, regulates and controls Polar Transport of Auxin, is dashed forward Variant causes Polar Transport of Auxin defect, and mutant shows roundleaf without calyx, female flower infertility, cotyledon heterauxesis and Root anormogenesis isophenous.It is above-mentioned b) in CsPID can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological table It reaches.It is above-mentioned b) in the encoding gene of CsPID can be by by SEQ ID NO in sequence table：In DNA sequence dna shown in 1 Carry out the nonsynonymous mutation of a base-pair.

The gene for encoding the CsPID also belongs to protection scope of the present invention.

CsPID genes particularly may be divided into gene 1) or 2) or 3)：

1) its nucleotide sequence is SEQ ID NO in sequence table：DNA molecular shown in 1；

It 2) under strict conditions can be with sequence table SEQ ID NO：1 DNA sequence dna limited hybridizes and encodes above-mentioned and cucumber The DNA molecular of roundleaf character-related protein；

3) there is more than 90% homology with gene 1), and encode the above-mentioned DNA with cucumber roundleaf character-related protein Molecule.

Sequence is by 1452 base compositions, and from 5 end, 1-3 are initiation codon, and coding has SEQ ID in sequence table NO：The protein of amino acid residue sequence shown in 2；The amino acid sequence is PID protein kinases, has 2 extrons and 1 Introne.The guanine (G) of the 1092bp positions script of gene base sequence is replaced by adenine (A), leads to V (the figured silk fabrics ammonia of coding Acid) sport M (methionine).

Recombinant vector, transgenic cell line and recombinant bacterium containing CsPID genes also belong to protection scope of the present invention.

The advantage of the invention is that using the method positioning regulation and control cucumber roundleaf of improved MutMap without calyx mutant Gene, it was found that a kind of leaf GAP-associated protein GAP of regulation and control cucumber and its encoding gene, amino acid sequence such as 2 institute of sequence table of the albumen Show, belong to agc kinase family, it is related to Polar Transport of Auxin；The encoding gene of the albumen such as sequence table SEQ ID NO：1 institute The base sequence shown, the base sequence are replaced in the guanine (G) of 1092bp by adenine (A), the amino acid residue of coding The Val (valine) of 346 of sequence sports Met (methionine), which inhibits Polar Transport of Auxin, cause to grow Element transport polarity defect, specific plant phenotype is leaf edge-smoothing, and middle part does not significantly protrude, and sawtooth is neat, and blade is near It is round；Main root is undeveloped, and main side root does not have significant difference；Male and female flowering is without calyx, female flower infertility etc..The hair of the gene and its albumen It is existing, can be the improvement of cucumber leaf type, yield increases and ideotype is cultivated and auxin signal path molecular regulation mechanism Research provides basis.

Description of the drawings

Cucumber roundleaf mutant rl and wild type WT phenotype comparison diagrams.Fig. 1 is maturity period plant type phenotype, and the left side is wild type WT, the right show whole strain roundleaf phenotype for mutant rl.Blade profile phenotype when Fig. 2 is a leaf phase, the left side is shown for wild type Palmate is shallow to be split, and the right shows subcircular for mutant；Fig. 3 shows cotyledon asymmetry for mutant；Fig. 4 is spent for female flower The developmental state of calyx, Fig. 5 are the developmental state of male flower calyx, compare left side wild type WT, the right mutant rl shows nothing Calyx phenomenon；Fig. 6 main roots and lateral root development situation, the left side are mutant rl, and the right side is wild type WT；

Specific embodiment

The present invention is described in further detail below in conjunction with attached drawing, embodiment.

The phenotype of 1 mutant of embodiment and genetic analysis

A cucumber is screened from the Chang Chun Mi Ci mutant library of EMS (ethyl methane sulfonate) mutagenesis Roundleaf mutant rl.The mutant leaf development initial stage just shows circle, and female flower and male flower are all without calyx, female flower infertility, cotyledon Heterauxesis (three pieces cotyledon is most common phenotype) and root anormogenesis isophenous.

Using mutant r1 as male parent, hybridize with wild type Chang Chun Mi Ci, obtain first filial generation F1 plant, all F1 offsprings have There is wild type phenotype.Since homozygous r1 mutant plants are female steriles, statistics is from r1 mutant and WT wild types The wild-type plant (RLRL or RLr1) in F2 generations of intermolecular hybrid and mutant plant (rlr1), in the F2 groups containing 78 plants of plants Only have 24 plants of plant performances to go out roundleaf phenotype, 3: 1 (χ of segregation ratio in body²=0.19, P=0.66), show the mutant by list Recessive gene regulates and controls, and it is CsPID to name the gene.

The positioning of 2 CsPID genes of embodiment

- CTAB methods extraction roundleaf the mutant of F2 segregating populations and wild-type leaves DNA according to a conventional method.DNA mixes pond use Sonicator is crushed to 150bp~250bp, to the small fragment built based on 2500 microarray datasets of Illumina HiSEQ DNA library carries out full-length genome and resurveys sequence.After removing low-quality reads by quality control, BWA (versions are utilized： ) and SAMtools software (versions 0.5.9rc1：0.1.19) by data and Cucumber germplasm sequence (" 9930 " reference gene group) It is compared, by SNP numbers divided by the sequencing depth calculation allele index in the site.It is filtered out in two mixed ponds identical SNP, by corresponding allele index subtraction calculations Δ allele index.It is wild to 1 plant at random using same method Type Chang Chun Mi Ci single plant carries out high-flux sequence, obtains the SNP site between wild type Chang Chun Mi Ci and 9930, makes wild type The SNP index maps in pond, these differences are mainly as caused by reference sequences sequencing mistake.Pond, which is mixed, in wild type filters out allele The SNP of index >=0.9 removes mutant and mixes the identical SNP of SNP that Chi Zhongyu is filtered out, and SNP index in these SNP≤ 0.3 or SNP of the sequencing depth less than or equal to 7 is considered incredible.In reliable SNP site, using 1Mb as window, 10kb is moved for step and is drawn line chart using SNPindex calculating SNPindices, and the highest regions of SNP indices are candidate The region of gene.Theoretically, 1 should be equal to by causing the mutational site SNP indexes of mutant character, as homozygous site；Due to losing Pass it is chain, near SNP site index should be equal or close to 1.The SNP positions of cluster distribution are found in first localization region Point, further according to the mutation that EMS mutagenesis generally produces, i.e. G → A or C → T mutation have found altogether 27 SNP sites.27 25 SNPs in SNPs are located at intergenic region or include sub-district, and only 2 SNPs are corresponding with code area (CDS) area.Wherein SNP (SNP-1G19389144) is a nonsynonymous mutation for being happened at protein coding region, and the guanine (G) of script is fast by gland Purine (A) replaces, and leads to the V (valine) of coding to the mutation of M (methionine), so that it is determined that leading mutagenic candidate gene CsPID。

3 CsPID gene clonings of embodiment and identification

CsPID gene mutations are located at second exons 1 092bp of Csa1M537400, and bases G sports A.For Verification sequencing as a result, to cDNA that number in Cucurbit Genomics Database is Csa1M537400 genes into Row sequencing, it was demonstrated that the guanine (G) of code area cDNA scripts sports adenine (A), and the V (valine) of coding is caused to sport M (methionine), is consistent with sequencing result, it was demonstrated that the mutation of the gene leads to generate roundleaf without calyx mutant, names the base Because CsPID.

The acquisition of CsPID full length genes cDNA：

The extraction of cucumber Chang Chun Mi Ci and roundleaf total serum IgE uses TRIzol methods, and DNA digestion is carried out under the conditions of 25 DEG C 30 minutes, it was the reverse transcription reagent box PrimeScript that template uses TaKaRa companies that 2mg is drawn from total serum IgE^TM RT Reagent Kit synthesize the first chain cDNA.Using this cDNA as template, with primer Csa1M537400-F (5 '- ATGTTACAACACCTTCCCCCAGA-3 ') and primer Csa1M537400-R (5 '-GAAATAATCGAACCCGGTCGACT-3 ') Using the high-fidelity enzyme of TaKaRa companiesGXL DNA polymerase carry out pcr amplification reaction, reaction condition It is as follows：

Reaction volume 26ul, wherein containing：

26ul volumes are complemented to distilled water.

Response procedures are as follows：It 95 DEG C, is denaturalized 5 minutes, is then denaturalized 30 seconds for 95 DEG C, 57.8 DEG C are annealed 30 seconds, 72 DEG C of extensions 90 seconds, expand 35 cycles；Finally extend 10 minutes at 72 DEG C.

Amplified production is purified with the DNA gel QIAquick Gel Extraction Kit of OMEGA companies by product description, is then purified Product is carried out with rTaq enzymes (TaKaRa) plus A reactions, reaction condition are as follows：

Reaction volume 10ul, wherein containing

Response procedures：72 DEG C connect 10 minutes.

The product after A will be added to be connected 4 hours at 16 DEG C with pMD19-T carriers (TaKaRa), build recombinant vector pMD19-CsPID.Recombinant vector pMD19-CsPID is converted bacillus coli DH 5 alpha (TaKaRa), conversion product in 70 seconds by 42 DEG C of thermal shocks It is grown on the LB plating mediums containing ampicillin, picked clones, extracts plasmid, deliver sequencing.Sequencing result shows The nucleotide sequence of the segment expanded such as sequence table SEQ ID NO：Shown in 1, which is named as CsPID genes, CsPID Full length gene 1425bp, the amino acid sequence of coding are shown in sequence SEQ ID NO in sequence table：Shown in 2.

Bibliography：

[1] Benjamins, R., Quint, A., Weijers, D., Hooykaas, P., and Offringa, R. (2001) The PINOID protein kinase regulates organ development in Arabidopsis by enhancing polar auxin transport.Development 128：4057-4067.

[2] Friml, J., Yang, X., Michniewicz, M., Weijers, D., Quint, A., Tietz, O., et al.(2004)A PINOID-dependent binary switch in apical-basal PIN polar targeting directs auxin efflux.Science 306：862-865.

[3] Morita, Y., and Kyozuka, J. (2007) Characterization of OsPID, the rice Ortholog of PINOID, and its possible involvement in the control of polar auxin transport.Plant Cell Physiol.48：540-549.

Claims (5)

1. a kind of cucumber roundleaf GAP-associated protein GAP, it is characterized in that following albumen a) or b)：

A) by SEQ ID NO in sequence table：The protein of amino acid sequence composition shown in 2；

B) the SEQ ID NO in sequence table：In amino acid sequence shown in 2 by substitution one amino acid and with the leaf phase of cucumber The protein as derived from a) closed.

2. a kind of coding gene of cucumber roundleaf GAP-associated protein GAP described in claim 1, it is characterized in that the gene be it is following 1) Or 2) gene or 3)：

1) SEQ ID NO in its nucleotide sequence such as sequence table：DNA molecular shown in 1；

It 2) under strict conditions can be with sequence table SEQ ID NO：Nucleotide sequence hybridization and the coding roundleaf shown in 1 is related The DNA molecular of albumen；

3) there is more than 90% homology with gene 1), and encode the DNA molecular of above-mentioned roundleaf GAP-associated protein GAP.

3. the recombinant expression carrier containing the gene described in claim 3.

4. transgenic cell line or recombinant bacterium containing the gene described in claim 3.

5. the cucumber roundleaf phase character described in claim 2 closes application of the encoding gene of albumen in change cucumber is leaf.