CN104789578B - Cotton glycosyltransferase gene GhUGT73C6 and its application in plant plant type is regulated and controled - Google Patents

Abstract

The invention discloses a kind of applications of land cotton glycosyltransferase gene GhUGT73C6 in plant plant type is regulated and controled, wherein described glycosyltransferase gene GhUGT73C6 nucleotide sequence is as shown in SEQ ID NO.2.Specific steps of the present invention include：A, GhUGT73C6 full length genes cDNA clone；B, overexpression vector pCAMBIA2301 CaMV35S GhUGT73C6 structure；C, functional verification is carried out by arabidopsis thaliana transformation, it was demonstrated that the GhUGT73C6 genes can make plant have the small phenotype of dwarfing, leaf area.The present invention has important value for the breeding wheat for semidwarfness of plant.

Description

Cotton glycosyltransferase gene GhUGT73C6 and its in plant plant type is regulated and controled Using

Technical field

The invention belongs to technical field of biological genetic engineering, is that cotton glycosyltransferase gene GhUGT73C6 plants in regulation and control Application in thing plant type.

Background technology

Cotton is important industrial crops, but in recent years due to the shortage of rural laborer, sown areas of cotton are significantly Decline, DEVELOPMENT OF COTTON entire mechanization turns into the inevitable direction of current Cotton Production, therefore cultivates the cotton strain of suitable for mechanization Type is wherein importance.It is relatively difficult that the phenotype of dwarfing is obtained by traditional breeding technique, and conventional breeding methods pair The cotton progress Genetic improvement cycle is long, effect is low.Recently as the rapid development of Protocols in Molecular Biology, transgenic technology into For a kind of good selection.

Brassinosteroid is a kind of important plant hormone, and the brassinosteroid with bioactivity is sent out in the growth of plant Educate and play an important role, it can promote the sprouting of seed, the elongation of stem root leaf, trichome development, pollen tube growth, Hua Heguo Real development etc..And study and show that the activity of brassinosteroid in plant is regulated and controled by multiple genes, wherein glycosyl shifts Some genes of enzyme family can inactivate brassinosteroid.Research to arabidopsis shows, in glycosyl transferase family C1, C2, C3, C4 can not inactivate brassinosteroid in 6 genes of UGT73C subfamilies, and C5, C6 can make in rape element Ester inactivates (Husar S, Berthiller F, Fujioka S, et al.Overexpression of the UGT73C6alters brassinosteroid glucoside formation in Arabidopsis thaliana,BMC Plant Biology,2011,11:51.doi:10.1186/1471-2229-11-51).Illustrate glycosyl transferase UGT73C Asias The function of family gene has differences.

Therefore the present invention is according to AtUGT73C6 (NM_129234.2) base that in arabidopsis brassinosteroid can inactivate Cause, compared in cotton redmond this cotton gene group and obtain the forecasting sequence of UGT73C6 genes, the amino acid of the predicted gene Sequence is disclosed in NCBI (KJB13655.1), but passes through retrieval, cotton glycosyltransferase gene GhUGT73C6 function And UGT73C6 gene orders are not reported in upland cotton.

The present invention obtains GhUGT73C6 genes in reviving cotton 18 from upland cotton cotton variety, and it is sub- with UGT73C in upland cotton GhUGT73C14 genes (JX846921.1) amino acid sequence similarity announced in family is less than 80%, and research is thought GhUGT73C14 genes participate in activity regulation (Gilbert MK, Bland JM, Shockey JM, the et al., A of abscisic acid transcript profiling approach reveals an abscisic acid-specific glycosyltransferase(UGT73C14)induced in developing fiber of Ligon lintless- 2mutant of cotton(Gossypium hirsutum L.).Plos One,2013,8(9):e75268).By turning base Because technology makes GhUGT73C6 genes overexpression in arabidopsis, the GhUGT73C6 gene plants that turn of acquisition have dwarfing, leaf The small phenotype of area.So the breeding wheat for semidwarfness that the clone of cotton GhUGT73C6 genes and gene function checking can be cotton carries Genetic resources is supplied.

The content of the invention

Cotton AA, DD diploid and the completion of AADD tetraploid genome sequencings, very polygenic forecasting sequence is obtained, But gene function and be not known.Therefore it is an object of the invention to provide a kind of cotton glycosyltransferase gene GhUGT73C6 to exist Regulate and control the application in plant plant type, the gene cDNA nucleotide sequence row are as shown in SEQ ID NO.2.

The protein of the cotton glycosyltransferase gene GhUGT73C6 gene codes of described regulation and control plant plant type, has Amino acid sequence in sequence table described in SEQ ID NO.3.

The invention provides the plant expression vector containing above-mentioned cotton glycosyltransferase gene GhUGT73C6.Will GhUGT73C6 gene clonings obtain pCAMBIA2301-CaMV35S-GhUGT73C6 to pCAMBIA2301.

Applications of the gene GhUGT73C6 of the present invention in dwarf plant is cultivated.Specifically GhUGT73C6 genes are led to Plant expression vector is crossed to be transferred in purpose plant.The plant is model plant arabidopsis.

Beneficial effects of the present invention：Using existing plant gene engineering technology, using electronic cloning and RT-PCR technology, Separation and identification cotton dwarf-related gene sequence information, and gene is transferred to by arabidopsis by Agrobacterium tumefaciems flower-dipping method, reflect Surely prove that the plant height of transfer-gen plant is significantly less than wild type (Fig. 6) significantly lower than wild type, blade, illustrate GhUGT73C6 bases Because regulation and control plant plant type has positive effect.

Brief description of the drawings

The amplification of Fig. 1 GhUGT73C6 full length gene cDNA sequences.

M：DL2000DNA marker, 1,2 be GhUGT73C6 full length gene cDNA PCR amplifications.

Fig. 2 and other UGT73C subfamily genes amino acid alignment.

Fig. 3 plant expression vectors pCAMBIA2301-CaMV35S-GhUGT73C6 structure.

(a) Escherichia coli pCAMBIA2301-CaMV35S-GhUGT73C6PCR detects electrophoresis result；

M：DL2000DNA marker, 1,2,3,4,5 be Escherichia coli pCAMBIA2301-CaMV35S-GhUGT73C6 bacterium Liquid PCR results ,+it is GhUGT73C6 gene PCR results ,-it is negative control.

(b) PCR of pCAMBIA2301-CaMV35S-GhUGT73C6 plasmids, digestion identification.

M：DL2000DNA marker, 1 is blank, and 2 be the PCR of pCAMBIA2301-CaMV35S-GhUGT73C6 plasmids As a result, 3 be pCAMBIA2301-CaMV35S-GhUGT73C6 plasmids digestion result.

The PCR identifications of Fig. 4 transfer-gen plants.

M：DL2000DNA marker；1st, 2,3 be transfer-gen plant."+" is pCAMBIA2301--CaMV35S- GhUGT73C6 plasmids, "-" are negative control.

The RT-PCR identifications of Fig. 5 transfer-gen plants.

M：DL2000DNA marker；WT is wildtype Arabidopsis thaliana, and 2,3 be to turn GhUGT73C6 gene plants 2,3.

Fig. 6 transgenic arabidopsis is compared with wildtype Arabidopsis thaliana plant type.

(a) florescence plant type；(b) maturity period plant type；(c) blade.

Transfer-gen plant plant height is significantly lower than wild type, and blade is significantly less than wild type.

The plant height of Fig. 7 transgenic arabidopsis strain and wildtype Arabidopsis thaliana.

WT is wildtype Arabidopsis thaliana, and GhUGT73C6-2 is transgenic line 2, and GhUGT73C6-3 is transgenic line 3.

Embodiment

The acquisition of embodiment 1, GhUGT73C6 genes

1.1 RNA extraction

(1) leaf tissue of the fresh upland cotton cotton variety Soviet Union cottons 18 of 0.5g is taken, adds 0.1g polyvinyl pyrrolidones (PVPP), be fully ground in liquid nitrogen to powder, by agar end be transferred in 10ml centrifuge tubes rapidly, add 5ml CTAB extract solutions and 500 μ l0.1M pH8.0 Tris-HCl, 65 DEG C of water-bath 20min, midway upset mix；

(2) plus isometric chloroform fully mixes, and ice bath stands 10min；

(3) 4 DEG C, 10000rpm centrifugations 20min.It is sub-packed in 4 1.5ml centrifuge tubes；

(4) clear, the 8M LiCl mixings of 1/3 volume of addition are sucted, -70 DEG C of 1h or -20 DEG C are stayed overnight；

(5) 4 DEG C, 10000rpm centrifugations 20min.Supernatant is abandoned, after 70% ethanol washes twice, drying is precipitated and dissolved in 30 μ l DEPC water；

(6) DNase and 25U RNase Inhabitor, 10 × buffer digestion 30mins of the 10U without RNase activity is added Afterwards plus isometric chloroform, extracting is once；

(7) supernatant is transferred in new pipe, adds the 3M pH 5.2NaAc and isometric isopropanol or 2.5 times of 1/10 volume The absolute ethyl alcohol of volume, -20 DEG C stand overnight or -70 DEG C of ice bath 3h；

(8) 4 DEG C, 10000rpm centrifugation 20min, supernatant is abandoned, 70% ethanol is dissolved in 30 μ l DEPC water after washing twice. Produce cotton RNA.

1.2 cDNA synthesis

System:

The clone of 1.3 GhUGT73C6 genes

According to arabidopsis UGT73C6 genes (NM_129234.2), Blast is compared in cotton redmond this cotton genome The UGT73C6 predictive genes sequence into cotton, its nucleotide sequence is as shown in SEQ ID NO.1.Upstream is designed in ORF sequences both sides - the CTCATATTCCTATGGGTT-3 ' of primer 5 ' (the SEQ ID NO.4) and-GACGTTATCTCGATTTTC-3 ' of anti-sense primer 5 ' (SEQ ID NO.5), enter performing PCR, PCR reaction systems (25 μ l) with the cDNA (being synthesized by step 1.2) of upland cotton Soviet Union cotton 18：10 ×EasyPfu buffer 2.5μl,50mM MgSO₄The μ l of 0.5 μ l, 2.5mM dNTP 2, the μ l of primer 1 (10 μM) 1, primer 2 (10 μM) 1 μ l, ddH₂The μ l of 16.5 1 μ l, EasyPfu DNA Polymerase of μ l, cDNA of O 0.5.PCR amplification programs：94℃ 3min；94 DEG C of 30sec, 48 DEG C of 45sec, 72 DEG C of 2min, 36 circulations；72℃10min；4 DEG C of insulations.

The PCR primer that recovery amplification obtains, is cloned into pEASY-T1 carriers (Beijing Quanshijin Biotechnology Co., Ltd), By PCR screening positive clones, the handsome company's sequencing in Shanghai is delivered to.Isolated upland cotton unnamed gene is by we GhUGT73C6, ORF total length 1494bp (SEQ ID NO.2) (Fig. 1), the amino acid sequence of coding is as shown in SEQ ID NO.3. The GhUGT73C6 genes that are obtained in upland cotton and the gene order similarity 99% predicted in redmond this cotton genome.Will Obtain GhUGT73C6 genes and acquired UGT73C Asias man in acquired GhUGT73C14 in cotton and other crops Gene (AtUGT73C6, AtUGT73C5 in arabidopsis, Tcglu1, TcUGT73C5 in cocoa, the orange of race CsUGT73C1, plum PmUGT73C1) discovery is compared, GhUGT73C6 gene amino acid sequences are sub- with other crop UGT73C The uniformity of the gene amino acid sequence of family is below 80% (Fig. 2).

The structure of embodiment 2, pCAMBIA2301-CaMV35S-GhUGT73C6 plant expression vectors

With the primer GhUGT73C6-KpnI containing restriction enzyme site：5’-CTCATATT CCTATGGGTT-3 ' (SEQ ID NO.6) and GhUGT73C6-XbaI:5'-GACG TTATCTCGATTTTC-3'(SEQ ID NO.7) enter performing PCR amplification, agarose gel electrophoresis is detected whether containing expected fragment. PCR response procedures are as follows：94℃5min；94 DEG C of 30sec, 48 DEG C of 45sec, 72 DEG C of 1min 30sec, 36 circulations；72℃ 10min；4 DEG C of insulations.Obtain the GhUGT73C6PCR products containing restriction enzyme site.

From Kpn I and Xba I respectively to plant expression vector pCAMBIA2301-CaMV35S plasmids (this laboratory preserve, Zheng Qing, the research [D] of resisting I-type Diabetes Gene transformation of tobacco, tomato, Yangzhou University's Master degree candidate's thesis, 2010) and GhUGT73C6PCR products containing restriction enzyme site carry out double digestion.

PCAMBIA2301-CaMV35S plasmid double digestion systems are as follows：

GhUGT73C6PCR product double digestion systems containing restriction enzyme site：

In 37 DEG C of digestions, reaction time >=3h.Electrophoresis detection is carried out to double digestion product with Ago-Gel, as a result sees figure 3(b).PCAMBIA2301-CaMV35S carriers large fragment and target gene fragment are reclaimed, large intestine is converted after being connected with T4 ligases Bacillus Trans1-T1 competent cells (are purchased from Beijing Quanshijin Biotechnology Co., Ltd), and band is obtained after identifying recon The plant expression vector of purposeful gene.

The coupled reaction system for the pCAMBIA2301 large fragments that gene obtains with digestion：

16 DEG C of connections are overnight.

Connection product converts competent escherichia coli cell, 37 DEG C of vibrations in the LB fluid nutrient mediums containing kan100mg/L After cultivating 1h.It is evenly coated on kan100mg/L LB solid mediums, 37 DEG C of overnight incubations.

Picking single bacterium colony is inoculated in 37 DEG C of shaken cultivations in LB fluid nutrient mediums of the 1.5ml containing kanamycins, uses GhUGT73C6 gene primer GhUGT73C6-Kpn I (SEQ ID NO.6) and GhUGT73C6-Xba I (SEQ ID NO.7) enter Performing PCR amplification identification, is as a result shown in Fig. 3 (a).

With the bacterium solution plasmid of AXYGEN plasmid extraction kits extraction PCR test positive, choose Xba I and the enzymes of Kpn I enter Row double digestion, agarose gel electrophoresis detect Fig. 3 (b).The recombinant expression carrier of acquisition is named as pCAMBIA2301- CaMV35S-GhUGT73C6。

The preparation and conversion of embodiment 3, Agrobacterium competence

The preparation of 3.1 Agrobacterium EHA105 competence

(1) picking EHA105 single bacterium colonies, it is inoculated in 5ml LB fluid nutrient mediums, 28 DEG C, 200rpm concussion and cultivates are stayed overnight It is 0.4 to OD600 values；

(2) with 1：100 are inoculated in 40-50ml LB culture mediums (in 50ml centrifuge tubes), shake bacterium to OD600 be 0.6- 0.8, ice bath 10min；

(3) 4 DEG C, 5000rpm, centrifuge 5min；

(4) supernatant is abandoned, precipitation is fully suspended with sterilized water, 4 DEG C, 5000rpm, centrifuges 5min；Repeat this process 3 times.

(5) 1ml (depending on thalline is how many) is added to contain 10% sterile glycerol resuspension cell into washed thalline.

(6) be distributed into 50 μ L often to manage, liquid nitrogen flash freezer, be placed in -80 DEG C it is standby.

3.2 electric shocking methods conversion Agrobacterium competent cell EHA105

1. Agrobacterium competent cell is taken out in freeze thawing on ice.

2. 1 μ l pCAMBIA2301-CaMV35S-GhUGT73C6 DNAs are added to use pipette tips in 50 μ l competent cells It is gently mixed mixing.

3. the mixture for taking out cell and plasmid is transferred in electric shock cup (- 20 DEG C of precoolings of electric shock cup), electric shock cup surface is blotted Water, will electric shock cup be put into the electrode of electric conversion instrument between, shock by electricity 4-5s under 2400V high pressures.

4. taking out electric shock cup, it is rapidly added 1ml LB fluid nutrient mediums and is free of antibiotic, mix and shift mixed liquor and arrive In 1.5ml centrifuge tubes, 28 DEG C, 200rpm shaken cultivations 3h.

5. taking 100ul bacterium solutions to be coated on containing on Rif (50mg/L) and Kan (100mg/L) LB flat boards, 28 DEG C are inverted culture 2- 3 days.

3.3 bacterium solution PCR are identified

The single bacterium colony that picking grows in Rif and Kan resistant panels enters performing PCR identification.Identification is contained into target gene Bacterium solution adds 50% glycerine, mixes, is stored in standby in -80 DEG C of refrigerator.

Embodiment 4, arabidopsis thaliana transformation and transgenosis functional verification

4.1 flower-dipping methods infect arabidopsis

1. growing the arabidopsis of about 20-30 titbit in selection basin, mature Fruit pod is cut, is poured within 2-3 days before conversion Water.

2. with oese by 3.3 preserve the gene containing GhUGT73C6 Agrobacterium inoculation into 1.5ml centrifuge tubes 28 DEG C, 200rpm shaken cultivations 1-2 days.

3. take in 100ul to 50ml LB (50mg/L Rif and 100mg/L Kan) culture medium, 28 DEG C, 200rpm vibrations Culture is until OD₆₀₀=0.6-1.0.

4. 4000g is centrifuged 5 minutes.

5. abandoning supernatant, thalline is collected, it is standby with 30ml 1/2MS conversion fluid resuspension thalline.

6. plant to be transformed is inverted in into 45s in 1/2MS conversion fluids, bud will be totally immersed into conversion fluid, and (conversion fluid adds Enter silwette-775 μ l/100ml).

7. plant bagging will be infected, it is placed in after half-light culture 24h under regular culture conditions.

8. being converted again once after one week, step is same as above.

It is T0 generations that 9. seed is harvested after maturation.

The identification of 4.2 transgenic positive plant

4.2.1 screening is with the transgenic arabidopsis seed for blocking that resistance

1. by the T0 of harvest in EB pipes of the seed loaded on 2ml, 70% ethanol (v/v) sterilization 1-3min, 15% NaClO (v/v) sterilizes 5min, and 9000rpm is centrifuged 1 minute, sterile water wash 3-5 times.

2. adding appropriate sterilized water, uniformly sowing is on the 1/2MS culture mediums containing 100mg/L Kan.

3. 4 DEG C of light cultures are put in incubator after 2-3 days.

4. being observed after 2 weeks, positive transgenic plant grows true leaf and in green, and phenotype is normal, and nontransgenic plants are given birth to Length rests on cotyledon period, and turns to be yellow.

5. true leaf will be grown and greeny Arabidopsis plant is transplanted in soil property matrix, it is placed in incubator and normally cultivates Several weeks.

4.2.2 performing PCR detection is entered to transfer-gen plant

The PCR that genome and transcript profile level are carried out to transfer-gen plant is verified.Using T1 for transfer-gen plant STb gene as mould Plate, using plasmid as positive control, using the STb gene of unconverted plant as negative control, with GhUGT73C6-F (SEQ ID NO.4), GhUGT73C6-R (SEQ ID NO.5) enters performing PCR amplification.Positive plasmid compare and transfer-gen plant in numbering be two of 2,3 Plant amplifies purpose fragment, and the transfer-gen plant that negative control and numbering are 1 is without purpose band (Fig. 4).

RNAs of the T1 for Arabidopsis leaf is extracted, using transgenic Arabidopsis plants cDNA as template, is planted with unconverted arabidopsis The cDNA of strain (WT) is negative control, carries out the semi-quantitative expressed analysis of GhUGT73C6 genes.According to GhUGT73C6 genes CDNA sequence designs RT-PCR primer, and primer is GhUGT73C6-RT-F GAAGCACGGTTACCAGAC (SEQ ID NO.8), GhUGT73C6-RT-R CTCCCAAGACAAGCATAA (SEQ ID NO.9), with actin genes (GenBank No：NM_ 112764.3) it is internal reference gene (460bp), primer actin-F:5′-TGCCAATCTACG AGGGTT-3′(SEQ ID NO.10), actin-R:5′-TCTTTGCTCATACGGTCA-3′(SEQ ID NO.11).The semidefinite scale of GhUGT73C6 genes Further confirmed up to result (Fig. 5), GhUGT73C6 genes have been imported in arabidopsis and expressed.

4.2.3 the phenotypic evaluation of transgenic arabidopsis

T2 is taken for homozygous transgenic GhUGT73C6 arabidopsis strain (GhUGT73C6-2, GhUGT73C6-3) and wild Type arabidopsis Col-0 (WT) seed, through 4 degree of vernalization 2-3 days after sterilizing, sow black after 22-23 degree, daily 16h illumination 8 hours Under dark condition, normal water and fertilizer management, peduncle-growing period for rapeseed and it is ripe when observe phenotype (Fig. 6), when ripe measure plant height (each 5 plants of strain) (Fig. 7).It can be seen that the Arabidopsis plant of overexpression GhUGT73C6 genes has the small phenotype of dwarfing, blade.

Claims (4)

1. cotton glycosyltransferase gene GhUGT73C6, it is characterised in that its sequence is as shown in SEQ ID NO.2.

2. applications of the cotton glycosyltransferase gene GhUGT73C6 in terms of plant plant type is regulated and controled described in claim 1, it is special Sign is that for the regulation and control plant plant type to reduce plant height, reducing blade, the plant is upland cotton cotton or arabidopsis.

3. the protein encoded as the cotton glycosyltransferase gene GhUGT73C6 described in claim 1, it is characterised in that it Amino acid sequence is as shown in SEQ ID NO.3.

4. the overexpression vector containing the cotton glycosyltransferase gene GhUGT73C6 described in claim 1, it is characterised in that The overexpression vector is pCAMBIA2301-CaMV35S-GhUGT73C6.