CN104789578A - Cotton glycosyl transferase gene GhUGT73C6 and application thereof for regulating plant type - Google Patents

Abstract

The invention discloses an application of an upland cotton glycosyl transferase gene GhUGT73C6 for regulating plant type. The nucleotide sequence of the glycosyl transferase gene GhUGT73C6 is shown in SEQ ID NO.2. The method comprises the following steps: A: cloning the GhUGT73C6 full-length gene cDNA; B: constructing an over-expression vector pCAMBIA2301-CaMV35S-GhUGT73C6; C: performing functional verification through transformed into arabidopsis thaliana and proving that the plant has dwarf and small-leaf area phenotype due to the GhUGT73C6 gene. The gene has an important value for the plant dwarf breeding.

Description

Cotton glycosyltransferase gene GhUGT73C6 and the application in regulating plant plant type thereof

Technical field

The invention belongs to technical field of biological genetic engineering, is the application of cotton glycosyltransferase gene GhUGT73C6 in regulating plant plant type.

Background technology

Cotton is important cash crop, but in recent years due to the shortage of rural laborer, sown areas of cotton significantly decline, and DEVELOPMENT OF COTTON entire mechanization becomes the inevitable direction of current Cotton Production, and the cotton plant type of therefore cultivating suitable for mechanization is wherein importance.Obtain by traditional breeding technique the phenotype downgraded more difficult, and conventional breeding methods carries out cotton, and the Genetic improvement cycle is long, effect is low.In recent years along with the develop rapidly of Protocols in Molecular Biology, transgenic technology becomes a kind of selection well.

Brassinolide is the important plant hormone of a class, and have bioactive brassinolide and play an important role growing of plant, it can promote the sprouting of seed, the elongation of stem root leaf, trichome development, pollen tube growth, flower and fruit development etc.And research shows that in plant materials, the activity of brassinolide is subject to the regulation and control of multiple gene, and wherein some gene of glycosyltransferase family can make brassinolide inactivation.The research of Arabidopis thaliana is shown, in glycosyltransferase family UGT73C subfamily 6 genes in C1, C2, C3, C4 can not make brassinolide inactivation, and C5, C6 can make brassinolide inactivation (Husar S, Berthiller F, Fujioka S, et al.Overexpression of the UGT73C6alters brassinosteroidglucoside formation in Arabidopsis thaliana, BMC Plant Biology, 2011,11:51.doi:10.1186/1471-2229-11-51).Illustrate that the function of glycosyltransferase UGT73C subfamily gene there are differences.

Therefore the present invention is according to AtUGT73C6 (NM_129234.2) gene that can make brassinolide inactivation in Arabidopis thaliana, in cotton Lei Mengdesi cotton gene group, comparison obtains the forecasting sequence of UGT73C6 gene, the aminoacid sequence open (KJB13655.1) in NCBI of this predicted gene, but be through retrieval, in the function of cotton glycosyltransferase gene GhUGT73C6 and upland cotton, UGT73C6 gene order is not reported.

The present invention obtains GhUGT73C6 gene from upland cotton cotton variety Soviet Union cotton 18, GhUGT73C14 gene (JX846921.1) amino acid sequence similarity announced in UGT73C subfamily in itself and upland cotton is lower than 80%, research thinks that GhUGT73C14 gene participates in activity regulation (the Gilbert MK of dormin, Bland JM, Shockey JM, et al., Atranscript 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).Make GhUGT73C6 gene overexpression in Arabidopis thaliana by transgenic technology, the GhUGT73C6 gene plant that turns of acquisition has dwarfing, phenotype that leaf area is little.So the clone of cotton GhUGT73C6 gene and gene function checking can provide genetic resources for the breeding wheat for semidwarfness of cotton.

Summary of the invention

Completing of cotton AA, DD diploid and AADD tetraploid genome sequencing, obtain very polygenic forecasting sequence, but gene function is not clear and definite.Therefore the object of this invention is to provide the application of a kind of cotton glycosyltransferase gene GhUGT73C6 in regulating plant plant type, described gene cDNA nucleotide sequence arranges as shown in SEQ ID NO.2.

The protein of the cotton glycosyltransferase gene GhUGT73C6 genes encoding of described regulating plant plant type, has the aminoacid sequence described in SEQ ID NO.3 in sequence table.

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

Gene GhUGT73C6 of the present invention is cultivating the application in dwarf plant.Specifically GhUGT73C6 gene is proceeded in object plant by plant expression vector.Described plant is model plant Arabidopis thaliana.

Beneficial effect of the present invention: utilize existing plant gene engineering technology, utilize electronic cloning and RT-PCR technology, Isolation and ldentification cotton dwarf-related gene sequence information, and by agrobacterium tumefaciens flower-dipping method, gene is proceeded to Arabidopis thaliana, the plant height of assay certificate transfer-gen plant is starkly lower than wild-type, blade is significantly less than wild-type (Fig. 6), illustrates that GhUGT73C6 gene regulating plant plant type has positive effect.

Accompanying drawing explanation

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

M:DL2000DNA marker, 1,2 is GhUGT73C6 full length gene cDNA pcr amplification result.

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

The structure of Fig. 3 plant expression vector pCAMBIA2301-CaMV35S-GhUGT73C6.

A () intestinal bacteria pCAMBIA2301-CaMV35S-GhUGT73C6PCR detects electrophoresis result;

M:DL2000DNA marker, 1,2,3,4,5 is intestinal bacteria pCAMBIA2301-CaMV35S-GhUGT73C6 bacterium liquid PCR result ,+be GhUGT73C6 gene PCR result ,-be negative control.

B PCR, the enzyme of () pCAMBIA2301-CaMV35S-GhUGT73C6 plasmid cut qualification.

M:DL2000DNA marker, 1 is blank, and 2 is the PCR result of pCAMBIA2301-CaMV35S-GhUGT73C6 plasmid, and 3 is that the enzyme of pCAMBIA2301-CaMV35S-GhUGT73C6 plasmid cuts result.

The PCR qualification of Fig. 4 transfer-gen plant.

M:DL2000DNA marker; 1,2,3 is transfer-gen plant."+" is pCAMBIA2301--CaMV35S-GhUGT73C6 plasmid, and "-" is negative control.

The RT-PCR qualification of Fig. 5 transfer-gen plant.

M:DL2000DNA marker; WT is wildtype Arabidopsis thaliana, and 2,3 for turning GhUGT73C6 gene plant 2,3.

Fig. 6 transgenic arabidopsis compares with wildtype Arabidopsis thaliana plant type.

(a) flowering period plant type; (b) ripening stage plant type; (c) blade.

Transfer-gen plant plant height is starkly 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, GhUGT73C6-3 is transgenic line 3.

Embodiment

The acquisition of embodiment 1, GhUGT73C6 gene

The extraction of 1.1 RNA

(1) leaf tissue of the fresh upland cotton cotton variety Soviet Union cotton 18 of 0.5g is got, add 0.1g polyvinyl pyrrolidone (PVPP), fully powder is ground in liquid nitrogen, agar end is proceeded to rapidly in 10ml centrifuge tube, add the Tris-HCl of 5ml CTAB extracting solution and 500 μ l0.1M pH8.0,65 DEG C of water-bath 20min, midway overturns mixing;

(2) add equal-volume chloroform fully to mix, ice bath leaves standstill 10min;

(3) 4 DEG C, the centrifugal 20min of 10000rpm.Be sub-packed in 4 1.5ml centrifuge tubes;

(4) suct clearly, the 8M LiCl adding 1/3 volume mixes, and-70 DEG C 1h or-20 DEG C is spent the night;

(5) 4 DEG C, the centrifugal 20min of 10000rpm.Abandon supernatant, after 70% washing with alcohol twice, dry up and be precipitated and dissolved in 30 μ lDEPC water;

(6) add DNase and the 25U RNase Inhabitor of 10U without RNase activity, 10 × buffer adds equal-volume chloroform after digesting 30min, and extracting once;

(7) supernatant is transferred in new pipe, adds the 3M pH 5.2NaAc of 1/10 volume and the dehydrated alcohol of isopyknic Virahol or 2.5 times of volumes, and-20 DEG C of placements are spent the night or-70 DEG C of ice bath 3h;

(8) 4 DEG C, the centrifugal 20min of 10000rpm, abandons supernatant, is dissolved in 30 μ l DEPC water after 70% washing with alcohol twice.Obtain cotton RNA.

The synthesis of 1.2 cDNA

System:

The clone of 1.3 GhUGT73C6 genes

Obtain UGT73C6 predictive genes sequence in cotton according to Arabidopis thaliana UGT73C6 gene (NM_129234.2) Blast comparison in the cotton genome of cotton Lei Mengdesi, its nucleotide sequence is as shown in SEQ ID NO.1.At ORF sequence both sides design upstream primer 5 '-CTCATATTCCTATGGGTT-3 ' (SEQ ID NO.4) and downstream primer 5 '-GACGTTATCTCGATTTTC-3 ' (SEQ ID NO.5), PCR is carried out with the cDNA (being synthesized by step 1.2) of upland cotton Soviet Union cotton 18, PCR reaction system (25 μ l): 10 × EasyPfu buffer 2.5 μ l, 50mM MgSO ₄0.5 μ l, 2.5mM dNTP 2 μ l, primer 1 (10 μMs) 1 μ l, primer 2 (10 μMs) 1 μ l, ddH ₂o 16.5 μ l, cDNA 1 μ l, EasyPfu DNA Polymerase 0.5 μ l.Pcr amplification program: 94 DEG C of 3min; 94 DEG C of 30sec, 48 DEG C of 45sec, 72 DEG C of 2min, 36 circulations; 72 DEG C of 10min; 4 DEG C of insulations.

Reclaim the PCR primer increasing and obtain, be cloned into pEASY-T1 carrier (Beijing Quanshijin Biotechnology Co., Ltd), by PCR screening positive clone, deliver to the order-checking of the handsome company in Shanghai.We are GhUGT73C6, ORF total length 1494bp (SEQ ID NO.2) (Fig. 1) by being separated the upland cotton unnamed gene obtained, and the aminoacid sequence of coding is as shown in SEQ ID NO.3.The GhUGT73C6 gene obtained in upland cotton and the gene order similarity 99% predicted in the cotton genome of Lei Mengdesi.By gene (AtUGT73C6, the AtUGT73C5 in Arabidopis thaliana of acquired UGT73C subfamily in acquired GhUGT73C14 in acquisition GhUGT73C6 gene and cotton and other crops, Tcglu1, TcUGT73C5 in cocoa, orange CsUGT73C1, plum PmUGT73C1) discovery of comparing, the consistence of the gene amino acid sequence of GhUGT73C6 gene amino acid sequence and other crop UGT73C subfamilies is all lower than 80% (Fig. 2).

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

With the primer GhUGT73C6-KpnI:5 ' containing restriction enzyme site- cTCATATTCCTATGGGTT-3 ' (SEQ ID NO.6) and GhUGT73C6-XbaI:5'- gACGTTATCTCGATTTTC-3'(SEQ ID NO.7) carry out pcr amplification, agarose gel electrophoresis detects whether contain expection fragment.PCR response procedures is as follows: 94 DEG C of 5min; 94 DEG C of 30sec, 48 DEG C of 45sec, 72 DEG C of 1min 30sec, 36 circulations; 72 DEG C of 10min; 4 DEG C of insulations.Obtain the GhUGT73C6PCR product containing restriction enzyme site.

To plant expression vector pCAMBIA2301-CaMV35S plasmid, (this laboratory is preserved respectively to select Kpn I and Xba I, Zheng Qing, the research [D] of resisting I-type Diabetes Gene transformation of tobacco, tomato, Yangzhou University Master degree candidate Diplomarbeit, 2010) the GhUGT73C6PCR product with containing restriction enzyme site carries out double digestion.

PCAMBIA2301-CaMV35S plasmid double digestion system is as follows:

GhUGT73C6PCR product double digestion system containing restriction enzyme site:

Cut in 37 DEG C of enzymes, reaction times >=3h.With sepharose, electrophoresis detection is carried out to double digestion product, the results are shown in Figure 3 (b).Reclaim pCAMBIA2301-CaMV35S carrier large fragment and goal gene fragment, transformation of E. coli Trans1-T1 competent cell (purchased from Beijing Quanshijin Biotechnology Co., Ltd) after connecting with T4 ligase enzyme, namely obtains the plant expression vector with goal gene after qualification recon.

Gene and enzyme cut the ligation system of the pCAMBIA2301 large fragment obtained:

16 DEG C of connections are spent the night.

Connect product conversion competent escherichia coli cell, in the LB liquid nutrient medium containing kan100mg/L after 37 DEG C of shaking culture 1h.Be evenly coated on the LB solid medium of kan100mg/L, 37 DEG C of overnight incubation.

Picking list colony inoculation is 37 DEG C of shaking culture in 1.5ml is containing the LB liquid nutrient medium of kantlex, carry out pcr amplification qualification with GhUGT73C6 gene primer GhUGT73C6-Kpn I (SEQ ID NO.6) and GhUGT73C6-Xba I (SEQ ID NO.7), the results are shown in Figure 3 (a).

Extract the bacterium liquid plasmid of PCR test positive with AXYGEN plasmid extraction kit, choose Xba I and Kpn I enzyme carries out double digestion, agarose gel electrophoresis detects Fig. 3 (b).By the recombinant expression vector called after pCAMBIA2301-CaMV35S-GhUGT73C6 obtained.

Embodiment 3, the competent preparation of Agrobacterium and conversion

The competent preparation of 3.1 Agrobacterium EHA105

(1) the mono-bacterium colony of picking EHA105, is inoculated in 5ml LB liquid nutrient medium, 28 DEG C, and it is 0.4 that 200rpm shakes overnight incubation to OD600 value;

(2) be inoculated in (in 50ml centrifuge tube) in 40-50ml LB substratum with 1:100, shaking bacterium to OD600 is 0.6-0.8, ice bath 10min;

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

(4) abandon supernatant, precipitation sterilized water fully suspends, 4 DEG C, 5000rpm, centrifugal 5min; Repeat this process 3 times.

(5) in washed thalline, 1ml (according to thalline how many and determine) is added containing 10% sterile glycerol resuspension cell.

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

3.2 electric shocking method transformation Agrobacterium competent cell EHA105

1. take out Agrobacterium competent cell in freeze thawing on ice.

2. add 1 μ l pCAMBIA2301-CaMV35S-GhUGT73C6 plasmid DNA in 50 μ l competent cells, with rifle head stirring and evenly mixing gently.

3. the mixture taking out cell and plasmid proceeds to and shocks by electricity (cup-20 DEG C of precoolings of shocking by electricity) in cup, blots the water on electric shock cup surface, and put between the electrode of electric conversion instrument by electric shock cup, shock by electricity 4-5s under 2400V high pressure.

4. take out electric shock cup, add rapidly 1ml LB liquid nutrient medium not containing microbiotic, mix and shift mixed solution in 1.5ml centrifuge tube, 28 DEG C, 200rpm shaking culture 3h.

5. getting 100ul bacterium liquid coats containing on Rif (50mg/L) and Kan (100mg/L) LB flat board, is inverted for 28 DEG C and cultivates 2-3 days.

3.3 bacterium liquid PCR identify

Single bacterium colony that picking grows in Rif and Kan resistant panel carries out PCR qualification.The bacterium liquid of qualification containing goal gene is added 50% glycerine, and mixing, is stored in the refrigerator of-80 DEG C for subsequent use.

Embodiment 4, arabidopsis thaliana transformation and transgenosis functional verification

4.1 flower-dipping methods infect Arabidopis thaliana

1. select the Arabidopis thaliana growing an about 20-30 titbit in basin, cut mature fruit pod, before transforming, within 2-3 days, water sufficient water.

2. with transfering loop preserve 3.3 containing the Agrobacterium inoculation of GhUGT73C6 gene in 1.5ml centrifuge tube 28 DEG C, 200rpm shaking culture 1-2 days.

3. get in 100ul to 50ml LB (Kan of Rif and 100mg/L of 50mg/L) substratum, 28 DEG C, 200rpm shaking culture is until OD ₆₀₀=0.6-1.0.

4. centrifugal 5 minutes of 4000g.

5. abandon supernatant, collect thalline, for subsequent use with the 1/2MS conversion fluid resuspension thalline of 30ml.

6. plant to be transformed is inverted in 45s in 1/2MS conversion fluid, bud all will immerse conversion fluid (conversion fluid adds silwette-775 μ l/100ml).

7. will infect plant bagging, under half-light cultivation 24h is placed on regular culture conditions.

8. transform once after one week, step is the same again.

9. ripe rear results seed is T0 generation.

The qualification of 4.2 transgenic positive plant

4.2.1 screening has the transgenic arabidopsis seed of that resistance of card

1. be loaded in the EB pipe of 2ml by the T0 of results for seed, the ethanol (v/v) of 70% is sterilized 1-3min, and the NaClO (v/v) of 15% sterilizes centrifugal 1 minute of 5min, 9000rpm, sterile water wash 3-5 time.

2. add appropriate sterilized water, evenly sowing is on the 1/2MS substratum containing 100mg/L Kan.

3. 4 DEG C after light culture 2-3 days, be put in incubator.

4. observe after 2 weeks, positive transgenic plant grows true leaf and in green, phenotype is normal, and nontransgenic plants growth rests on cotyledon period, and turns to be yellow.

5. will grow true leaf and greeny Arabidopsis plant is transplanted in soil property matrix, and be placed in incubator and normally cultivate several weeks.

4.2.2 PCR detection is carried out to transfer-gen plant

Transfer-gen plant is carried out to the PCR checking of genome and transcript profile level.With T1 for transfer-gen plant STb gene for template, take plasmid as positive control, with the STb gene of unconverted plant for negative control, with GhUGT73C6-F (SEQ ID NO.4), GhUGT73C6-R (SEQ ID NO.5) carries out pcr amplification.Two plant being numbered 2,3 in positive plasmid contrast and transfer-gen plant amplify object fragment, and negative control be numbered the transfer-gen plant of 1 without object band (Fig. 4).

Extract T1 for the RNA of Arabidopsis leaf, with transgenic Arabidopsis plants cDNA for template, with the cDNA of unconverted Arabidopsis plant (WT) for negative control, carry out the semi-quantitative expressed analysis of GhUGT73C6 gene.According to the cDNA sequence design RT-PCR primer of GhUGT73C6 gene, primer is GhUGT73C6-RT-F GAAGCACGGTTACCAGAC (SEQID NO.8), GhUGT73C6-RT-R CTCCCAAGACAAGCATAA (SEQ ID NO.9), with actin gene (GenBankNo:NM_112764.3) for internal reference gene (460bp), primer is actin-F:5 '-TGCCAATCTACGAGGGTT-3 ' (SEQ ID NO.10), actin-R:5 '-TCTTTGCTCATACGGTCA-3 ' (SEQ ID NO.11).The semi-quantitative expressed result (Fig. 5) of GhUGT73C6 gene confirms further, and GhUGT73C6 gene to have imported in Arabidopis thaliana and expressed.

4.2.3 the phenotypic evaluation of transgenic arabidopsis

Get T2 for the Arabidopis thaliana strain (GhUGT73C6-2, GhUGT73C6-3) of homozygous transgenic GhUGT73C6 and wildtype Arabidopsis thaliana Col-0 (WT) seed, through 4 degree of vernalization 2-3 days after sterilizing, after planting under 22-23 degree, 16h illumination 8 every day h dark condition, normal water and fertilizer management, observe phenotype (Fig. 6) when peduncle-growing period for rapeseed and maturation, time ripe, measure plant height (strain of each strain 5) (Fig. 7).Can find out that the Arabidopsis plant of overexpression GhUGT73C6 gene has dwarfing, phenotype that blade is little.

Claims (5)

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

2. the application of cotton glycosyltransferase gene GhUGT73C6 according to claim 1 in regulating plant plant type.

3. application according to claim 2, is characterized in that: described regulating plant plant type is for reducing plant height, reducing blade.

4. the protein of being encoded by cotton glycosyltransferase gene GhUGT73C6 according to claim 1, is characterized in that its aminoacid sequence is as shown in SEQ ID NO.3.

5. the overexpression vector containing cotton glycosyltransferase gene GhUGT73C6 according to claim 1 is pCAMBIA2301-CaMV35S-GhUGT73C6.