CN104975033B

CN104975033B - Applications of the arabidopsis glycosyltransferase gene UGT76D1 in plant surface wax is reduced

Info

Publication number: CN104975033B
Application number: CN201510319744.4A
Authority: CN
Inventors: 侯丙凯; 黄戌戌; 李燕洁
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2015-06-11
Filing date: 2015-06-11
Publication date: 2017-10-27
Anticipated expiration: 2035-06-11
Also published as: CN104975033A

Abstract

The invention discloses a kind of applications of arabidopsis glycosyltransferase gene UGT76D1 in plant wax is reduced, wherein the nucleotide sequence of the glycosyltransferase gene UGT76D1 is as shown in SEQ ID No.1.The present invention builds plant over-express vector using gene UGT76D1, carries out plant transgene operation, obtains genetically modified plants.Detection shows that the genetically modified plants wax is substantially reduced, the indication present invention implement after by the enhancing micro- fertile absorption efficiency of plant leaf surface, enhancing light energy absorption and plant photosynthesis efficiency, increase yield, improve mouthfeel of the vegetables during edible, increase the emerald green color of ornamental plant and improve perception, beneficial effect will be all brought, it is significant for new variety of plant Development and Production.

Description

Arabidopsis glycosyltransferase gene UGT76D1 is in plant surface wax is reduced Using

Technical field

The present invention relates to a kind of application of glycosyltransferase gene, more particularly to a kind of arabidopsis glycosyltransferase gene Applications of the UGT76D1 in plant surface wax is reduced, belongs to genetic engineering field.

Background technology

Wax is one layer of hydrophobic protective layers of plant surface, is usually coated on blade, the stalk surface of plant.Plant surface Wax wax in the wax outside the cuticula of plant epidermis and cuticula is constituted.Wax is different in different plant constituents, It is main that alkane, primary and secondary alcohol, aldehyde, ketone and terpene and micro secondary generation are included by speciality chain fatty acid and its derivative Thank to the compound groups such as product such as flavonoids into (Pollard et al., 2008；Samuels et al.,2008；Li-Beisson et al.,2013)。

Wax as plant surface hydrophobic barrier, although moisture loss can be reduced to a certain extent, disease pest is resisted Do harm to and prevent ultraviolet radioactive, but also bring many drawbacks to agricultural production simultaneously.For example, plant surface wax have impact on blade face Micro- fertile absorption efficiency, the presence of wax have impact on the absorption of light, reduce photosynthetic efficiency, and the wax on vegetable waste surface can also Mouthfeel, reduction digestive efficiency are influenceed, in addition, for ornamental plant, surface wax has covered the emerald green color of plant in itself, It has impact on perception, etc..Therefore, plant surface wax how is reduced, the different demands of agricultural production are preferably met, is also mesh One of the problem of preceding urgent need to resolve.However, retrieval finds that current transgenic technology does not find a good solution party also Case.

During glycosyl transferase is the enzyme for being responsible for being catalyzed glycosylation modified reaction specially, arabidopsis glycosyl transferase family 1 Have 119 possible glycosyl transferases.Although the sequence of these glycosyltransferase genes has been disclosed in GenBank In, but the specific function of these glycosyltransferase genes is also far from being revealed, in such as arabidopsis glycosyl transferase family 1 The function and applications of the UGT76D1 genes genetically modified plants that can create surface wax reduction have no report at present.

The content of the invention

In view of the shortcomings of the prior art, it is an object of the invention to provide a kind of arabidopsis glycosyltransferase gene Applications of the UGT76D1 in plant surface wax is reduced.

Applications of the arabidopsis glycosyltransferase gene UGT76D1 of the present invention in plant surface wax is reduced.

Wherein：The nucleotide sequence of the glycosyltransferase gene UGT76D1 is as shown in SEQ ID No.1.The plant Preferably crucifer, the crucifer is preferably arabidopsis, rape, Chinese cabbage, wild cabbage or leaf mustard.

The present invention is southern from intending by RT-PCR technology using the primer sequence shown in SEQ ID No.3 and SEQ ID No.4 Glycosyltransferase gene UGT76D1 is cloned in mustard, then using the gene constructed plant over-express vector, plant transgene is carried out Operation, obtains genetically modified plants.Detection shows that the genetically modified plants surface wax is significantly reduced (see Fig. 1, Fig. 2, Fig. 3, Fig. 4).

The beneficial effect that the present invention may be brought after implementing：

It is experimentally confirmed that carrying out plant transgene operation using glycosyltransferase gene UGT76D1 of the present invention, it can subtract The surface wax of few genetically modified plants.This new technology is for the enhancing micro- fertile absorption efficiency of plant leaf surface, enhancing light energy absorption With plant photosynthesis efficiency, increase yield, improve mouthfeel of the vegetables during edible, increase the emerald green color of ornamental plant and carry High perception, will all bring beneficial effect, significant for new variety of plant Development and Production.

Brief description of the drawings

The photo in kind (one of evidence) of Fig. 1 .UGT76D1 genetically modified plants surface wax reduction.

Wherein WT is check plant, OE-1 and the overexpression strain that OE-2 is two UGT76D1 transgenosis.Check plant and Strain is overexpressed normally after culture 6 weeks, finding to be overexpressed strain, to show cauline leaf surface emerald green, and wax is reduced.

The photo in kind (the two of evidence) of Fig. 2 .UGT76D1 genetically modified plants surface wax reduction.

Blade is after aniline blue dyeing, and two are overexpressed strain and are easier coloring, further prove that surface wax matter subtracts It is few, add the infiltration of coloring agent.

Photo in kind --- --- cane surface Scanning Electron of Fig. 3 .UGT76D1 genetically modified plants surface wax reduction is micro- Mirror result (the three of evidence).

Wherein WT is check plant, and OE-1 and OE-2 are two overexpression strains.Experiment material is the normal growth plan of 2 weeks Southern mustard check plant and two overexpression strains.As a result showing two plants, to be overexpressed be that cane surface wax substantially reduces that (surface is white Coloured particles shape structure).

Fig. 4 chlorophyll recovery rates result figure (the four of the evidence of UGT76D1 genetically modified plants surface wax reduction).

Chlorophyll extracts experiment, and wherein WT is check plant, and OE-1 and OE-2 are two overexpression strains.Carried by chlorophyll The rate result of taking shows, due to the reduction of surface wax, and the chlorophyll recovery rates of two overexpression strains is apparently higher than control.Illustrate UGT76D1 genes have new opplication in terms of wax is reduced.

Embodiment

The clone's arabidopsis glycosyltransferase gene of embodiment 1 UGT76D1

1. arabidopsis glycosyltransferase gene UGT76D1 clone

Pass through open website http://www.cazy.org obtains the cDNA sequence of UGT76D1 genes.According to cDNA sequence Primer is designed, forward primer is 76D1-F:5 '-CGGGATCCATGGCAGAGATTCGCCAG-3 ', reverse primer is 76D1-R: 5’-CGAGCTCTCATTGTTCGTCAATTTGCATC-3’.Arabidopsis RNA, RT-PCR method are extracted using TRIzol kits Expand the full length cDNA sequence of UGT76D1 genes.It is to first pass through BamHI and the digestions of Sac I, Zhi Houlian by the process of cDNA clone In pBluescript II SK (+) carrier for entering corresponding digestion, sequencing intermediate carrier, referred to as pK76D1, then with load are built into Body carries out full length gene PCR amplifications and BamHI and the digestion verifications of Sac I, finally carries out sequencing, checking cloned sequence is just True property.

2. arabidopsis glycosyltransferase gene UGT76D1 sequence information and specificity analysis

The code area cDNA of UGT76D1 genes is 1359bp (nucleotide sequence is as shown in SEQ ID No.1), coding 452 The 57.6kDa albumen of individual amino acid (amino acid sequence is as shown in SEQ ID No.2), C-terminal has the PSPG boxes of 44 amino acid, The conserved sequence having jointly for Plant Secondary Metabolites glycosyl transferase.

The arabidopsis glycosyltransferase gene UGT76D1 of embodiment 2 transgenosis application

1. the structure containing UGT76D1 code areas cDNA expression vectors

Sequencing vector obtains the total length with digestion cohesive end after BamHI and the double digestions of Sac I in the middle of pK76D1 CDNA sequence.This genetic fragment is connected to the pBI121 carrier parts after corresponding enzyme digestion, obtains starting with CaMV 35S The plant expression vector that son driving glycosyltransferase gene is overexpressed, referred to as pBI76D1.

2. Agrobacterium-mediated plant is converted

Agrobacterium GV3101 has the ability for infecting plant and metastatic gene, therefore the UGT76D1 plants of structure are expressed and carried Body (pBI76D1) is transferred to Agrobacterium, then enters performing PCR checking and digestion verification.Utilize a kind of flower-dipping method (disclosed routine side Method), the Agrobacterium GV3101 containing plant expression vector is contaminated arabidopsis bud.Treat after its silique maturation grown, receive Collection T1 is screened for seed and on screening and culturing medium (the additional 30mg/L kanamycins of MS culture mediums), is possible to normal growth Green conversion transplantation of seedlings cultivate into Nutrition Soil, harvest respectively its T2 carried out again for seed next round kanamycins screen, Pick out green seedling：Bai Miaowei 3：1 culture dish.By the green transplantation of seedlings on this culture dish, individual plant harvest seed (T3 generations).To each The seed fraction of individual plant is screened for kanamycins plate, is complete green strain until selecting on screening and culturing medium, as pure Close transgenic line.

3. transfer-gen plant Molecular Identification

The detection of gene expression dose is carried out to above-mentioned transfer-gen plant.Transfer-gen plant and WT lines are extracted respectively RNA, RT-PCR amplifications are carried out after reverse transcription, analysis is overexpressed the gene expression difference of plant and WT lines.UGT76D1 Expression quantity in plant is overexpressed is all apparently higher than WT lines.Using the high strain of two UGT76D1 expression quantity, i.e., OE-1, OE-2, to carry out follow-up work.

4.UGT76D1 gene reduces plant surface wax functional verification

(1) one of evidence of UGT76D1 genetically modified plants surface wax reduction.

After check plant and overexpression strain are normally cultivated 6 weeks, naked eyes can be observed overexpression strain and show cauline leaf Surface is emerald green, and wax is reduced.It is check plant, OE-1 and the mistake that OE-2 is two UGT76D1 transgenosis to see Fig. 1, wherein WT Express strain.

(2) the two of the evidence of UGT76D1 genetically modified plants surface wax reduction.

Blade is after aniline blue dyeing, and two are overexpressed strain and are easier coloring, further prove that surface wax matter subtracts It is few, add the infiltration of coloring agent.See Fig. 2.

(3) the three of the evidence of UGT76D1 genetically modified plants surface wax reduction.

Cane surface is scanned electron microscope observation, and experiment material is the normal growth arabidopsis check plant of 2 weeks With two overexpression strains.Choose growth conditions consistent with height stem section, SEM detection cane surface wax, knot Fruit finds that two plants of overexpression are that cane surface wax is substantially reduced, and sees Fig. 3, and wax is superficial white nutty structure, and WT is pair According to plant, OE-1 and OE-2 are two overexpression strains.

(4) the four of the evidence of UGT76D1 genetically modified plants surface wax reduction.

Chlorophyll extracts experiment, and experiment material is the normal growth arabidopsis check plant of 2 weeks and two overexpression strains. Choose the consistent plant of growth conditions, clip ground leaf, be immersed in 80% ethanol, every 20min in 647nm and 664nm Place determines the absorbance of solution, calculates chlorophyll recovery rate.As a result Fig. 4 is seen, due to the reduction of surface wax, two overexpression strains The chlorophyll recovery rate of system is apparently higher than control.WT is check plant, and OE-1 and OE-2 are two overexpression strains.Demonstrate again that New function of the UGT76D1 genes in terms of wax is reduced.

Claims

1. arabidopsis glycosyltransferase geneUGT76D1Application in crucifer surface wax is reduced, its feature exists In：The glycosyltransferase geneUGT76D1Nucleotide sequence as shown in SEQ ID No.1；The crucifer is Arabidopsis, rape, Chinese cabbage, wild cabbage or leaf mustard.