CN104975033A

CN104975033A - Use of arabidopis thaliana glycosyl transferase gene UGT76D1 in reduction of plant surface wax

Info

Publication number: CN104975033A
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: 2015-10-14
Anticipated expiration: 2035-06-11
Also published as: CN104975033B

Abstract

The invention discloses a use of an arabidopis thaliana glycosyl transferase gene UGT76D1 in reduction of plant surface wax. The arabidopis thaliana glycosyl transferase gene UGT76D1 has a nucleotide sequence shown in the formula of SEQ ID No.1. A plant overexpression vector is constructed by the gene UGT76D1, and a plant transgenosis process is carried out so that a transgenic plant is obtained. A detection result shows that the transgenic plant has substantially less wax and the use provides beneficial effects of improving plant blade face microelement fertilizer absorption efficiency, luminous energy absorption, plant photosynthetic efficiency, a yield, a taste of vegetables in eating, an ornamental plant jade green color and appearance and has an important meaning for plant new specie development.

Description

Arabidopis thaliana glycosyltransferase gene UGT76D1 is reducing the application in plant surface wax

Technical field

The present invention relates to a kind of application of glycosyltransferase gene, particularly relating to a kind of Arabidopis thaliana glycosyltransferase gene UGT76D1 reducing the application in plant surface wax, belonging to genetically engineered field.

Background technology

Wax is plant surface one deck hydrophobic protective layers, is usually coated on the blade of plant, stem stalk surface.Plant surface wax is made up of the outer wax of the stratum corneum being positioned at plant epidermis and stratum corneum wax.Wax is different in different plant moietys, primarily of speciality chain fatty acids and derivative thereof comprise alkane, primary and secondary alcohol, aldehyde, ketone and terpene and trace secondary metabolite as compound composition (Pollard et al., 2008 such as flavonoids; Samuels et al., 2008; Li-Beisson et al., 2013).

Wax, as the hydrophobic barrier of plant surface, although can reduce moisture loss to a certain extent, resist disease and pest and prevent uv-radiation, also brings a lot of drawback to agriculture production simultaneously.Such as, plant surface wax have impact on the assimilated efficiency of leaf surface trace fertilizer, the existence of wax have impact on light absorption, reduce photosynthetic efficiency, the wax on vegetable waste surface can also affect mouthfeel, reduce digestive efficiency, in addition, for ornamental plant, surperficial wax has covered the emerald green color of plant itself, have impact on perception, etc.Therefore, how reducing plant surface wax, meet the different demands of agriculture production better, is also one of problem needing solution badly at present.But retrieval finds that current transgenic technology does not also find a good solution.

Glycosyltransferase is the enzyme being responsible for the glycosylation modified reaction of catalysis specially, has 119 possible glycosyltransferases in Arabidopis thaliana glycosyltransferase family 1.Although the sequence of these glycosyltransferase genes has been disclosed in GenBank, but the specific function of these glycosyltransferase genes is also far from revealed, the function and application that can create the transgenic plant that surperficial wax reduces as the UGT76D1 gene in Arabidopis thaliana glycosyltransferase family 1 has no report at present.

Summary of the invention

For the deficiencies in the prior art, the object of this invention is to provide a kind of Arabidopis thaliana glycosyltransferase gene UGT76D1 and reduced the application in plant surface wax.

Arabidopis thaliana glycosyltransferase gene UGT76D1 of the present invention is reducing the application in plant surface wax.

Wherein: the nucleotide sequence of described glycosyltransferase gene UGT76D1 is as shown in SEQ ID No.1.Described plant optimization is cress, and described cress is preferably Arabidopis thaliana, rape, Chinese cabbage, wild cabbage or leaf mustard.

The present invention utilizes the primer sequence shown in SEQ ID No.3 and SEQ ID No.4, from Arabidopis thaliana, glycosyltransferase gene UGT76D1 is cloned by RT-PCR technology, then utilize this gene constructed plant over-express vector, carry out plant transgene operation, obtain transgenic plant.Detection shows that this transgenic plant surface wax obviously reduces (see Fig. 1, Fig. 2, Fig. 3, Fig. 4).

The beneficial effect that may bring after the invention process:

Experiment confirms, applies glycosyltransferase gene UGT76D1 of the present invention and carries out plant transgene operation, can reduce the surperficial wax of transgenic plant.This new technology for strengthen plant leaf surface micro-fertilizer assimilated efficiency, strengthen light energy absorption and plant photosynthesis efficiency, increase yield, improve the mouthfeel of vegetables in edible process, increase the emerald green color of ornamental plant and improve perception, all will bring beneficial effect, significant for new variety of plant Development and Production.

Accompanying drawing explanation

The photo in kind (one of evidence) that Fig. 1 .UGT76D1 transgenic plant surface wax reduces.

Wherein WT is control plant, OE-1 and OE-2 is two genetically modified process LAN strains of UGT76D1.After control plant and process LAN strain normally cultivate 6 weeks, it is emerald green that discovery process LAN strain all shows cauline leaf surface, and wax reduces.

The photo in kind (evidence two) that Fig. 2 .UGT76D1 transgenic plant surface wax reduces.

Blade is after aniline blue dyeing, and two process LAN strains are more easily painted, and further proof list mercolized wax matter reduces, and adds the infiltration of staining agent.

Photo in kind------cane surface Scanning Electron microscopy images (evidence three) that Fig. 3 .UGT76D1 transgenic plant surface wax reduces.

Wherein WT is control plant, OE-1 and OE-2 is two process LAN strains.Experiment material is the normal growth Arabidopis thaliana control plant of 2 weeks and two process LAN strains.Result shows two strain process LAN system cane surface waxs and significantly reduces (superficial white grainy texture).

Fig. 4. chlorophyll extraction yield result figure (evidence four that UGT76D1 transgenic plant surface wax reduces).

Chlorophyll extracts experiment, and wherein WT is control plant, OE-1 and OE-2 is two process LAN strains.Shown by chlorophyll extraction yield result, due to the minimizing of surperficial wax, the chlorophyll extraction yield of two process LAN strains is apparently higher than contrast.Illustrate UGT76D1 gene, in minimizing wax, there is new opplication.

Embodiment

Embodiment 1 clones Arabidopis thaliana glycosyltransferase gene UGT76D1

1. the clone of Arabidopis thaliana glycosyltransferase gene UGT76D1

The cDNA sequence of UGT76D1 gene is obtained by open website http://www.cazy.org.According to cDNA sequence design primer, forward primer is 76D1-F:5 '-CGGGATCCATGGCAGAGATTCGCCAG-3 ', and reverse primer is 76D1-R:5 '-CGAGCTCTCATTGTTCGTCAATTTGCATC-3 '.TRIzol test kit is utilized to extract Arabidopis thaliana RNA, the full length cDNA sequence of RT-PCR method amplification UGT76D1 gene.The process of being cloned by cDNA is first cut through BamHI and Sac I enzyme, be connected in pBluescript II SK (+) carrier that corresponding enzyme cuts afterwards, be built into order-checking intermediate carrier, be called pK76D1, then full length gene pcr amplification and BamHI and Sac I digestion verification is carried out with carrier, finally carry out sequencing, the exactness of checking cloned sequence.

2. the sequence information of Arabidopis thaliana glycosyltransferase gene UGT76D1 and specificity analysis

The coding region cDNA of UGT76D1 gene is 1359bp (nucleotide sequence is as shown in SEQ ID No.1), to encode the 57.6kDa albumen of 452 amino acid (aminoacid sequence is as shown in SEQ ID No.2), C end has 44 amino acid whose PSPG boxes, is the conserved sequence that Plant Secondary Metabolites glycosyltransferase has jointly.

The transgenosis application of embodiment 2 Arabidopis thaliana glycosyltransferase gene UGT76D1

1. the structure containing UGT76D1 coding region cDNA expression vector

In the middle of pK76D1, sequencing vector is after BamHI and Sac I double digestion, obtains the full length cDNA sequence cutting sticky end with enzyme.By this gene fragment to cut with corresponding enzyme enzyme after pBI121 carrier part be connected, obtain the plant expression vector driving glycosyltransferase gene process LAN with CaMV 35S promoter, be called pBI76D1.

2. Agrobacterium-mediated plant transforms

Agrobacterium GV3101 has the ability infecting plant and metastatic gene, therefore the UGT76D1 plant expression vector (pBI76D1) built is proceeded to Agrobacterium, then carries out PCR checking and digestion verification.Utilize flower-dipping method (a kind of disclosed ordinary method), make the Agrobacterium GV3101 containing plant expression vector contaminate Arabidopis thaliana bud.After treating its angle fruit maturation grown, collect T1 for seed and at screening culture medium (MS substratum adds 30mg/L kantlex) enterprising row filter, the green transformation seedlings of normal growth can transplant and cultivate to Nutrition Soil, gather in the crops its T2 carries out next round again kantlex screening for seed respectively, pick out green seedling: the culture dish of Bai Miaowei 3:1.By the green transplantation of seedlings on this culture dish, individual plant results seed (T3 generation).The screening of kantlex plate is used for the seed fraction of each individual plant, until selecting in screening culture medium is entirely green strain, is Transgenic wheat line.

3. transfer-gen plant Molecular Identification

Above-mentioned transfer-gen plant is carried out to the detection of gene expression dose.Extract the RNA of transfer-gen plant and WT lines respectively, after reverse transcription, carry out RT-PCR amplification, analyze the gene expression difference of process LAN plant and WT lines.The expression amount of UGT76D1 in process LAN plant is all apparently higher than WT lines.Utilize the strain that two UGT76D1 expression amounts are high, i.e. OE-1, OE-2, carry out follow-up work.

4.UGT76D1 gene reduces the functional verification of plant surface wax

(1) one of the evidence that reduces of UGT76D1 transgenic plant surface wax.

After control plant and process LAN strain normally cultivate 6 weeks, namely naked eyes can be observed process LAN strain, and all to show cauline leaf surface emerald green, and wax reduces.See Fig. 1, wherein WT is control plant, OE-1 and OE-2 is two genetically modified process LAN strains of UGT76D1.

(2) evidence two of UGT76D1 transgenic plant surface wax minimizing.

Blade is after aniline blue dyeing, and two process LAN strains are more easily painted, and further proof list mercolized wax matter reduces, and adds the infiltration of staining agent.See Fig. 2.

(3) evidence three of UGT76D1 transgenic plant surface wax minimizing.

Sem observation is carried out on cane surface, and experiment material is the normal growth Arabidopis thaliana control plant of 2 weeks and two process LAN strains.Choose growth conditions consistent co-altitude stem section, scanning electronic microscope detects cane surface wax, found that two strain process LAN system cane surface waxs significantly reduce, see Fig. 3, wax is superficial white grainy texture, and WT is control plant, OE-1 and OE-2 is two process LAN strains.

(4) evidence four of UGT76D1 transgenic plant surface wax minimizing.

Chlorophyll extracts experiment, and experiment material is the normal growth Arabidopis thaliana control plant of 2 weeks and two process LAN strains.Choose the plant that growth conditions is consistent, clip is leaf on the ground, is immersed in the ethanol of 80%, measures the absorbancy of solution every 20min at 647nm and 664nm place, calculates chlorophyll extraction yield.The results are shown in Figure 4, due to the minimizing of surperficial wax, the chlorophyll extraction yield of two process LAN strains is apparently higher than contrast.WT is control plant, OE-1 and OE-2 is two process LAN strains.Again prove that UGT76D1 gene is reducing the new function in wax.

Claims

1. Arabidopis thaliana glycosyltransferase gene UGT76D1 is reducing the application in plant surface wax.

2. apply as claimed in claim 1, it is characterized in that: the nucleotide sequence of described glycosyltransferase gene UGT76D1 is as shown in SEQ ID No.1.

3. apply as claimed in claim 1, it is characterized in that: described plant is cress.

4. apply as claimed in claim 3, it is characterized in that: described cress is Arabidopis thaliana, rape, Chinese cabbage, wild cabbage or leaf mustard.