CN102796762B - Application of arabidopsis glycosyl transferase gene UGT 76C2 in improving plant drought resistance - Google Patents
Application of arabidopsis glycosyl transferase gene UGT 76C2 in improving plant drought resistance Download PDFInfo
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- CN102796762B CN102796762B CN 201210334363 CN201210334363A CN102796762B CN 102796762 B CN102796762 B CN 102796762B CN 201210334363 CN201210334363 CN 201210334363 CN 201210334363 A CN201210334363 A CN 201210334363A CN 102796762 B CN102796762 B CN 102796762B
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Abstract
The invention discloses an application of an arabidopsis glycosyl transferase gene UGT 76C2 in improving plant drought resistance, wherein the nucleotide sequence of the glycosyl transferase gene UGT 76C2 is shown as SEQ ID No.1, and is cloned from arabidopsis through a reverse transcription-polymerase chain reaction (RT-PCR) technology. The application utilizes the gene UGT 76C2 to structure a plant over expression carrier, the plant transgenic operation is carried out, and a transgenic plant is obtained. Tests prove that drought resistance of the transgenic plant is obviously improved, and indicate that a novel drought-resistant plant can be created after the glycosyl transferase gene UGT 76C2 is implemented; and the glycosyl transferase gene UGT 76C2 can be used for subsequent crop variety improvement, and has an important meaning to agricultural production of China.
Description
Technical field
The present invention relates to a glycosyltransferase gene and application thereof, relate in particular to the application of a kind of Arabidopis thaliana glycosyltransferase gene UGT76C2 in improving drought resistance in plants, belong to the genetically engineered field.
Background technology
Glycosyltransferase is the enzyme of being responsible for the glycosylation modified reaction of catalysis specially, and it is transferred to active glycosyl on acceptor molecule from donor (normally UDP-glucose).The glycosylation modified biological activity that tends to change plant molecular, water-soluble, in cell and the transport features of whole plant, Subcellular Localization and with mutual identification and the binding characteristic of acceptor, can also reduce or eliminate in addition endogenous and toxicity (Lim and Bowles, 2004 allogenic material; Bowles et al., 2006; Wang and Hou, 2009).Therefore, glycosyltransferase gene in regulating plant cellular metabolism balance, to keep the aspects such as plant normal growth growth significant.For example, have been reported the synthetic and plant signal transduction of the adjusting of glycosyltransferase gene involved in plant hormonal equilibrium, plant defense response, Plant Secondary Metabolites etc. (Wang and Hou, 2009).
The glycosyltransferase that organic sphere exists adheres to 94 different families separately according to substrate properties and the serial correlation of institute's catalysis.Wherein the number of members that comprises of family 1 is maximum, and is the closest with the relation of plant.Most of gene C ends have a conserved sequence that is comprised of 44 amino acid in family 1, i.e. PSPG box (plant secondary product glycosyltransferase box).Along with completing of Arabidopis thaliana (Arabidopsis thaliana) genome sequencing, by the sequential analysis of PSPG box is found to have 119 possible glycosyltransferases in Arabidopis thaliana, in these glycosyltransferases, most function is not clear.
UGT76C2 is a member in Arabidopis thaliana glycosyltransferase family 1, and its gene order is disclosed in GenBank at present.But through retrieval, the application of Arabidopis thaliana glycosyltransferase gene UGT76C2 in strengthening drought resistance in plants has no report at present.
Summary of the invention
(1) purpose of the present invention
For the deficiencies in the prior art, the purpose of this invention is to provide the application of a kind of Arabidopis thaliana glycosyltransferase gene UGT76C2 in improving drought resistance in plants.
(2) realize concrete technical scheme of the present invention
The application of Arabidopis thaliana glycosyltransferase gene UGT76C2 of the present invention in improving drought resistance in plants.
Wherein: the nucleotide sequence of described glycosyltransferase gene UGT76C2 is as shown in SEQ ID No.1.Described plant optimization is cress, and described cress is Arabidopis thaliana, leaf mustard, rape, Chinese cabbage or wild cabbage preferably.
The present invention utilizes the primer sequence shown in SEQ ID No.3 and SEQ ID No.4, clone glycosyltransferase gene UGT76C2 from Arabidopis thaliana by the RT-PCR technology, then utilize this gene constructed plant over-express vector, carry out the plant transgene operation, obtain transgenic plant.Detection shows that the drought tolerance of transgenic plant is significantly improved.
The beneficial effect that (3) may bring after the invention process
Experiment confirm is used Arabidopis thaliana glycosyltransferase gene UGT76C2 of the present invention and is carried out the plant transgene operation, can significantly improve the drought tolerance (seeing accompanying drawing 1, accompanying drawing 2 and accompanying drawing 3) of transgenic plant.Will create novel drought-enduring plant after the indication the invention process, can be used for follow-up improvement of crop cultivar, China's agriculture production is significant.
Description of drawings
Fig. 1. arid is processed experiment.Wherein Col-0 is the Arabidopis thaliana control plant, and ugt76c2 is mutant, and UGT76C2OE4-2 and UGT76C2OE19-2 are two and cross the expression strain.Then Arabidopis thaliana control plant, mutant and cross that the expression strain is normal cultivated for 2 weeks stopped watering 12 days, and wilting almost all appears in mutant, and wilting appears in wild-type contrast only part, and two crossed the expression strain almost without the wilting phenomenon.Obviously strengthened the drought resistance of plant after this explanation UGT76C2 gene overexpression.
Fig. 2. the survival rate statistics after arid is processed.Wherein Col-0 is the Arabidopis thaliana control plant, and ugt76c2 is mutant, and UGT76C2OE4-2 and UGT76C2OE19-2 are two and cross the expression strain.Then Arabidopis thaliana control plant, mutant and cross that the expression strain is normal cultivated for 2 weeks stopped watering 12 days, then recover to water 3 days, the survival rate of statistics plant.Two mistakes of result are expressed the survival rate of strain apparently higher than wild-type contrast and mutant.The survival rate of mutant ugt76c2 is minimum.This illustrated that the drought tolerance of expressing strain was the strongest, had obviously strengthened the drought resistance of plant after the UGT76C2 gene overexpression.
Fig. 3. the percentage of water loss that exsomatizes experiment.Wherein Col-0 is the Arabidopis thaliana control plant, and ugt76c2 is mutant, and UGT76C2OE4-2 and UGT76C2OE19-2 are two and cross the expression strain.Experiment material is Arabidopis thaliana control plant, mutant and two mistake expression strains in 2 weeks of normal growth.Choose the consistent plant of growth conditions, the clip over-ground part is put on the table top under room temperature, and the fresh weight every 20min measures plant calculates the stripped percentage of water loss of each time period.2 mistakes stripped percentage of water loss of expressing strain is minimum as a result, illustrates that expression strain moisture loss was slower, and water-retentivity is better than wild-type contrast and mutant.The effect of UGT76C2 gene aspect the enhancing drought resistance has been described.
Embodiment
Embodiment 1 clone's Arabidopis thaliana glycosyltransferase gene UGT76C2
1. the clone of Arabidopis thaliana glycosyltransferase gene UGT76C2
Obtain the cDNA sequence of UGT76C2 gene by open website http://www.cazy.org.According to the cDNA primers, forward primer is 76C2-F:5 '-CGCGGATCCGCCGCCATGGAGGAGAAGAGAAATGG-3 ', and reverse primer is 76C2-R:5 '-CGGGAGCTCTTACAACAATAGTATATGATTAGCT-3 '.Utilize the TRIzol test kit to extract Arabidopis thaliana RNA, the full length cDNA sequence of RT-PCR method amplification UGT76C2 gene.First to cut through BamHI and Sac I enzyme with cDNA clone's process, be connected into afterwards in pBluescript II SK (+) carrier that corresponding enzyme cuts, be built into the order-checking intermediate carrier, be called pK76C2, then carry out full length gene pcr amplification and BamHI and Sac I enzyme with carrier and cut checking, carry out at last sequencing, the exactness of checking cloned sequence.
2. the sequence information of Arabidopis thaliana glycosyltransferase gene UGT76C2 and specificity analysis
The coding region cDNA of UGT76C2 gene is 1353bp, and 450 the amino acid whose 57.6kDa albumen of encoding, C end have 44 amino acid whose PSPG boxes, the conserved sequence that is jointly had for the Plant Secondary Metabolites glycosyltransferase.
3. the expression analysis of Arabidopis thaliana glycosyltransferase gene UGT76C2
Tissue specific expression: each histoorgan to the wild-type Arabidopis thaliana Col-0 of maturation comprises that root, stem, lotus throne leaf, stem leaf, flower, angle fruit etc. carry out RT-PCR and analyze, and finds that UGT76C2 has expression in each histoorgan.
UGT76C2 promoters driven GUS expresses: in order to locate the meticulous position of UGT76C2 genetic expression, the fragment of UGT76C2 promoter region 2000bp is building up to pBI121 (GUS expression vector), replaces the 35S promoter on this carrier.By Agrobacterium GV3101 arabidopsis thaliana transformation, screening at last obtains the pUGT76C2 promotor:: GUS is sheerly plant.The GUS staining analysis is found, the Seedling Stage within 4 days, and UGT76C2 has than strongly expressed in root, hypocotyl, cotyledon.At growth and development stage subsequently, UGT76C2 has than strongly expressed in titbit handle, titbit, seed, root.
The transgenosis of embodiment 2 Arabidopis thaliana glycosyltransferase gene UGT76C2 is used
1. the structure that contains UGT76C2 coding region cDNA expression vector
After sequencing vector process BamHI and Sac I double digestion, obtain to cut with enzyme the full length cDNA sequence of sticky end in the middle of pK76C2.With this gene fragment with cut with corresponding enzyme enzyme after the pBI121 carrier part be connected, obtain driving glycosyltransferase gene with the CaMV 35S promoter and cross the plant expression vector of expression, be called pBI76C2.
2. agriculture bacillus mediated Plant Transformation
Agrobacterium GV3101 has the ability that infects plant and metastatic gene, therefore change the UGT76C2 plant expression vector (pBI76C2) that builds over to Agrobacterium, then carries out PCR checking and enzyme and cuts checking.Utilize flower-dipping method (a kind of disclosed universal method), make the Agrobacterium GV3101 that contains plant expression vector contaminate the Arabidopis thaliana bud.After treating its angle fruit maturation that grows, collect T1 for seed and at the enterprising row filter of screening culture medium (the MS substratum adds the 30mg/L kantlex), green transformation seedlings that can normal growth is transplanted to Nutrition Soil and is cultivated, gather in the crops respectively its T2 and carry out again the kantlex screening of next round for seed, pick out green seedling: the culture dish of Bai Miaowei 3:1.With the green transplantation of seedlings on this culture dish, individual plant results seed (T3 generation).Kind subdivision to each individual plant is used for the screening of kantlex plate, is complete green strain until select on screening culture medium, is Transgenic wheat line.
3. transfer-gen plant Molecular Identification
Above-mentioned transfer-gen plant is carried out the detection of gene expression dose.Extract respectively the RNA of transfer-gen plant and wild-type plant, carry out the RT-PCR amplification after reverse transcription, analyzed the gene expression difference of expressing plant and wild-type plant.The expression amount of UGT76C2 in excessively expressing plant is all apparently higher than the wild-type plant.Utilize two strains that the UGT76C2 expression amount is high, namely UGT76C2OE4-2, UGT76C2OE19-2, carry out follow-up work.
4.UGT76C2 the drought resistance functional verification of gene
(1) arid of seedling is processed experiment.To Arabidopis thaliana control plant (Col-0), mutant (ugt76c2), two mistake expression strains (UGT76C2OE4-2, UGT76C2OE19-2) in 2 weeks of normal growth stop watering 12 days in soil, found that wilting almost all appears in mutant, the wild-type contrast has part to occur wilting, and two mistake expression strains are almost without wilting phenomenon (seeing accompanying drawing 1).And then recover to water 3 days, the survival rate of statistics plant.Two mistakes of result are expressed the survival rate of strain apparently higher than wild-type contrast and mutant.The minimum (see figure 2) of the survival rate of mutant ugt76c2.These results can obviously strengthen the drought resistance of plant after showing the UGT76C2 gene overexpression.
(2) motion of the Stoma of Leaves under arid treatment condition.When plant was subject to drought stress, in order to reduce the moisture loss in body, plant can start the defense mechanism of self, for example regulate the aperture of pore and resist stress conditions, so experiment test the motion conditions of pore under the drought stress.Under the normal growth condition, the stomatal aperture of Col-0, ugt76c2, UGT76C2OE4-2, UGT76C2OE19-2 does not show any difference, after the blade arid is processed 0.5 and 1 hour, variation has in various degree occured in the pore opening degree of above four strains, the aperture that showed as expression body UGT76C2OE4-2, UGT76C2OE19-2 is significantly less than wild-type Col-0, and mutant ugt76c2 stomatal aperture is greater than wild-type.Illustrate that UGT76C2 can reduce scattering and disappearing of moisture by reducing stomatal aperture after crossing expression, improves water retention capacity.
(3) stripped percentage of water loss experiment.Experiment material is Arabidopis thaliana control plant, mutant and two mistake expression strains in 2 weeks of normal growth.Choose the consistent plant of growth conditions, the clip over-ground part is put on the table top under room temperature, and the fresh weight every 20min measures plant calculates the stripped percentage of water loss of each time period.2 mistakes stripped percentage of water loss of expressing strain is minimum as a result, illustrates that expression strain moisture loss was slower, and water-retentivity is better than wild-type contrast and mutant.The effect (see accompanying drawing 3) of UGT76C2 gene aspect the enhancing drought resistance has been described again.
Claims (1)
1. the application of Arabidopis thaliana glycosyltransferase gene UGT76C2 in improving drought resistance in plants, the nucleotide sequence of wherein said glycosyltransferase gene UGT76C2 is as shown in SEQ ID No.1, and described plant is the cress Arabidopis thaliana.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195150A (en) * | 2014-09-25 | 2014-12-10 | 山东大学 | Application of arabidopsis glycosyl transferase gene UGT79B2 in improving salt resistance and drought resistance of plants |
| CN104975033A (en) * | 2015-06-11 | 2015-10-14 | 山东大学 | Use of arabidopis thaliana glycosyl transferase gene UGT76D1 in reduction of plant surface wax |
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| CN107012155A (en) * | 2016-01-28 | 2017-08-04 | 刘春生 | Participate in UGT genes and its coded product and the application of glycyrrhizic acid biosynthesis |
| CN106591338B (en) * | 2016-12-14 | 2019-07-19 | 南京工业大学 | Method for increasing soluble heterologous expression of plant-derived glycosyltransferase gene |
| CN107164396A (en) * | 2017-06-26 | 2017-09-15 | 吉林大学 | Uridine 5'-diphosphate glycosyltransferase gene Ugt366 clone and application |
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| CN116445446B (en) * | 2023-05-10 | 2024-05-03 | 天津科润农业科技股份有限公司 | Wild cabbage glycosyltransferase BoUGT C2 gene and application |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104195150A (en) * | 2014-09-25 | 2014-12-10 | 山东大学 | Application of arabidopsis glycosyl transferase gene UGT79B2 in improving salt resistance and drought resistance of plants |
| CN104975033A (en) * | 2015-06-11 | 2015-10-14 | 山东大学 | Use of arabidopis thaliana glycosyl transferase gene UGT76D1 in reduction of plant surface wax |
| CN104975033B (en) * | 2015-06-11 | 2017-10-27 | 山东大学 | Applications of the arabidopsis glycosyltransferase gene UGT76D1 in plant surface wax is reduced |
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