CN105802976A - Gene for regulating rice resistance to drought and salt/alkali and application thereof - Google Patents
Gene for regulating rice resistance to drought and salt/alkali and application thereof Download PDFInfo
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Abstract
The invention discloses an application of a rice ERF family member OsERF2 in regulating abiotic stress of rice. The OsERF2 expression or gene silencing influences the carbohydrate accumulation of rice and the rice resistance to drought and salt/alkali. Transgenic rice with OsERF2 gene silencing is created by the transgenic technology, and the obtained transgenic plant has resistance to drought and salt/alkali; and therefore, the OsERF2 gene is of a great application value in modifying the plant stress resistance and crop varieties.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of adjusting and controlling rice to the gene of arid and saline and alkaline patience and in the reaction of plant control environment-stress and the application in crops transgene improvement.
Background technology
Shortage of water resources and the soil salinization are that Global Agriculture produces the severe fact faced.The arable land of China has the soil of nearly 600,000,000 mu by the most saliferous impact, soil salination be cause China western and in the area such as Coastal beach, low-yield land and large area soil resource be difficult to the immediate cause that effectively utilizes.Owing to drought is frequent, China's annual area suffered from drought reaches 3.27 hundred million mu, and along with global warming, drought will be on the rise, and is threatened farmland area can exceed that 700,000,000 mu by drought.Therefore water resources shortage and Saline Alkali Stress have become the important restriction factor affecting rice high yield, stable yields.
Under the abiotic stress such as high salt and arid, plant accumulates and reduces loss of water by intercellular infiltration material, and then obtains Thief zone state and maintain cell rigidity.Crop response environment Stress responses includes the change of the levels such as form, physiology, biochemistry, cytology and molecular biology.When plant suffers from adverse circumstance, many stress responsive gene are induced to express differential protein, regulation plant physiology and biochemistry and the change of metabolism, thus adapt to external stress.Plant by after arid, cell rapid perception outer signals, by the signal transduction of series of complex and activate specific transcription regulatory factor, then by specific cis acting element, the transcriptional expression of a large amount of relevant target gene of regulation and control, the drought tolerance of final raising plant.Crop is all the composite defense reaction participated in by polygenes for the degeneration-resistant reaction of the abiotic stress such as low temperature, arid, high salt and disease and biotic, therefore, in improving the crop molecular breeding to environment-stress, certain limitation is there is in the transgenic strategy of single resistance functional gene to the improvement of cropper resistance, and the regulation and control strengthening a crucial transcription factor can regulate and control the expression of multiple downstreams resistance functional gene simultaneously, improve the more effectively method of crop anti-adversity.
In Oryza sativa L. at least 186 transcription factor genes by arid or high Salt treatment.The ethylene response factor (ERF) contains a conservative ERF domain, has more than 170 ERF gene in Oryza sativa L., regulates and controls the processes such as degeneration-resistant and growth promoter.Rice Os DREB1A overexpression can improve transgenic paddy rice to arid, high salt and the patience of freeze injury；Overexpression OsDREB1G and OsDREB2B improves transfer-gen plant to arid, salt and the patience of low temperature.Oryza sativa L. ERF factor S ub1A and Sub1C play an important role during metabolic regulation etc. at flooding stress responsing reaction, growth promoter.Therefore, utilizing transcription factor to cultivate resistance to rice varieties against the current by biotechnology is to make full use of China salt-soda soil, alleviation shortage of water resources, the effective way of guarantee rice high yield stable yields, to ensureing that agricultural sustainable development has important practical significance.
Summary of the invention
The present inventor creates the transgenic paddy rice of rice Os ERF2 gene silencing, and resistance phenotype analytical finds that this gene silencing can strengthen Oryza sativa L. to arid and the patience of high-salt stress, shows the OsERF2 adjusting and controlling rice responsing reaction to the environment stress such as arid and saline and alkaline.Under normal growth state, glucose activated donor UDPG(uridine diphosphoglucose in the transgenic paddy rice of OsERF2 gene silencing) content higher than non-transgenic Oryza sativa L., show OsERF2 may by the accumulation of UDPG in regulation Oryza sativa L. body thus improve transgenic paddy rice to arid and saline and alkaline patience.
Present invention provide the technical scheme that a kind of Oryza sativa L. ERF gene, its nucleotide sequence is as shown in SEQ ID NO:1, and referred to as OsERF2 gene, its encoding amino acid sequence is as shown in SEQ ID NO:2.This gene silencing improves rice seedling to arid and saline and alkaline patience.
The present invention also provides for comprising the expression vector of described gene, preferably plant expression vector, more preferably pCAMBIA5300-AmiERF2 expression vector.
The present invention also provides for described gene and is cultivating the application of degeneration-resistant crop varieties, is to import in plant by the expression vector including described gene, and screening obtains the plant that resistance improves, and is preferably Oryza sativa L..
The method have the advantages that the present invention studies to show the environment stresses such as OsERF2 is arid in Oryza sativa L. response, saline and alkaline reaction plays an important role.Created the transgenic paddy rice of OsERF2 gene silencing by transgenic technology, transfer-gen plant has patience to relevant coercing, and then cultivates the transgenic plant kind that resistance strengthens.Experiment shows, the DNA sequencing fragment Introduced into Rice of encoding regulator plant salt stress inducible gene OsERF2 of the present invention will make the reticent process LAN part of OsERF2 gene suppress (by the expression of RNAi method suppression rice Os ERF2) by artificial tiny RNA method (artificial microRNA), it is possible to increase transgenic paddy rice salt tolerance and drought tolerance.
Accompanying drawing explanation
In Fig. 1 gene silencing transgenic paddy rice, OsERF2 gene expression dose reduces, and is followed successively by WT, Ami-957, Ami-958, Ami-991 from left to right.
Fig. 2 grows does not has notable difference between transgenic paddy rice and the wild rice of 2 weeks.
The salt tolerance of the transgenic paddy rice of Fig. 3 OsERF2 gene silencing strengthens.
The drought tolerance of the transgenic paddy rice of Fig. 4 OsERF2 gene silencing strengthens.
The accumulation of Fig. 5 OsERF2 regulation Oryza sativa L. UDPG.
Fig. 6 pCAMBIA5300-AmiERF2 expression vector.
Detailed description of the invention
It is further elucidated with the present invention below by the detailed description of detailed description of the invention, but is not limitation of the present invention, only illustrate.
The establishment of embodiment one transgenic paddy rice material
The present inventor creates the transgenic paddy rice of OsERF2 gene silencing by Agrobacterium infestation method.
Gene silencing methods is as follows:
Its nucleotide sequence gene (OsERF2 gene) 3 ' end non-coding area sequence AGCATCATTTAGAGCATAA as shown in SEQ ID NO:1 is utilized as aim sequence, to design about 21 bases amiRNA by bioinformatic analysis；Within miRNAs precursor (pre-miRNA) skeleton be template, use over-lap PCR build amiRNA gene；AmiRNA gene is cloned on corresponding plant conversion carrier；By transgenic technology, amiRNA is imported in plant, utilize the miRNA elaboration system of plant to express amiRNA silencing of target genes.Building process is as follows: utilize WMD3 software Designer module (http://wmd3.weigelworld.org) to design amiRNA sequence first against ERF2 gene, 4 amiRNA overlapping PCR primers are designed with pNW55 for carrier with Oligo module, i.e. PI miR-s1th agTTTATGCTCTAAATGATGCTGcaggagattc agtttga, PII miR-a1th tgCAGCATCATTTAGAGCATAAActgctgctgctacagcc, PIII miR*s1th
CtCAGCAACATATAGAGCATAAAttcctgctgctaggctg and PIV miR*a1th
aaTTTATGCTCTATAT GTTGCTGagagaggcaaaagtgaa.In conjunction with universal primer G-4368 (5 '-CTGCAAGGCGATTAAGTT
GGGTAAC-3 ') and G-4369 (5 '-GCGGATAACAATTTCACACAGGAAACAG-3 ') with the plasmid pNW55 precursor miRNA sequence of 254bp of endogenous miR-96 gene osa-MIR528 of Oryza sativa L. (clone have) as template, use 3 to primer G-4368+PII, P I+P IV, P III+G-4369 carry out PCR amplification.Mix as template after respectively PCR primer being reclaimed, use primer that (G-4368+G-4369) carries out fusion DNA vaccine and obtain the product of 554b, plant conversion carrier pCAMBIA5300 is proceeded to after using BamHI/KpnI double digestion, obtain expression vector (pCAMBIA5300-AmiERF2) (referring to Fig. 6), import in Agrobacterium tumefaciems AGL0.
Method by the transgenic paddy rice of Agrobacterium infestation method establishment OsERF2 gene silencing is as follows:
1) induction of EMBRYO IN RICE wound healing and subculture: shelled by rice paddy seed (more light), by 75% soak with ethanol 1min, soaks 30min, aseptic water washing with liquor natrii hypochloritis, repeats immersion 15min with sodium hypochlorite, use aseptic water washing.Rice paddy seed after sterilizing is dried on sterilizing filter paper, plants in inducing culture.28 DEG C of light culture 2 weeks.Separate callus and be transferred to 28 DEG C of subcultures of inducing culture 3 times.Picking color is yellowish and quality 28 DEG C of light culture of loose wound healing granule 3 days.
2) actication of culture and expanding propagation: under the conditions of 28 DEG C, by the Agrobacterium containing destination gene expression carrier (pCAMBIA5300-AmiERF2) on YEB solid medium streak culture 2 days.
3) the co-culturing of Agrobacterium and callus: take appropriate thalline and be suspended in 100 μMs of AS+AA fluid mediums, when bacterium solution OD value reaches 0.3, make callus immerse in bacterium solution, jog 20 minutes.Abandon bacterium solution, take out wound healing, be filtered dry unnecessary bacterium solution, transfer on NB solid medium 22 DEG C of light culture 3 days.
4) screening kanamycin-resistant callus tissue: selecting the callus co-cultured, rinsed with sterile water, be laid in screening culture medium after being filtered dry, 2 weeks follow-up generations of 28 DEG C of light culture are once.
5) pre-differentiation and differentiation: choosing grows the vigorous callus being creamy white and goes on pre-division culture medium, 28 DEG C of light culture 1 week, 28 DEG C of illumination cultivation 2 weeks, when seeing at green, subculture is once, seedling differentiation after 4 weeks.
6) take root and strong sprout: preferable for growing way seedling is moved on root media, transplant after cultivating 2 weeks.
7) results to seed are cultivated by growing normal transgenic seedling intermediate house.
8) seedling of the transgenic positive plant obtained, obtains T1 and the seed of advanced lines through adding generation.
Embodiment two
In different transgenic lines
OsERF2
Expression identify
TRIzol method extracts 2 week old wild types and OsERF2 rotaring gene plant blade total serum IgE, takes 2 μ g total serum IgE, with 2 μ RNase-free DNase
(Promega) 37 DEG C process 30 min, with polyA as primer, M-MLV reverse transcription 42 DEG C is utilized to synthesize cDNA the first chain, then with cDNA as template, with Ubiquitin (primer 5-CCATCCTCAAGCTGCTTACC-3 and 5-GACTGGCAAGACC ATTACCC-3) as internal reference, PCR method detection OsERF2 gene (primer 5-AGATCTCCTGCTGA TGTGCC 3 and 5-CACAAGCTGCTCCAGAACTC-3) is expressed, and result is as shown in Figure 1.Relative to Wild type control plants, the expression of OsERF2 is reduced to the 30%-50% of wild type in transfer-gen plant (Ri-1, Ri-2).This shows that the expression of OsERF2 in transfer-gen plant is reticent by part.
Embodiment three
OsERF2
Gene silencing can improve Oryza sativa L. to arid and saline and alkaline patience
After wild type and OsERF2 transgenic paddy rice seed 42 DEG C process 3 days, Yu Shuizhong soaks seed 24 hours, 37 DEG C of accelerating germination, choose the consistent seed of rudiment in equipped with in the little basin of Nutrition Soil, cover one layer of preservative film moisturizing, after coming up, remove preservative film, after 2 weeks, the little basin of rice cultivation Seedling is put into containing 100 mM
Soaking 1 week in the water of NaCl, then poured out by saline, change a water every day, the physiological phenotype after normal 3 weeks, as in figure 2 it is shown, do not have obvious phenotypic difference between transgenic paddy rice and wild rice, shows that this hidden silence does not affect the normal growth of Oryza sativa L..But 2 weeks seedling are early by saline treatment rehydration after 5 days after 1 week, the almost all of Wild type control plants is dead, and the plant major part of transgenic paddy rice strain Ami957 of OsERF2 gene silencing, Ami958 and Ami991 is also survived normally, it is significantly higher than wild type control (seeing Fig. 3).For Osmotic treatment, little for the rice seedlings of 2 weeks basin carries out naturally arid guttation process, after 6 days, wild type starts slight leaf rolling, and the transgenic paddy rice blade of gene silencing is the most normally unfolded, the blade of major part WT lines extremely curling or withered and yellow after 8 days, and transgenic paddy rice blade is the most mostly unfolded, result shows compared with non-transgenic wild type control, and the drought tolerance of the transgenic paddy rice strain of OsERF2 gene silencing significantly improves (seeing Fig. 4).
Embodiment four
In transgenic paddy rice
UDPG
The change of content
UDPG is the glucose glycosyl donor of a kind of activation, plant carbohydrate metabolism, cellulosic electrode, nourishing and growing plays important regulating and controlling effect in reproductive growth conversion and the physiological process such as stress response reaction.The present inventor compares the accumulation of UDPG between wild rice and the transgenic paddy rice of gene silencing by the method for liquid chromatograph, result is as shown in Figure 5, in the transgenic paddy rice of gene silencing, UDPG accumulation is higher than wild rice, and the accumulation of UDPG may have important regulating and controlling effect in terms of transgenic paddy rice resistance of reverse raising.
Embodiment five
In transgenic paddy rice
OsERF2
Expression analysis
Gene expression analysis analysis method: take 2 weeks transgenic paddy rices and the blade of wild rice, extracts test kit (MagMAX Total RNA Isolation Kit) with RNA and extracts RNA.Use M-MLV test kit (Promega)
Synthesis cDNA.On ABI PRISM7000, the expression of candidate gene is analyzed with SYBR Premix Ex TaqTM (TaKaRa).PCR condition is 95 DEG C of 4 min, 95 DEG C of 15 s, 59 DEG C of 30s, 72 DEG C of 30s, totally 40 circulations, using ubiquitin as internal reference, analyzes gene expression dose by 2-CT method.Relative to wild rice, the expression of OsERF2 reduces in transgenic paddy rice.
<110>Biological Technology institute, Chinese Academy of Agricultural Sciences
<120>a kind of adjusting and controlling rice is to the gene of arid and saline and alkaline patience and application thereof
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ATGGCGCGGC
CGCAGCAGCG GTATCGCGGCGTGCGGCAGCGCCACTGG GGCTCATGGGTCTCCGAGATCC GCCACCCTCT
CCTGAAGACGAGGATCT GGCTGGGCACGTT CGAGACGGCGGAGGACGCGG CACGCGCGTACGACG AGGCG
GCGCGGATCA TGTGCGGCCC GCGCGTGCGCACCAACTTCCCCC ACGACGT CGCCGACGAG GCCGCGCCGC
CGCCGCCGCC GCACAGCGCCG CCGCAGCCT CCTCGTCGTT CCTCTCCGCG GCGCTCGTCGCCAAGCTCCA
CCGCTTCAACCTCGCCTCCG TCCAGGCTGC GCAGCGCGGCAACAGCAA CGACGACGACTCCACCACCTCCT
CCTCCGCCG CCGCGTCGTCGCGCGCCG
TGATTCCGTCCCTTCCCGCCGCCGCCGGCGCATTGGGCAATGCGGCGGCGA CGGCGGAGTGG
AGCGGCGGGTTCCTCGAGGAGCAGTACGTGGACCAGAT GA TCGAGGAGCT CCTCGACTCC AACTTCTCCA
TGGAGATCTC CTGCTGA ATGTGCCCTG TTCATCTACC TGTTCTTCTC TGCTCCATCT CCTGTTTCTTTC
TCTCTCTCTTTTTTTTCTTTCTTTTTTTTTGTTTTTTTAGTTTAGTAAGCTATGTGAG GAAGAACTCTGATCGAGGTT
AGTTTGGTCA CAGTGAGTTCTGGA GCAGCT TGTGTATACG GTAGCATCATTTAGAGCATA ATAGGGTTGC
AGTT GAGACC TTC
<210>
2
<211>
178
<212>aminoacid
<400>
2
MARPQQRYRGVRQRHWGSWVSEIRHPLLKTRIWLGTFETAEDAARAYDEAARIMCGPRVRTNFPHDVADEAAPPPPPHSAAAASSSFLSAALVAKLHRFNLASVQAAQRGNSNDDDSTTSSSAAASSRAVIPSLPAAAGALGNAAATAEWSGGFLEEQYV
DQMIEELLDSNFSMEISC
Claims (8)
1. an Oryza sativa L. ERF gene, its nucleotide sequence is as shown in SEQ ID NO:1, or its degenerate sequence.
2. comprise the carrier of gene described in claim 1.
3. carrier as claimed in claim 2, it is plant expression vector.
4. carrier as claimed in claim 3, it is pCAMBIA5300-AmiERF2 expression vector.
5. the method cultivating the crop varieties that resistance improves, it is characterised in that the gene as claimed in claim 1 in plant is carried out silence by gene engineering method, to obtain the plant that resistance improves.
6. method as claimed in claim 5, described plant is Oryza sativa L..
7. method as claimed in claim 5, described resistance rises to arid and/or saline and alkaline patience and improves.
8. the method as described in any one of claim 5-7, it is characterised in that described gene engineering method is RNAi interference method.
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| CN201510439176.1A CN105802976B (en) | 2015-07-23 | 2015-07-23 | Gene for regulating and controlling drought and saline-alkali tolerance of rice and application thereof |
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| CN105802976B CN105802976B (en) | 2020-04-17 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109354614A (en) * | 2018-11-30 | 2019-02-19 | 中国农业科学院生物技术研究所 | OsCSLD4 albumen is improving the application in plant salt stress tolerance |
| CN109385431A (en) * | 2017-08-09 | 2019-02-26 | 中国农业科学院生物技术研究所 | The application of OsERF2 gene regulation rice grain size |
-
2015
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| KIKUCHI S.等: "AK108355.1", 《GENBANK》 * |
| MATSUMOTO T.等: "BAF18895.1", 《GENBANK》 * |
| MISHRA M.等: "Plant omics:genome-wide analysis of ABA repressor1(ABR1) related genes in rice during abiotic stress and development.", 《OMICS》 * |
| 丰锦等: "抗逆相关AP2 /EREBP 转录因子研究进展", 《生物技术通报》 * |
| 杨德鑫: "水稻ERF蛋白OsERF109的特性及功能分析", 《中国优秀硕士学位论文全文数据库》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109385431A (en) * | 2017-08-09 | 2019-02-26 | 中国农业科学院生物技术研究所 | The application of OsERF2 gene regulation rice grain size |
| CN109385431B (en) * | 2017-08-09 | 2021-10-08 | 中国农业科学院生物技术研究所 | Application of OsERF2 gene in regulation and control of rice grain size |
| CN109354614A (en) * | 2018-11-30 | 2019-02-19 | 中国农业科学院生物技术研究所 | OsCSLD4 albumen is improving the application in plant salt stress tolerance |
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| CN105802976B (en) | 2020-04-17 |
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