CN105112381A - Kiwi fruit AdADH1 gene and application - Google Patents
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Abstract
The invention discloses a kiwi fruit AdADH1 gene and application. Kiwi fruit AdADH1 is protein provided with an amino acid sequence represented by SEQIDNO.2 in a sequence table, and the encoding gene of the kiwi fruit AdADH1 is provided with a DNA sequence of SEQIDNO.1 representing the AdADH1 gene. Waterlogging damage, low temperature or salt damage induces expression of the kiwi fruit AdADH1 gene. Compared with wild type groups, the waterlogging resistance, salt resistance and low temperature resistance capacity of transgenic arabidopsis thaliana having AdADH1 over-expression is remarkably improved. It is shown that the kiwi fruit AdADH1 plays an important role on plants in the process of improving waterlogging damage resistance, salt damage resistance and low temperature resistance. Therefore, the AdADH1 gene and a recombinant expression vector containing the AdADH1 gene can be applied to development of novel germplasm resistant to low temperature, waterlogging and salt and/or variety improvement of the plants.
Description
Technical field
The present invention relates to plant genetic engineering field, be specifically related to Kiwifruit AdADH1 gene and application.
Background technology
Actinidia is in Actinidiaceae (Actinidiaceae) Actinidia (Actinidia) fallen leaves property vine fruit, and whole world actinidia has 21 mutation of 54 kinds, altogether about 75 taxonomical units, and germ plasm resource is extremely abundant.1904, New Zealand introduced a fine variety Kiwifruit from China, domestication, started breed breeding work and commercially produced.The principal item of cultivating in the market is selected from Kiwifruit and A.chinensis Planch. mostly, is selected from tara vine on a small quantity in addition.Kiwifruit is owing to containing extremely abundant nutritive value, and especially Vc content is very high, is described as " king of Vc ", so be more and more subject to the favor of people.
Jiangsu Province's wet season of summer is longer, and rainwater is more, produces bring stern challenge to Kiwi growth.2011, because rainwater in rainy season is more, damage or crop failure caused by waterlogging was serious, and Yangzhou a large amount of adult rhesus peach plant is dead, causes huge financial loss to orchard worker.Therefore, the waterlogging problem of Kiwifruit is one of principal element of restriction Jiangsu Province's Kiwifruit industry development.Seed selection resistance to overhead flooding injury stock and variety source solve the main path of this problem beyond doubt.The application in fruit tree field along with molecular biology and genetic engineering technique, using gene engineering technique means transform the resistance of Kiwifruit, are the targets of Resistence research from now on.Therefore, excavating and study the waterlogging gene of Kiwifruit will be cultivate Kiwifruit resistance to overhead flooding injury stock and variety source based theoretical by genetic engineering technique means.
At present, the research of concerned plant waterlogging genescreen aspect is less, and the research in waterlogging gene expression and regulation is very little especially.Kiwifruit Cultivars ' Jin Kui ' is one of Kiwifruit kind of China's seed selection, and drought resisting, waterlogging, freezing tolerance is stronger.Research in the waterlogging gene excavating of Kiwifruit have not been reported.Therefore clone and study Kiwifruit anti contravariance related gene, fruit tree adversity gene exploitation to Chinese independent intellectual property right has great importance.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of Kiwifruit AdADH1 albumen is provided.
Another object of the present invention is to provide the encoding gene of this Kiwifruit AdADH1 albumen.
Another object of the present invention is to provide the application of this gene, albumen.
Object of the present invention realizes by following technical scheme:
Kiwifruit AdADH1 albumen provided by the present invention, derive from Kiwifruit improved seeds ' Jin Kui ', aminoacid sequence is as shown in SEQIDNO.2.
The encoding gene of Kiwifruit AdADH1 albumen of the present invention, its cDNA sequence as shown in SEQIDNO.1, the maximum open reading frame containing 1143bp, coding 380 shown in a SEQIDNO.2 amino acid residue sequence.
The expression vector of the encoding gene containing AdADH1 albumen of the present invention, the expression vector of the encoding gene preferably containing the AdADH1 albumen shown in SEQIDNO.1.
The described expression vector further preferred encoding gene by described Kiwifruit AdADH1 albumen is inserted into gained between the Kpn I of pCAMBIA1301 carrier and Sac I restriction enzyme site.
The Host Strains of the encoding gene containing AdADH1 albumen of the present invention.
The agrobacterium tumefaciens EHA105. that pCAMBIA1301-AdADH1 preferably proceeds to by described Host Strains
The primer of amplification AdADH1cDNA total length is:
AdADH1-ORFsense：5'-GGAGTTTGTGAGAGAAGTTAGAG-3'
AdADH1-ORFantisense：5'-AGATTTCACCAGCAAACTCAC-3'
The qPCR primer of the AdADH1 related in real-time fluorescence quantitative RT-PCR analysis is:
AdADH1-qPCRsense：5'-TGGAGTGTACTGGAAGTGTCAATG-3'
AdADH1-qPCRantisense：5'-AGCAGGGAGGTCGGAACG-3'
Above-mentioned Kiwifruit AdADH1 albumen, its encoding gene, the expression vector containing encoding gene, the Host Strains containing this encoding gene are cultivating the application in waterlogging, low temperature resistant or salt-tolerant plant.
Beneficial effect:
The present invention is cloned into the encoding gene of an AdADH1 albumen in Jinkui kiwifruit, and AdADH1 participates in waterlogging, the low temperature resistant and salt tolerant stress procedure of Kiwifruit.
The encoding gene of AdADH1 is coerced in damage or crop failure caused by waterlogging, express under low temperature stress and salt damage stress-inducing condition.The encoding gene of overexpression AdADH1 in transgenic arabidopsis, compared with control group, transgenic line waterlogging, the salt tolerant of performance and low temperature resistant characteristic.The result shows that the encoding gene involved in plant of AdADH1 is resisted in damage or crop failure caused by waterlogging, low temperature or salt damage stress procedure and play very important effect.
The encoding gene of AdADH1 of the present invention for cultivation waterlogging, low temperature resistant or salt-tolerant plant kind or plant improvement kind significant, be with a wide range of applications in crop breeding.
Utilize plant expression vector of the present invention, the encoding gene of AdADH1 is imported in plant materials, transfer-gen plant that is waterlogging, low temperature resistant and salt tolerant can be obtained.
Accompanying drawing explanation
The expression characterization of Figure 1A dADH1 gene under Kiwifruit damage or crop failure caused by waterlogging (A), low temperature (B) and high salt (C) are coerced
Fig. 2 transgenic arabidopsis and the Phenotypic Observation of wildtype Arabidopsis thaliana under damage or crop failure caused by waterlogging Stress treatment condition
ADH1-3: transgenic arabidopsis strain; WT: WT strain; A: phenotype before process; B: damage or crop failure caused by waterlogging process is phenotype after 2 weeks; C: restoration ecosystem is phenotype after 1 week.
The statistical study of Fig. 3 transgenic arabidopsis and the wildtype Arabidopsis thaliana root of restoration ecosystem after 1 week long (A), fresh weight (B) and dry weight (C) after damage or crop failure caused by waterlogging Stress treatment
ADH1-3: transgenic arabidopsis strain; WT: WT strain;
Fig. 4 transgenic arabidopsis and the Phenotypic Observation of wildtype Arabidopsis thaliana under low temperature (-20 DEG C) Stress treatment condition
ADH1-3: transgenic arabidopsis strain; WT: WT strain; A: phenotype before process; B:-20 DEG C of process 5h, phenotype after restoration ecosystem 16h.
Fig. 5 transgenic arabidopsis and wildtype Arabidopsis thaliana at low temperature (-20 DEG C) Stress treatment 5h, the wilted percent after restoration ecosystem 16h
ADH1-3: transgenic arabidopsis strain; WT: WT strain;
The germination analysis under salt stress treatment condition of Fig. 6 transgenic arabidopsis and wildtype Arabidopsis thaliana
ADH1-3: transgenic arabidopsis strain; WT: WT strain;
The root of hair situation analysis under salt stress treatment condition of Fig. 7 transgenic arabidopsis and wildtype Arabidopsis thaliana
ADH1-3: transgenic arabidopsis strain; WT: WT strain;
Percentage of germination under salt stress treatment condition of Fig. 8 transgenic arabidopsis and wildtype Arabidopsis thaliana and the long statistical study of root
ADH1-3: transgenic arabidopsis strain; WT: WT strain;
Embodiment
Methods all in following Examples, without special instruction, is ordinary method
The cloning and identification of embodiment 1 Kiwifruit AdADH1 gene
Experiment material is Kiwifruit Cultivars ' Jin Kui ' cuttage seeding, carries out damage or crop failure caused by waterlogging process 0 day, 1 day to it, 2 days, after 4 days, get its root tissue, with reference to CTAB method (Cai Binhua, Zhang Jiyu, Gao Zhihong that Cai Binhua etc. improves, in the canal careful spring, Tong Zhaoguo, wastes woods, Qiao Yushan, Zhang Zhen. a kind of method [J] of extraction Fragaria blade total serum IgE of improvement. Jiangsu's agriculture journal, 2008,24 (6): 875-877) RNA is extracted, reverse transcription cDNA.According to the sequence reporting species, design primer AdADH1-ORFsense:5'-GGAGTTTGTGAGAGAAGTTAGAG-3'(SEQIDNO.3 respectively at the maximum open reading frame two ends of ADH1) and AdADH1-ORFantisense:5'-AGATTTCACCAGCAAACTCAC-3'(SEQIDNO. 4).Take cDNA as template, carry out PCR, reaction conditions is: 94 DEG C of 5min thermally denatures; 94 DEG C of 45s, 58 DEG C of 45s, 72 DEG C of 90s, totally 35 circulations; 72 DEG C extend 15min.After PCR primer glue being reclaimed test kit (Genscript) recovery, connect with pMD19-T carrier (Takara, Japan), then transform colibacillus DH5 α, select positive colony, check order.The fragment length obtained that checks order is 1283bp (SEQIDNO.5), the maximum open reading frame (SEQIDNO.1) containing 1143bp, 380 amino acid residue sequences (SEQIDNO.2) of encoding.
The expression characterization of embodiment 2 Kiwifruit AdADH1 gene under damage or crop failure caused by waterlogging, low temperature and high-salt stress condition
With Kiwifruit Cultivars ' Jin Kui ' for material, damage or crop failure caused by waterlogging, low temperature (4 DEG C), high salt (200mMNaCl) process are carried out to it, its blade is got after process, extract RNA, the synthesis test kit eliminating residual DNA in RNA of cDNA, PrimeScrioptRTreagentkitwithgDNAEraser (Takara, Japan), concrete steps reference reagent box specification sheets carries out.Real-time fluorescence quantitative RT-PCR is utilized to study the expression characterization of Kiwifruit AdADH1 gene under damage or crop failure caused by waterlogging, low temperature and high-salt stress condition.Internal reference uses actin gene, and primer sequence is ACTIN-F:5'-TGCATGAGCGATCAAGTTTCAAG-3'(SEQIDNO.6), ACTIN-R:5'-TGTCCCATGTCTGGTTGATGACT-3'(SEQIDNO.7).According to AdADH1 gene order design detection of expression primer AdADH1-qPCRsense:5'-TGGAGTGTACTGGAAGTGTCAATG-3'(SEQIDNO. 8) and AdADH1-qPCRantisense:5'-AGCAGGGAGGTCGGAACG-3'(SEQIDNO.9).Real-time fluorescence quantitative RT-PCR reaction conditions is: reaction system is dilution 10 × cDNA1 μ L, and upstream and downstream primer is respectively 0.15pmolL
-1, 10 μ L
premixExTaq
tM(TaKaRa) 10 μ L, ddH2O complement to 20 μ L.Response procedures is: 95 DEG C, 4min; 95 DEG C of 20s, 57 DEG C, 20s, 72 DEG C, 40s, 40 circulations.Data analysis adopts 7300system software and 2
-△ △ Ctmethod (LivakKJ, SchmittgenTD.Analysisofrelativegeneexpressiondatausingre al-timequantitativePCRandthe2-DDCtmethod.Methods, 2001,25:402-408)
Interpretation of result shows (Fig. 1), and damage or crop failure caused by waterlogging, low temperature (4 DEG C), high Ficus caricaL all can induce the expression of Kiwifruit AdADH1 gene.After damage or crop failure caused by waterlogging process, the expression amount of Kiwifruit AdADH1 gene first increases rear reduction, reaches maximum (Figure 1A) after the 48h of process.After 4 DEG C of subzero treatment, the expression amount of Kiwifruit AdADH1 gene first increases rear reduction, reaches maximum (Figure 1B) after the 4h of process.In the 48h of process, NaCl induces the expression amount of Kiwifruit AdADH1 gene to continue to increase (Fig. 1 C).
The resistance to overhead flooding injury qualification of embodiment 3AdADH1 gene
Extract the T-carrier containing AdADH1 encoding gene object fragment and expression vector pCAMBIA1301 plasmid DNA, with restriction enzyme Kpn I and Sac I double digestion, cut glue and reclaim, then connect with T4DNA ligase enzyme, transformation of E. coli.Extract plasmid, PCR and enzyme cut qualification.Escherichia coli plasmid freeze-thaw method is proceeded to Agrobacterium (EHA105) competent cell, bacterium liquid PCR identifies positive colony.Obtain AdADH1 overexpression expression vector pCAMBIA1301-AdADH1.By Overexpression vector by pCAMBIA1301-AdADH1 being proceeded in agrobacterium tumefaciens strain EHA105 (Avsian-Kretchmeretal, 2004, PlantPhysiology, 135:1685-1696) with freeze-thaw method.PCAMBIA1301-AdADH1 is by the mediation of Agrobacterium strain EHA105, and arabidopsis thaliana transformation, utilizes antibiotic-screening positive transgenic plant.Broadcast in cultivation matrix for seed and wildtype Arabidopsis thaliana seed by the transgenosis T2 identified, culture conditions is simultaneously: relative humidity 80%, intensity of illumination 80-200 μm of ol/M2/S, and the temperature photoperiod is 16h illumination, and 22 DEG C/8h is dark.
The Arabidopis thaliana strain of cultivating 35d is carried out damage or crop failure caused by waterlogging Stress treatment, and treatment condition are as follows: nutrition pot is put into processing tank, flood soil surface 0.5cm with tap water, and the temperature in processing tank controls at about 25 DEG C.Then observe transgenic line and the phenotypic characteristic of WT strain under flooding condition, after seeing phenotype, recover under Arabidopis thaliana strain is proceeded to normal growing conditions, observe its phenotypic characteristic.And to its fresh weight, dry weight and the statistical study of root progress row.
Result shows: damage or crop failure caused by waterlogging process is after 2 weeks, and the growing state of transgenic arabidopsis strain is significantly better than wildtype Arabidopsis thaliana (Fig. 2 B).After recovering 1 week under transgenic line after damage or crop failure caused by waterlogging process and WT strain are proceeded to normal growing conditions, the growing state of transgenic arabidopsis strain is significantly better than wildtype Arabidopsis thaliana (Fig. 2 B).To the fresh weight of transgenic line and WT strain, dry weight and the statistical study of root progress row, fresh weight, the dry weight of transgenic line and root is long is significantly higher than WT strain (Fig. 3).Illustrate that the Arabidopis thaliana strain turning Kiwifruit AdADH1 gene improves the Flood resistance of plant.
The Evaluation of Cold Tolerance of embodiment 4AdADH1 gene
Broadcast in cultivation matrix for seed and wildtype Arabidopsis thaliana seed by the transgenosis T2 identified, culture conditions is simultaneously: relative humidity 80%, intensity of illumination 80-200 μm of ol/M2/S, and the temperature photoperiod is 16h illumination, and 22 DEG C/8h is dark.The Arabidopis thaliana strain of cultivating 35d is carried out damaging to plants caused by sudden drop in temperature Stress treatment, treatment condition are as follows: nutrition pot is placed on 5h in the refrigerator of-20 DEG C, then taken out and be placed on regular culture conditions (relative humidity 80%, intensity of illumination 80-200 μm of ol/M2/S, the temperature photoperiod is 16h illumination, 22 DEG C/8h is dark) under carry out renewal cultivation, observe after cultivating 16h.
Result shows: damage to plants caused by sudden drop in temperature process 5h at-20 DEG C, after renewal cultivation 16h, WT strain is all wilted, and transgenic line growth better (Fig. 4).And the wilted percent of transgenic line is significantly lower than WT strain (Fig. 5).Illustrate that the Arabidopis thaliana strain turning Kiwifruit AdADH1 gene improves the winter resistance of plant.
The Salt-Tolerance Identification of embodiment 5AdADH1 gene
The transgenosis T2 identified is seeded into MS respectively for seed and wildtype Arabidopsis thaliana seed, on the substratum of MS+NaCl (0.1M), MS+NaCl (0.2M), then relative humidity 80% is placed on, intensity of illumination 80-200 μm of ol/M2/S, the temperature photoperiod is 16h illumination, cultivate under the condition of 22 DEG C/8h dark, after 7 days, add up percentage of germination.In addition by the transgenosis T2 that identifies for seed and wildtype Arabidopsis thaliana planting seed on MS minimum medium, after its germination shows money or valuables one carries unintentionally, transfer to containing MS, on the substratum of MS+NaCl (0.1M), MS+NaCl (0.15M), place cultivation vertically, cultivate after 7 days, to its root progress row statistical study.
Result shows: on MS minimum medium, the percentage of germination of transgenosis and wildtype Arabidopsis thaliana seed and root length do not have difference (Fig. 6, Fig. 7, Fig. 8), and containing on the substratum of NaCl, the percentage of germination of transgenic arabidopsis and root is long is significantly higher than WT strain (Fig. 6, Fig. 7, Fig. 8).Illustrate that the Arabidopis thaliana strain turning Kiwifruit AdADH1 gene improves the salt resistance of plant.
Claims (10)
1. Kiwifruit AdADH1 aminoacid sequence is as shown in SEQIDNO.2.
2. the encoding gene of Kiwifruit AdADH1 according to claim 1.
3. encoding gene according to claim 2, is characterized in that the cDNA gene nucleotide series of described Kiwifruit AdADH1 is as shown in SEQIDNO.1.
4. the expression vector of the encoding gene containing the Kiwifruit AdADH1 described in Claims 2 or 3.
5. expression vector according to claim 4, is characterized in that described expression vector is that the encoding gene of described Kiwifruit AdADH1 is inserted into gained between the Kpn I of pCAMBIA1301 carrier and Sac I restriction enzyme site.
6. the Host Strains containing gene described in claim 2 and 3.
7. Host Strains according to claim 6, is characterized in that described Host Strains is that the expression vector described in claim 4 or 5 is proceeded to agrobacterium tumefaciens EHA105 gained.
8. Kiwifruit AdADH1 according to claim 1 is cultivating the application in waterlogging, salt tolerant or low temperature resistant plant.
9. the encoding gene of the Kiwifruit AdADH1 described in Claims 2 or 3 is cultivating the application in waterlogging, salt tolerant or low temperature resistant plant.
10. the expression vector described in claim 4 or 5, the Host Strains described in claim 6 or 7 are cultivating the application in waterlogging, salt tolerant or low temperature resistant plant.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN117586349A (en) * | 2024-01-19 | 2024-02-23 | 齐齐哈尔大学 | Corn gluten source antioxidant active peptide and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117586349A (en) * | 2024-01-19 | 2024-02-23 | 齐齐哈尔大学 | Corn gluten source antioxidant active peptide and preparation method and application thereof |
| CN117586349B (en) * | 2024-01-19 | 2024-04-05 | 齐齐哈尔大学 | Corn gluten source antioxidant active peptide and preparation method and application thereof |
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