CN104946663B - A kind of cotton anti-drought gene GhSNAC1 and its application - Google Patents
A kind of cotton anti-drought gene GhSNAC1 and its application Download PDFInfo
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Abstract
The invention discloses a kind of cotton anti-drought gene GhSNAC1, its nucleotides sequence is classified as S EQ ID NO.1, the protein of described cotton anti-drought gene GhSNAC1 codings, its amino acid sequence is SEQ ID NO.2, and present invention additionally comprises the expression vector pCAMBIA3301 GhSNAC1 containing the anti-drought gene GhSNA C1.Anti-drought gene GhSNAC1 of the present invention and expression vector pCAMBIA3301 GhSNAC1 is used for the genetically modified plants for cultivating GhSNAC1 gene overexpressions, the results showed that the transfer-gen plant of overexpression GhSNAC1 genes resistance under drought stress to strengthen.
Description
Technical field
The invention belongs to gene engineering technique field, and in particular to a kind of cotton anti-drought gene GhSNAC1 and its application.
Background technology
Arid has a strong impact on growing for crop, in many natural calamities, is lost caused by arid the most serious.I
The area of state's half is arid and semi-arid lands, also can be often by drought even if non-arid area.Especially
Its in the last few years, with the continuous propulsion of urbanization and the increase of population, cultivated area is constantly reduced, and freshwater resources are also increasingly deficient
It is weary, how plant water use efficiency is improved under drought condition, ensure crop production to greatest extent, turn into agricultural production
The key subjects of urgent need to resolve.Studies have found that, plant, can be by adjusting from figure during drought stress is adapted to
State, physiology or metabolic alterations resist arid.At present, in arabidopsis and rice isotype plant, numerous sound have been cloned
The functional gene of drought stress is answered, wherein transcription factor enjoys due to that can adjust the expression of multiple stress response functional genes
Concern.
China is global maximum Cotton Production state and country of consumption, economic development of the cotton industry in national economy and cotton region
In have the function that it is very important.But China cotton region is also main grain-production area, grain and cotton is striven particularly thorny.In order to
Ensure the effective supply of cotton, our cottons to salt-soda soil, nonirrigated farmland and barren are developed.Therefore, the drought resisting of cotton is improved
Salt resistance ability has more importantly realistic meaning.Cotton NAC transcription factor research is started late, and correlative study is mainly concentrated
In China scientist.2009, the Guo Wangzhen professor seminars of Agricultural University Of Nanjing utilized bioinformatics and RT-PCR etc.
Method takes the lead in having cloned 6 NAC genes from upland cotton, is named as GhNAC1-GhNAC6.GhNAC1-GhNAC6 is mainly in leaf
Expressed in piece, GhNAC2 and GhNAC3 are expressed by low temperature induction, GhNAC5 by low temperature and drought-induced expression, GhNAC4 and
GhNAC6 is by low temperature, arid and salt stress induced expression.Central China Normal University Li Xuebao professors seminar has cloned 7 within 2013
Adverse circumstance correlation NAC genes, are named as GhNAC7-GhNAC13, and qPCR analyses find this 7 GhNAC genes mainly table in root
Reach, and by ABA and high salt induced expression.Yu Shuxun academician seminar of the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute clones within 2013
10 NAC genes, are named as GhNAC8-GhNAC17, and qPCR analyses find them by leaf senile, arid, high salt, low temperature, height
The induced expressions such as temperature, ABA and ethene.2014 Nian Yushuxun academician seminars utilize bioinformatics and RT-PCR method from land
61 NAC genes are identified in cotton, are named as GhNAC18-GhNAC78, finding the expression of most GhNAC genes has group
Specificity is knitted, the expression of part GhNAC genes is by induced expressions such as ethene, ABA, GA, arid, salt marsh and agings.Up to now,
China's scientists have cloned nearly 80 upland cotton NAC genes, and analyze it in Various Tissues, a variety of hormones and adverse circumstance
Expression change under processing, but concrete function research is there is not yet report.We using the conserved sequence of NAC domains as probe,
Cotton EST and nucleotide sequence is searched for, obtained matching sequence is spliced and evolutionary analysis, excavation adverse circumstance are related
NAC sequences, cotton adverse circumstance correlation NAC genes then are cloned using RT-PCR, and analyze it and withered in arid, salt damage, low temperature and Huang
Expression under disease stress changes, the NAC genes of screening stress-inducing expression, structure Overexpression vector and suppression expression vector,
Its effect in environment stress is studied by transformation of tobacco and cotton.
The content of the invention
It is an object of the invention to provide a kind of cotton anti-drought gene GhSNAC1, the water conservation energy of the gene overexpression strain
Power strengthens, while the extent of the root system of transfer-gen plant is increased, and illustrates transgene tobacco resistance under drought stress
Enhancing.The present invention can be utilized gene constructed into various plant expression vectors, be made applied to agricultural biotechnologies breeding with improving
Thing drought-resistant character.
The present invention realizes especially by following technical scheme：
The present invention relates to Cloning Plant Genes and functional analysis, there is provided a kind of cotton anti-drought gene GhSNAC1, its core
Nucleotide sequence is SEQ ID NO.1.
Present invention also offers the protein that described cotton anti-drought gene GhSNAC1 is encoded, its amino acid sequence is SEQ
ID NO.2。
Further, present invention additionally comprises the expression vector pCAMBIA3301- containing the anti-drought gene GhSNAC1
GhSNAC1, described expression vector are built by pCAMBIA3301, and the primer in building process is respectively：SEQ ID NO.3
With SEQ ID NO.4.
The invention provides described anti-drought gene GhSNAC1 and expression vector pCAMBIA 3301-GhSNAC1 to cultivate
Application in the genetically modified plants of GhSNAC1 gene overexpressions.
Described plant is monocotyledon or dicotyledon, wherein being more preferably tobacco or cotton.
Beneficial effects of the present invention are：Will it is demonstrated experimentally that simulating drought (18% PEG6000) coerce 2h after,
The expression quantity of GhSNAC1 genes significantly improves, and indicates that it may work in drought stress is resisted.Further by building table
Up to carrier pCAMBIA3301-G hSNAC1, and convert Agrobacterium LBA4404, the results showed that overexpression GhSNAC1 genes
Transfer-gen plant resistance under drought stress strengthens.
Fig. 1 is the structure of GhSNAC1 genes；
Fig. 2 is the expression analysis of the lower GhSNAC1 genes of Different stress processing；
Fig. 3 is the expression change of GhSNAC1 genes under different drought stress time；
Fig. 4 is the PCR detections of transfer-gen plant；
Fig. 5 is the qPCR analyses of transfer-gen plant；
Fig. 6 is that wild type compares with the excised leaf percentage of water loss for turning GhSNAC1 genetic tobaccos；
Fig. 7 is that the root growth of transgene tobacco under artificial drought conditions compares；
Fig. 8 is the character mutation for turning GhSNAC1 genetic tobaccos under drought condition.
Embodiment
With reference to embodiment, the present invention is described further, as described below, is only the preferable implementation to the present invention
Example, is not limited the present invention, any person skilled in the art is possibly also with the disclosure above
Technology contents be changed to the equivalent embodiment changed on an equal basis.It is every of the invention without departing from the present invention program content, foundation
Technical spirit any simple modification that following examples are made or equivalent variations, all fall within protection scope of the present invention.
The GhSNAC1 of embodiment 1 clone
1.1 vegetable material
Shandong cotton, which grinds 32, to be passed through after grinding No. 18 hybridization with Shandong cotton by the system of Shandong 1565 (368 × Australia of stone A93-14 combines offspring and selects system)
What systematic breeding formed, there is anti-blight, resistance to verticillium wilt and Salt And Alkali Tolerance.Cottonseed is disappeared with 1% sodium hypochlorite surface
Malicious 15min, sterilized water are fully rinsed, and 12h is soaked in distilled water, seed is fully absorbed water, and the seed after imbibition is layered on wash water
Germinateed in cotton under the conditions of dark, 28 DEG C, be transferred in Hoagland nutrient solutions and grow after germination, it is true to treat that seedling grows 2-3 pieces
Following handle is carried out during leaf respectively：CK (culture of Hoagland nutrient solutions), PEG (10%PEG6000 solution), Salt
(verticillium wilt bacterium solution infects, using the defoliation verticillium wilt fungus strain with highly pathogenicity by (150mmol/L Na Cl solution), Wilt
VD8, spore suspension concentration about 1 × 107Individual/mL) and LT (4 DEG C of low-temperature treatments), processing time is 2h, and clip root system is used as
Experiment material, liquid nitrogen flash freezer, it is standby to be stored in -80 DEG C of refrigerators.
1.2 cotton root system RNA are extracted and GhSNAC1 gene clonings
Root system RNA extractions use improved CTAB methods, and cDNA synthesis is according to TaKaRa companies reverse transcription synthetic agent box
Specification step is carried out.GhSNAC1 gene clonings primer be GhSNAC1F (5 '-ATGGTAGTCCCGGAAACTGAT-3 ') and
GhSNAC1R(5’-AATTCCATACCTCTTTCCCCC-3’).RT-PCR amplifications are with reference to TaKaRa companies kit specification step
It is rapid to carry out.The PCR primer of acquisition is cloned into carrier T, and positive colony send Services Co., Ltd of Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Sequencing.Sequencing result retrieves sequence library through BLAST, and it is cotton NAC genoid coded sequences to determine institute's cloned sequence.
Diploid Lei Mengdeshi cottons (Gossypium raimondii) gene order-checking has been completed, Jin etc. (2014) profits
266 NAC genes (PlantTFDB 3.0, http are predicted from G.raimondii genomes with bioinformatics method://
planttfdb.cbi.pku.edu.cn/index.php).We have found that Gorai.009G433200.1 genes turn with drought resisting NAC
The sequence identity for recording factors A NAC072, ANAC019, ANAC055 and SNAC1 is up to 63% (E≤8e-142), 58% respectively
(E≤1e-130), 68% (E≤6e-128) and 57% (E≤5e-85), thus speculate that it may be in plant responding drought stress
In work.
Using Gorai.009G433200.1 gene orders as template, we using gene specific primer GhSNAC1F and
GhSNAC1R, in Shandong, cotton grinds No. 32 and has cloned a cDNA sequence homologous with NAC genoids, is named as GhSNAC1, the sequence
Length is 1075bp, has single entire open reading frame, thus it is speculated that encoding proteins contain 342 amino acid, and molecular weight is
38.3kDa, isoelectric point 8.85.InterProScan 5(http://www.expasy.org/) software analysis found the
NAC conserved domains (Fig. 1) between 14-139 amino acid be present.
The expression analysis of the GhSNAC1 genes of embodiment 2
With the expression of qPCR analysis GhSNAC1 genes.QPCR is according to Takara companiesPremix Ex
TaqTM kit specifications are carried out.Sense primer is 5 '-GTTCCCAGTGGACAAACTCAGAC-3 ', anti-sense primer 5 '-
CTTAAACACAAACCCACCCGAC-3’.With cotton ubiquitin protein (Ubiquitin) gene UBQ7 (Acc.No.AY-189972)
For reference gene, reaction system is 25 μ L.QPCR carries out 40 circulations altogether：95 DEG C of 30s, 95 DEG C of 5s, 60 DEG C of 30s, finally run
Melt program.Relative quantitative assay is with reference to Δ Δ Ct methods.
We analyze expression of the GhSNAC1 genes under Different stress processing using qPCR and changed, as shown in Fig. 2
After simulating drought (18% PEG6000) stress 2h, the expression quantity of GhSNAC1 genes significantly improves, and indicates that it may resisted
Worked in drought stress.
In order to further examine or check the relation of GhSNAC1 genes and drought stress time, we grind No. 32 cottons to Shandong cotton and entered
Gone the simulating drought processing of 6 different times, qPCR analysis shows, in Osmotic treatment early stage, the expression of GhSNAC1 genes by
It is cumulative strong, after handling 6h, expression quantity highest, gradually reduce (Fig. 3) later.
Embodiment 3GhSNAC1 functional analysis
We construct the Overexpression vector of GhSNAC1 genes using pCAMBIA3301, build plant expression vector
Upstream and downstream primer is respectively：5’-AGTCTAGAATGGTAGTCCCGGAAACTGAT-3 ' (underlined sequences are XbaI sites) and
5’-TT GGATCCAATTCCATACCTCTTTCCCCC-3 ' (underscore is BamHI sites).By the plant expression vector of structure
PCAMBIA3301-GhSNAC1 conversion Agrobacteriums LBA4 404, converts Ben Shi cigarette SR1 using leaf disc method, obtains 12 altogether
Individual T0Generation conversion individual plant.
From 12 T0For a small amount of blade of clip on individual plant, the genomic DNA of blade is extracted using CTAB methods, utilizes structure
GhSNAC1 plant expression vectors special primer (5 '-AGTCTAGAATGGTAGTCCCGGAAACTGAT-3 ' and 5 '-
TTGGATCCAATTCCATACCTCTTTCCCCC-3 ') enter performing PCR detection, as a result as shown in figure 4, in 12 T0For in individual plant,
There are 10 T0For individual plant amplify with positive control fragment of the same size, show that GhSNAC1 genes have been integrated into tobacco gene
In group.
By 10 T of residue0Harvested for the seed of individual plant by individual plant, each T0The seed of 200 or so is chosen for individual plant to be used for
Screening, screening and culturing medium is the 1/2MS culture mediums containing 50 μ g/ml, the ratio of positive plant and negative plant is counted, through card side
After test, reservation meets 3:The plant of 1 segregation ratio.By the positive plant (T after screening1) be transplanted in compost, plant to be planted
By individual plant harvest seed after maturation, it is continuing with the 1/2MS culture mediums containing 50 μ g/ml and is screened, T2Do not divide for seedling
From positive plant be homozygote.
The pure and mild family of tobacco and wild type SR 1 of 5 overexpression GhSNAC1 genes are chosen, using improved CTAB methods
Extract blade RNA, using GhSNAC1 gene expression analysis special primer (5 '-GTTCCCAGTGGACAAACTCAGAC-3 ' and
5 '-CTTAAACACAAACCCACCCGAC-3 ') qPCR analyses are carried out, as a result find GhSNAC1 genes in 5 transgenic lines
In all have expression, the expression quantity highest especially in G8 familys, and in wild type SR1 is all to fail because without GhSNAC1 genes
Detect expression signal (Fig. 5).
The excised leaf dehydration of embodiment 4 is analyzed
The moisture holding capacity of the aerial rate-of-loss of coolant reflection blade of Vitro Plant blade.We plant in soil respectively
The most strong G8 familys of GhSNAC1 gene expressions and wild-type tobacco SR1, when plant to be planted grows to 5-6 piece true leaves, clip near
The intact leaf on top, natural dehydration at room temperature, the fresh weight of blade is weighed in 1h, 2h, 3h, 4h, 5h and 6h respectively, calculates every section
The water content of time intra vane, water content=(fresh weight-dry weight)/fresh weight × 100%, converses leaves water loss rate, in triplicate,
Average.
As a result as shown in fig. 6, in 1-6h, the excised leaf percentage of water loss for turning the G8 familys of GhSNAC1 genes is significantly lower than
Wild type SR1, illustrate that overexpression GhSNAC1 genetic tobaccos water holding capacity strengthens, particularly in 6h, the guarantor of transgene tobacco
Outlet capacity improves 1.69 times compared with wild type.
The root growth analysis of the transgene tobacco of embodiment 5
The growth of plant roots is more sensitive to osmotic stress.Using Drought stress simulation solid medium, we observe
The root elongation situation of wild type and transgene tobacco.The SR1 and overexpression GhSNAC1 genes of 200 or so is chosen first
Transgenic Tobacco Seeds, soak seed 15 minutes with 0.5% sodium hypochlorite (NaClO) solution, vibrate repeatedly, it is ensured that all
Seed shows that thorough disinfection can be obtained, then adds sterilized water and rinse seed (4-6 times) repeatedly, remove the NaClO of residual.Will
Tobacco seed after sterilization is seeded in MS minimal mediums, is put into HPG-280B illumination boxs, and incubator is arranged to 16h light
According to/8h dark.After 3-4 days, seedling is transferred to after seed sprouting on simulating drought solid medium, it is ensured that root is straight and tight
Paste culture medium.Culture dish is vertically disposed in illumination box, the tobacco tip of a root is downward, and root elongation situation is measured after one week.
As a result find, under 200mM mannitol artificial drought conditions, overexpression GhSN AC1 genetic tobacco root systems are put down
Equal length is 3.48cm, and the average length of wild type SR1 root systems is 2.24cm (Fig. 7), illustrates that transgene tobacco is coerced in arid
Compel lower resistance enhancing.
We use method for potted simultaneously, plant the tobacco of wild type SR1 and overexpression GhSNAC1 genes respectively, treat
Seedling is grown to stopping watering, Follow-up observation tobacco leaf dehydration wilting situation during 4-5 piece leaves.As a result find after control water 25 days,
The substantially all wilting of blade of wild type SR1 tobaccos, and the tobacco drought-resistant ability of overexpression GhSNAC1 genes is remarkably reinforced,
Blade wilting lesser extent (Fig. 8).
Application of the embodiment 6GhSNAC1 genes in genetically modified plants
1) cultivate and configure：
Seed germination medium：1/2MS culture mediums
Leaf dish differential medium：MS+1mg/L 6-BA+0.1mg/L NAA
Leaf dish Selective agar medium：The herbicides of MS+1mg/L 6-BA+0.1mg/L NAA+500mg/L Cef+5 ‰
Multiple Buds root media：The herbicides of 1/2MS+500mg/L Cef+5 ‰
2) breeding method
A kind of method of the genetically modified plants of cultivation GhSNAC1 gene overexpressions, comprises the following steps：
(1) 75% alcohol disinfecting tobacco SR1 seed 1min, aseptic water washing 3~5 times are utilized；Then with 0.1% mercuric chloride
5~8min of disinfection seed, aseptic water washing 5~8 times；Aseptic seed is uniformly seeded in 1/2MS culture mediums, in 28 DEG C, 16h light
Sprouted according under the conditions of.
(2) after seed is sprouted 2 weeks, transfer them in the triangular flask containing 1/2MS culture mediums and cultivate, plant to be planted length to 4
Take healthy and strong blade to make explant during~5 leaf and carry out genetic transformation.
(3) picking carries the Agrobacterium inoculation of pCAMBIA3301-GhSNAC1 recombinant plasmids in that is mould containing 50 μ g/ml cards
Element, 50 μ g/ml rifampins YEP fluid nutrient mediums in, 200rpm shaken cultivations are to exponential phase under the conditions of 28 DEG C.
(4) bacterium solution 6000rpm centrifuges 5min, outwells supernatant, and it is standby that thalline is resuspended with MS fluid nutrient mediums.
(5) in the sterile tobacco leaf of superclean bench clip, blade is cut into the leaf dish of about 0.5cm2 sizes, immerses and is resuspended
Agrobacterium bacterium solution in, 5min is infected in gently vibration, aseptic filter paper suck dry moisture is used after taking out leaf dish, by its proper alignment in MS
On differential medium, light culture 2 days under the conditions of 28 DEG C.After 2 days, leaf dish is placed on MS Selective agar mediums, 28 DEG C, 16h illumination
Under the conditions of cultivate, 3 weeks subcultures are once.
(6) after growing 3~5cm Multiple Buds after leaf dish periphery, access root media is cut, promotees its seedling of taking root.
(7) after root system development is intact, triangle bottle closure is opened, in hardening in greenhouse 1~2 day, carefully by seedling from culture
Extracted in base, clean root culture medium, moved into the flowerpot for filling sterile soil, overlay film culture 3 days, thereafter greenhouse Routine Management.
Claims (4)
- A kind of 1. cotton anti-drought gene GhSNAC1, it is characterised in that：Described anti-drought gene GhSNAC1 nucleotides sequence is classified as SEQ ID NO.1。
- 2. the expression vector of the cotton anti-drought gene GhSNAC1 described in claim 1, it is characterised in that：Described expression vector Built by pCAMBIA3301, the primer in building process is respectively：SEQ ID NO.3 and SEQ ID NO.4.
- 3. the cotton anti-drought gene GhSNAC1 described in claim 1 is cultivating the transgenic dicot of GhSNAC1 gene overexpressions Application in plant.
- 4. the answering in the transgenic dicots for cultivating GhSNAC1 gene overexpressions of the expression vector described in claim 2 With.
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| CN106699858B (en) * | 2017-02-27 | 2019-10-25 | 中国农业科学院棉花研究所 | GhNAC79 and its application in regulation plant drought resistance |
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| CN102399789A (en) * | 2010-09-16 | 2012-04-04 | 创世纪转基因技术有限公司 | Cotton ghNAC2 transcription factor gene and use thereof |
| CN102399790A (en) * | 2010-09-16 | 2012-04-04 | 创世纪转基因技术有限公司 | Cotton ghNAC4 transcription factor gene and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102399789A (en) * | 2010-09-16 | 2012-04-04 | 创世纪转基因技术有限公司 | Cotton ghNAC2 transcription factor gene and use thereof |
| CN102399790A (en) * | 2010-09-16 | 2012-04-04 | 创世纪转基因技术有限公司 | Cotton ghNAC4 transcription factor gene and application thereof |
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