CN103865930A - Cold inducible expression promoter Poscold3 of plant stem leaf and application of promoter - Google Patents
Cold inducible expression promoter Poscold3 of plant stem leaf and application of promoter Download PDFInfo
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- CN103865930A CN103865930A CN201410123835.6A CN201410123835A CN103865930A CN 103865930 A CN103865930 A CN 103865930A CN 201410123835 A CN201410123835 A CN 201410123835A CN 103865930 A CN103865930 A CN 103865930A
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Abstract
The invention provides a cold inducible expression promoter Poscold3 of a plant stem leaf and an application of the promoter. The invention further provides an expression box with the promoter, a plant expression vector, a host bacterium and a converter. Particularly, the invention obtains the promoter and uses the promoter in plant transgenetic engineering. The promoter provided by the invention can drive an exogenous gene to be specifically expressed in stems and leaves of a plant under a cold inducible condition. The promoter is suitable for monocotyledon cereal, and particularly can drive the exogenous gene to be expressed under induction in stems and leaves of rice plant, so that the promoter can be used for enhancing and improving the growth characteristic and cold resistance of rice, thereby cultivating an ideal rice variety.
Description
Technical field
The present invention relates to biotechnology and plant gene engineering technology field.Particularly, the present invention relates to the cold inducible gene expression promotor of a kind of plant stem-leaf and application thereof, this promotor can drive target gene to express in plant in plant transgene adjustment and control system under cold inductive condition.
Background technology
Paddy rice is the important food crop of China, and damaging to plants caused by sudden drop in temperature is one of critical limitation factor affecting Rice Production.Chilling injury is that setting percentage reduces on the main manifestations that affects of rice yield, and pollen development postpones, and anther dehiscence is not smooth, affects fertilization, causes yield reducation.Rice breeding men thirst for by breeding technique, and the strong resistance to cold of local resources is transferred on Cultivar, to improve resistance to cold, reduce and damage to plants caused by sudden drop in temperature the Rice Yield Loss Caused causing.But local cold-resistant Rice Resources often unfavorable proterties is more, improvement difficulty is very big.Therefore, in the urgent need to excavating rice cold tolerance germ plasm resource and excellent gene, thereby cultivate rice cold tolerance new variety.
So far, researchist both domestic and external has carried out insistent exploration and research, has carried out the anti-cold breeding of new variety work of coercing, and has obtained certain achievement.Zhong Kang etc. (2007), by the proline content rising in overexpression render transgenic paddy rice in paddy rice of zinc finger protein albuminoid OsCOIN gene, improve the resistance to cold of plant.Zhang Hongsheng etc. (2009) are by paddy rice zinc finger protein ZFP245 gene overexpression in paddy rice, find that overexpression ZFP245 trans-genetic hybrid rice makes the expression amount of Proline Accumulation genes involved OsP5CS and proline(Pro) transporter gene OsProT gene improve under cold coercing, impel the amount of proline(Pro) to increase, strengthen the resistance to cold of plant.Remaining virtuous and beautiful grade (2010) is found can survive after MYBS3 overexpression paddy rice can be processed under 4 ℃ of conditions, and does not affect its Other Main Agronomic Characters.In addition, some are comprised to the overexpression of the transcription factor such as DREB/CBF, NAC can improve the resistance to cold of paddy rice.
At present, along with the develop rapidly of molecular biology, information biology and reaching its maturity of transgenic technology, carry out molecular breeding by modern genetic engineering technology and become a kind of high effective way of improvement rice cold tolerance.
China's paddy rice aboundresources, excavates, locates, clones cold-resistant genes involved and cold induced promoter thereof from rice seed, will the seed selection of rice cold tolerance kind be had to important theory and practical significance, will have wide application and market outlook at agriculture field.
Summary of the invention
The object of this invention is to provide a kind of drive foreign gene under cold inductive condition specific expressed promotor, obtain the transformant that contains this promoter sequence and the application of this promotor.Wherein, related " plant " refers to monocotyledons herein, and for example paddy rice, wheat, corn, barley, Chinese sorghum or oat, be preferably paddy rice.
To achieve these goals, on the one hand, the invention provides the cold abduction delivering promotor of a kind of plant stem-leaf Poscold3, the cold abduction delivering promotor of described plant stem-leaf Poscold3 comprises the DNA sequence dna shown in SEQ ID No:1 in sequence table.In sequence table, the DNA sequence dna shown in SEQ ID No:1, for deriving from the cold abduction delivering promotor of the stems and leaves of rice Poscold3 of Japanese fine paddy rice (Oryza sativa L cv.Nipponbare), is called Poscold3 or promotor Poscold3 herein.
Preferably, the DNA sequence dna of the cold abduction delivering promotor of plant stem-leaf provided by the invention Poscold3 is the sequence shown in SEQ ID No:1, i.e. Poscold3 or promotor Poscold3.
On the other hand, the invention provides the cold abduction delivering promotor of a kind of plant stem-leaf Poscold3, the DNA sequence dna shown in its DNA sequence dna and SEQ ID No:1 has at least 90% homology; Or the cold abduction delivering promotor of described plant stem-leaf Poscold3 add, replace, insert or delete mutant or allelotrope or the derivative that one or more Nucleotide generate in the DNA sequence dna shown in SEQ ID No:1; Or the cold abduction delivering promotor of described plant stem-leaf Poscold3 has the product with the DNA sequence dna hybridization shown in SEQ ID No:1.DNA sequence dna shown in the cold abduction delivering promotor of these plant stem-leafs Poscold3 sequence and SEQ ID No:1 has identical function, drives target gene specific expressed in plant stem-leaf under cold inductive condition.
The cold induction that the present invention mentions refers to the low temperature (for example, 4 degrees Celsius) that transfer-gen plant is applied to for some time, thereby expresses by promotor induction specific gene of the present invention.
On the other hand, the present invention also provides a kind of expression cassette that comprises the cold abduction delivering promotor of above-mentioned plant stem-leaf Poscold3.
Another aspect, the present invention also provides a kind of recombinant expression vector, described recombinant expression vector comprises the cold abduction delivering promotor of above-mentioned plant stem-leaf Poscold3, in described recombinant expression vector, the cold abduction delivering promotor of described plant stem-leaf Poscold3 is connected in the upstream of gene order to be expressed; Preferably, described gene to be expressed is Gus gene, described recombinant expression vector is pCAMBIA1391-Poscold3, this recombinant expression vector is to be that Poscold3 or promotor Poscold3 are implemented in the recombinant expression vector obtaining in pCAMBIA1391 by the sequence shown in SEQ ID No:1, is called pCAMBIA1391-Poscold3 herein.More preferably, described gene is the gene with resistance to cold.
On the other hand, the present invention also provides a kind of Host Strains, and described Host Strains comprises the cold abduction delivering promotor of above-mentioned plant stem-leaf provided by the invention Poscold3, above-mentioned expression cassette or above-mentioned recombinant expression vector; Preferably, described Host Strains is agrobacterium tumefaciens.
On the other hand, the invention provides a kind of transformant, described transformant comprises the cold abduction delivering promotor of above-mentioned plant stem-leaf provided by the invention Poscold3, above-mentioned expression cassette, above-mentioned recombinant expression vector.Wherein, described transformant is preferably transgenic cell line, callus or plant.
Again on the one hand, the invention provides the cold abduction delivering promotor of above-mentioned plant stem-leaf Poscold3 in the application of cultivating in transgenic plant.Described application comprise the cold abduction delivering promotor of above-mentioned plant stem-leaf provided by the invention Poscold3 is connected in to carrier gene order upstream to be expressed (for example, before described promoter sequence is placed in to target gene), thereby structure recombinant expression vector, is transformed into described recombinant expression vector in vegetable cell, tissue or organ and cultivates.Gene to be expressed in the present invention is preferably the gene with resistance to cold.
And preferably, described application can be for improving plant growth characteristic, and described plant is monocotyledons, and for example paddy rice, wheat, corn, barley, Chinese sorghum or oat, be preferably paddy rice.
The DNA sequence dna of the promotor providing in the present invention is (with identical in SEQ ID No:1 in sequence table):
It should be noted that: in the DNA sequence dna of above-mentioned promotor, the retention sequence of the forward primer that the sequence " aacgagcata acccgagcca ga " that sequence beginning represents take italic overstriking is used in obtaining promotor process, 22bp altogether; The retention sequence (the corresponding sequence complementation of this retention sequence and reverse primer) of the reverse primer that the sequence " cagcgagag aacacaaact cac " that sequence end represents take italic overstriking is used in obtaining promotor process, altogether 22bp; In this DNA sequence dna, remaining part is available from the DNA sequence dna in the fine paddy rice of Japan.It is emphasized that the promotor mentioned both can refer to above-mentioned whole DNA sequence dna herein, also can refer to remove above-mentioned primer and retain the DNA sequence dna after sequence.
In sum, the present inventor's separating clone from the fine paddy rice of Japan (Oryza sativa L cv.Nipponbare) obtains the DNA sequence dna of the 1833bp of structure including transcription initiation site, and by the SEQ ID No:1 in its called after Poscold3(sequence table).This sequence after cutting, enzyme is connected on plant binary expression vector pCAMBIA1391, obtain corresponding recombinant plasmid (being recombinant expression vector), utilize this recombinant plasmid transformed agrobacterium tumefaciens bacterial strain EHA105, then carry out the conversion of paddy rice by agriculture bacillus mediated method, obtain transgenic rice plant.The transgenic paddy rice obtaining is carried out to histological chemistry and detect discovery, transfer-gen plant is after cold induction is processed, the relatively high and aobvious blueness of Gus gene expression dose on the whole, thereby the sequence that proves this 1833bp has the activity that drives genetic expression, and the Gus gene of this promoters driven is specific expressed after Rice Cold induction is processed.
Promoter sequence of the present invention can be connected with plant binary expression vector, for replacing constitutive promoter.And, this promoter sequence can link with required target gene, build recombinant plant expression vector, after transforming, specific expressed can drive the cauline leaf of target gene plant after cold induction is processed in, thereby improve the expression amount of external source target gene in the cauline leaf of plant, increase genetically modified effect.
Technique effect
The rice starter Poscold3 that the present invention clones can concentrate and express by regulatory gene in the cauline leaf of plant under predetermined condition, has in actual applications remarkable value.By this promotor, variety of crops is carried out to genetic modification, as expressed in the cauline leaf of plant by this promoter regulation target gene, replace the constitutive promoters such as 35S, thereby cultivate the cold-resistant transgenic plant kind that desirable biological safety is high.
Accompanying drawing explanation
Below, describe by reference to the accompanying drawings embodiment of the present invention in detail, wherein:
Poscold3 promotor is implemented in the schematic diagram in pCAMBIA1391 vector plasmid by Fig. 1, wherein in Fig. 1, A is pCAMBIA1391 schematic diagram, in Fig. 1, B is pCAMBIA1391-Poscold3 schematic diagram, wherein shows the gus gene that utilizes Poscold3 promoters driven to be positioned at its downstream and expresses;
Fig. 2 is the Poscold3::GUS transfer-gen plant tissue staining figure sprouting after 21 days.The rice plant of normal growth under 28 ℃ of conditions, after dyeing in 24 hours, root (A), stem (B), leaf (C) are all without GUS activity, and through 4 ℃ of deepfreezes 24 hours, after dyeing, in root (D), express without GUS, but all have GUS strong expression (scale=10mm) in stem (E), leaf (F).
Fig. 3 is the result schematic diagram of promotor of the present invention being carried out enzyme and cut checking.
Embodiment
Referring to specific embodiment, the present invention is described.It will be appreciated by those skilled in the art that these embodiment are only for the present invention is described, the scope that it does not limit the present invention in any way.
Experimental technique in following embodiment, if no special instructions, is ordinary method.Medicinal raw material, reagent material etc. used in following embodiment, if no special instructions, is commercially available purchase product.
The acquisition of the Poscold2 promotor that contains restriction enzyme site
The design of step 1, primer
According to the rice varieties Japan providing in NCBI fine (Oryza sativa L cv.Nipponbare) whole genome sequence, according to the sequences Design amplimer of paddy rice PCole1 gene, and according to the feature of the carrier of selecting and target gene, the restriction enzyme site of design primer.
In the present embodiment with the A part in paddy rice binary expression vector pCAMBIA1391(Fig. 1, this carrier comes from CAMBIA, for open use carrier, in genetically modified organism product composition supervision and inspection center of the Academy of Agri-Science and Technology Anhui Province Ministry of Agriculture, paddy rice group is preserved) be example, target gene is Gus gene, the primer of specific design is: forward primer (SEQ ID No:2) 5 ' end band SalI, restriction enzyme site (GTCGAC), reverse primer (SEQ ID No:3) 5 ' end band EcoRI, restriction enzyme site (GAATTC), primer sequence is as follows:
Forward primer: GTCGACAACGAGCATAACCCGAGCCAGA SalI
Reverse primer: GAATTCGTGAGTTTGTGTTCTCTCGCTG EcoRI
Synthetic by Shenzhen Hua Da genome company.
The acquisition of step 2, promotor Poscold3
Take the fine DNA of rice varieties Japan as template, utilize forward primer, reverse primer amplification promotor Poscold3, PCR system routinely, adopts following amplification program:
95 ℃ of denaturation 5min; 95 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ are extended 2min30s, carry out 35 circulations from 95 ℃ of denaturation to 72 ℃ extensions; Last 72 ℃ are extended 10min.
Reclaim the object fragment of pcr amplification, object fragment length 1833bp, be connected to PGEM-T-Easy carrier (purchased from Promega company, mix in the ratio in carrier specification sheets) on, transform after intestinal bacteria XL-Blue competent cell according to heat shock method, competent cell is activated, and then object fragment is transferred in the competent cell of activation, then, obtain positive colony through bacterium colony PCR screening, picking mono-clonal shakes bacterium liquid upgrading grain, carries out double digestion checking with SalI and EcoRI, as shown in Figure 3.The order-checking of Invitrogen company will be delivered through the positive colony of identifying.Verify that correct clone is the promotor Poscold3 that will obtain, its nucleotide sequence is as shown in SEQ ID No:1.
The structure of plant expression vector and the conversion of Agrobacterium
In the positive colony obtaining " acquisition of promotor Poscold3 " process from above, extract plasmid, with SalI and EcoRI double digestion, reclaim promotor Poscold3 fragment.Utilize SalI and EcoRI to carry out linearization process to pCAMBIA1391 simultaneously, reclaim pCAMBIA1391, above-mentioned Poscold3 fragment is connected with T4 ligase enzyme for pCAMBIA1391 fragment (being purchased from TaKaRa company), obtain plant expression vector pCAMBIA1391-Poscold3(Figure 1B of promotor Poscold3 and Gus gene fusion), utilizing freeze-thaw method that plant expression vector is proceeded to agrobacterium tumefaciens (Agrobacterium tumefaciens) EHA105(Academy of Agri-Science and Technology Anhui Province genetically modified organism product composition supervision and inspection center of Ministry of Agriculture paddy rice group preserves), from freeze-thaw method products therefrom, extract positive plasmid, carry out enzyme with SalI and EcoRI and cut checking, the result as shown in Figure 3.
Utilize promotor Poscold3 to drive Gus reporter gene to express in paddy rice
Step 1: agriculture bacillus mediated rice transformation
Ripe rice paddy seed is removed after clever shell, with 70% alcohol-pickled seed 1min, outwell alcohol.With 50% clorox that contains 1 Tween20 (stoste effective chlorine density is greater than 4%) solution soaking seed 40min(150r/min).Outwell clorox, aseptic washing 5 times is to solution clarification, without clorox taste.Sterilized water soaks seed and spends the night.Seed is peeled embryo along aleurone layer with scalper, embryo is inoculated on calli induction media.Dark cultivation after 11 days callus and endosperm and germ separation at 30 ℃, by go bud in good condition, divide vigorous elementary callus carry out preculture after 3～5 days for Agrobacterium-mediated Transformation.
Adopt the agrobacterium tumefaciens that has proceeded to recombinant expression vector in above-mentioned " structure of plant expression vector and the conversion of Agrobacterium " process to carry out agriculture bacillus mediated genetic transformation, this genetic transformation, transformant screening and transgenic plant regeneration etc. are with reference to Yongbo Duan(Yongbo Duan, Chenguang Zhai, et al.An efficient and high-throughput protocol for Agrobacterium mediated transformation based on phosphomannose isomerase positive selection in Japonica rice (Oryza sativa L.) [J] .Plant Cell Report, method 2012.DOI10.1007/s00299-012-1275-3.) etc. proposing.
Obtain altogether 28 strain pCAMBIA1391-Poscold3 plant (Poscold3::gus transgenic rice plant).
Step 2, deepfreeze and GUS histochemical stain
Plant carries out GUS dyeing through 4 ℃ of deepfreezes after 24 hours, dyeing process with reference to Jefferson (the people .GUS fusion such as Jefferson RA: β-Glucuronidase as a sensitive and versatile gene fusion marker in higher plant[J] .EMBO J., 1987, method 6:3901-3907) etc. proposing, the tissue of needs dyeing is vacuumized, then immerse in staining fluid, 37 ℃ are dyeed 24 hours.When decolouring, under 37 ℃ of conditions, use 95% Ethanol Treatment, extremely negative control material is white in color.
The Poscold3::GUS transfer-gen plant tissue of sprouting after 21 days is dyeed.As shown in Figure 2, wherein, A-C is respectively root, stem, the result of leaf after dyeing in 24 hours of the rice plant of normal growth under 28 ℃ of conditions, and as can be seen from the figure, root (A), stem (B), leaf (C) are all without GUS activity.In Fig. 2, D-F is root, stem, the leaf result that deepfreeze is dyeed for 24 hours again at 4 ℃ to rice plant, as shown in the figure, expresses, but all have GUS strong expression after dyeing in root (D) without GUS in stem (E), leaf (F).
Can find out from above experimental result, promotor of the present invention, under cold inductive condition, can drive the stem of transfer-gen plant and leaf specific expressed, and other positions can be not specific expressed.
Specific description of embodiments of the present invention above does not limit the present invention, and those skilled in the art can make according to the present invention various changes or distortion, only otherwise depart from spirit of the present invention, all should belong to the scope of claims of the present invention.
Claims (9)
1. the cold abduction delivering promotor of a plant stem-leaf Poscold3, is characterized in that, the cold abduction delivering promotor of described plant stem-leaf Poscold3 comprises the DNA sequence dna shown in SEQ ID No:1.
2. the cold abduction delivering promotor of plant stem-leaf according to claim 1 Poscold3, is characterized in that, the DNA sequence dna of the cold abduction delivering promotor of described plant stem-leaf Poscold3 is the sequence shown in SEQ ID No:1.
3. the cold abduction delivering promotor of plant stem-leaf according to claim 1 Poscold3, is characterized in that, the DNA sequence dna shown in DNA sequence dna and the SEQ ID No:1 of the cold abduction delivering promotor of described plant stem-leaf Poscold3 has at least 90% homology;
Or the cold abduction delivering promotor of described plant stem-leaf Poscold3 add, replace, insert or delete mutant or allelotrope or the derivative that one or more Nucleotide generate in the DNA sequence dna shown in SEQ ID No:1;
Or the cold abduction delivering promotor of described plant stem-leaf Poscold3 has the product with the DNA sequence dna hybridization shown in SEQ ID No:1.
4. an expression cassette, is characterized in that, described expression cassette comprises the cold abduction delivering promotor of the plant stem-leaf described in any one Poscold3 in claim 1-3.
5. a recombinant expression vector, it is characterized in that, described recombinant expression vector comprises the cold abduction delivering promotor of the plant stem-leaf described in any one Poscold3 in claim 1-3, in described recombinant expression vector, the cold abduction delivering promotor of described plant stem-leaf Poscold3 is connected in the upstream of gene order to be expressed in plant expression vector;
Preferably, described gene to be expressed is Gus gene, described recombinant expression vector is pCAMBIA1391-Poscold3, wherein pCAMBIA1391 is that plant binary expression vector or described gene to be expressed are the gene with resistance to cold, and the cold abduction delivering promotor of described plant stem-leaf Poscold3 drives described genetic expression to be expressed under cold inductive condition.
6. a Host Strains, it is characterized in that, described Host Strains comprises the cold abduction delivering promotor of the plant stem-leaf described in any one Poscold3, expression cassette claimed in claim 4 or recombinant expression vector according to claim 5 in claim 1-3, wherein, described Host Strains is agrobacterium tumefaciens.
7. a transformant, it is characterized in that, described transformant comprises the cold abduction delivering promotor of the plant stem-leaf described in any one Poscold3, expression cassette claimed in claim 4 or recombinant expression vector according to claim 5 or Host Strains claimed in claim 6 in claim 1-3.
8. the application in cultivation transgenic plant according to the cold abduction delivering promotor of the plant stem-leaf Poscold3 described in any one in claim 1-3, it is characterized in that, described application comprises: will be connected in gene order upstream to be expressed in carrier according to the cold abduction delivering promotor of the plant stem-leaf Poscold3 described in any one in claim 1-3, thereby build recombinant expression vector; Described recombinant expression vector is transformed in vegetable cell, tissue or organ and is cultivated.
9. application according to claim 8, is characterized in that, described application is for improving plant growth characteristic, and described plant is monocotyledons: paddy rice, wheat, corn, barley, Chinese sorghum or oat.
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Cited By (3)
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| CN104059919A (en) * | 2014-07-08 | 2014-09-24 | 安徽省农业科学院水稻研究所 | Plant thermal-activation inducible promoter Posheat3 and application thereof |
| CN104845973A (en) * | 2015-06-11 | 2015-08-19 | 安徽省农业科学院水稻研究所 | Cloning and applications of low-temperature strong inducible promoter POscold6 of rice |
| CN104928295A (en) * | 2015-06-11 | 2015-09-23 | 安徽省农业科学院水稻研究所 | Rice seedling stage cold inducible expression promoter POscold7 |
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| CN101768590A (en) * | 2010-03-11 | 2010-07-07 | 中国农业大学 | Cold induced promoter p-LTT7 for rice and application thereof |
| CN102676572A (en) * | 2011-03-14 | 2012-09-19 | 中国科学院遗传与发育生物学研究所 | Plant disease resistant associated protein xa5PG1, coding genes thereof and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104059919A (en) * | 2014-07-08 | 2014-09-24 | 安徽省农业科学院水稻研究所 | Plant thermal-activation inducible promoter Posheat3 and application thereof |
| CN104845973A (en) * | 2015-06-11 | 2015-08-19 | 安徽省农业科学院水稻研究所 | Cloning and applications of low-temperature strong inducible promoter POscold6 of rice |
| CN104928295A (en) * | 2015-06-11 | 2015-09-23 | 安徽省农业科学院水稻研究所 | Rice seedling stage cold inducible expression promoter POscold7 |
| CN104845973B (en) * | 2015-06-11 | 2017-10-13 | 安徽省农业科学院水稻研究所 | The strong evoked promoter POscold6 of paddy rice low clone and application |
| CN104928295B (en) * | 2015-06-11 | 2018-06-12 | 安徽省农业科学院水稻研究所 | The cold-induced expression promoter POscold7 of rice seedling |
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