CN1417337A - Dehydrating gene BcDh2 and the application of its promoter in raising drought tolerant plant - Google Patents

Abstract

The present invention clones one new 420 bp long dehydrating gene of Boea crassifolia Hemsl. and its 5' end regulatory sequence 1084 bp. The gene, named BcDh2, is drought and cold inducing expressed, and its encode protein dehydrate can produce cooperation with sugar alcohol molecule cell to protect cell membrane and plant macro molecule in case of drought and this helps plant cell in resisting drought and raises plant's drought resistance. The gene has in its 5' end element reacting to drought, cold, heat and ABA, named BcDh2P, which may be used as stress inducing gene regulatory element and is used in resisting gene engineering breeding. The plant expression vector for the gene is constituted, and the drought tolerant gene is induced into various plants to raise the drought and cold tolerance.

Description

The application in cultivating drought-enduring plant of dehydrin gene BcDh2 and promotor thereof

The present invention has cloned long dehydrin gene and this gene 5 ' ending regulating sequence 1084bp of thick leaf new 402bp of outstanding capsule lettuce tongue (Boea crassifolia Hemsl.).Be the plain YSK2 of the family type member that dewaters, called after BcDh2, its promotor called after BcDh2P.This gene is subjected to arid and cold abduction delivering; the dehydration of its proteins encoded plain under the plant arid situation with cell in some glycitols small molecules synergies; protection cytolemma and biomacromolecule, thus vegetable cell opposing drought stress helped, improve the drought tolerance of plant.5 ' ending regulating sequence of this gene contains elements such as arid, cold, heat shock and ABA reaction, can be used as environment stress inductive gene regulating element, is used for the resistance genetic engineering breeding.We have made up the plant expression vector of this gene, and by agriculture bacillus mediated and method particle gun this drought-enduring gene are imported in all kinds of plants, have improved the drought-enduring winter hardiness of plant.

The dehydration element is a kind of arid of higher plant, cold inducible protein of extensively being present in.We adopt the DDRT-PCR method to amplify a 300bp and plant dehydration plain gene homologous sequence from the total RNA of boea crassifolia according to known plant dehydration plain gene conservative region design primer.Utilize 5 ' RACE technology to obtain the full-length cDNA (402bp) of a newcomer BcDh2 of plant dehydration plain gene family, and this expression of gene characteristic is analyzed.The Southem hybridization analysis shows that the BcDh2 gene exists with single copy form in the boea crassifolia genome.The Northern results of hybridization shows that this expression of gene is subjected to inducing of arid, cold and ABA.The BcDh2 gene is placed under the CaMV35S promoter regulation, transform soil Agrobacterium LBA4404, utilize leaf dish method transformation of tobacco to obtain transgenic tobacco plant again by freeze-thaw method.The BcDh2 gene is placed under the Ubiquitin promoter regulation, transform turfgrass and herbage, obtain transfer-gen plant, and tentatively show drought-enduring cold tolerance raising by particle gun.Method by ligation-mediated PCR, be cloned into the sequence B cDh2P of one section 1084bp of BcDh2 upstream region of gene, it is connected among the medial expression vector pCAMBIA1350 that contains gus reporter gene, to the analysis revealed that GUS in the transgene tobacco expresses, this can make a response BcDh2P to ABA, arid and cold.

The point of main innovation of the present invention is that (1) cloned a dehydrin gene BcDh2 first from the outstanding capsule lettuce tongue of thick leaf; (2) from the outstanding capsule lettuce tongue of thick leaf, clone 5 ' of dehydrin gene BcDh2 first and held control region BcDh2P; (3) made up the dicotyledonous and monocotyledonous expression vector that is suitable for of BcDh2 gene, made the proteins encoded of this gene of expression of plants by agriculture bacillus mediated and approach particle gun, thereby improve the drought-enduring cold tolerance of these species.

One of technical essential of this invention is a clone BcDh2 gene from the outstanding capsule lettuce tongue of thick leaf.Conserved regions EKKGIMD (E) KIKEKLPG that is rich in Methionin according to the plain genoid of dehydration designs degenerated primer, with the anchor primer 43,218 5 of GSP1 and mRNA 3 ' end ' GCG GCC GCT (18) 3 '.Adopt the boea crassifolia after TRIzol reagent is handled from drought stress to extract the total RNA of blade, add DNaseI digestion and remove remaining DNA, with carrying out the PCR reaction with primer GSP1 and 43128 behind synthetic cDNA first chain of primer 43128 reverse transcriptions.Sample carries out PCR:94 ℃ of 30s according to following parameters then through 94 ℃ of pre-sex change 5min, 53 ℃ of 30s, 72 ℃ of 20s, 30 circulations; 72 ℃ are extended 10min.After agarose gel electrophoresis detected, purifying reclaimed specific fragment, is connected into pGEM -T Easy carrier, carries out sequential analysis with the ABI377 automatic analyser.Obtain 5 ' end of BcDh2 gene for method by 5 ' RACE, reference literature (Kimura Y et al, 1996) synthetic upstream primer (46121) 5 ' GGC CCG ACGTCG CAT G 3 ' and anchor primer (46122) 5 ' GGC CCG ACG TCG CAT GAA TTC (12) 3 ', gene specific primer (GSP2) 5 ' TTG GAT TCT CAG TGG CAT GC 3 ' and (43514) 5 ' TAC ACT TCC TCG GTC TTC TTG 3 '.Adopt the boea crassifolia after TRIzol reagent is handled from drought stress to extract the total RNA of blade, add DNaseI digestion and remove remaining DNA, with synthetic total cDNA first chain of primer GSP1 reverse transcription, with phosphotransferase polyA is added to 5 ' end of cDNA chain behind the glass milk purifying, then the cDNA chain with tailing is the template pcr amplification.After agarose gel electrophoresis detected, purifying reclaimed specific fragment, is connected into pGEM -T Easy carrier, carries out sequential analysis with the ABI377 automatic analyser.

Two of the main points of this invention are clones of 5 ' end control region BcDh2P promotor of BcDh2 gene.The total DNA of boea crassifolia cuts with BamH I, Bgl II, Bcl I, Xho I and Sal I enzyme respectively, connect corresponding joint, have only the total DNA of boea crassifolia that handles through Xho I can amplify special segment behind the two-wheeled PCR, this fragment agarose gel electrophoresis recovery is connected on pGEM -T Easy carrier screening positive clone and order-checking.The BcDh2 upstream region of gene regulation and control head of district that chromosome walking method obtains is 1084bp.This promoters driven gus reporter gene is made up plant expression vector,, detect the activity of gus gene, find that this promotor can be subjected to inducing of arid, cold and ABA by different treatment such as arid, cold and ABA induce.

Three of the main points of this invention are structures of plant expression vector pBI121BcDh2 and pAHC-BcDh2.The BcDh2 gene coding region that is connected on pGEM -T Easy carrier is scaled off with Nco I and Sac I, be connected on the prokaryotic expression carrier pET-30a that handled with Nco I and Sac I, the proteic plasmid pET-30aBcDh2 of construction expression BcDh2, transformed into escherichia coli E.coli DH5 α bacterial strain screens correct clone.With Bgl II and Sac I digested plasmid p30aBcDh2, reclaim the BcDh2 fragment, it is connected among the plasmid pBI121 that digested with BamH I and Sac I, forms pBI121BcDh2, it is connected among the plasmid pAHC25 that digested with BamH I and Sac I forms pAHC-BcDh2.The plant expression vector construction flow process is seen Fig. 1: the structure flow process of plant expression vector pAHC-BcDh2.

In one embodiment of the invention, the BcDh2 gene places under the driving of CaMV35S promotor, is marker gene with the NPTII gene, makes up plant expression vector pBI121BcDh2.This plasmid is transformed Agrobacterium LBA4404, obtain containing the Agrobacterium of this plasmid, and infect the tobacco leaf, cultivated altogether two days, change screening culture medium over to, wound, 2 week back has callus to form, and differentiates seedling from callus successively about 4 weeks, when seedling grows to the 2cm left and right sides, seedling downcut moved into to contain in the antibiotic root media take root.At early growth period, extract the total DNA of tobacco leaf, utilize the specific fragment of pcr amplification BcDh2 gene, obtain positive plant.

In one embodiment of the invention, the BcDh2 gene places under the driving of Ubiquitin promotor, is marker gene with the bar gene, makes up plant expression vector pAHC-BcDh2.After a large amount of extractions of pAHC-BcDh2 plasmid, be used for particle gun and transform monocotyledonss such as turfgrass, herbage.Conversion process comprises that seed or young fringe evoked callus, particle gun bombardment, kanamycin-resistant callus tissue screening, the differentiation of seedling, plant identify.The step of seed callus induction: after mature seed is sterilized with 0.1% mercuric chloride, be connected to earlier on the MS minimum medium, allow it sprout 3 days, when exposing budlet, to transfer after the embryo rip cutting on the evoked callus substratum, after forming callus (about about 30 days), select color and luster aquatic foods, fast, the hard callus succeeding transfer culture of quality of propagation, the subculture callus can transform for particle gun at every turn; The step of children's fringe callus induction: get the young fringe that is about about 0.2～1cm, after 70% ethanol surface sterilization, strip out young fringe in super clean bench, be inoculated in and induce on the inducing culture, after the formation callus, per 20 days succeeding transfer culture once transform for particle gun; The conversion of particle gun and the differentiation of seedling: plasmid: the plasmid concentration of extraction is adjusted into 1 μ g/ μ L.The bullet preparation: 30mg bronze or tungsten powder add 1mL 100% ethanol vortex washing 15 minutes, aseptic washing 3 times, and it is standby to add 500 μ L, 50% glycerine.Get above-mentioned tungsten or bronze suspension 50 μ L, add 5 μ L DNA, add 50 μ L 2.5M CaCl2, add 20 μ L 0.1M spermidines, vortex left standstill the centrifugal several seconds on ice 15 minutes, 70% ethanol, 142 μ L wash once, the centrifugal several seconds, 100% 140 μ L ethanol are washed once the centrifugal several seconds, add 50 μ L, 100% ethanol, use for 5 rifles.Film can be split for examination particle gun: PDS-1000/He (Bio Rad Laboratories) particle gun and film can be split with 1350Psi, vacuum tightness 25InHg, the 6cm shooting distance, every ware is shot a rifle.Callus before the gunslinging was cultivated on the subculture medium that contains 0.4M N.F,USP MANNITOL 4～8 hours, and gunslinging moves into normal subculture medium after 16 hours.Callus after the gunslinging goes to the subculture screening 2～4 that contains weedicide 2-5mg/L and takes turns every the wheel 20 days after one week.The resistant calli that obtains after the screening changes in the substratum that contains 50g sugar, and illumination cultivation 20 days changes over to then and removes 2, seedling differentiation in the division culture medium of 4-D.When seedling length arrived 4-5 sheet leaf, the blade that takes a morsel extracted the total DNA of plant, carried out PCR and detected.Seedling grows to 5～10cm when size, moves into vermiculite: pine soil is that after plastics bag was incubated a week, seedling promptly survived in 1: 1 the soil.

In one embodiment of the invention, the BcDh2 gene places under the driving of BcDh2P promotor, is marker gene with the bar gene, makes up plant expression vector pAHCP-BcDh2.After a large amount of extractions of pAHCP-BcDh2 plasmid, be used for particle gun and transform monocotyledonss such as turfgrass, herbage.Conversion process, resistant plant screening and PCR detection method are the same.

The bacterial classification that this patent relates to is in China Committee for Culture Collection of Microorganisms's common micro-organisms center preservation, Zhong Guan-cun, unit address BeiJing, China, this microorganism classification name is called colon bacillus (Ecoli.), the preservation code name is pT-BcDh2, deposit number is CGMCC NO.0838, and preservation date is on November 22nd, 2002.

Claims (6)

1, long dehydrin gene (BcDh2) and this gene 5 ' ending regulating sequence 1084bp (BcDh2P) of thick leaf 402bp of outstanding capsule lettuce tongue (Boea crassifolia Hemsl.).The total length of this gene and 5 ' ending regulating sequence thereof and partial sequence are all within claim.

2, according to claim 1.Use this gene constructed plant expression vector, promotor is BcDh2P, Ubiquitin, actin, CaMV35S or other plant promoter, within this patent claim.

3, according to claim 1.Use the gene constructed plant expression vector of all or part of sequence driving purposes of this gene promoter BcDh2P, goal gene is resistance genes involveds such as drought-enduring, cold-resistant, salt tolerant alkali, within this patent claim.

4, according to claim 1.The plant expression vector that uses this gene or its promotor to make up, marker gene are plants such as bar, hptII, NPT II marker gene commonly used, within this patent claim.

5, according to claim 1.This gene is made suitably sudden change or the improved gene of preference codon, also within this patent claim.

6, according to claim 1.The plant expression vector that this gene or its promotor are made up obtains the method for drought-enduring cold-resistant plant by particle gun or agriculture bacillus mediated genetic transformation, also within this patent power requires.