CN101503703A - Use of cotton steroids 5 alpha-reductase gene and expression vector including the same - Google Patents

Abstract

The invention relates to application of cotton steroid 5alpha-reductase gene GhDET2 in promoting the development of plant roots. A GhDET2 gene expressed in plants promotes the elongation of transgenic plant radicle, the occurrence and elongation of lateral roots, the growth of root hairs, the growth of adventitious roots of transgenic plant stems, as well as the vegetative growth of the transgenic plants, and increases the yield of the transgenic plants and the size of seeds. The gene GhDET2 can be used for breeding crops and forest trees, increasing the vegetative growth of the transgenic plants and increasing the biological yield of the plants and seed yield. The invention also provides a plant expression vector containing the gene GhDET2 and a method for preparing the transgenic plants containing the gene.

Description

The purposes of cotton steroid 5 gene and contain its expression vector

Technical field

The invention belongs to plant genetic engineering field, specifically, relate to the purposes of cotton steroid 5 gene, contain the coded protein and the plant expression vector of this gene; The invention still further relates to the method for preparing the transgenic plant that contain said gene.

Background technology

The plant particularly root system of farm crop and forest is they absorb moisture and nutrient inorganic salt from soil a major organs, also be the major organs of plant set simultaneously, therefore, plant root system development plays crucial effects to the growth of plant shoot branch and the output of farm crop.The root morphology of each kind of plant is different, but the basic function of root is identical.Root is made up of main root, lateral root or adventive root.Main root is developed into by seminal root (radicle), links to each other with stem, downwards growth.From main root to around the root that bears be called lateral root, the lateral root lateral root of can also regenerating.The root that directly grows on stem or the leaf is called adventive root.All root stack ups of one strain plant are root system, and root system can be divided into taproot system (as the root of dicotyledons) and fibrous root system (as monocotyledonous) two big classes.The main position of root absorption moisture and inorganic salt is root hairs, and the root hair is the outwards outstanding hair of tip of a root epidermic cell.Quantity is a lot, and collection is born in the root-hair zone of the tip of a root.The life-span of root hair is very short, and wilting at once about about 1 week comes off.Along with the growth of the tip of a root, bear new root hair again at new position.

The growth of root system of plant equally also is subjected to the regulation and control of plant hormone, and the effect that plant hormone that research history is long such as growth hormone, phytokinin, dormin etc. are grown plant root growth has had more deep research.(Brassinosteroids BRs) is the novel plant hormone of a class to the plain steroid substances of rape, and research history is shorter.But because it has vital role in plant growth and development process, the investigator carries out extensive studies in many aspects.Many results of study all confirm the outer BL (Brassinolide that executes, the highest BRs of a kind of biological activity) can promote plant root system development (Brassinosteroids interact with auxin topromote lateral root development in arabidopsis.Plant Physiology, 2004,134).But these reports all are the results that external source is used BL, utilize plant gene engineering technology, obtain overexpression or suppress the transgenic line of BRs biosynthetic enzyme genes, study certain concrete BRs and synthase gene that certain the is concrete also rare report of influence the plant root growth growth with this.

DET2 (de-etiolated2, the Arabidopis thaliana steroid 5) is the enzyme that catalysis is early reacted in the BRs biosynthetic pathway, the catalysis campesterol changes into campestanol (The Arabidopsisde-etiolated2 mutant is blocked early in brassinosteroid biosynthesis.Plant Cell, 1997,9).Detailed afterwards (24R)-24-methyl-cholest-in this conversion process of the DET2 catalysis-4-alkene-3-ketone that studies show that arrives 5 α reduction reactions (the Arabidopsis det2is defective in the conversion of (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one in brassinosteroid biosynthesis.PlantPhysiology of (24R)-24-methyl-5 α-cholestane-3-ketone, 1999,120).Result of study shows subsequently, and the catalytic reaction of DET2 is a rate-limiting step main in the BRs biosynthetic pathway.The transgenic arabidopsis of overexpression DET2 is grown obviously big than wild-type plant type under light, and the biological growth amount increases 100%-150% than wild-type; And the Arabidopis thaliana plant that changes the DET2 inverted defined gene has obtained a series of phenotypes, wild-type (the Brassinosteroid actions in plants.Journal of Experimental Botany of plant height from the extreme dwarf-type of similar det2 to normal plant height, 1999,50), illustrate that Arabidopis thaliana steroid 5 expression of gene level and growth and development of plant have substantial connection.

In order to study the effect of steroid 5 gene in cotton fiber grows, by screening and Arabidopis thaliana steroid 5 (DET2) homologous cotton est sequence, and candidate's est sequence spliced and cloning and sequencing, after obtaining one section sequence, carry out the complete sequence that 3 '-RACE obtains cotton steroid 5 gene again, at last clone's cotton steroid 5 gene (calling the GhDET2 gene in the following text) from cotton fiber.Utilize external restoring system to prove that GhDET2 has the steroid 5 activity, and then make up the plant expression vector of overexpression and Antisense Suppression GhDET2 gene and carry out the genetic transformation of cotton, result's proof suppresses the expression render transgenic cotton of GhDET2 in cotton growth is subjected to the intensive inhibition, fibrocellular starting quantity reduces, elongation of fiber is suppressed, and is difficult to obtain sophisticated seed and fiber; On the contrary, in fiber, suitably improve the expression of GhDET2, can promote the initial sum elongation of fiber, illustrate that the GhDET2 gene has vital role (GhDET2 in cotton fiber grows, a steroid 5a-reductase, plays an important role in cotton fiber cellinitiation and elongation.Plant Journal, 2007,51).

Up to the present, Arabidopis thaliana, tomato, pea, lead a cow and cotton in the steroid 5 gene cloned and identified that but DET2 does not also see the report of the influence of root growth.

Summary of the invention

One object of the present invention is to provide cotton steroid 5 gene GhDET2 (purposes of Gossypium hirsutum steroid 5 α-reductase), i.e. application in promoting plant root system development.

Another purpose of the present invention is to provide the plant expression vector that contains this GhDET2 gene.

Another object of the present invention is to provide the method for preparation based on the transgenic plant of plant expression vector of the present invention.

According to an aspect of the present invention, described coding cotton steroid 5 gene GhDET2 has following nucleotide sequence:

1) nucleotide sequence shown in SEQ ID NO.1; Perhaps

2) nucleotide sequence with SEQ ID NO.1 has more than 80%, preferred 90% above homology, and the proteinic Nucleotide of coding identical function.

By the protein of above-mentioned GhDET2 genes encoding, have the amino acid residue sequence shown in the SEQ ID NO.2 or with the amino acid residue sequence of SEQ ID NO.2 through replacement, disappearance or the interpolation of one or several amino-acid residue and have identical bioactive by this sequence deutero-protein with the amino acid residue sequence of SEQ ID NO.2.

According to a further aspect in the invention, the plant expression vector that provides comprises Nucleotide and the promoter sequence of coding cotton steroid 5 GhDET2 at least, and described plant expression vector is operably connected with expression vector and makes up by the cotton steroid 5 gene GhDET2 that will encode, promoter sequence.For the needs that screen and express, also in expression vector, comprise the various restriction enzyme sites that screening-gene sequence, reporter gene sequence and other the needs for the genetically engineered operation insert alternatively, screening-gene and reporter gene can be selected from the gene order that this area is used always, preferably, expression of plants of the present invention has structure as shown in Figure 1.

The promotor that is used to make up plant expression vector of the present invention is preferably constitutive promoter or tissue-specific promoter, and for example, the special-shaped promotor of Gent more preferably derives from the plant constitutive promoter CaMV35S of cauliflower mosaic virus.Usually, with the gene constructed downstream of GhDET2 at CaMV35S.

In a specific embodiments of the present invention, at first make up the expression vector pBI121-35S-NOS double base plasmid vector that contains the CaMV35S promoter sequence, then GhDET2 gene forward is inserted in the plant expression vector, start expression with the CaMV35S promotor, made up the plant expression vector pBI121-35S-GhDET2-NOS that contains the GhDET2 gene, it has structure as shown in Figure 4, this expression vector has comprised reporter gene GUS sequence simultaneously, screening-gene sequence (kalamycin resistance) and be used for each restriction enzyme site of genetic manipulation, it will be understood by those skilled in the art that, above-mentioned reporter gene, screening-gene and each genetic manipulation sequence all are interchangeable, and the present invention does not limit this.

According to a further aspect in the invention, provide a kind of transformant, utilize methods such as electric shocking method or freeze-thaw method to handle, plant expression vector transfecting host of the present invention is obtained transformant, this transformant can be used for transforming plant and obtains transgenic plant.Preferred host is an agrobacterium strains.

In accordance with a further aspect of the present invention, provide preparation to contain the method for the transgenic plant of GhDET2 gene.GhDET2 gene of the present invention and promotor are made up plant expression vector, described plant expression vector is transformed the transformant that the host obtains to contain the GhDET2 gene, transform plant with described transformant and obtain transgenic plant.

The invention provides the new purposes of GhDET2 gene, successfully made up the plant expression vector that comprises GhDET2 gene and promotor, and transform plant acquisition transgenic plant with this expression vector, the GhDET2 gene is by overexpression in transgenic plant.By analysis, find that overexpression GhDET2 gene can promote the elongation growth of main root, the generation of lateral root and elongation growth, and the growth of the growth of adventive root and root hair to the transgene tobacco phenotypic variation.Therefore GhDET2 gene pairs whole plants root growth has promoter action; Simultaneously, the overexpression of this gene also can increase the biological growth amount and the fruit and seed output of transgene tobacco, and this has important theory and practical significance for cultivating good crop varieties.

Description of drawings

Fig. 1: contain the structure iron of the plant expression vector of GhDET2 gene, wherein

35S represents the CaMV35S promotor; GhDET2: represent the GhDET2 gene cDNA; Term represents terminator; LB represents the T-DNA left margin; RB represents the T-DNA right margin.

Fig. 2: the structure schema of pBI121-35S-NOS double base plasmid vector, wherein

NPTII, neomycin phosphotransferase gene; GUS, β-gluconic acid glycoside enzyme gene; CaMV35S derives from the plant constitutive promoter of cauliflower mosaic virus; Nosterm, the Nos terminator; LB, the T-DNA left margin; RB, the T-DNA right margin.

Fig. 3: the structure schema of the cotton steroid 5 gene GhDET2 expression vector under the constitutive promoter CaMV35S regulation and control

Amp, ampicillin resistance gene; NPTII, neomycin phosphotransferase gene; GUS, β-gluconic acid glycoside enzyme gene; CaMV 35S derives from the plant composition promotor of cauliflower mosaic virus; Nosterm, the Nos terminator; LB, the T-DNA left margin; RB, the T-DNA right margin.The skeleton carrier that is used to make up plant expression vector is the pBI121-35S-NOS carrier of transforming on the pBI121 basis, has CaMV 35S promoter regulation and control gus gene down, is convenient in the process of plant genetic conversion transformant be carried out the painted screening of GUS.

Fig. 4: the preferred plant expression vector P6-GhDET2 of the present invention structure iron;

Fig. 5: the RT-PCR of GhDET2 gene in transgene tobacco analyzes

Blade with wild-type tobacco and transgenic tobacco plant is a material, extracts total RNA, and by the synthesizing single-stranded cDNA of reverse transcription, (on Fig. 5) increases to use the special primer D6P1 of GhDET2 and D6P2 (SEQ ID NO.3 and SEQ ID NO.4) then.Be to determine each sample strand cDNA synthetic quality and concentration, (among Fig. 5) increases with the Ubiquitin special primer Ubi-up of tobacco and Ubi-down (SEQ ID NO.9 and SEQ ID NO.10).Be getting rid of the pollution of transgene tobacco genomic dna, is template increase (under Fig. 5) with total RNA directly.WT, wild-type tobacco; UBI, Ubiquitin; 1,2,3,4 2#, 13#, 18# and the 19# plant that are respectively transgene tobacco.

Fig. 6: the main root growth of transgene tobacco and wild-type tobacco

A, 4 days main root growing state behind the wild-type tobacco planting seed.B, 4 days main root growing state behind transgene tobacco (TOD2) planting seed.C, 6 days main root growing state behind the wild-type tobacco planting seed.D, 6 days main root growing state behind transgene tobacco (TOD2) planting seed.E, the main root length that transgene tobacco (TOD2) and wild-type tobacco were grown in sand 30 days.F, the main root length behind transgene tobacco seed and the wild-type tobacco planting seed 6 days the time.WT, wild-type tobacco; TOD2～TOD19, the transgene tobacco strain of overexpression GhDET2 gene is 2# to 19#.G, the speed of growth of transgene tobacco (TOD2) and wild-type tobacco main root.WT, wild-type tobacco; TOD2, the transgene tobacco strain of overexpression GhDET2 gene is 2#.

Fig. 7: the lateral root of transgene tobacco is grown

A, after planting 10 days wild-type tobacco root system, the long 1cm of scale.

B, 10 days transgenic tobacco root after planting, the long 1cm of scale.

Arrow is represented the happening part of lateral root on the main root among the figure.

Fig. 8: the root hair of transgene tobacco is educated

A, the wild-type tobacco seed of sprouting (soaking seed 60 hours); B, the transgenosis of sprouting (TOD2) tobacco seed (soaking seed 60 hours); C, the root hair of wild-type tobacco (after planting 10 days); D～F, the root hair (after planting 10 days) that different transgene tobacco strains are.

Fig. 9: the growth of transgene tobacco adventive root

Get transgene tobacco T ₁For the stem section of aseptic seedling and wild-type tobacco aseptic seedling, in the MS substratum, take root, grow and add up the quantity and the length of adventive root after 20 days.A and B, the growing state of adventive root on transgene tobacco (TOD2) and the wild-type tobacco stem section; C, the mean length of adventive root; D, the average quantity of adventive root on each stem section

Figure 10: the plant height of transgene tobacco and wild-type tobacco and blade are relatively

A, after this bright tobacco plant comes to the ripening period, the plant forms of transgene tobacco and wild-type tobacco and plant height.TOD, the transgene tobacco of overexpression GhDET2 gene; WT, wild-type tobacco.B, the coordination mature leaf of transgene tobacco and wild-type tobacco.TOD, the blade of the transgene tobacco of overexpression GhDET2 gene; WT, the blade of wild-type tobacco.

Figure 11: the flower of transgene tobacco and wild-type tobacco, fruit and seed A, the mode of appearance of transgene tobacco and wild-type tobacco flower and Fruit Development Process.WT, wild-type tobacco; TOD: the transgene tobacco of overexpression GhDET2.B, the fruit development of transgene tobacco and wild-type tobacco (having peeled off the rest part of floral organ).WT, wild-type tobacco; TOD, the transgene tobacco of overexpression GhDET2.C, the seed size of transgene tobacco and wild-type tobacco, the long 1cm of scale.WT, wild-type tobacco; TOD, the transgene tobacco of overexpression GhDET2.D, the thousand seed weight of this bright tobacco of transgenosis and this bright tobacco seed of wild-type.WT, this bright tobacco seed of wild-type; TOD1～TOD19, the transgenic line 1～19 of overexpression GhDET2 gene.E, with respect to the weight of this bright tobacco seed of wild-type, the increase ratio of this bright seed weight of tobacco of transgenosis.WT, this bright tobacco seed of wild-type; TOD1～TOD19, the transgenic line 1～19 of overexpression GhDET2 gene, seed weight unit is gram.

Embodiment

Below in conjunction with accompanying drawing the present invention is further described in detail, but following explanation does not limit the present invention, any to distortion of the present invention and change, only otherwise break away from spirit of the present invention, all should belong to the defined scope of claims of the present invention.

It is common commercially available that reagent chemicals in the example of the present invention is not done being of specifying, and the material method is not done the equal reference " molecular cloning experiment guide " (Sambrook and Russell, 2001) that specifies.

In following example of the present invention, used cotton experiment material is Xuzhou 142 (Gossypiumhirsutum cv Xuzhoul42), and used tobacco experiment material is this bright cigarette (Nicotianabenthamiana)

[embodiment one] The clone of GhDET2 gene order

1. the extraction of cotton RNA

Choose about 3g fresh cotton floral material, in liquid nitrogen, wear into fine powder rapidly, the 50mL centrifuge tube of packing into, RNA extracting solution (2% CTAB (W/V) of 65 ℃ of preheatings of adding 15mL, 2% PVP (W/V), 100mmol/L Tris-HCl (pH8.0), 0.5g/L Spermidine, 2,0mol/L NaCl, 2% mercaptoethanol (V/V adds before using)), put upside down mixing.65 ℃ of water-bath 3～10min, during mixing 2～3 times.Chloroform: primary isoamyl alcohol (24:1) extracting 2 times (10,000r/min, room temperature, 5min).Get supernatant, add 1/4 volume 10mol/L LiCl solution, place 6h for 4 ℃, with chloroform: each extracting of primary isoamyl alcohol (25:24:1) 1 time (10,000r/min, room temperature, 5min).The dehydrated alcohol that adds 2 times of volumes is more than-70 ℃ of refrigerator precipitation 30min.12,000r/min, 4 ℃ of centrifugal 20min abandon supernatant.Precipitation is dissolved with the DEPC treating water of 200 μ L.Phenol (pH4.5): chloroform: primary isoamyl alcohol (25:24:1), chloroform: each extracting of primary isoamyl alcohol (24:1) 1 time (10,000r/min, room temperature, 5min).The dehydrated alcohol that adds 1/10 volume 3mol/L NaAc solution and 2.5 times of volumes is more than-70 ℃ of refrigerator precipitation 30min.12,000r/min, 4 ℃ of centrifugal 20min abandon supernatant.Precipitation with 70% alcohol rinsing once, and is air-dry.Add the DEPC treating water dissolving of 200 μ L.Detect the quality of RNA with non-sex change agarose gel electrophoresis and ultraviolet spectrophotometer scanning.

2.cDNA the pcr amplification of synthetic and GhDET2 gene cDNA sequence

Analyze according to the EST integration sequence, at the upstream from start codon sequences Design primer D6P1 (SEQ ID NO.3) that infers, with 3 '-the increase cDNA sequence of cotton steroid 5 gene of the method for RACE.Operation steps by 3 '-RACE test kit (TaKaRa) specification sheets carries out.Concrete grammar is as follows:

(1) cDNA one chain is synthetic

Get in the amplification pipe that the total RNA of about 10 μ g handles to DEPC-, after 65 ℃ of water-bath 10min make the RNA sex change, ice bath 3min immediately.In the amplification pipe, add 2 μ L, 10 * RNA reaction buffer successively then, 4 μ L 25mmol/L MgCl2,2 μ L 10mmol/L dNTPs, 5U AMV RTase, 0.5 μ L RNaseinhibitor (20U) and 1 μ L, 2.5 μ mol/L Oligo-dT, 3 ' site adaptor, the water that adds the DEPC processing is to final volume 20 μ L.The insulation program of reverse transcription reaction is: 30 ℃, and 10min; 50 ℃, 45min; 95 ℃, 5min; 5 ℃, 5min.After the EP (end of program), a chain product is frozen in-20 ℃.

(2) amplification

The cDNA amplification system of 25 μ L contains 2.5 μ L, 10 * Ex PCR buffer (Mg ²⁺Free), 2 μ L 2.5mmol/L dNTPs, 2 μ l 25mmol/L MgCl ₂, 1 μ L special primer D6P1 (5 μ mol/L), 1 μ L3 '-site primer (5 μ mol/L), 0.2 μ L Ex Taq archaeal dna polymerase, 1 μ L cDNA, one chain product.Amplification program is: 94 ℃, and 5min; 94 ℃, 30sec, 56 ℃, 30sec, 72 ℃, 1min, 30 circulations; 72 ℃ are extended 10min.

3.cDNA fragment reclaims, and connects transformed into escherichia coli DH5a.

(1) electrophoresis

The cDNA amplified production is carried out electrophoretic separation in 1.2% (W/V) sepharose.

(2) reclaim

Use and reclaim test kit: recycling step carries out according to the test kit specification sheets, and it is quantitative to reclaim fragment electrophoresis on sepharose.

(3) clone and order-checking

The fragment that reclaims is quantitative through agarose gel electrophoresis.Press the test kit specification sheets, fragment transforms with the intestinal bacteria that are connected, connect product of cloning vector by reclaiming, the cultivation and the plasmid enzyme restriction of positive bacterium colony are verified, will reclaim fragment cloning to pUCm-T (Shanghai Sangon) carrier.Sequencing is finished by Shanghai Bo Ya company.

Fragment that reclaims and pUCm-T (worker is given birth in Shanghai) carrier is set up following linked system:

Supply the linked system of volume to 10 μ L with distilled water

The carrier DNA fragment is connected product D NA fragment mol ratio with external source be 1:3, and 16 ℃ connect 12h.To connect product transformed into escherichia coli DH5a afterwards.

[embodiment two] The structure of pBI121-35S-NOS double base plasmid vector

PBI121 double base plasmid is a kind of commercial plant expression vector, and this laboratory is bought and preserved.The expression of plants element of one cover Nos promotor control NPTII gene and the expression of plants element of a cover CaMV 35S promoter control gus gene are arranged in the border, the left and right sides in T-DNA zone.In order to express target gene, we have added the expression of plants element of a cover by CaMV 35S promoter controlled target gene in this plasmid vector.This plant expression vector can be realized Kan (kalamycin resistance) and the active double-tagging screening of GUS.(Multiple cloning site MCS) inserts foreign gene, can realize the overexpression (Fig. 2) of foreign gene in multiple clone site.

At first, in order to obtain CaMV 35S promoter fragment, (sequence of p6-1 is seen SEQ ID NO.5 to have the primer p6-1 of BamHI and SmaI restriction enzyme site and p6-2 according to the CaMV35S promoter sequence on pBI121 double base plasmid design, the sequence of p6-2 is seen SEQ ID NO.6), with pBI121 double base plasmid DNA is that template increases, with BamHI and SmaI double digestion amplified production and pBI121 plasmid, by reclaiming, connect, transform and verify the intermediate carrier that obtains to insert the CaMV35S promoter fragment.(primer p6-3 sequence is seen SEQ ID NO.7 to obtain Nos terminator fragment according to the sequences Design amplimer of Nos terminator on the pBI121 double base plasmid then, primer p6-4 sequence is seen SEQ ID NO.8), this segmental end has XbaI, BamHI and KpnI restriction enzyme site, and the other end has the BglII restriction enzyme site.By reclaiming, connect, transform and checking having obtained to contain the skeleton (Fig. 2) of the pBI121-35S-NOS plant expression vector of CaMV 35S promoter.

[embodiment three] The structure of overexpression carrier and tobacco genetic transformation

1. the structure of overexpression carrier

The pUC-GhDET2 carrier is to make up when clone GhDET2 gene, and the GhDET2 fragment on it checks order.PBluescript-SK is the commercialization carrier, is bought by this laboratory and preserves, and is used for purpose fragment conversion direction in this experiment or uses new restriction enzyme site.The skeleton pBI121-35S-NOS of plant expression vector preserves for this laboratory makes up according to embodiment two methods.

For overexpression GhDET2 gene in transgenic plant, GhDET2 gene forward need be inserted in the plant expression vector, and start expression with suitable promotor.We have designed vector construction route as shown in Figure 3 according to direction of insertion and the multiple clone site on the plant expression vector (pBI121-35S-NOS) and the direction of CaMV35S promotor of restriction enzyme site on the pUC-GhDET2 carrier and GhDET2 for this reason.According to this plant expression vector construction route, made up the plant expression vector pBI121-35S-GhDET2-NOS of overexpression GhDET2 gene, carrier structure such as Fig. 4.

2. tobacco genetic transformation

(1) with electrization the plant expression carrier plasmid that makes up is imported Agrobacterium LBA4404 and tobacco genetic transformation.

With reference to Bio-RAD MicroPulser instruction manual book, above-mentioned carrier is swashed conversion method by electricity import Agrobacterium LBA4404.

Above-mentioned plant expression vector imports tobacco by the method for agriculture bacillus mediated leaf disk infection.Concrete grammar is as follows:

Tobacco seed with the sterilization of 1% clorox after, on the MSB solid medium, sprout, culture condition is 25 ℃, photoperiod of 16hr illumination/8hr dark.The aseptic seedling of robust growth promptly can be used as the conversion explant after about one month.

Adopt agriculture bacillus mediated leaf disc transformation method: the agrobacterium strains that contains plant expression vector is inoculated in liquid YEB, and 28 ℃ of 200rpm shaking table overnight incubation are drawn 1～2ml activation once more in 20～25ml liquid YEB again from the bacterium liquid that has shaken.Treating that bacterium liquid shakes to OD600 is about at 0.8 o'clock, take out be diluted to YEB 0.05～0.2 standby.

With the healthy and strong blade of aseptic seedling, be cut into the big leaflet dish of about 0.5cm * 0.5cm, put into the OD600 value and be 0.05～0.2 Agrobacterium bacterium liquid, after 5min is infected in vibration gently, the bacterium liquid that inclines at once inserts the surface with explant and is covered with on the common substratum of one deck filter paper, 24 ℃ of dark 3d that cultivate.

After cultivation was finished altogether, explant inserted in the screening culture medium and carries out differentiation culture, and per 2～3w subculture once.After the green bud of regeneration occurring, its cutting-out changed in the root media take root.

When root growth to the 2～3cm of resistance seedling is long, clean the agar of root, water planting hardening 2～3d is transplanted to nutrition pot, grows under natural condition.

Table 1: Agrobacterium tumefaciens mediated tobacco genetic transformation substratum

MS：Murashige?&?Skoog，1962

B5：Gamborg，1986

(2) evaluation of transformed plant

Histological chemistry identifies: transformed plant is being taken root on the root media and long during to 2～3cm, seedling is taken out clean from culturing bottle, changes water planting hardening 2～3d on the triangular flask over to.Simultaneously, get a fritter blade and a bit of and carry out GUS dyeing.The GUS male directly is transplanted in the nutrition pot by plant.

PCR identifies: treat the tobacco plant transplant survival and grow into a certain size, get the 0.5g blade and extract tobacco gene group DNA, increase with primer D6P1 and the D6P2 (SEQ ID NO.3 and SEQ IDNO.4) of GhDET2.The amplification system of 25 μ L contains 2.5 μ L, 10 * PCR buffer, 2 μ L 2.5mmol/LdNTPs, 1.5 μ L 25mmol/L MgCl2, each 1 μ L primer D6P1 and D6P2 (5 μ mol/L), 1U TaqDNA polysaccharase, 1 μ L tobacco gene group DNA (50ng).Amplification program is: 94 ℃, and 5min; 94 ℃, 30sec, 56 ℃, 30sec, 72 ℃, 60sec, 25 circulations; 72 ℃ are extended 10min.Make positive control with the P6-GhDET2 vector plasmid, make negative control with water and wild-type tobacco genomic dna.

[embodiment four] Detect the expression level of GhDET2 gene in transgene tobacco

Expression with goal gene in the sxemiquantitative RT-PCR methods analyst transfer-gen plant.The GhDET2 gene increases with primer D6P1 and D6P2 (SEQ ID NO.3 and SEQ ID NO.4), the amplification system of 25 μ L contains 2.5 μ L, 10 * PCR buffer, 2 μ L 2.5mmol/L dNTPs, 1.5 μ L 25mmol/L MgCl2, each 1 μ L primer D6P1 and D6P2 (5 μ mol/L), 1U Taq archaeal dna polymerase, 1 μ L cDNA, one chain synthetic product.Amplification program is: 94 ℃, and 5min; 94 ℃, 30sec, 56 ℃, 30sec, 72 ℃, 60sec, 20～25 circulations; 72 ℃ are extended 10min.Do interior mark with the Ubiquitin gene, primer is Ubi-up and Ubi-down.

In order to determine the expression of GhDET2 gene in transgene tobacco, we extract total RNA of transgene tobacco and wild-type tobacco blade.Strand cDNA with these total RNA reverse transcriptions is that template is carried out pcr amplification, and the amplified production of tobacco Ubiquitin special primer Ubi-up and Ubi-down shows that the total RNA amount and the strand cDNA synthetic quality of each sample is similar with concentration.The special primer D6P1 of GhDET2 and D6P2 in transgene tobacco, amplify one with the expection special band of the same size, and in the wild-type tobacco without any amplified production.In order to get rid of the pollution that has genomic dna among total RNA, be that template increases with total RNA simultaneously, without any special band (Fig. 5), illustrate that the GhDET2 gene expresses in the transgene tobacco that detects in the amplified production, certain difference is arranged on expression amount between the different plants.

[embodiment five] The comparison of transgene tobacco and wild-type tobacco root growth and development

1. transgene tobacco and wild-type tobacco T1 are for the comparison of seedling main root growth

Be the influence of research GhDET2 gene pairs root system development, transgene tobacco T0 sows after sterilizing on vertical flat board for seed, regularly main root length.Concrete operation is as follows:

(1) tobacco seed sterilization and rinsing

With transgene tobacco T ₀Seed for seed and wild-type tobacco soaked 8 minutes in 1% aqueous sodium hypochlorite solution, ceaselessly rocked therebetween so that seed-coat fully contacts with thimerosal.Outwell thimerosal then, add an amount of sterile water wash 4 times, each 3-5 minute.

(2) prepare flat board

The preparation agar concentration is 0.8% 1/2MSB substratum, is divided under aseptic condition behind the high-temperature sterilization in the culture dish that diameter is 12cm, and each culture dish is contained the 50ml substratum.Cooled and solidified is stand-by.

(3) sowing and cultivation

The stroke straight line is used at middle part, the back side at each culture dish, so that the tobacco seed marshalling is sealed culture dish in order to avoid the moisture loss in the substratum with sealing film (Parafilm) at last.Culture dish after planting vertically is placed in the fixed support, and makes the straight line maintenance level of drawing.All culture dish all are placed on culturing room and cultivate (28 ℃, illumination condition).

(4) growth conditions observation

After planting, observe seed germination, root and hypocotylar growth every day.Approximately sowing is after 6 days, and the root of tobacco seedling and hypocotyl have grown into certain-length, measure root and hypocotylar length for the first time with ruler.Simultaneously make mark of marking pen, measured root and hypocotylar extended length then every 24 hours in the corresponding position of the tip of a root and cotyledon inserted part.Last statistical study is changeed the tobacco of GhDET2 gene and wild-type tobacco seedling under the same conditions, main root and hypocotylar difference in length and growth velocity difference.

[embodiment six] Transgene tobacco seed germination and Sha Pei experiment

Though sprout the process of growth that tobacco seed helps directly observing root with vertical flat band method, because vertical dull and stereotyped space and the restriction of substratum nutrition and the factor of microbial contamination can't compare for a long time to the elongation situation of root.For the growth length at longer time detecting transgene tobacco root, Sha Peifa has been adopted in this experiment.And observed the sprouting situation of transgene tobacco seed in the presprouting of seeds stage.The concrete operations step is as follows:

(1) presprouting of seeds and germination rate detect

With the vernalization that is soaked in water of an amount of transgene tobacco and wild-type tobacco seed, the transgene tobacco seed is immersed in the water that contains 100mg/L kanamycin, the seed of wild-type tobacco is immersed in the water that does not contain kanamycin, rocks 2d with the speed of 100rpm on the shaking table of plant tissue culture chamber.In this process, randomly draw a quantity of seeds with a big mouthful suction pipe, the statistics seed germination rate.

(2) prepare husky training matrix

Clean up mud in the sand with tap water, the river sand of the equal height of in the foraminous culturing pot of bottom, packing into then, all culturing pots of single test are placed in the tray that can be filled with water.And it is drenched by the river sand in the alms bowl to add water.

(3) sowing and cultivation

The tobacco seed that will show money or valuables one carries unintentionally is broadcast the river sand surface at each alms bowl.Cultivate in the greenhouse, added water once every 5 days therebetween, water can not directly water on the river sand in culturing pot, can only be added in the chassis.

(4) get root and measurement

After tobacco seedling is cultivated 30d, culturing pot is placed in the cardinal principle ponding, jog alms bowl body makes river sand softening loose, then with the complete taking-up of tobacco seedling.Each transgenic line and wild-type tobacco are chosen representational 10 strain seedling and are measured the long and hypocotyl length of its root (Fig. 6-Fig. 8).

[embodiment seven] T1 analyzes for transgene tobacco adventive root growth variability

In order to detect the influence of transforming gene to transgene tobacco adventive root growth, on screening culture medium, cultivate aseptic seedling with the T0 after the sterilization for seed, cut the aseptic seedling stem sections experiment of taking root, observe the growth variability situation of adventive root.Concrete operation is as follows:

(1) sterilization of transgene tobacco seed and cleaning

The tobacco T0 that changes the GhDET2 gene is soaked 8min for the seed of seed and wild-type tobacco in the aqueous sodium hypochlorite solution of 1% (V/V), ceaselessly rock therebetween so that seed-coat fully contacts with thimerosal.Outwell thimerosal then, add an amount of sterile water wash 4 times, each 3～5min.

(2) sowing

The transgene tobacco seed of sterilization is placed on the 1/2MSB substratum that contains 50mg/L kanamycin equably, and the seed of wild-type tobacco is broadcast and is not being contained on the 1/2MSB substratum of kanamycin.Be placed on then in the culturing room, make seed germination.

(3) cutting tobacco seedling stem section takes root

Cut 30d days aseptic tobacco seedling stem section of growth, be inserted on the 1/2MS substratum, observe the generation and the growing state of adventive root.When the adventive root for the treatment of wild-type tobacco stem section grows into to a certain degree, seedling is taken out, the quantity of adventive root on statistics transgene tobacco stem section and the wild-type tobacco stem section, and measure the adventive root length (Fig. 9) of length greater than 5mm.

[embodiment eight] The comparison of transgene tobacco and wild-type tobacco plant height and blade

The transgene tobacco of overexpression GhDET2 gene and wild-type tobacco are planted in the culturing pot simultaneously, through normal cultivation management, and after plant comes to the ripening period, the upgrowth situation (Figure 10) of observation transgene tobacco and wild-type tobacco.

[embodiment nine] The comparison of transgene tobacco and wild-type tobacco fruit and seed development

Overexpression GhDET2 gene can promote nourishing and growing of transgene tobacco.In order to understand fully the influence of GhDET2 to the tobacco reproductive development, we have observed flower organ morphology, fruit and the seed size (Figure 11) of the transgene tobacco of overexpression GhDET2 gene.

SEQUENCE?LISTING

＜110〉Southwestern University

＜120〉purposes of cotton steroid 5 gene and contain its expression vector

<130>09P103582

<160>10

<170>PatentIn?version?3.5

<210>1

<211>899

<212>DNA

<213>Gossypium?hirsutum

<220>

<221>misc_feature

<222>(1)..(899)

＜223〉nucleotide sequence of cotton steroid 5 gene

<400>1

<210>2

<211>258

<212>PRT

＜213〉artificial sequence

<220>

<221>MISC_FEATURE

<222>(1)..(258)

＜223〉cotton steroid 5 gene GhDET2 encoded protein matter sequence

<400>2

<210>3

<211>21

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(21)

＜223〉the special primer D6P1 of amplification GhDET2 gene

<400>3

<210>4

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉the special primer D6P2 of amplification GhDET2 gene

<400>4

<210>5

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉the special primer p6-1 (having the BamHI restriction enzyme site) of amplification CaMV35S promotor

<400>5

<210>6

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉the special primer p6-2 (having the SmaI restriction enzyme site) of amplification CaMV35S promotor

<400>6

<210>7

<211>39

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(39)

＜223〉the special primer p6-3 (having XbaI, BamHI and KpnI restriction enzyme site) of amplification Nos terminator

<400>7

<210>8

<211>27

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(27)

＜223〉the special primer p6-3 (having the BglII restriction enzyme site) of amplification Nos terminator

<400>8

<210>9

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉amplification tobacco Ubiquitin special primer Ubi-up

<400>9

<210>10

<211>20

<212>DNA

＜213〉artificial sequence

<220>

<221>misc_feature

<222>(1)..(20)

＜223〉amplification tobacco Ubiquitin special primer Ubi-down

<400>10

Claims (9)

1, the application of cotton steroid 5 gene GhDET2 in promoting plant root system development, wherein said cotton steroid 5 gene GhDET2 has following nucleotide sequence:

1) nucleotide sequence shown in SEQ ID NO.1; Or

2) nucleotide sequence with SEQ ID NO.1 has 80% above homology, and the proteinic nucleotide sequence of coding identical function.

2, the described application of claim 1, the nucleotide sequence that wherein said cotton steroid 5 gene GhDET2 has with SEQ ID NO.1 has 90% above homology, and the proteinic nucleotide sequence of coding identical function.

3, the application of cotton steroid 5 gene GhDET2 in crop breeding or forest genetics, wherein said cotton steroid 5 gene GhDET2 has following nucleotide sequence:

1) nucleotide sequence shown in SEQ ID NO.1; Or

2) nucleotide sequence with SEQ ID NO.1 has 80% above homology, and the proteinic nucleotide sequence of coding identical function.

4, the described application of claim 3, the nucleotide sequence that wherein said cotton steroid 5 gene GhDET2 has with SEQ ID NO.1 has 90% above homology, and the proteinic nucleotide sequence of coding identical function.

5, the plant expression vector that contains cotton steroid 5 gene GhDET2 and composing type or tissue-specific promoter.

6, plant expression vector according to claim 5, it has structure as shown in Figure 1.

7, plant expression vector according to claim 5, it has structure as shown in Figure 4.

8, a kind of transformant transforms the host with the described plant expression vector of claim 5 and obtains.

9, a kind of preparation method who contains the transgenic plant of cotton steroid 5 gene GhDET2 comprises following step:

1) gene GhDET2 is operably connected with composing type or tissue-specific promoter;

2) make up the plant expression vector that contains gene GhDET2 and constitutive promoter or tissue-specific promoter;

3) transform the host with described plant expression vector, obtain transformant;

4) transform plant with described transformant, obtain transgenic plant.

Images